Septal cholinergic input to CA2 hippocampal region controls social novelty discrimination via nicotinic receptor-mediated disinhibition

Domenico Pimpinella; Valentina Mastrorilli; Corinna Giorgi; Silke Coemans; Salvatore Lecca; Arnaud L Lalive; Hannah Ostermann; Elke C Fuchs; Hannah Monyer; Andrea Mele; Enrico Cherubini; Marilena Griguoli

doi:10.7554/eLife.65580

. 2021 Oct 26;10:e65580. doi: 10.7554/eLife.65580

Septal cholinergic input to CA2 hippocampal region controls social novelty discrimination via nicotinic receptor-mediated disinhibition

Domenico Pimpinella ¹, Valentina Mastrorilli ², Corinna Giorgi ^1,³, Silke Coemans ¹, Salvatore Lecca ⁴, Arnaud L Lalive ⁴, Hannah Ostermann ⁵, Elke C Fuchs ⁵, Hannah Monyer ⁵, Andrea Mele ², Enrico Cherubini ¹, Marilena Griguoli ^1,^6,^✉

Editors: Serena M Dudek⁷, Laura L Colgin⁸

PMCID: PMC8547952 PMID: 34696824

Abstract

Acetylcholine (ACh), released in the hippocampus from fibers originating in the medial septum/diagonal band of Broca (MSDB) complex, is crucial for learning and memory. The CA2 region of the hippocampus has received increasing attention in the context of social memory. However, the contribution of ACh to this process remains unclear. Here, we show that in mice, ACh controls social memory. Specifically, MSDB cholinergic neurons inhibition impairs social novelty discrimination, meaning the propensity of a mouse to interact with a novel rather than a familiar conspecific. This effect is mimicked by a selective antagonist of nicotinic AChRs delivered in CA2. Ex vivo recordings from hippocampal slices provide insight into the underlying mechanism, as activation of nAChRs by nicotine increases the excitatory drive to CA2 principal cells via disinhibition. In line with this observation, optogenetic activation of cholinergic neurons in MSDB increases the firing of CA2 principal cells in vivo. These results point to nAChRs as essential players in social novelty discrimination by controlling inhibition in the CA2 region.

Research organism: Mouse

Introduction

Acting in the brain as a neurotransmitter and a neuromodulator, acetylcholine (ACh) controls neuronal circuits involved in attention, learning, and memory (for review see Hasselmo, 2006). Dysfunction of the cholinergic system contributes to cognitive impairments associated with several neuropsychiatric diseases including neurodevelopmental and neurodegenerative disorders (Deutsch et al., 2016; Hampel et al., 2018; Perez-Lloret and Barrantes, 2016). The medial septum/diagonal band of Broca complex (MSDB) in the basal forebrain provides the major cholinergic innervation to the hippocampus through the fimbria–fornix pathway (for review see Dutar et al., 1995). ACh, released from cholinergic terminals via both wired and volume transmission, targets nicotinic and muscarinic receptors (nAChRs and mAChRs) differently distributed in subcellular domains and cell types across the hippocampal layers (Umbriaco et al., 1995; Sarter et al., 2009). Activation of nAChRs and mAChRs, which relies on local ACh concentration controlled by the hydrolytic action of acetylcholinesterase (Vijayan, 1979), leads to complex effects on neuronal excitability, synaptic plasticity, rhythmic oscillations, brain states, and behavior (for review see Teles-Grilo Ruivo and Mellor, 2013; Dannenberg et al., 2017; Haam and Yakel, 2017).

Recently, the dorsal CA2 and the ventral CA1 hippocampal regions have been associated with social memory, which in rodents can be assessed as the capacity of the animal to discriminate between novel and familiar individuals (Stevenson and Caldwell, 2014; Hitti and Siegelbaum, 2014; Okuyama et al., 2016; Raam et al., 2017; for review see Dudek et al., 2016; Piskorowski and Chevaleyre, 2018). Like other hippocampal subfields, the CA2 receives substantial cholinergic innervation from the MSDB (Cui et al., 2013; Hitti and Siegelbaum, 2014; Kohara et al., 2014); however, the potential role of ACh in social memory has not been explored yet. Here, we report that ChAT⁺ neurons in the MSDB are activated in response to social stimuli, and that selective inhibition of cholinergic neurons in the MSDB or cholinergic fibers in the dorsal CA2 hippocampal region impairs social novelty discrimination. This effect is mimicked by local delivery of nAChRs but not mAChRs blockers, suggesting a key role of ACh in controlling social novelty discrimination, via nAChRs.

Results

ACh released from cholinergic neurons of the MSDB is required for social novelty discrimination

To evaluate the extent of MSDB cholinergic fibers targeting the hippocampus, we injected an AAV-DIO-ChR2-mCherry virus into the MSDB of 1-month-old male mice-expressing Cre recombinase in acetylcholine transferase-positive neurons (ChAT-Cre) (Figure 1A). mCherry was expressed in cells bodies of neurons in the MSDB (Figure 1B) and in cholinergic fibers targeting different hippocampal areas (Figure 1C). Macroscopic inspection and quantitative analysis revealed a high cholinergic fiber density in molecular layer (ML), CA2 and CA3 regions as compared to CA1 and DG (Figure 1D-E).

Figure 1. — (A) Schematic drawing showing the injection site of DIO-AAV-ChR2mCherry delivered in the MSDB of ChAT-Cre mice. (B) Confocal fluorescent image showing ChR2-mCherry expression in ChAT⁺ neurons in the MSDB. (C) Confocal fluorescent image showing the distribution of ChAT⁺ axon fibers, which express ChR2-mCherry and innervate different hippocampal regions. (D) High magnification images showing ChAT⁺ axon fibers in the DG, molecular layer (ML), CA3, CA2, and CA1 regions. (E) Quantification of septal cholinergic axon densities in the DG, ML, CA3, CA2, and CA1 regions (n = 4 animals; four hippocampal slices/animal; DG: 8.73 ± 1.1; ML: 42.3 ± 8.8; CA3: 46.3 ± 5.3; CA2: 48.2 ± 8.6; CA1: 14.6 ± 3.5; p = 0.0006; one-way ANOVA). Open circles are values from single animals and bars are mean ± SEM. *: p < 0.05; **: p < 0.01.

To validate the hypothesis that MSDB ChAT⁺ neurons are activated during social tasks, the expression of the activity-dependent gene c-Fos was assessed by immunofluorescence analysis. Counting of MSDB c-Fos⁺ nuclei was evaluated in home-caged control (HCC) mice or in animals exposed to social interaction (SI) by performing sociability and social novelty discrimination tasks in the three-chamber test (Moy et al., 2004). Minimal c-Fos⁺ labeling was detected in MSDB cells of HCC animals while a strong increase in c-Fos⁺ nuclei, including a subset of ChAT⁺ neurons, was observed 1 h after SI (Figure 2A-C). To understand whether this was triggered by the environment, the expression of c-Fos was measured in MSDB neurons of animals exposed to the empty arena (EA). In this condition, there was also a significant increase in the percentage of MSDB c-Fos⁺ nuclei compared to HCC (Figure 2D-F). However, the stimulation of c-Fos expression induced by the exposure to EA was significantly less than that observed in response to social stimuli (Figure 2G). Furthermore and most interestingly, in the EA condition, none of the c-Fos⁺ cells were ChAT⁺ indicating a selective activation of MSDB cholinergic neurons in response to social stimuli (Figure 2H).

Figure 2. — (**A–F**) Confocal images of MSDB coronal slices, immunolabeled for ChAT (green) and c-Fos (red) to detect behavior-dependent activation of cholinergic neurons. (A) No c-Fos⁺ neurons were detected in the MSDB of home-caged controls (HCCs). (B) c-Fos labeling of the MSDB from animals subjected to the three-chamber test (social interaction, SI) and sacrificed 1 h later, revealed sparse activation of cells including ChAT⁺ neurons. (C) High magnification of insets in (B) showing representative c-Fos⁺ nuclei of cholinergic neurons (white arrows). (D) No c-Fos⁺ neurons were detected in the MSDB of HCCs. (E) c-Fos labeling of the MSDB from animals subjected to three-chamber exploration (empty arena, EA) and sacrificed 1 h later, revealed sparse activation of cells not including ChAT⁺ neurons. (F) High magnification of insets in (E) showing representative c-Fos⁺ nuclei of MSDN ChAT⁻ neurons. (G) Aligned dot plot showing the mean percentage of c-Fos⁺ nuclei detected in the MSDB of HCC, SI, and EA experimental groups (n = 4 animals/group; HCC: 0.72% ± 0.2%; SI: 6.83% ± 0.6%, p < 0.0001; HCC: 0.26% ± 0.1%; EA: 2.73% ± 0.8%, p = 0.02; SI vs EA, p = 0.0004, one-way ANOVA). (H) Aligned dot plot showing the mean percentage of c-Fos⁺ nuclei detected in ChAT⁺ neurons of HCC, SI and EA experimental groups (n = 4 animals/group; HCC: 0.29% ± 0.3%; SI: 18.3% ± 3.9%, p = 0.03; Mann–Whitney test). Open circles are values from single animals and bars are mean ± SEM. *: p < 0.05; ***: p < 0.001; ****: p < 0.0001.

Figure 2—source data 1. Raw data of c-Fos quantification in the MSDB.

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To assess whether endogenous ACh released from MSDB nuclei is involved in the animal’s ability to discriminate between novel and familiar individuals, multiple strategies were used. Firstly, we sought to inactivate ChAT⁺ neurons in the MSDB. To this end, the MSDB of ChAT-Cre mice was injected stereotactically with an AAV carrying a floxed eYFP, as control, or the Tetanus toxin light chain fused to GFP (TeNT). TeNT contains a zinc-endopeptidase domain that selectively cleaves VAMP/synaptobrevin on synaptic vesicles, hence blocking the neuroexocytosis process (Schiavo et al., 1992; Rossetto et al., 1995). This approach allows to block the release of ACh from cholinergic terminals. TeNT was efficiently expressed by ChAT⁺ neurons in the MSDB (Figure 3—figure supplement 1A). Patch clamp recordings from MSDB ChAT⁺ neurons in acute slices allowed to investigate the effect of TeNT expression on intrinsic membrane properties (Figure 3—figure supplement 1B-D). As shown in the figure, all ChAT⁺ neurons were spontaneously active. No significant differences in the resting membrane potential (V_m) and in the spontaneous firing frequency between eYFP- and TeNT-expressing neurons were observed (Figure 3—figure supplement 1B-C). In addition, as already reported in cultured neurons (Dimpfel, 1979), TeNT-expressing neurons exhibited a significant reduction in their spike half-width and in their input resistance (R_in) (Figure 3—figure supplement 1D).

Mice-expressing eYFP or TeNT were subjected to the three-chamber test for both sociability and social novelty evaluation (Figure 3A-D). During the sociability task, TeNT-expressing mice were comparable to eYFP mice and showed a preference for the animal over the object (Figure 3A-B). To evaluate social novelty discrimination, after 1 h the mouse was subjected to a social novelty task consisting in the exposition to a previously encountered familiar mouse or a novel one. TeNT mice did not show the typical preference for the novel subject compared to the familiar one, as was the case in the control group (Figure 3C-D), indicating that the release of ACh is required for social novelty discrimination.

Figure 3—figure supplement 1. — (A) Top: schematic illustration of the sociability task in the three-chamber test. Bottom: representative heat map showing the time spent by an eYFP (left) or a TeNT (right) mouse in exploring the animal (left) and the object (right). (B) Left: aligned dot plot showing interaction time spent to explore the animal and the object during sociability task in eYFP (control, green, n = 9) and TeNT mice (orange, n = 11) (eYFP: 187 ± 12 vs 121 ± 7.7 s, p = 0.018; TeNT: 197 ± 20 vs 127 ± 12 s, p = 0.005; one-way ANOVA). Right: aligned dot plot showing the sociability score in eYFP (control, green, n = 9) and TeNT mice (orange, n = 11) (eYFP: 66.8 ± 15 s; TeNT: 70.5 ± 21 s, p = 0.94; Mann–Whitney test). (C) Top: schematic illustration of the social novelty task in the three-chamber test. Bottom: representative heat map showing the time spent by an eYFP (left) or a TeNT (right) mouse in exploring the novel animal (left) and the familiar one (right). (D) Left: aligned dot plot showing interaction time spent to explore the novel and the familiar animal in the social novelty task in eYFP (control, green, n = 9) and TeNT (orange, n = 11) mice (eYFP: 145 ± 10 vs 81.2 ± 10 s, p = 0.002; TeNT: 79.8 ± 13 vs 101 ± 10 s, p = 0.47; one-way ANOVA). Right: aligned dot plot showing the social novelty score in eYFP (control, green, n = 9) and TeNT (orange, n = 11) mice (eYFP: 64.1 ± 12 s; TeNT: −21.7 ± 5.8 s, p < 0.0001; Mann–Whitney test). (E) Top: schematic illustration of the novel object recognition (NOR) test. Bottom: representative heat map showing the time spent by an eYFP (left) or a TeNT (right) mouse in exploring the novel (left) and the familiar (right) object. (F) Left: aligned dot plot showing the exploration time spent to explore the familiar and the novel object during NOR task in eYFP (control, green, n = 11) and TeNT (orange, n = 12) mice (eYFP: 76.6 ± 8.8 vs 39 ± 3.6 s, p = 0.03; TeNT: 99.9 ± 16 vs 37.6 ± 9.8 s, p = 0.0002; one-way ANOVA). Right: aligned dot plot showing the exploration score in eYFP (control, green, n = 11) and TeNT (orange, n = 12) mice (eYFP: 37.6 ± 9.8 s; TeNT: 62.7 ± 17 s, p = 0.38; Mann–Whitney test). Open circles are values from single animals and bars are mean ± SEM. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 3—source data 1. Interaction times and scores for three-chamber and novel object recognition tests.

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To investigate whether this effect was selective for social recognition, TeNT and control mice were subjected to a general learning and memory test, named novel object recognition (NOR). During NOR, the test animal was first exposed to two identical objects. After 1 h, one object was replaced by a novel one (Figure 3E). TeNT mice did not show any impairment in the preference for the novel object as compared to eYFP group (Figure 3F). Thus, the NOR task is not dependent on MSDB ChAT⁺ neurons.

To assess whether the TeNT-induced impairment in social novelty discrimination was associated with deficits in motor behavior and anxiety, mice carrying eYFP or TeNT were tested in the open-field paradigm (Figure 3—figure supplement 2A). No differences in distance traveled, speed, or time spent in the outer or inner zone were observed between eYFP and TeNT mice (Figure 3—figure supplement 2B-D). Moreover, no difference in distance traveled or speed was found between the two groups when analyzed during the execution of the social novelty task in the three-chamber apparatus (Figure 3—figure supplement 2E-F).

TeNT is persistently expressed for months after stereotaxic injection (Kaspar et al., 2002), thus inducing a long lasting block of ACh release from MSDB cholinergic neurons that could account for the observed effect on social novelty task. To rule out this hypothesis and to gain better temporal control of cholinergic inhibition, we sought to corroborate the results of the three-chamber test using the DREADD tool. Thus, an AAV carrying the floxed sequence of the human muscarinic receptor subtype four fused to the mCherry florescent protein (hM4) was injected in the MSDB. hM4 was engineered to be selectively activated by N-clozapine (CNO), a pharmacologically inert metabolite of the antipsychotic drug clozapine, inducing a hyperpolarization of the neuronal membrane via activation of a G_i-mediated potassium conductance (for review see Sternson and Roth, 2014). This effect relies on the time of CNO availability in the brain that lasts up to 200 min, depending on the concentration used and on the receptor desensitization rate (for review see Roth, 2016). hM4 was efficiently expressed by ChAT⁺ neurons in the MSDB (Figure 4—figure supplement 1A). Patch clamp recordings from hM4-expressing neurons in acute slices allowed to evaluate the effect of bath application of CNO (10 µM) on membrane potential and spontaneous firing frequency of ChAT⁺ neurons. CNO application hyperpolarized the membrane potential and reduced the firing frequency (Figure 4—figure supplement 1B-C). It was shown that in the absence of hM4, this CNO concentration does not alter the firing and membrane properties of hippocampal neurons (Zhu et al., 2014).

When tested for sociability in the three-chamber paradigm, hM4 mice exhibited a preference for the animal over the object as was the case for the control group (Figure 4A-B). As control group, we used eYFP mice treated with CNO to exclude possible side effects of this drug on social behavior that might result from its conversion into the psychoactive drug clozapine (MacLaren et al., 2016; Gomez et al., 2017). Before social novelty evaluation (30 min), CNO (3 mg/kg, dissolved in saline, for review see Sternson and Roth, 2014) was injected intraperitoneally (i.p.) in both eYFP and hM4 mice. This CNO concentration is effective in activating hM4 in mice without inducing unspecific behavioral effects due to clozapine conversion (Jendryka et al., 2019). Following CNO treatment, hM4 mice did not show the typical preference for the novel mouse compared to the familiar one as observed in the control group (Figure 4C-D). These data corroborate the results obtained with TeNT, overall indicating that ChAT⁺ neurons in the MSDB participate to, and are required for, social novelty discrimination.

Figure 4. — (A) Top: schematic illustration of the sociability task in the three-chamber test. Bottom: representative heat map showing the time spent by an eYFP (left) or a hM4 (right) mouse in exploring the animal (left) and the object (right). (B) Left: aligned dot plot showing interaction time spent to explore the animal and the object during sociability task in eYFP (control, green, n = 12) and hM4 mice (orange, n = 13) (eYFP: 144 ± 12 vs 99.1 ± 7.8 s, p = 0.02; hM4: 160 ± 13 vs 109 ± 8.2 s, p = 0.006; one-way ANOVA). Right: aligned dot plot showing the sociability score in eYFP (control, green, n = 12) and hM4 mice (orange, n = 13) (eYFP: 45 ± 12 s; hM4: 51 ± 15 s, p = 0.69; Mann–Whitney test). (C) Top: schematic illustration of the social novelty task in the three-chamber test performed 30 min after i.p. injection of CNO (3 mg/kg). Bottom: representative heat map showing the time spent by an eYFP (left) or a hM4 (right) mouse in exploring the novel (left) and the familiar (right) mouse. (D) Left: aligned dot plot showing interaction time spent to explore the novel and the familiar animal in the social novelty task in eYFP (control, green, n = 12) and hM4 (orange, n = 13) mice (eYFP: 75.5 ± 7.3 vs 46.8 ± 5.8 s, p = 0.01; hM4: 71.5 ± 11 vs 71.4 ± 5.4 s, p = 0.99; one-way ANOVA). Right: aligned dot plot showing the social novelty score in eYFP (control, green, n = 12) and hM4 (orange, n = 13) mice (eYFP: 28.7 ± 8.2 s; hM4: 0.15 ± 12 s, p = 0.03; Mann–Whitney test). (E) Confocal images showing (from left to right): Pcp4 (CA2 marker, in green), c-Fos (in red), nuclear-staining DAPI (blue), and merge images of CA2 hippocampal neurons. One hour after social novelty test, mice-expressing eYFP or hM4 in MSDB ChAT⁺ neurons were sacrificed. The behavioral-dependent (social interaction, SI) activation of CA2 (detected by c-Fos immunostaining) was observed in eYFP mice but not in hM4 mice. Data were compared with those obtained from home-caged controls (HCCs). (F) Aligned dot plot showing the average percentage of c-Fos⁺ nuclei detected in the CA2 region in the experimental groups (n = 3–4 animals/group; HCC: 4.9% ± 0.4%; SI: 12.2% ± 2.3%; hM4: 5.02% ± 0.7%; p = 0.015, one-way ANOVA); open circles are values from single animals and bars are mean ± SEM. *:p < 0.05; ***: p < 0.001.

Figure 4—source data 1. Interaction times and scores for three-chamber and c-Fos quantification in CA2.

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Figure 4—figure supplement 1. — (A) Top: schematic illustration of the sociability task in the three-chamber test. Bottom: representative heat map showing the time spent by an eYFP (left) or a hM4 (right) mouse in exploring the animal (left) and the object (right). (B) Left: aligned dot plot showing interaction time spent to explore the animal and the object during sociability task in eYFP (control, green, n = 12) and hM4 mice (orange, n = 13) (eYFP: 144 ± 12 vs 99.1 ± 7.8 s, p = 0.02; hM4: 160 ± 13 vs 109 ± 8.2 s, p = 0.006; one-way ANOVA). Right: aligned dot plot showing the sociability score in eYFP (control, green, n = 12) and hM4 mice (orange, n = 13) (eYFP: 45 ± 12 s; hM4: 51 ± 15 s, p = 0.69; Mann–Whitney test). (C) Top: schematic illustration of the social novelty task in the three-chamber test performed 30 min after i.p. injection of CNO (3 mg/kg). Bottom: representative heat map showing the time spent by an eYFP (left) or a hM4 (right) mouse in exploring the novel (left) and the familiar (right) mouse. (D) Left: aligned dot plot showing interaction time spent to explore the novel and the familiar animal in the social novelty task in eYFP (control, green, n = 12) and hM4 (orange, n = 13) mice (eYFP: 75.5 ± 7.3 vs 46.8 ± 5.8 s, p = 0.01; hM4: 71.5 ± 11 vs 71.4 ± 5.4 s, p = 0.99; one-way ANOVA). Right: aligned dot plot showing the social novelty score in eYFP (control, green, n = 12) and hM4 (orange, n = 13) mice (eYFP: 28.7 ± 8.2 s; hM4: 0.15 ± 12 s, p = 0.03; Mann–Whitney test). (E) Confocal images showing (from left to right): Pcp4 (CA2 marker, in green), c-Fos (in red), nuclear-staining DAPI (blue), and merge images of CA2 hippocampal neurons. One hour after social novelty test, mice-expressing eYFP or hM4 in MSDB ChAT⁺ neurons were sacrificed. The behavioral-dependent (social interaction, SI) activation of CA2 (detected by c-Fos immunostaining) was observed in eYFP mice but not in hM4 mice. Data were compared with those obtained from home-caged controls (HCCs). (F) Aligned dot plot showing the average percentage of c-Fos⁺ nuclei detected in the CA2 region in the experimental groups (n = 3–4 animals/group; HCC: 4.9% ± 0.4%; SI: 12.2% ± 2.3%; hM4: 5.02% ± 0.7%; p = 0.015, one-way ANOVA); open circles are values from single animals and bars are mean ± SEM. *:p < 0.05; ***: p < 0.001.

Figure 4—source data 1. Interaction times and scores for three-chamber and c-Fos quantification in CA2.

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We next sought to investigate whether social behavior-dependent activation of hippocampal CA2 neurons is affected by MSDB cholinergic inhibition. To this aim, animals expressing either control eYFP or hM4 were subjected to the three-chamber test and treated with CNO 30 min before the social novelty task. One hour later, animals were sacrificed and brain slices encompassing the CA2 region were immunolabeled for c-Fos. As shown in Figure 4E-F, social behavior induced activation of CA2 hippocampal neurons in eYFP control animals but not in hM4 mice. Interestingly, the majority of c-Fos⁺ neurons were PCP4⁻ (Figure 4—figure supplement 2A-B) suggesting a restricted activation of a subpopulation of PCP4⁻ principal neurons or local GABAergic interneurons. Immunolabeling with parvalbumin (PV), the marker of a subpopulation of GABAergic interneurons, revealed that the vast majority of c-Fos⁺ neurons was PV⁻ (Figure 4—figure supplement 2A-C).

To exclude the possibility that c-Fos activation was induced merely by the animals exploring the environment, we analyzed c-Fos expression in the CA2 region of animals exploring EA. We did not observe a significant difference between HCC and EA conditions (Figure 4—figure supplement 3A-B).

We next evaluated whether CNO-mediated inhibition of c-Fos expression in the CA2 of hM4 group that underwent SI extended to the neighboring CA3 and CA1 regions. No significant changes in c-Fos activation were observed in either CA3 or CA1 in mice of the three experimental groups (Figure 4—figure supplement 4A-D). Altogether, these data reveal that MSDB cholinergic neuron activation is elicited by social recognition tasks and is required for the downstream activation of the CA2 hippocampal region.

To assess whether cholinergic signaling is specifically relevant to social novelty discrimination, as compared to other forms of hippocampal-dependent cognitive tasks such as spatial novelty, we performed the object location test (OLT). During OLT, the test animal was first exposed to two identical objects. After 1 h, the animal was tested in the same arena with one of the two objects relocated to a new position (Figure 4—figure supplement 5A). hM4 and control eYFP animals received CNO i.p. injection 30 min before the test phase. hM4 mice showed a reduced interaction time with the object in the novel location as compared to the eYFP group (Figure 4—figure supplement 5B). These results suggest that the inhibition of MSDB cholinergic neurons also affects spatial novelty.

Local release of ACh in the CA2 hippocampal region is necessary for social novelty discrimination

It is known that cholinergic projections from MSDB nuclei target other brain areas such as the prefrontal cortex, the olfactory bulb, and the entorhinal cortex (Li et al., 2018; Desikan et al., 2018), which may also be involved in social novelty control. To test whether ACh release in CA2 is sufficient to control social novelty discrimination, CNO (100 µM) was locally applied through a cannula (Figure 5A) stereotactically implanted into the CA2 region. According to previous studies, the concentration of CNO for in vivo local application was tenfold higher than that used in slice recordings, which was effective in inducing neuronal silencing (Meira et al., 2018). Both eYFP (n = 15) and hM4 (n = 14) animal groups showed a preference for the animal rather than the object (Figure 5B). CNO was administered in the CA2 region 30 min before the social novelty test. In contrast to the control group (eYFP) hM4 animals did not show any significant difference in interaction time spent with the novel and the familiar mice, indicating that local release of ACh in the CA2 is necessary for social novelty discrimination (Figure 5C).

Figure 5. — (A) Schematic representations of cannula placements in the dorsal hippocampus of eYFP (control, green, n = 15) and hM4 (orange, n = 14) mice. (B) Left: aligned dot plots showing interaction time spent to explore the animal and the object during sociability task in eYFP (control, green, n = 15) and hM4 (orange, n = 14) mice (eYFP: 185 ± 10 vs 85.3 ± 7.3 s, p < 0.0001; hM4: 207 ± 17 vs 85 ± 3.7 s, p < 0.0001; one-way ANOVA). Right: aligned dot plot showing the sociability score in eYFP (control, green, n = 15) and hM4 (orange, n = 14) mice (eYFP: 99.4 ± 12 s; hM4: 122 ± 17 s, p = 0.27; Mann–Whitney test). (C) Left: aligned dot plots showing interaction time spent to explore the novel and the familiar animal in the social novelty task in eYFP (control, green, n = 15) and hM4 (orange, n = 14) mice following CNO (100 µM) delivery to the CA2 area of the hippocampus (30 min before social novelty task) (eYFP: 53 ± 6.6 vs 100 ± 14 s, p = 0.0009; hM4: 82.2 ± 10 vs 90.9 ± 13 s, p = 0.9; one-way ANOVA). Right: aligned dot plot showing the social novelty score in eYFP (control, green, n = 15) and hM4 (orange, n = 14) mice (eYFP: 47.4 ± 8.7 s; hM4: 8.7. ± 11 s, p = 0.3; Mann–Whitney test). Open circles are values from single animals and bars are mean ± SEM. **: p < 0.01; ***p < 0.001; ****: p < 0.0001.

Figure 5—source data 1. Interaction times and scores for three-chamber test.

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Figure 5—figure supplement 1. — (A) Schematic representations of cannula placements in the dorsal hippocampus of eYFP (control, green, n = 15) and hM4 (orange, n = 14) mice. (B) Left: aligned dot plots showing interaction time spent to explore the animal and the object during sociability task in eYFP (control, green, n = 15) and hM4 (orange, n = 14) mice (eYFP: 185 ± 10 vs 85.3 ± 7.3 s, p < 0.0001; hM4: 207 ± 17 vs 85 ± 3.7 s, p < 0.0001; one-way ANOVA). Right: aligned dot plot showing the sociability score in eYFP (control, green, n = 15) and hM4 (orange, n = 14) mice (eYFP: 99.4 ± 12 s; hM4: 122 ± 17 s, p = 0.27; Mann–Whitney test). (C) Left: aligned dot plots showing interaction time spent to explore the novel and the familiar animal in the social novelty task in eYFP (control, green, n = 15) and hM4 (orange, n = 14) mice following CNO (100 µM) delivery to the CA2 area of the hippocampus (30 min before social novelty task) (eYFP: 53 ± 6.6 vs 100 ± 14 s, p = 0.0009; hM4: 82.2 ± 10 vs 90.9 ± 13 s, p = 0.9; one-way ANOVA). Right: aligned dot plot showing the social novelty score in eYFP (control, green, n = 15) and hM4 (orange, n = 14) mice (eYFP: 47.4 ± 8.7 s; hM4: 8.7. ± 11 s, p = 0.3; Mann–Whitney test). Open circles are values from single animals and bars are mean ± SEM. **: p < 0.01; ***p < 0.001; ****: p < 0.0001.

Figure 5—source data 1. Interaction times and scores for three-chamber test.

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To unveil the mechanism by which ACh released in the hippocampus controls CA2 circuits, we performed whole-cell patch clamp experiments from CA2 pyramidal cells in hippocampal slices obtained from naive and ChAT-Cre mice-expressing hM4 in the MSDB. Firstly, we characterized the firing and membrane properties of the CA2 principal neurons. As shown in Figure 5—figure supplement 1, values similar to those previously published (Chevaleyre and Siegelbaum, 2010) were detected. Hence, pharmacologically isolated spontaneous inhibitory and excitatory postsynaptic currents (sIPSCs and sEPSCs) were recorded before and after CNO (10 μM) application in the presence of CNQX (10 µM), gabazine (10 µM), and physostigmine (3 μM) to block AMPA, GABA-A receptors, and acetylcholinesterase, respectively.

In slices obtained from naive mice not expressing hM4, bath application of CNO (10 μM) had no effect on either frequency or amplitude of sIPSCs or sEPSCs (Figure 5—figure supplement 2A-D). In contrast, in slices obtained from hM4 mice, CNO decreased the frequency, but not the amplitude of sIPSCs (Figure 5—figure supplement 2E-F). Furthermore, CNO decreased both the frequency and the amplitude of sEPSCs (Figure 5—figure supplement 2G-H). Possible off-target effects of CNO could be excluded since CNO differently affected the frequency and amplitude of synaptic currents recorded from naive and hM4 animals (Figure 5—figure supplement 2I-J). These data clearly demonstrate that ACh controls synaptic transmission in CA2.

ACh controls social novelty discrimination via nAChR activation in CA2 neurons

Evidence has emerged that in the hippocampus and cortex ACh and GABA can be released from ChAT⁺ fibers (Takács et al., 2018; Saunders et al., 2015; Desikan et al., 2018). To test whether ACh is sufficient to control social novelty discrimination, we selectively blocked ACh receptors locally. Specifically, nAChRs antagonist dihydro-β-erythroidine (DHβE) 50 mM or saline was administered in the CA2 area of the hippocampus in ChAT-Cre mice 30 min before the social novelty task (Figure 6A-C). The two groups of mice did not show differences in the sociability task (Figure 6B). However, when compared to saline-treated animals, DHβE-treated mice were impaired in the social novelty task (Figure 6C), indicating that nicotinic receptors are crucial for social novelty discrimination. In another set of experiments, mAChR antagonist atropine (1 mM) or saline was administered using the same paradigm of delivery (Figure 6D-F). Both groups of mice did not show differences in the sociability (Figure 6E) and social novelty tasks (Figure 6F), indicating that muscarinic receptor activation is not required for social novelty discrimination.

Figure 6. — (A) Schematic representations of cannula placements in the dorsal hippocampus of mice receiving a solution containing saline (control, green, n = 10) or dihydro-β-erythroidine (DHβE) (50 mM, orange, n = 11). (B) Left: aligned dot plots showing interaction time spent to explore the animal and the object during sociability task in the two groups of mice (green, n = 10 and orange, n = 11) (green: 126 ± 8 vs 87.3 ± 6.3 s, p = 0.03; orange: 128 ± 13 vs 79 ± 9 s, p = 0.005; one-way ANOVA). Right: aligned dot plot showing the sociability score in the two groups of mice (green, n = 10 and orange, n = 11) (green: 38.7 ± 7.3 s; orange: 48.8 ± 12 s, P = 0.6; Mann–Whitney test). (C) Left: aligned dot plot showing interaction time spent to explore the novel and the familiar animal in the social novelty task in saline- (control, green, n = 10) and DHβE-treated (orange, n = 11) mice (saline: 93.2 ± 14 vs 54.2 ± 7.7 s, p = 0.05; DHβE: 51.7 ± 10 vs 70.3 ± 7.5 s, p = 0.54; one-way ANOVA). Right: aligned dot plot showing the social novelty score saline- (control, green, n = 10) and DHβE-treated (orange, n = 11) mice (saline: 39 ± 15 s; DHβE: –18.6 ± 13 s, p = 0.016; Mann–Whitney test). (D) Schematic representations of cannula placements in mice that received saline solution (control, green, n = 8) or atropine (1 mM, orange, n = 8). (E) Left: aligned dot plot showing interaction time spent to explore the animal and the object during sociability task in the two groups of mice (green, n = 8 and orange, n = 8) (green: 159 ± 5.2 vs 97.3 ± 8.9 s, p = 0.0003; orange: 167 ± 14 vs 92.4 ± 8 s, p < 0.0001; one-way ANOVA). Right: aligned dot plot showing the sociability score in the two groups of mice (green, n = 8 and orange, n = 8) (green: 61.7 ± 11 s; orange: 74.4 ± 14 s, p > 0.99; Mann–Whitney test). (F) Left: aligned dot plot showing interaction time spent to explore the novel and the familiar animal in the social novelty task in saline- (control, green, n = 8) and atropine-treated (orange, n = 8) mice (saline: 97.9 ± 5.9 vs 57.4 ± 7.04 s, p = 0.009; atropine: 102 ± 11 vs 60.6 ± 7.6 s, p = 0.007; one-way ANOVA). Right: aligned dot plot showing the social novelty score in saline- (control, green, n = 8) and atropine-treated (orange, n = 8) mice (saline: 40.5 ± 10 s; atropine: 41.5 ± 9 s, p = 0.96; Mann–Whitney test). Open circles are values from single animals and bars are mean ± SEM. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 6—source data 1. Interaction times and scores for three-chamber test.

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Figure 6—figure supplement 1. — (A) Schematic representations of cannula placements in the dorsal hippocampus of mice receiving a solution containing saline (control, green, n = 10) or dihydro-β-erythroidine (DHβE) (50 mM, orange, n = 11). (B) Left: aligned dot plots showing interaction time spent to explore the animal and the object during sociability task in the two groups of mice (green, n = 10 and orange, n = 11) (green: 126 ± 8 vs 87.3 ± 6.3 s, p = 0.03; orange: 128 ± 13 vs 79 ± 9 s, p = 0.005; one-way ANOVA). Right: aligned dot plot showing the sociability score in the two groups of mice (green, n = 10 and orange, n = 11) (green: 38.7 ± 7.3 s; orange: 48.8 ± 12 s, P = 0.6; Mann–Whitney test). (C) Left: aligned dot plot showing interaction time spent to explore the novel and the familiar animal in the social novelty task in saline- (control, green, n = 10) and DHβE-treated (orange, n = 11) mice (saline: 93.2 ± 14 vs 54.2 ± 7.7 s, p = 0.05; DHβE: 51.7 ± 10 vs 70.3 ± 7.5 s, p = 0.54; one-way ANOVA). Right: aligned dot plot showing the social novelty score saline- (control, green, n = 10) and DHβE-treated (orange, n = 11) mice (saline: 39 ± 15 s; DHβE: –18.6 ± 13 s, p = 0.016; Mann–Whitney test). (D) Schematic representations of cannula placements in mice that received saline solution (control, green, n = 8) or atropine (1 mM, orange, n = 8). (E) Left: aligned dot plot showing interaction time spent to explore the animal and the object during sociability task in the two groups of mice (green, n = 8 and orange, n = 8) (green: 159 ± 5.2 vs 97.3 ± 8.9 s, p = 0.0003; orange: 167 ± 14 vs 92.4 ± 8 s, p < 0.0001; one-way ANOVA). Right: aligned dot plot showing the sociability score in the two groups of mice (green, n = 8 and orange, n = 8) (green: 61.7 ± 11 s; orange: 74.4 ± 14 s, p > 0.99; Mann–Whitney test). (F) Left: aligned dot plot showing interaction time spent to explore the novel and the familiar animal in the social novelty task in saline- (control, green, n = 8) and atropine-treated (orange, n = 8) mice (saline: 97.9 ± 5.9 vs 57.4 ± 7.04 s, p = 0.009; atropine: 102 ± 11 vs 60.6 ± 7.6 s, p = 0.007; one-way ANOVA). Right: aligned dot plot showing the social novelty score in saline- (control, green, n = 8) and atropine-treated (orange, n = 8) mice (saline: 40.5 ± 10 s; atropine: 41.5 ± 9 s, p = 0.96; Mann–Whitney test). Open circles are values from single animals and bars are mean ± SEM. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 6—source data 1. Interaction times and scores for three-chamber test.

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Analysis of locomotor activity in the three-chamber apparatus did not reveal any changes between eYFP and hM4 mice that received CNO injection (both i.p. and locally; Figure 6—figure supplement 1A-B), or between pharmacologically treated mice (i.e. DHβE and atropine, Figure 6—figure supplement 1C-D), excluding possible off-target effects of the drugs that were used.

nAChR-mediated increase of glutamatergic transmission in CA2 principal cells via disinhibition

To understand how nicotinic ACh receptor activation impacts synaptic transmission in the CA2, we recorded spontaneous miniature inhibitory and excitatory postsynaptic currents (mIPSCs and mEPSCs) from CA2 principal neurons in acute hippocampal slices in the presence of tetrodotoxin (1 μM) to block sodium channels and action potentials propagation, CNQX (10 µM) or gabazine (10 µM) to block AMPA or GABA-A receptors, respectively (Figure 7—figure supplement 1A-D). Bath application of nicotine (1 μM) induced a significant decrease in the frequency of mIPSCs without affecting their amplitude (Figure 7—figure supplement 1A-B). No effect was observed in either frequency or amplitude of mEPSCs (Figure 7—figure supplement 1C-D), indicating that nAChR activation controls mainly GABAergic transmission in the CA2 region. These data are in agreement with previous studies showing a preferential expression of nAChRs by GABAergic interneurons (for review see Griguoli and Cherubini, 2012; Pancotti and Topolnik, 2021). To assess whether nicotine could indirectly control the activity of excitatory cells by acting on GABAergic interneurons, we recorded spontaneous action potential-dependent excitatory postsynaptic currents (sEPSCs) at the reversal potential of Cl⁻ (E_Cl⁻ = −65 mV) without blocking GABAergic-mediated synaptic transmission. Under this condition, nicotine significantly increased the frequency of sEPSCs without affecting their amplitude (Figure 7A-B). In 6 out of 13 cells a partial recovery of the control frequency was obtained during nicotine washout (frequency: Nic: 1.45 ± 0.3 Hz, washout: 0.9 ± 0.1 Hz; p = 0.03; Wilcoxon test), ruling out a possible desensitizing effect of nicotine. These results suggest that nAChRs activation augments the glutamatergic drive to CA2 principal cells via disinhibition.

Figure 7. — (A) Sample traces showing spontaneous excitatory postsynaptic currents (sEPSCs) of a CA2 pyramidal neuron recorded at E_Cl⁻ in control (black) and in the presence of nicotine (1 μM; orange). (B) Aligned dot plots showing the mean frequency (left) and amplitude (right) of sEPSCs recorded from CA2 pyramidal neurons in control (black) and in the presence of nicotine (1 μM; orange) (n = 13 cells; frequency: control: 0.81 ± 0.1 Hz, Nic: 1.5 ± 0.2 Hz; p = 0.0005; amplitude: control: 19.9 ± 1.4 pA, Nic: 19.7 ± 0.8 pA; p = 0.76; Wilcoxon test). (C) Sample traces showing sEPSCs of a CA2 pyramidal neuron recorded at E_Cl⁻ in the presence of α7 nAChR antagonist methyllycaconitine (MLA, (10 nM; black)) and in the presence of MLA plus nicotine (1 μM; orange). (D) Aligned dot plots showing the mean frequency (left) and amplitude (right) of sEPSCs recorded from CA2 pyramidal neurons in the presence of MLA (10 nM; black) and in the presence of MLA plus nicotine (1 μM; orange) (n = 9 cells; frequency: MLA: 1.9 ± 0.3 Hz, MLA+ Nic: 1.7 ± 0.2 Hz; p = 0.34; amplitude: MLA: 15.9 ± 1.1 pA, MLA+ Nic: 17.3 ± 1.0 pA; p = 0.36; Wilcoxon test). (E) Sample traces showing sEPSCs of a CA2 pyramidal neuron recorded at E_Cl⁻ in the presence of non-α7 nAChR antagonist dihydro-β-erythroidine (DHβE, 0.5 μM; black) and in the presence of DHβE plus nicotine (1 μM; orange). (F) Aligned dot plots showing the mean frequency (left) and amplitude (right) of sEPSCs recorded from CA2 pyramidal neurons in the presence of DHβE (0.5 μM; black) or DHβE plus nicotine (1 μM; orange) (n = 9 cells; frequency: DHβE: 0.9 ± 0.1 Hz, DHβE + Nic: 1.05 ± 0.3 Hz; p > 0.99; amplitude: DHβE: 16.3 ± 1.5 pA, DHβE + Nic: 18.2 ± 1.2 pA; p = 0.25; Wilcoxon test). Open or closed circles represent values from single cells. Laterally located circles represent mean ± SEM. ***: p < 0.001.

Figure 7—source data 1. Frequency and amplitude of spontaneous excitatory postsynaptic currents .

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Figure 7—figure supplement 1. — (A) Sample traces showing spontaneous excitatory postsynaptic currents (sEPSCs) of a CA2 pyramidal neuron recorded at E_Cl⁻ in control (black) and in the presence of nicotine (1 μM; orange). (B) Aligned dot plots showing the mean frequency (left) and amplitude (right) of sEPSCs recorded from CA2 pyramidal neurons in control (black) and in the presence of nicotine (1 μM; orange) (n = 13 cells; frequency: control: 0.81 ± 0.1 Hz, Nic: 1.5 ± 0.2 Hz; p = 0.0005; amplitude: control: 19.9 ± 1.4 pA, Nic: 19.7 ± 0.8 pA; p = 0.76; Wilcoxon test). (C) Sample traces showing sEPSCs of a CA2 pyramidal neuron recorded at E_Cl⁻ in the presence of α7 nAChR antagonist methyllycaconitine (MLA, (10 nM; black)) and in the presence of MLA plus nicotine (1 μM; orange). (D) Aligned dot plots showing the mean frequency (left) and amplitude (right) of sEPSCs recorded from CA2 pyramidal neurons in the presence of MLA (10 nM; black) and in the presence of MLA plus nicotine (1 μM; orange) (n = 9 cells; frequency: MLA: 1.9 ± 0.3 Hz, MLA+ Nic: 1.7 ± 0.2 Hz; p = 0.34; amplitude: MLA: 15.9 ± 1.1 pA, MLA+ Nic: 17.3 ± 1.0 pA; p = 0.36; Wilcoxon test). (E) Sample traces showing sEPSCs of a CA2 pyramidal neuron recorded at E_Cl⁻ in the presence of non-α7 nAChR antagonist dihydro-β-erythroidine (DHβE, 0.5 μM; black) and in the presence of DHβE plus nicotine (1 μM; orange). (F) Aligned dot plots showing the mean frequency (left) and amplitude (right) of sEPSCs recorded from CA2 pyramidal neurons in the presence of DHβE (0.5 μM; black) or DHβE plus nicotine (1 μM; orange) (n = 9 cells; frequency: DHβE: 0.9 ± 0.1 Hz, DHβE + Nic: 1.05 ± 0.3 Hz; p > 0.99; amplitude: DHβE: 16.3 ± 1.5 pA, DHβE + Nic: 18.2 ± 1.2 pA; p = 0.25; Wilcoxon test). Open or closed circles represent values from single cells. Laterally located circles represent mean ± SEM. ***: p < 0.001.

Figure 7—source data 1. Frequency and amplitude of spontaneous excitatory postsynaptic currents .

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To identify the nAChR subtypes responsible for nicotine-induced disinhibition, we repeated the experiments in the presence of methyllycaconitine (MLA, 10 nM) or DHβE (0,5 μM) which, at these concentrations selectively block α7- and non-α7-nAChR subtypes, respectively. In the presence of either MLA or DHβE nicotine did not change the frequency of sEPSCs (Figure 7C-F), indicating that both α7- and non-α7-nAChR subtypes are involved in nicotine-induced CA2 disinhibition. To elucidate whether the effect of nicotine was due to nicotinic modulation of neighboring hippocampal areas, we recorded sEPSCs from CA3 and CA1 principal neurons. In contrast to what observed in CA2, in both CA3 and CA1 regions, nicotine significantly reduced the frequency of sEPSCs recorded at E_Cl⁻ without blocking GABAergic transmission (Figure 7—figure supplement 2A-D). We then performed additional experiments to manipulate endogenous release of ACh via activation of the excitatory DREADD hM3, which leads to membrane depolarization when activated by CNO (Alexander et al., 2009). Patch clamp recordings from hM3-expressing neurons (Figure 8—figure supplement 1A) in acute slices allowed to evaluate the effect of CNO (10 µM) bath application on membrane potential and spontaneous firing of ChAT⁺ neurons. CNO application depolarized the membrane and increased the frequency of spontaneous action potentials (Figure 8—figure supplement 1B-C). We then evaluated the effect of CNO application on sEPSCs recorded from CA2 principal neurons in the presence of physostigmine (3 μM) and atropine (1 μM) to block the acetylcholinesterase and muscarinic receptors, respectively. In these conditions CNO increased the frequency, but not the amplitude of sEPSCs confirming the results obtained with nicotine (Figure 8A-B). In addition, as observed with nicotine, CNO decreased the frequency and the amplitude of sEPSCs recorded from CA3 principal neurons (Figure 8C-D). In contrast with results obtained using nicotine, CNO did not affect either the frequency or the amplitude of sEPSCs recorded from CA1 principal neurons (Figure 8E-F). This could be explained by a smaller number of cholinergic terminals expressing hM4 in CA1, as compared to CA2 and CA3 regions.

Figure 8. — (A) Sample traces showing spontaneous excitatory postsynaptic currents (sEPSCs) of a CA2 pyramidal neuron recorded at E_Cl⁻ in control (black) and in the presence of clozapine N-oxide (CNO, 10 μM; orange). (B) Aligned dot plots showing the mean frequency (left) and amplitude (right) of sEPSCs recorded from CA2 pyramidal neurons in control (black) and in the presence of CNO (10 μM; orange) (n = 8 cells; frequency: control: 0.98 ± 0.2 Hz, CNO: 1.3 ± 0.3 Hz; p = 0.02; amplitude: control: 17.7 ± 0.8 pA, CNO: 17.7 ± 1.3 pA; p = 0.94; Wilcoxon test). (C) Sample traces showing sEPSCs of a CA3 pyramidal neuron recorded at E_Cl⁻ in control (black) and in the presence of CNO (10 μM; orange). (D) Aligned dot plots showing the mean frequency (left) and amplitude (right) of sEPSCs recorded from CA3 pyramidal neurons in control (black) and in the presence of CNO (10 μM; orange) (n = 11 cells; frequency: control: 4.7 ± 0.8 Hz, CNO: 2.5 ± 0.3 Hz; p = 0.002; amplitude: control: 23.6 ± 1.2 pA, CNO: 21.7 ± 1.2 pA; p = 0.03; Wilcoxon test). (E) Sample traces showing sEPSCs of a CA1 pyramidal neuron recorded at E_Cl⁻ in control (black) and in the presence of CNO (10 μM; orange). (F) Aligned dot plots showing the mean frequency (left) and amplitude (right) of sEPSCs recorded from CA1 pyramidal neurons in control (black) and in the presence of CNO (10 μM; orange) (n = 12 cells; frequency: control: 1.36 ± 0.4 Hz, CNO: 1.3 ± 0.4 Hz; p = 0.35; amplitude: control: 19.4 ± 1.1 pA, CNO: 18.6 ± 0.9 pA; p = 0.2; Wilcoxon test). Open or closed circles represent values from single cells. Laterally located circles represent mean ± SEM. *: p < 0.05; **: p < 0.01.

Figure 8—source data 1. Frequency and amplitude of spontaneous excitatory postsynaptic currents.

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Figure 8—figure supplement 1. — (A) Sample traces showing spontaneous excitatory postsynaptic currents (sEPSCs) of a CA2 pyramidal neuron recorded at E_Cl⁻ in control (black) and in the presence of clozapine N-oxide (CNO, 10 μM; orange). (B) Aligned dot plots showing the mean frequency (left) and amplitude (right) of sEPSCs recorded from CA2 pyramidal neurons in control (black) and in the presence of CNO (10 μM; orange) (n = 8 cells; frequency: control: 0.98 ± 0.2 Hz, CNO: 1.3 ± 0.3 Hz; p = 0.02; amplitude: control: 17.7 ± 0.8 pA, CNO: 17.7 ± 1.3 pA; p = 0.94; Wilcoxon test). (C) Sample traces showing sEPSCs of a CA3 pyramidal neuron recorded at E_Cl⁻ in control (black) and in the presence of CNO (10 μM; orange). (D) Aligned dot plots showing the mean frequency (left) and amplitude (right) of sEPSCs recorded from CA3 pyramidal neurons in control (black) and in the presence of CNO (10 μM; orange) (n = 11 cells; frequency: control: 4.7 ± 0.8 Hz, CNO: 2.5 ± 0.3 Hz; p = 0.002; amplitude: control: 23.6 ± 1.2 pA, CNO: 21.7 ± 1.2 pA; p = 0.03; Wilcoxon test). (E) Sample traces showing sEPSCs of a CA1 pyramidal neuron recorded at E_Cl⁻ in control (black) and in the presence of CNO (10 μM; orange). (F) Aligned dot plots showing the mean frequency (left) and amplitude (right) of sEPSCs recorded from CA1 pyramidal neurons in control (black) and in the presence of CNO (10 μM; orange) (n = 12 cells; frequency: control: 1.36 ± 0.4 Hz, CNO: 1.3 ± 0.4 Hz; p = 0.35; amplitude: control: 19.4 ± 1.1 pA, CNO: 18.6 ± 0.9 pA; p = 0.2; Wilcoxon test). Open or closed circles represent values from single cells. Laterally located circles represent mean ± SEM. *: p < 0.05; **: p < 0.01.

Figure 8—source data 1. Frequency and amplitude of spontaneous excitatory postsynaptic currents.

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Optogenetic activation of ChAT⁺ neurons in the MSDB increases the firing of CA2 neurons in vivo

To study the effects of endogenously released ACh on CA2 principal cells output in vivo, juxtacellular recordings were performed from neurons in the CA2 region of anesthetized (tiletamine/zolazepam-xylazine) ChAT-Cre mice, expressing ChR2-eYFP in the MSDB (Figure 9A). Principal cells were identified based on their bursting behavior (Csicsvari et al., 1999; Ding et al., 2020; Figure 9B-C). Brief light pulses delivered at low frequency (5 ms duration, at 1 Hz for 30 s) to cholinergic neurons in the MSDB by an optical fiber, increased the firing rate of CA2 neurons (Figure 9B-D) without altering the intraburst frequency (Figure 9D). This suggests that ACh release during light-induced ChR2 activation strongly affects the output of CA2 principal neurons. No effect was observed when light was delivered to not injected mice (Figure 9—figure supplement 1A-D). These data were independent from the anesthetic used, as a similar effect was observed when the animals were anesthetized with ketamine–xylazine (Figure 9—figure supplement 1E-H).

Figure 9. — (A) Schematic illustration showing the experimental settings of in vivo juxtacellular recordings combined with light stimulation of MSDB ChAT⁺ neurons expressing channelrhodopsin (ChR2). (B) Representative trace showing spontaneous firing from a CA2 bursting neuron in control (black) and during ChR2 activation (blue) via light pulses (below the trace). (C) Individual bursts in (B) (asterisk and hashtag for control and light activation, respectively) shown on an expanded time scale. (D) Left: aligned dot plot showing the frequency of spikes in control (black) and during light activation of ChAT⁺ neurons in the MSDB (blue) (n = 9 cells; control: 3.62 ± 1.0 Hz; light: 4.26 ± 0.97 Hz; p = 0.012, Wilcoxon test); right: aligned dot plot showing the frequency of spikes within the bursts in control and during light activation of ChAT⁺ neurons in the MSDB (n = 9 cells; control: 118 ± 19 Hz; light: 114 ± 20 Hz; p = 0.36, Wilcoxon test). Open circles represent values from single cells. Closed circles represent mean ± SEM. **: p = 0.01.

Figure 9—source data 1. Spike analysis.

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Figure 9—figure supplement 1. — (A) Schematic illustration showing the experimental settings of in vivo juxtacellular recordings combined with light stimulation of MSDB ChAT⁺ neurons expressing channelrhodopsin (ChR2). (B) Representative trace showing spontaneous firing from a CA2 bursting neuron in control (black) and during ChR2 activation (blue) via light pulses (below the trace). (C) Individual bursts in (B) (asterisk and hashtag for control and light activation, respectively) shown on an expanded time scale. (D) Left: aligned dot plot showing the frequency of spikes in control (black) and during light activation of ChAT⁺ neurons in the MSDB (blue) (n = 9 cells; control: 3.62 ± 1.0 Hz; light: 4.26 ± 0.97 Hz; p = 0.012, Wilcoxon test); right: aligned dot plot showing the frequency of spikes within the bursts in control and during light activation of ChAT⁺ neurons in the MSDB (n = 9 cells; control: 118 ± 19 Hz; light: 114 ± 20 Hz; p = 0.36, Wilcoxon test). Open circles represent values from single cells. Closed circles represent mean ± SEM. **: p = 0.01.

Figure 9—source data 1. Spike analysis.

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Additional experiments were performed to study the effect of MSDB light stimulation on both CA3 and CA1 regions. Photostimulation of ChAT⁺ neurons in the MSDB induced no significant change in the firing frequency of both CA3 and CA1 bursting cells (Figure 9—figure supplement 2A-H).

In line with slices recordings, these in vivo data suggest that ACh released from MSDB enhances CA2 principal cells firing via disinhibition, by activating nAChRs localized on GABAergic interneurons.

Discussion

Here, we show that cholinergic inputs from the MSDB support social memory mediated by the CA2 region of the hippocampus. Among social areas in the brain, the CA2 has recently emerged as a key structure for social cognition (for review see Dudek et al., 2016; Piskorowski and Chevaleyre, 2018). Hitti and Siegelbaum, 2014 clearly demonstrated that genetically targeted inactivation of CA2 principal cells leads to a loss of social memory, namely the ability of an animal to recognize a conspecific. More recently, evidence was provided that consolidation of social memory strictly depends on reactivation of CA2 pyramidal cell ensembles during sharp-wave ripples (Oliva et al., 2020). However, despite the increasing information regarding the circuits that are involved, the underlying mechanisms are still largely unknown. Among possible candidates taking part in social cognition, cholinergic signaling may play a key role. This signaling pathway is known to be involved in several cognitive processes (for review see Ballinger et al., 2016; Solari and Hangya, 2018), and deficits in cholinergic transmission are associated with cognitive impairments in various forms of neuropsychiatric disorders (for review see Dineley et al., 2015; Terry and Callahan, 2020). Our results indicate that social memory requires the activation of cholinergic neurons in the MSDB. In particular, c-Fos immunolabeling revealed a selective activation of ChAT⁺ neurons in response to social stimuli. Furthermore, the inhibition of cholinergic neurons in the MSDB with TeNT severely impaired social novelty discrimination, indicating that ACh is involved in this task. Notably, this effect was not related to the novelty per se since TeNT-expressing mice did not show an impairment in the NOR test. This result is in agreement with findings demonstrating that an increased level of ACh in the hippocampus during object exploration was not related to object familiarity or novelty (Stanley et al., 2012). Furthermore, ACh released in the perirhinal cortex seems to play a major role in the novel object discrimination task by acting via mAChRs (Winters et al., 2006; Balderas et al., 2012) rather than nAChRs (Tinsley et al., 2011).

Social memory impairment induced by TeNT was also observed in hM4-expressing mice treated with CNO, known to block the firing of targeted neurons via membrane hyperpolarization. These results strongly support the role of ACh in social novelty discrimination. However, ACh seems not to play an exclusive role in social novelty discrimination as hM4 mice, subjected to OLT, also showed an impairment in spatial novelty discrimination. This result is in agreement with a previous study, whereby ChAT⁺ neurons in the MSDB were selectively eliminated using a genetic cell targeting technique (Okada et al., 2015). Deficits in OLT were also observed in mice lacking the M2 mAChR subtype suggesting the involvement of these receptors in this task (Romberg et al., 2018).

We focused on the role of cholinergic signaling in the CA2 region that is key to social memory formation and is highly innervated by cholinergic fibers originating from ChAT⁺ cells in the MSDB. Analysis of c-Fos expression following social behavior unveiled an increased learning-dependent activation of neurons in the CA2 region, which was reduced upon hM4-dependent inhibition of MSDB cholinergic neurons. Previous studies (Wintzer et al., 2014; Alexander et al., 2016) demonstrated that exposure to a novel context elicits learning-dependent transcription of the immediate-early gene Arc in the CA2 region of rodents. In our experiments, animals exposed to the three-chamber arena in the absence of social stimuli did not show a significant increase in c-Fos expression in the CA2 region. However, we cannot state that the increase in c-Fos staining in CA2 is selective for social stimuli since a similar response to a novel object has not been assessed (Alexander et al., 2016).

The notion that social memory depends on MSDB cholinergic inputs to the CA2 region relies on evidence that local CNO-triggered inhibition of cholinergic fibers significantly impaired this task. Although the volume of drugs injected locally in CA2 was very small, the possibility of spillover to adjacent CA3 and CA1 areas cannot be excluded. Hence, this finding warrants further support that ideally would be based on another experimental approach. Furthermore, we provided evidence that social memory requires the activation of nicotinic, but not muscarinic AChRs. Previous work suggested the involvement of nAChRs in social interactions. In particular, the lack of β2 nAChR subtype was shown to affect social interaction during aggressive behavior (Granon et al., 2003), an effect likely involving the medial prefrontal cortex (mPFC; Avale et al., 2011). The lack of β4 nAChR subunit that is highly expressed in the olfactory bulb and in the lateral habenula (Salas et al., 2003), leads to a decreased interaction between a resident and juvenile intruder mouse (Salas et al., 2013). Furthermore, a single nucleotide polymorphism in the α5 subunit of nAChRs, observed in patients affected by schizophrenia (Schizophrenia Working Group of the Psychiatric Genomics Consortium, 2014), leads to deficits in sociability when engineered in mice (Koukouli et al., 2017).

The effect of nAChRs activation on glutamatergic and GABAergic terminals impinging on CA2 pyramidal cells is not known. Our ex vivo patch clamp recordings of miniature synaptic currents from CA2 principal cells in hippocampal slices did not unveil any effects of nicotine on spontaneous glutamatergic events in comparison to GABAergic ones, suggesting that ACh receptors are preferentially expressed on GABAergic interneurons. nAChR-mediated modulation seems to be region specific since the activation of nAChRs increases GABA release in CA1 (Rosato-Siri et al., 2006; Tang et al., 2011) that may account for the observed reduction in the frequency of sEPSCs. Similarly, in CA3 principal cells, activation of nAChRs by low concentration of nicotine enhances GABA release either directly (Hajós et al., 2005) or indirectly, via NMDA receptors localized on GABAergic interneurons (Mann and Mody, 2010; Wang et al., 2015). Furthermore, previous studies showed that brain states associated with high cholinergic activity and theta oscillations resulted in CA3 principal neuron inhibition via activation of interneurons (Malezieux et al., 2020; Dannenberg et al., 2015). Thus, enhanced GABAergic tone may lead, as in CA1, to a reduced glutamate release from CA3 principal cells. Our results on nicotine-mediated control of glutamatergic transmission in CA2, CA3, and CA1 regions were confirmed by ACh released from hM3-expressing hippocampal fibers, supporting a circuit-specific regulation of CA2 by nAChRs.

The inhibitory effect of CNO on spontaneous EPSCs and IPSCs in mice-expressing hM4 in MSDB ChAT⁺ neurons may be related to its action on both nicotinic and muscarinic receptors, the latter known to be present on CA2 neurons (Robert et al., 2020).

In our experiments, the decrease of GABAergic transmission induced by nicotine in CA2 was associated with an indirect increase in frequency of spontaneous glutamatergic events, recorded at the equilibrium potential for chloride, suggesting a disinhibitory mechanism. Interestingly a disinhibitory effect of ACh was also observed in the prelimbic area of mPFC, where α5 nAChR-dependent activation of VIP⁺ interneurons inhibited downstream SOM⁺ cells, which in turn led to an enhancement of principal neuron firing in layer 2/3 (Koukouli et al., 2017). In accordance with results based on slice recordings, our in vivo experiments showed that optogenetic activation of MSDB ChAT⁺ neurons enhanced the firing frequency of CA2 pyramidal neurons identified by their bursting behavior (Csicsvari et al., 1999; Ding et al., 2020), indicating that ACh strongly controls the CA2 output. This seems to be selective for the CA2 region, since optogenetic activation of MSDB ChAT⁺ neuron did not modify the firing frequency of neighboring CA3 and CA1 putative principal neurons. The lack of an effect in CA3 and CA1 may be related to the concomitant activation of mAChRs, probably differently distributed among hippocampal areas. Altogether, our results are commensurate with a scenario in which ACh controls social memory through nAChR activation by decreasing GABAergic signaling and thus enhancing the excitatory drive to CA2 principal cells. Recordings from GABAergic interneurons will be critical to identify how different subtypes contribute to social memory via nAChR-mediated disinhibition.

Materials and methods

Animals

All experiments were performed in accordance with the Italian Animal Welfare legislation (D.L. 26/2014) that were implemented by the European Committee Council Directive (2010/63 EEC) and were approved by local veterinary authorities, the EBRI ethical committee and the Italian Ministry of Health (565/PR18). All efforts were made to minimize animal suffering and to reduce the number of animals used. At least four to five male mice were used for a given experiment. We used B6;129S6-Chat tm2(cre)Lowl/J (ChAT-Cre), purchased from Jackson Laboratory (Stock No: 006410). Experiments were performed on male off-spring derived from homozygous mating. Only male mice were used in this study to limit the effects of estrous cycle on cholinergic signaling (Gibbs, 1996). Mice were housed in 4–5 per cage at constant temperature (22°C) and humidity (30–50%) and were kept on a regular circadian cycle (12 h:12 h light:dark cycle, lights on at 7:00 a.m.). Mice were provided with food and water ad libitum.

Viruses

Adeno-associated virus (AAV) containing Tetanus toxin light chain [AAV-DJ CMV DIO eGFP-2A-TeNT] was purchased from Stanford University Gene Vector and Virus core (CA, USA) with genomic titer of 1.4 × 10¹³ particles/ml. AAV serotype 2/9 containing channelrhodopsin-2 [ChR2; AAV-DIO-ChR2(H134R)-enhanced yellow fluorescent protein (eYFP) or -mCherry] or AAV-Ef1a-DIO eYFP with genomic titers of 1.49 × 10¹³ and 3.95 × 10¹³ particles/ml, respectively, AAV serotype 2/8 containing human muscarinic receptor 3 and 4 DREADD [hM3D(Gq) and hM4D(Gi); pAAV-hSyn-DIO-hM3D(Gq)-mCherry and pAAV-hSyn-DIO-hM4D(Gi)-mCherry] with genomic titers of 4.0 × 10¹² particles/ml and 1.4 × 10¹³ particles/ml were purchased from Addgene (MA, USA).

In vivo stereotactic injections

ChAT-Cre mice (postnatal (P) day P25–30; weight 16–20 g) were anesthetized with an intraperitoneal injection (i.p.) of mixture of tiletamine/zolazepam (zoletyl, 80 mg/kg body weight) and xylazine (rompun, 10 mg/kg body weight). Viral vectors were injected in the MSDB (anteroposterior [AP], 0.8 mm; mediolateral [ML], 0.5 mm; dorsoventral [DV], −4.5 mm; all coordinates were relative to Bregma) through a 26 G needle lowered at an angle of 6.5° (in the ML axis) relative to the vertical plane in order to avoid sagittal sinus (Boyce et al., 2016). The injection volume and flow rate (500 nl at 60 nl/min) were controlled with an injection pump (UMP3 UltraMicroPump, World Precision Instruments, USA). After a minimum of 4 weeks that allowed for protein expression, mice were used for experiments.

Cannula implantation for local drug delivery

Guide cannulas were placed at specific coordinates for targeting the dorsal hippocampal CA2. To this aim, a small hole was drilled bilaterally following the coordinates: AP = −1.6 mm, ML = ±1.7 mm, DV = −1.3 mm (all coordinates were relative to Bregma). Bilateral guide cannulas (7 mm length, 0.5 mm outer diameter with 0.25 mm inner diameter; Unimed, Switzerland) were slowly lowered into the brain through the holes until the target DV coordinate was reached. Quickly, the cannulas were fixed with acrylic dental cement (Riccardo Ilic, Italy) to be stably held on the calvarium at the DV coordinate established. Mice were let to recover from surgery for 1 week.

Drug and injection procedure

Clozapine N-oxide (CNO, 100 µM), DHβE (50 mM) or atropine (1 mM), all dissolved in saline, were injected bilaterally (150 nl/hemisphere, 75 nl/min injection rate) in the dorsal hippocampus (CA2) 30 min before the social novelty test. To this end, mice were gently restrained to insert one of the guide cannulas, the injection needle (length, 7.6 mm; diameter, 0.25 mm) connected with a plastic tube to a 2 μl Hamilton syringe (Hamilton 7002 N – G 0.5/70 mm/pts2; Hamilton Company, USA). The needle was left in place for an additional 1 min to allow diffusion of the preparation. Immediately after, the injection was repeated in the other hemisphere using the same procedure. During the injections, mice were awake and free to move in the holding cage. CNO injected in mice-expressing control virus (eYFP) was used as control, ACSF was used as vehicle in the experiments with DHβE and atropine.

Behavioral experiments

Three-chamber test

Sociability and social novelty skills were tested using the three-chamber test, adapted from Moy et al., 2004 in a homemade rectangular, clear Plexiglas three-chambered box (each chamber was 20 × 40 × 21 cm in size). Dividing walls included rectangular openings (6 × 8.5 cm) allowing access to each chamber. The light intensity (6 lux) was distributed equally in the apparatus. Between trials, the chambers of the arena were cleaned with 70% ethanol (EtOH) to eliminate lingering smells. Mice were handled 5 min a day for 5 days before the test. On the day before the test, mice were habituated to the empty chamber for 30 min. On the test day, after a 10 min habituation phase in the empty apparatus, during the first two trials (sociability task) the test mouse was placed in the middle compartment and allowed to explore for 10 min between a wire cup (ø 10.5 cm × 10.5 cm h) with an unfamiliar juvenile (P30) C57BL7/6J male mouse (stranger one) and an identical empty wire cup. The position of stranger one was alternated between the first and second trial, to prevent side preference. The interaction time was recorded by the video-tracking system (ANY-maze, StoeltingCo, IL, USA), and the score was calculated as the difference between the investigation time for the novel mouse and that for the empty cup. After a 1 h intersession interval a second unfamiliar C57BL7/6J male mouse (stranger two or novel) was placed into the previously empty wire cup, while stranger one (familiar) remained inside its cup. The subject mouse was given 10 min to explore all three chambers. The score for social novelty was calculated as the difference between the investigation time for the novel mouse and that for the familiar mouse.

Open-field exploration test

Open field was used to test general locomotor activity, anxiety, and willingness to explore. The experimental apparatus consisted of a black rectangular open field (60 × 60 × 30 cm; Panlab, USA). The arena was cleaned with 70% EtOH between trials to eliminate lingering smells. During the test, each animal was placed in the center of the arena and allowed to freely move for 10 min while being recorded with an overhead camera. The mouse activity was then analyzed by an automated tracking system (ANY-maze, StoeltingCo, IL, USA) for the following parameters: total ambulatory distance and velocity. A series of 12 × 12 cm zones were identified and used to evaluate the time spent in the center (inner zone) or peripheral zones (outer zone). The outer zone consisted of 12 blocks close to walls while the inner zone consisted of 9 blocks in the center. Greater time spent in the outer zones of the maze was indicative of amplified anxiety-related behavior (Seibenhener and Wooten, 2015).

NOR test

The NOR test was slightly adapted as described in Leger et al., 2013. The experimental apparatus consisted of a black rectangular open field (60 × 60 × 30 cm; Panlab, USA). Prior to training, mice were handled for 5 min a day for 5 days. On the day before the test, mice were placed in the empty chamber and allowed to explore for 30 min. During the training phase mice were placed in the experimental apparatus in the presence of two identical objects and were allowed to explore for 10 min. After 1 h, mice were placed again for 10 min in the apparatus, where one of the objects had been replaced by a novel one. The objects consisted of two plastic boxes with different shapes, both approximately of the same height. In both tasks, the arena was cleaned with 70% EtOH to eliminate lingering smells. The interaction time with the familiar object and the novel one was recorded by the video-tracking system (ANY-maze, Stoelting Co, IL, USA) and manually analyzed.

Object location test

Animals were tested by using OLT adapted from Denninger et al., 2018. This test is based on the spontaneous tendency of rodents to recognize when an object has been relocated. The experimental apparatus and the objects were the same used in the NOR task. Visual cues were inserted into the arena to help mice in spatial orientation. Prior to training, mice were handled for 5 min a day for 5 days. The day before the test, mice were placed in the empty chamber, and allowed to explore for 30 min. During the training phase mice were placed in the experimental apparatus in the presence of two identical objects and allowed to explore for 10 min. After 1 h, the location of one object was changed and the animal was free to explore the two objects for 10 min. In both tasks, the arena was cleaned with 70% EtOH to eliminate lingering smells. The interaction time with the familiar location and the novel location was recorded by the video-tracking system (ANY-maze, Stoelting Co, IL, USA) and manually analyzed.

Slice preparation

Transverse hippocampal slices (320 μm tick) were obtained from P60–P70-old animals, using a standard protocol (Bischofberger et al., 2006). Briefly, after being anesthetized with an intraperitoneal injection of a mixture of tiletamine/zolazepam (zoletyl, 80 mg/kg body weight) and xylazine (rompun, 10 mg/kg body weight), mice were decapitated. The brain was quickly removed from the skull, placed in artificial cerebrospinal fluid (ACSF) containing (in mM): sucrose 75, NaCl 87, KCl 2.5, NaH₂PO₄ 1.25, MgCl₂ 7, CaCl₂ 0.5, NaHCO₃ 25, and glucose 25. After recovery, an individual slice was transferred to a submerged recording chamber and continuously perfused at room temperature with oxygenated ACSF at a rate of 3 ml/min. ASCF saturated with 95% O2 and 5% CO2 and contained in mM: NaCl 125, KCl 2.5, NaH₂PO₄ 1.25, MgCl₂ 1, CaCl₂ 2, NaHCO₃ 25, and glucose 10.

Electrophysiological recordings in slices

Cells were visualized with a 60 × water immersed objective mounted on an upright microscope (Nikon, eclipse FN1) equipped with a CCD camera (Scientifica, UK). Whole-cell patch clamp recordings, in voltage and current clamp modes, were performed with a MultiClamp 700B amplifier (Axon Instruments, Sunnyvale, CA, USA). Patch electrodes were pulled from borosilicate glass capillaries (WPI, Florida, US); they had a resistance of 3–4 MΩ when filled with an intracellular solution containing (in mM): K gluconate 70, KCl 70, HEPES 10, EGTA 0.2, MgCl₂ 2, MgATP 4, MgGTP 0.3, Na-phosphocreatine 5; the pH was adjusted to 7.2 with KOH; the osmolarity was 295–300 mOsm. Membrane potential values were not corrected for liquid junction potentials. Miniature GABA_A-mediated inhibitory postsynaptic currents (mIPSCs) and AMPA-mediated excitatory postsynaptic currents (mEPSCs) were recorded in the CA2 region of the hippocampus from a holding potential of −70 mV in the presence of tetrodotoxin (TTX 1 μM) and 1 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM) or gabazine (10 μM), respectively. In patch clamp experiments performed to record spontaneous AMPA-mediated excitatory postsynaptic currents (sEPSCs) the electrodes were filled with an intracellular solution containing: K gluconate 127, KCl 6, HEPES 10, EGTA 1, MgCl₂ 2, MgATP 4, MgGTP 0.3; the pH was adjusted to 7.2 with KOH; the osmolarity was 290–300 mOsm. sEPSCs were recorded at the equilibrium potential for chloride (E_Cl⁻) that was approximately −65 mV based on the Nernst equation. Membrane potential values were not corrected for liquid junction potentials. The stability of the patch was checked by repetitively monitoring the input and series resistance during the experiments. Series resistance (10–20 MΩ) was not compensated. Cells exhibiting 15% changes were excluded from the analysis. Drugs were applied in the bath and the ratio of flow rate to bath volume ensured complete exchange within 2–3 min.

Data analysis

Data were transferred to a computer hard disk after digitization with an A/D converter (Digidata 1550, Molecular Devices, Sunnyvale, CA, USA). Data acquisition (digitized at 10 kHz and filtered at 3 kHz) was performed with pClamp 10.4 software (Molecular Devices, Sunnyvale, CA, USA). Input resistance and cells capacitance were measured online with the membrane test feature of the pClamp software. Spontaneous and miniature EPSCs and IPSCs were analyzed with pClamp 10.4 (Molecular Devices, Sunnyvale, CA, USA). This program uses a detection algorithm based on a sliding template. The template did not induce any bias in the sampling of events because it was moved along the data trace by one point at a time and was optimally scaled to fit the data at each position.

Drugs

Drugs were applied in the bath by gravity by changing the superfusion solution to one differing only in its content of drug(s). The following drugs were used: CNQX, DL-APV, and picrotoxin purchased from Tocris (UK), nicotine (SML1236), atropine, and DHβE purchased from Sigma (USA), CNO purchased from Abcam (UK), physostigmine hemisulfate purchased from Santa Cruz (USA). Stock solutions were made in distilled water and then aliquoted and frozen at −20 °C. Picrotoxin and CNQX were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in the bathing solution was 0.1%. At this concentration, DMSO alone did not modify the membrane potential, input resistance, or the firing properties of CA2 neurons.

Electrophysiological recordings in vivo

Mice (P60–70) were anesthetized with i.p. injection of a mixture of tiletamine/zolazepam (zoletyl; 80 mg/kg) and xylazine (rompun, 10 mg/kg body weight ) to induce anesthesia before surgery and during recordings. A subset of animals was injected with i.p. injection of a mixture of ketamine (lobotor; 100 mg/kg body weight) and xylazine (rompun, 10 mg/kg body weight). Temperature was maintained between 36°C and 37°C using a feedback-controlled heating pad. Two craniotomies for optogenetic stimulation (MSDB) and for recording (CA2) sites were drilled at +0.8 mm AP and +0.5 L for MSDB and −1.6 mm AP and +1.7 L for CA2 all relative to Bregma. Extracellular mapping allowed to locate the depth of CA2 pyramidal cell layer. Extracellular recordings of field potentials for activity mapping were obtained with glass electrodes (Hingelberg, Malsfeld, Germany) prepared with a vertical puller PP-830 (Narishige, Japan), and the tip was broken to obtain a resistance between 1 and 2 MΩ. Electrodes were filled with a standard Ringer’s solution containing the following (in mM): NaCl 135, KCl 5.4, HEPES 5, CaCl₂ 1.8, and MgCl₂ 1.

Juxtacellular recordings of spontaneous neuronal firing were obtained using glass electrodes (7–10 MΩ) filled with potassium-based solution. Activation of ChAT⁺ neurons in the MSDB was achieved with a 50 mW, 473 nm laser (NovaPro Lasersytems, Germany) delivered through an optic fiber (200 µm, 0.22 numerical aperture) lowered into the MSDB. Light power measured at the tip of the fiber outside the brain was 3–4 mW. Pulses of blue light (5 ms) at 1 Hz (30 stimuli) were externally triggered using pClamp (Molecular Devices). Recordings were obtained with a Multiclamp 700B amplifier connected to the Digidata 1550 system. Data were acquired with pClamp 10 (Molecular Devices, Sunnyvale, CA, USA), digitized at 10 kHz, filtered at 3 kHz, and analyzed off-line with Clampfit 10.4 (Molecular Devices, Sunnyvale, CA, USA). Traces were high pass filtered (300 Hz) and events were detected using a threshold search function in clampfit. A burst was defined as a sequence of two or more action potentials occurring at >20 Hz (Zucca et al., 2017). A clampfit algorithm was used to detect bursts from the total events. The interval for burst detections ranged from 20 to 60 ms.

Tissue preparation for immunohistochemistry

Tissue preparation and immunohistochemistry procedures were performed as previously described (Modi et al., 2019). Mice (aged P60–70) were anesthetized with i.p. injection of a mixture of tiletamine/zolazepam (zoletyl; 80 mg/kg body weight) and xylazine (rompun, 10 mg/kg body weight) and perfused transcardially with ice-cold oxygenated ACSF (pH 7.4) for 2 min (Notter et al., 2014). Brains were rapidly dissected and fixed for 48 h in 4% paraformaldehyde phosphate-buffered saline (PBS) solution (Santa Cruz, USA). After rinsing in PBS, brains were incubated with 30% (wt/vol) sucrose in PBS at 4 °C overnight, frozen with dry ice-cold isopentane and stored at −80 °C. Brains were embedded in the OCT compound (Leica, Germany) and sectioned by cryostat (Leica CM1850 UV, Germany).

Histological verification of the cannula placements

Coronal brain sections (90-μm thick) were cut with a freezing microtome (Leica Microsystem, Germany). Serial slices were collected on gelatinized slides and stained with Cresyl Violet (Sigma-Aldrich, Italy). For the staining, slices were kept for 2 or 3 min in a 0.5% Cresyl Violet solution in distillated water. Immediately after, cleaning and dehydrating steps followed in this order: 1 min in distillated water for two times; 1 min in 50% EtOH, 1 min in 75% EtOH, 1 min in 100% EtOH, and 1 min in xylene substitute (Sigma-Aldrich, USA). Immediately after, slices were covered with cover slices and limonene (Sigma-Aldrich, Italy) as mounting medium. With the use of a stereomicroscope (Zeiss), the position of cannulas and injectors was verified and the most ventral point of the placement left by the injector during the administration was identified. Only animals with correct placements (within 350 µm from the CA2 border to CA1) were included in the statistical analysis. Illustration of coronal sections from animals were then represented for each pharmacology experiment.

Immunohistochemistry

Free-floating coronal sections (60-μm thick) were rinsed in PBS 1×, permeabilized for 2 h at room temperature in blocking solution (1× DPBS; 0.3% Triton X-100; 5% normal donkey serum) and incubated overnight at 4 °C with combined primary antibodies in blocking solution. Antibody dilutions were as follows: anti-c-Fos (mouse, 1:150; Santa Cruz C-10, sc271243); anti-PCP4 (rabbit, 1:200; SIGMA, HPA005792); anti-ChAT (goat, 1:200; Millipore, AB144P); anti-PV (guinea pig, 1:1000; ImmunoStar, 24428). Following three 10 min washes in 1× DPBS/0.1% Triton X-100, slices were incubated with the appropriate donkey-raised secondary antibodies (1:500 dilution; Alexa Fluor, Thermo Fisher) for 2 h at room temperature, washed twice in 1× DPBS/0.1% Triton X-100 for 20 min, once in 1× DPBS/ DAPI for 20 min and then mounted with Aqua-Poly/Mount (Polysciences cat: 18606).

Image acquisition

Z-stack images (16 optical sections, 0.4 µm step size) were recorded of all specimens using a spinning disk (X-Light V2, Crest Optics) microscope Olympus IX73 equipped with a LED light source (Spectra X light Engine, Lumencore, USA) and an Optimos camera (QImaging, Canada). Images were acquired using a ×40 or ×20 objective with numerical aperture of 1.35 and 1.30, which had a pixel size of 108.3 × 108.3 and 106.2 × 106.2 nm², respectively. The Z-stacks were done with a motorized stage (HLD117, Prior Scientific, UK) controlled by MetaMorph software (Molecular Devices) and maximum intensity projections created for subsequent analyses. All imaging parameters were kept constant among samples and among experiments. The count of c-Fos⁺ nuclei (% of c-Fos⁺/DAPI⁺ nuclei) was performed manually and the experimenter was not blinded to the treatment. A second experimenter repeated the analysis in blind and the results obtained were comparable. In hippocampi c-Fos⁺ count was restricted to the CA2 area identified by the PCP4 marker. For each biological sample, the percentage of CA2 Fos⁺ nuclei was calculated as a total from two hippocampal slices. In the MSDB, 2–3 coronal sections and a minimum of 3 fields/section were analyzed for each biological sample to identify an average of 79 ChAT⁺ neurons/animal and calculate the percentage of c-Fos⁺ cholinergic neurons.

Analysis of axon density

Images were taken using a Zeiss LSM 700 confocal microscope with a 20× objective. For quantification of axonal densities, we analyzed Z-stacks of seven focal planes with a distance of 2 µm from each other. To reduce as much as possible the probability to count individual axons more than once we counted the number of axons in bins of 50 × 50 µm. We analyzed 10 bins per hippocampal area in one individual section. The sum of these 10 bins represents the axon density for one individual section. Four sections from each animal were analyzed. The final value of axon density for one hippocampal area from one mouse represents the mean of axon density from the four analyzed sections for this specific hippocampal area.

Statistical analysis

Details of specific statistical designs and appropriate tests are described in each figure legend. Values are given as the mean ± SEM of n experiments. No statistical methods were used to predetermine sample sizes, but our samples were in agreement with similar published studies. Significance of differences was assessed mainly by Student’s paired or unpaired t-test and one-way ANOVA, as indicated. Wilcoxon or Mann–Whitney test was used for comparison of two groups and Tukey’s test was used for more than two groups. Outliers were identified using ROUT method (Q = 1%). Statistical differences were considered significant at P < 0.05. Statistical analysis was performed with GraphPad Prism 9.0 software (GraphPad, CA, USA).

Acknowledgements

We thank M Mameli, R Pizzarelli, and M Rosato-Siri for comments on the manuscript. This work was supported by grants from the Veronesi’s Foundation (Postdoctoral Fellowship 2016 and 2019 to MG), from Telethon (GGP 16083 to EC), from the European Union’s Horizon 2020 Framework Programme for Research and Innovation under the Specific Grant Agreement No. 785,907 (Human Brain Project SGA2 to EC), from Sovena Foundation (Fellowship 2020 to DP), from Fondo Ordinario Enti (FOE D.M 865/2019) funds in the framework of a collaboration agreement between the Italian National Research Council and EBRI (2019–2021).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Marilena Griguoli, Email: m.griguoli@ebri.it.

Serena M Dudek, National Institute of Environmental Health Sciences, United States.

Laura L Colgin, University of Texas at Austin, United States.

Funding Information

This paper was supported by the following grants:

Fondazione Telethon GGP 16083 to Enrico Cherubini.
Fondazione Umberto Veronesi Fellowship 2016-2019 to Marilena Griguoli.
Fondazione Sovena Fellowship 2020 to Domenico Pimpinella.
Horizon 2020 - Research and Innovation Framework Programme 785907 to Enrico Cherubini.

Additional information

Competing interests

No competing interests declared.

Author contributions

Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing.

Data curation, Formal analysis, Investigation, Methodology.

Data curation, Investigation, Methodology, Writing – review and editing.

Investigation.

Formal analysis, Investigation.

Funding acquisition, Writing – review and editing.

Funding acquisition, Writing – original draft, Writing – review and editing.

Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Supervision, Writing – original draft, Writing – review and editing.

Ethics

Additional files

Transparent reporting form

elife-65580-transrepform1.pdf^{(231KB, pdf)}

Data availability

Data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2–9.

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eLife. doi: 10.7554/eLife.65580.sa1

Decision letter

Editor: Serena M Dudek¹

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Septal cholinergic input to CA2 hippocampal region controls social memory via nicotinic receptor-mediated disinhibition" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The reviewers have opted to remain anonymous.

We are sorry to say that, after consultation with the reviewers, we have decided that your work will not be considered further for publication by eLife. That said, the reviewers were excited about the experimental question and the potential of the results, and so if you choose to expand the study to include new experiments according to their suggestions, you can submit it again as a new manuscript. In this case, please refer to the name of the editor, Laura Colgin, and your prior manuscript number, and every attempt will be made to recruit the same reviewers. Either way, we hope that you will find the reviewer comments useful in revising your manuscript for future submission(s).

Reviewer #1:

Pimpinella, et al. describe results of experiments aimed at determining the role of septal cholinergic inputs into hippocampal area CA2 in social memory. On one level, the authors show compelling evidence, using two different methods, that disruption of cholinergic neuron activity or output impairs social memory as assessed by social novelty preference. However, the primary weakness is that the authors have not yet proven a specific role for CA2 or CA2 interneurons in the behavior; area CA3 seems to have just as much MS axonal coverage as does area CA2, and the local drug infusions in the hippocampus would not appear to be selective to CA2. At the least, the authors should analyze the Fos staining in CA1 and CA3, which appear to have much less (in the case of CA1, or similar in the case of CA3) MS axonal coverage. In addition, some key control experiments and details are missing. The experiments address important aspects of both social behavior and cholinergic signaling, however more experiments in vitro and in vivo, performed in CA1 and CA3 really should be performed, or else the conclusions narrowly implicating CA2 will need to be modified to include the other CA regions.

The experiments and analyses appear to be performed to a high standard, and the manuscript is well written and the data nicely presented. The main findings, that inhibition of MSDB cholinergic neurons impairs social memory, are an important contribution to the field. Other conclusions, such a specific role for ChAT+ neuron influence on CA2 pyramidal neurons, through CA2 interneurons, impacting social memory, are less well supported by the data presented. Although generally supportive of the authors' hypothesis, little data was presented to test whether the behavioral effects of ACh release inhibition are due to the influence on hippocampal areas with similar (CA3) or less (CA1) MS axonal input, as their data would also support similar conclusions involving those areas (Fos staining blocked by CNO and electrophysiological effects in vivo and in vitro, for example, were not tested for other areas). Therefore, a number of additional experiments/analyses, listed below, would be required to make the case.

1. The whole justification for studying CA2 without comparing it to CA1 and CA3 rests on the reported role for CA2 in social memory and not say, a distinctive input from the MSDB. However, the authors' Figure 1D does seem to show some differences in ChAT axon patterns in the different areas. The authors could make a stronger justification for the focus on CA2 if they present some type of quantification of the terminal labeling in CA1/CA2/CA3 stratum pyramidale vs. radiatum vs. lucidum, for example.

2. The authors found that social interaction elicited a strong increase in c-Fos-positive cells in the MS and in CA2, which was prevented in DREADD silenced mice (Figure 3E). CA2 typically has the least amount of Fos stain of all the hippocampal subfields, and it looks from the images presented that there is a strong increase in Fos in what may be CA1, which also looked to be inhibited by CNO. As MS projects strongly to CA3, and to a lesser extent, CA1, the authors will need to present the Fos data from CA3 and CA1 as similar increases and disruptions by MS silencing would weaken the idea that CA2 is substantially different in MS influence on hippocampal neuronal activity (this not necessarily problematic, as CA1 could be activated by CA2, but it would need to be discussed in this context if so).

3. Also, are the Fos+ cells in CA2 the ones also positive for PCP4? This is unclear from the images (and almost looks like they are not). If not, did the authors also co-stain for inhibitory markers? (this would be important given that the authors are proposing a role for inhibitory neurons, and an increase in Fos in them would not support the authors' proposal that MS input to interneurons is decreasing their firing).

4. The authors should consider determining whether MSDB Fos induction (Figure 3 suppl 2) is specific for social interaction. Does novel object and/or novel environment similarly increase Fos staining? Same could be asked regarding CA2 Fos.

5. The synaptic effects of MSDB axonal silencing and nicotine in vitro (Figure 6 and Figure 4 suppl) in CA2 are very interesting, but the results are lacking context in relation to the axonal coverage shown in Figure 1 (again, comparing CA1 and/or CA3). One would expect a dramatically smaller effect in CA1 and a similar effect in CA3. Additionally, the conclusions could be strengthened by experiments using excitatory DREADDs or optogenetic stimulation.

6. Likewise, the optogenetic activation of cholinergic neurons in MSDB increases the firing of CA2 principal cells in vivo is an exciting result, but data from CA3 and/or CA1 would reveal whether there is anything fundamentally different between the areas (such as what might be expected if the ACh receptor distribution is different, like chrm3, and perhaps chrna4. See http://mouse.brain-map.org/gene/show/12456)

7. The conclusions based on the data in Figure 7 would be stronger if the authors would have included light-only controls, ideally in Chat-cre-negative- littermates. Although the light in MS is unlikely to be directly affecting CA2 neurons, heat generated in the MS but could be activating ChAT- neg neurons projecting to the hippocampus.

Further comments

1. In some places, the authors use 'impairs social novelty discrimination", which is a good description of what was actually measured. This should be used throughout the manuscript instead of 'memory', which is an interpretation (better brought up in the Discussion).

2. Methods: please expand on the methods used to count Fos+ nuclei (software for counting or manually counted? If manually, was the experimenter blinded to treatment?)

3. Methods: please include a statement justifying use of male mice only.

4. Results heading (line 72): "ACh released from cholinergic neurons in the MSDB is required for social novelty" should be '… social novelty discrimination'.

5. Legend in S2. (C and D) (and throughout) are not really a scatter plots.

6. Fos images are labeled with 'social behavior'. More accurate would be 'social interaction'.

7. The axis labels for the Fos staining in Figure 3 suppl 2 (D) is unclear. In the methods, the authors state "for each biological sample, the percentage of CA2 Fos+ nuclei was calculated as a total from two hippocampal slices and normalized to that obtained in the HCC condition" but the axis is labeled "% of fos+ cholinergic neurons". Do the authors mean "% of cholinergic neurons that are Fos+"? Similarly for Figure 3 F "normalized % of Fos+ nuclei". Should be "% of nuclei that are Fos+ (normalized to HCC)".

8. The graphs in Supp 3-2 'D' and Figure 3F are low resolution.

9. Please label CA1/CA2/CA3 on images.

10. Wording on lines 183-4: "In slices obtained from naive mice not expressing hM4, CNO did not affect the frequency and the amplitude of both sIPSCs (Figure 4-supplement 2C) and sEPSCs (Figure 4-supplement 2D)." should read: ".. CNO had no effect on either frequency or amplitude of sIPSCs or sEPSCs." Or something similar.

11. Methods, line 494: spelling should be Xylazine, not Xilazine (unless brand name? please confirm).

12. Methods, line 432: please clarify or be consistent with usage of 'spontaneous' synaptic currents vs. 'miniature' currents. (stated: "Miniature GABAA-mediated inhibitory postsynaptic currents…", but abbreviate with sIPSCs)". If TTX was used in both cases, why the difference in nomenclature? (should be mEPSC and mIPSC in that case, no?).

13. Methods, line 498: "frost" should be "frozen".

14. References: should cite Raam, et al., 2017, showing role for CA2 in social discrimination.

Reviewer #2:

In this manuscript, the authors investigate the role of acetylcholine in social memory and sociability. They combine pharmacological experiments with chemogenetics, in-vivo experiments and different behavior paradigms. In general, the proposed question of how a common neuromodulator such as ACh regulate social memory is of general interest. The authors find that social novelty recognition it is mediated by ACh release from Medial Septum, which specifically activates nicotinic receptors in local interneurons, what contributes to disinhibition of CA2 pyramidal neurons. The main caveat of the study is the lack of temporal resolution for most of the points regarding the mechanisms by which ACh controls different aspects of social memory. The nature of these experiments assumes social memory as a whole part, while it has been shown that social (and in general any type of memory) it is further subdivided in different processes (encoding, consolidation, recall…) which in turn involve different brain mechanisms. Yet, I think that in general the authors made a good effort combining different techniques to dissect the present mechanisms that could potentially relate ACh with social memory. Some specific points that could strengthen the main conclusions are detailed below.

1. In Supplemental Figure 1 (panel C), the authors show that after applying TeNT the frequency of spiking is not affected, however, there is a clear trend of a lower frequency with TeNT conditions, suggesting that there might be a masked effect due to low N.

2. In general, it could be nice to clarify in every part how long after infusion of different drugs were the animals tested, I couldn't find it for example for the TeNT experiments.

3. The cFos experiments are nice but lack the temporal resolution to correlate this activity dependent marker with any specifics of the task, i.e., social interactions/memory. It cannot be rule out that the cFos activity is free movement of the animal in any task (since there are no controls with any other behavioral paradigm). For this and in general for all group of experiments, it would be helpful to compare it with the responses obtained in the object recognition test (novel/familiar).

4. In line 358 the authors claim that "local release of ACh in the CA2 is sufficient for social memory's encoding". I think that what it can be proved with their experiments is that local release of ACh in the CA2 is necessary for social memory's encoding. In addition, the title of this part states "Local release of ACh in the CA2 hippocampal region is necessary for social novelty", shouldn't it be "for social novelty encoding/detection/recognition"?

5. In the main text, it is stated that n=14/n=13 animals were used for optogenetics groups, while in the figure 4 legend it is n=12/n=10. Were some animals excluded maybe due to virus or canula mistargeting?

6. Could the authors provide any insight with immunostaining (or even just speculate/discuss it), on which type of interneurons might be being involved in disinhibiting CA2 cells?

Reviewer #3:

The current study by Pimpinella et al. investigates an important and timely question regarding the contribution of the neuromodulator acetylcholine to social memory. By conducting behavioral experiments with chemogenetic, pharmacological, and optogenetic manipulations of cholinergic neurons in the medial septum or cholinergic release sites in the hippocampal CA2 region in mice, the authors demonstrate that cholinergic signaling is critically important for social memory and also for social novelty-related neuronal activity in CA2. Cholinergic neuromodulation in the hippocampus has long been implicated in novelty encoding but the dependence of social memory on cholinergic modulation has not been shown so far. The authors further show that cholinergic modulation of social memory depends on nicotinic acetylcholine receptors as opposed to muscarinic receptors, an important step towards deciphering the mechanism of cholinergic action contributing to social memory encoding. The authors claim that they have identified the mechanism of cholinergic action underlying social memory formation as disinhibition of CA2 principal neurons via activation of nicotinic acetylcholine receptors on CA2 interneurons. However, this claim is currently only poorly supported by their experimental data and the authors do not rule out alternative hypotheses that may account for the observed effects. The authors further claim that the inhibition of cholinergic modulation in the CA2 region specifically affects social memory but no other types of memory such as spatial memory. However, they do not present convincing evidence that spatial memory is spared and that inhibition of cholinergic signaling is constrained to the CA2 region. In summary, the manuscript presents very interesting data on the importance of cholinergic signaling for social memory. However, the manuscript in its current form lacks critical evidence supporting the authors' claims regarding the proposed mechanism of cholinergic modulation of social memory.

1. With respect to the TeNT experiments: The authors convincingly demonstrate that cholinergic MSDB neurons are important for social memory. However, they then further claim in lines 102-107 that the memory effect is specific for social memory based on comparing the effect on social memory with effects on novel object recognition. I am not convinced that this is the correct control experiment because social memory is hippocampus-dependent, while novel object recognition is not (or at least there is no strong evidence for novel object recognition being hippocampus-dependent). It is odd that the authors chose the NOR test as opposed to a hippocampus-dependent test such as the novel object location task. If the authors want to make the point that cholinergic signaling is specifically important for social memory, they would need to show that other forms of hippocampal-dependent memory such as spatial memory is unaffected or less affected by the same manipulations. Given the current data, it seems that hippocampal acetylcholine release from MSDB cholinergic projection neurons is important for hippocampus-dependent novelty tasks including-but not necessarily specific-to CA2-dependent social memory.

2. Lines 89-91, Figure 2—figure supplement 1C: "No changes in the (…) spontaneous firing frequency between eYFP and TeNT expressing neurons were observed." The authors only compared n = 5 TeNT cells with n = 6 eYFP cells using a non-parametric test that is underpowered at a sample size of 5. Nevertheless, they observe a more than threefold reduction in firing rate with a p-value of p = 0.08. This is very close to significance despite the low sample size and the test being underpowered. It is very likely that the authors would find a significant difference if they increase the sample size to n > 10. I am concerned that TeNT does not only affect synaptic release of vesicles but also basic firing properties of the cells. The authors may argue that, even if that is the case, the main conclusion of the paper may not change because the overall effect of TeNT expression is an inhibition of cholinergic activity. However, it should be discussed that TeNT may not be specifically affecting only synaptic acetylcholine release.

3. With respect to systemic CNO experiments: It has been shown by Gomez et al. (2017) that CNO is very likely not the active component responsible for DREADD-effects but that CNO is instead converted to clozapine which then acts on DREADD receptors. The authors cite this paper and do the correct control experiments by comparing their results with CNO-injected eYFP mice. However, I am missing a more detailed discussion when introducing the method (line 120) and a short discussion about potential off-target effects that has been previously shown for the CNO concentrations used in the current study (10 µM for in vitro experiments and 100 µM or 3 mg/kg for in vivo experiments).

4. Lines 145-147: "(…) social behavior elicited strong increase in c-Fos-positive cells (in the MSDB)". The authors do not provide evidence that c-Fos induction is caused specifically by social novelty as opposed to general novelty or exposure to the three-chamber test apparatus. The authors mention in the Discussion section that "(…) to rule out the contribution of c-Fos expression induced by a novel context, the animals were exposed to the three-chamber apparatus 24 hours before the social test" (lines 269-271). However, a single exposure to the test chamber one day before the test does very likely not reduce c-Fos expression on the next day. If the authors want to make that claim, they need to provide data showing that a single exposure to the same environment on the previous day significantly reduces c-Fos activation. As it stands, the current data do not show that social memory is related to c-Fos activation in the MSDB or hippocampus in addition to the c-Fos activation generally observed when taking mice out of their home cage and placing them in a test chamber.

5. With respect to experiments using local injections of CNO or nicotine into the CA2 region, how can the authors be sure that CNO and nicotine (both injected at relatively high concentrations) do not diffuse to the surrounding areas CA1 and CA3? A caveat of those experiments is that the inhibition of cholinergic signaling may not be constrained to the CA2 region. Observed effects of cholinergic inhibition may therefore be completely or partially caused by blocking cholinergic action in surrounding areas CA1 and/or CA3. This point becomes even more important in the light of the results of the local CNO injection, which-contrary to what the authors argue in the manuscript-did not show a significant difference between eYFP and hM4 animals.

Related to this point, the authors claim that "local release of ACh in the CA2 is sufficient for social memory's encoding" (line 168). However, this claim is not supported by their data. The data shown in Figure 4C clearly show that there is NO significant difference in the difference score between eYFP mice and hM4 mice (right panel). It would be wrong to draw that conclusion simply because there is a significant difference between Familiar and Novel in the eYFP group but not the hM4 group (see Nieuwenhuis et al., 2011). The correct statistical comparison is the one shown in the right panel (as acknowledged by the authors themselves earlier in the manuscript when introducing the difference score).

A similar consideration applies to data presented in Figure 4 —figure supplement 2. Since CNO can have off-target effects, the authors should statistically compare effects of CNO in hM4 mice to effects of CNO in naïve mice.

6. Regarding experiments on nicotinic control of neuronal activity in CA2: The authors claim that the mechanism of nicotinic receptor-dependent social memory is disinhibition of principal neurons in area CA2 via nicotinic activation of interneuron-selective interneurons. While this is an intriguing model consistent with their data, the authors do not provide convincing evidence for such a model or mechanism. In particular, recordings of interneurons are missing to support the author's conclusion and alternative hypotheses have not been addressed.

7. Previous studies (Malezieux et al., 2020 and Dannenberg et al., 2015) have shown that brain states associated with high cholinergic activity and theta oscillations result in reduced firing of CA3 principal neurons via activation of interneurons. If the net effect of acetylcholine release in CA2 is activation of principal neurons via disinhibition as opposed to inhibition of principal neurons as shown in CA3, that would be a very interesting finding. However, the authors should discuss the effect of tiletamine/xylazine anesthesia in their experiments. Moreover, the authors could address alternative explanations. For example, cholinergic stimulation could result in a general increase of network activity resulting in higher firing rates in both interneurons and principal neurons. Data on interneurons would help distinguish between those hypotheses.

8. I couldn't find details on optogenetics experiments in the Methods section (e.g., light power, wavelength, viral construct).

References:

Dannenberg, H., Young, K., and Hasselmo, M. (2017). Modulation of Hippocampal Circuits by Muscarinic and Nicotinic Receptors. Frontiers in Neural Circuits, 11, 102. https://doi.org/10.3389/fncir.2017.00102

Haam, J., and Yakel, J. L. (2017). Cholinergic modulation of the hippocampal region and memory function. Journal of Neurochemistry, 142 Suppl 2, 111-121. https://doi.org/10.1111/jnc.14052

Nieuwenhuis, S., Forstmann, B. U., and Wagenmakers, E.-J. (2011). Erroneous analyses of interactions in neuroscience: A problem of significance. Nature Neuroscience, 14(9), 1105-1107. https://doi.org/10.1038/nn.2886

McQuiston, A. R. (2014). Acetylcholine release and inhibitory interneuron activity in hippocampal CA1. Frontiers in Synaptic Neuroscience, 6. https://doi.org/10.3389/fnsyn.2014.00020

Malezieux, M., Kees, A. L., and Mulle, C. (2020). Theta Oscillations Coincide with Sustained Hyperpolarization in CA3 Pyramidal Cells, Underlying Decreased Firing. Cell Reports, 32(1). https://doi.org/10.1016/j.celrep.2020.107868

Dannenberg, H., Pabst, M., Braganza, O., Schoch, S., Niediek, J., Bayraktar, M., Mormann, F., and Beck, H. (2015). Synergy of direct and indirect cholinergic septo-hippocampal pathways coordinates firing in hippocampal networks. The Journal of Neuroscience, 35(22), 8394-8410. https://doi.org/10.1523/JNEUROSCI.4460-14.2015

Further comments:

1. Title and elsewhere (e.g., line 69): "Septal cholinergic input (…) controls social memory (…)." The wording "controls social memory" is very vague. Can the authors describe the major finding of the manuscript more precisely?

2. Lines 29-30 and elsewhere: Patch clamp recordings in hippocampal slices are usually referred to as "in vitro" experiments as opposed to "ex vivo". Is there any specific reason why the authors have chosen "ex vivo" as opposed to "in vitro"?

3. Lines 59: The authors could cite more recent reviews of the cholinergic system in addition to the reference to Teles-Grilo Ruivo and Mellor (2013), for example Dannenberg et al. (2017) and Haam and Yakel (2017).

4. Figure 2: I find the way the symbols and colors are used confusing. For example, the circle symbol is used for Animal, Object, Novel, and Familiar. It is further confusing that in E, orange and blue colors are used to indicate Novel and Familiar, but in B, D, and F, orange and blue colors are used to indicate eYFP and TeNT.

5. Line 173: "isolated pharmacologically". How?

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Septal cholinergic input to CA2 hippocampal region controls social novelty discrimination via nicotinic receptor-mediated disinhibition" for further consideration by eLife. Your revised article has been evaluated by Laura Colgin (Senior Editor) and a Reviewing Editor.

Essential revisions:

The authors describe results of experiments aimed at determining whether septal cholinergic inputs into hippocampal area CA2 play a role in social memory, a topic that is important and timely for a broad audience of neuroscientists interested in mechanisms of learning and memory and social behavior. The authors show that disruption of cholinergic neuron output impairs social memory as assessed by social novelty preference and that cholinergic input into the hippocampus, particularly in CA2, are playing substantial role in this finding. The revised manuscript is much improved from the first submission and the authors present some exciting new data that contrasts CA2 with the other hippocampal subregions. Nevertheless, there are still several issues that should be addressed in the text and relating to the figures before it is acceptable for publication.

Reviewer #1:

This study is an important contribution to the field and is likely of interest to a broad readership of neuroscientists. The revised manuscript is much improved from the first submission and presents some exciting new data. Although the authors addressed most of the reviewer concerns, there are still a number of issues that should be addressed in the text before it is acceptable for publication. These, along with some minor issues, are listed below.

1. The authors now include data from Novel Object Recognition and Object Location tests (Figure 3E, F), however they still did not show whether investigation of a Novel Object induces Fos in a way similar to that induced by the social stimulus. The data now included showing Fos in response to exploration of an empty arena and home cage controls are important additions, but the possibility that an increase in Fos staining in CA2 may not be selective for a social stimulus should be clearly acknowledged and discussed since a similar response to a novel object has not been ruled out. This is an important point given that place field remapping occurs with novel objects to a similar degree as that occurring with a social stimulus (Alexander, 2016).

2. The authors should address the locations of the cannulas, several of which look like they were in CA1. In this case, they should acknowledge in the discussion the limitations of the method.

3. It was interesting that most of the Fos+ neurons in CA2 were likely interneurons, and so it is somewhat surprising that the authors still do not show recordings from interneurons. The findings do not appear to be overstated ( "…provides insight into the mechanism" in the abstract), but some discussion stating the limitations of the study is warranted. These recordings would be critical for supporting the authors model of cholinergic regulation of CA2 output, and so the authors are encouraged to submit those recordings as an eLife "Research Advance".

4. The graphic is confusing in light of (1) the new data showing the increase in Fos in the interneurons, (2) no recordings from interneurons, and (3) there is some discrepancy with the observation of increased firing of CA2 pyramidal cells in response to opto-stimulation. I suggest deleting the graphic unless it can be revised to better reflect the findings.

Reviewer #2:

In the revised version of the manuscript, the authors have added new experimental support to their previous point, which substantially helped to strengthen the points made. Increasing the n numbers for several measures also helped to make sure the conclusions are solid. I am more convinced now about the mechanism that they proposed, stating that ACh, via nicotinic receptors, is necessary for encoding social novelty. However, although is interesting that they provide several possible models at the end, I still regret the lack of more specific data in relation to the type of interneurons. Overall, I think the authors made a decent job tackling most of the points raised in the previous review round and the information provided in this manuscript will be altogether of interest for the community.

Figure 1

– Units in figure 1 for axon density? Should it be "fluorescence density (AU, X20)"?

– 676 line reads: "stacks of 7 focal planes with a distance of 2?m" should it be "stacks of 7 focal planes with a distance of 2mm"

– No background subtraction was used I assume?

Figure 2

– This figure, still compares "stimulus" versus "non-stimulus" condition, without assessing whether the responses cFos+ are exclusive for an animal (social) or could be anything, object, spatial trajectory, foraging…

Figure 7

– Scales missing in A and C traces (or are they all the same one?), please clarify.

Figure 8

– Scales missing in A and C traces.

Figure 9

– Duration of the bursts would also be informative.

– Number of bursts would also be informative.

Figure 5—figure supplement 2

– Temporal scale missing in traces showed in A.

– Scales missing in traces showed in E.

– Y labels in I and J are confusing (shown in "%"), sIPSCs frequency is shown in Hz (in the above plots), so perhaps the authors meant difference between conditions or ratio compare to control in these plots?

Figure 7—figure supplement 1

– Temporal scale missing in trace showed in A.

Figure 7—figure supplement 2

– Temporal scale missing in trace showed in A.

Figure 9—figure supplement 1

– Temporal scale missing in trace showed in B.

Figure 9—figure supplement 2

– It is interesting that the spiking frequency of CA3 neurons is not affected at all by optical stimulation of MSDB, despite the frequency of sEPSCs being affected by nicotine.

– Temporal scale missing in trace showed in B.

Reviewer #3:

The revised manuscript entitled "Septal cholinergic input to CA2 hippocampal region controls social novelty discrimination via nicotinic receptor-mediated disinhibition" by Pimpinella et al. now presents additional experimental data and analyses that address previous concerns raised by reviewers. In particular, the authors now demonstrate that local delivery of CNO to the CA2 region of hM4-expressing mice impairs social novelty detection. Furthermore, the authors added in vitro experimental data that demonstrate an increase in spontaneous EPSCs in the presence of nicotine in CA2 but not in CA1 and CA3. In fact, a decrease in spontaneous EPSCs is observed in CA3. These new data provide evidence that nicotinic cholinergic receptors differentially affect network activity in hippocampal subregions and support the authors' hypothesis that cholinergic disinhibition of CA2 via nicotinic receptors is important for social novelty discrimination. Furthermore, the authors now report experimental results that demonstrate that social interaction increases cFos expression in cholinergic neurons in the medial septal complex further highlighting a significant activation and contribution of the septal cholinergic system during social interaction. I have few remaining comments, mostly addressing statistical analyses and the clarity of data presentation.

1. Regarding major point 2 (TeNT experiments): Do the authors now interpret their findings as an effect of TeNT on intrinsic firing properties? To more clearly distinguish between the alternative and null hypotheses that TeNT has or has not an effect on intrinsic firing properties, I suggest a statistical analysis using Bayes factor (Keysers, Christian, Valeria Gazzola, and Eric-Jan Wagenmakers. "Using Bayes Factor Hypothesis Testing in Neuroscience to Establish Evidence of Absence." Nature Neuroscience 23, no. 7 (July 2020): 788-99. https://doi.org/10.1038/s41593-020-0660-4.; Dienes, Zoltan. "Using Bayes to Get the Most out of Non-Significant Results." Frontiers in Psychology 5 (2014): 781. https://doi.org/10.3389/fpsyg.2014.00781). The Bayes factor will indicate whether (a) the data support the null hypothesis or (b) the data support the alternative hypothesis, or (c) the sample size is too low to draw a conclusion.

2. Why does the eYFP virus has a different serotype (2/9) than the TeNT carrying AAV (DJ serotype)? The possibility that diverse tropism of eYFP carrying AAV vs TeNT carrying AAV may target different subtypes of cholinergic neurons – as stated by the authors in the rebuttal letter – should also be discussed in the manuscript itself.

3. To Figure 3C and Figure 4C: It is not immediately clear from looking at the figure if Novel and Familiar refers to an object or to an animal. I suggest to replace the circles with symbols that make this immediately clear to the reader (e.g., a drawing of a mouse in Figure 3C and drawing of an object in Figure 3A).

4. Figure 4 —figure supplement 5: In my opinion, this figure should be part of main Figure 4 because it is an important control experiment and shows that chemogenetic inactivation of cholinergic neurons in the medial septal complex also affects spatial memory in addition to social memory, though the effect size appears smaller for effects on spatial memory compared to the larger effects on social memory. But I leave the final decision to the authors.

5. Line 211: "necessary" instead of "sufficient"?

eLife. 2021 Oct 26;10:e65580. doi: 10.7554/eLife.65580.sa2

Author response

[Editors’ note: The authors appealed the original decision. What follows is the authors’ response to the first round of review.]

Reviewer #1:

Pimpinella, et al. describe results of experiments aimed at determining the role of septal cholinergic inputs into hippocampal area CA2 in social memory. On one level, the authors show compelling evidence, using two different methods, that disruption of cholinergic neuron activity or output impairs social memory as assessed by social novelty preference. However, the primary weakness is that the authors have not yet proven a specific role for CA2 or CA2 interneurons in the behavior; area CA3 seems to have just as much MS axonal coverage as does area CA2, and the local drug infusions in the hippocampus would not appear to be selective to CA2. At the least, the authors should analyze the Fos staining in CA1 and CA3, which appear to have much less (in the case of CA1, or similar in the case of CA3) MS axonal coverage. In addition, some key control experiments and details are missing. The experiments address important aspects of both social behavior and cholinergic signaling, however more experiments in vitro and in vivo, performed in CA1 and CA3 really should be performed, or else the conclusions narrowly implicating CA2 will need to be modified to include the other CA regions.

The experiments and analyses appear to be performed to a high standard, and the manuscript is well written and the data nicely presented. The main findings, that inhibition of MSDB cholinergic neurons impairs social memory, are an important contribution to the field. Other conclusions, such a specific role for ChAT+ neuron influence on CA2 pyramidal neurons, through CA2 interneurons, impacting social memory, are less well supported by the data presented. Although generally supportive of the authors' hypothesis, little data was presented to test whether the behavioral effects of ACh release inhibition are due to the influence on hippocampal areas with similar (CA3) or less (CA1) MS axonal input, as their data would also support similar conclusions involving those areas (Fos staining blocked by CNO and electrophysiological effects in vivo and in vitro, for example, were not tested for other areas). Therefore, a number of additional experiments/analyses, listed below, would be required to make the case.

1. The whole justification for studying CA2 without comparing it to CA1 and CA3 rests on the reported role for CA2 in social memory and not say, a distinctive input from the MSDB. However, the authors' Figure 1D does seem to show some differences in ChAT axon patterns in the different areas. The authors could make a stronger justification for the focus on CA2 if they present some type of quantification of the terminal labeling in CA1/CA2/CA3 stratum pyramidale vs. radiatum vs. lucidum, for example.

We followed the Reviewer’s suggestion and a quantification of cholinergic terminals labeling in CA1/CA2/CA3 was included in the New figure 1 (Panel E).

2. The authors found that social interaction elicited a strong increase in c-Fos-positive cells in the MS and in CA2, which was prevented in DREADD silenced mice (Figure 3E). CA2 typically has the least amount of Fos stain of all the hippocampal subfields, and it looks from the images presented that there is a strong increase in Fos in what may be CA1, which also looked to be inhibited by CNO. As MS projects strongly to CA3, and to a lesser extent, CA1, the authors will need to present the Fos data from CA3 and CA1 as similar increases and disruptions by MS silencing would weaken the idea that CA2 is substantially different in MS influence on hippocampal neuronal activity (this not necessarily problematic, as CA1 could be activated by CA2, but it would need to be discussed in this context if so).

A quantification of c-Fos-positive nuclei in CA3 and CA1 was performed in both eYFP and hM4expressing mice that were subjected to social behavior and that received 30 minutes prior to the social novelty task a systemic injection of CNO. Home-caged animals, not exposed to behavioral tasks, were used as controls. c-Fos quantification in CA3 and CA1 was performed on the same sections analyzed for CA2 (CA2 results in Figure 3E of the previous manuscripts’ version are now shown in Figure 4E-F). No significant changes in the % of nuclei expressing c-Fos were detected among the three experimental groups. These results suggest that under these conditions there is not a selective activation of c-Fos in response to social interaction in CA3 and CA1 (New Figure 4figure supplement 4).

3. Also, are the Fos+ cells in CA2 the ones also positive for PCP4? This is unclear from the images (and almost looks like they are not). If not, did the authors also co-stain for inhibitory markers? (this would be important given that the authors are proposing a role for inhibitory neurons, and an increase in Fos in them would not support the authors' proposal that MS input to interneurons is decreasing their firing).

We thank the Reviewer for this suggestion and we proceeded to analyze whether c-Fos positive cells are also positive for the PCP4 marker. Indeed, in our sections (n=3 animals, average n=8 c-Fos⁺ nuclei/animal) 94.1 ± 3 % of c-Fos expressing cells were PCP4^- (Figure 4—figure supplement 2A-B), confirming the Reviewer’s observation. Previous studies showed that the PCP4 marker overlaps with different CA2 markers expressed by principal neurons (Kohara et al., 2014). It was shown that PCP4-expressing neurons are putative pyramidal cells as they express CAMKII, a glutamatergic marker (Kohara et al., 2014). However, we cannot exclude the possibility that there is a subpopulation of PCP4^- principal cells within the CA2 boundaries. To investigate whether PCP4^- neurons were GABAergic interneurons, we performed double immunostaining for parvalbumin (PV) and c-Fos. The use of this marker was justified by the fact that PV⁺ neurons frequently have large pyramid shaped or fusiform somas that are typically localized within or in immediate vicinity to stratum pyramidale (Pelkey et al., 2017). Furthermore, PV⁺ neurons contributing to cortical disinhibitory circuits (Pi et al., 2013; Pfeffer et al., 2013). Our analysis revealed that the majority of c-Fos⁺ cells were PV^- (91.5 ± 4.5 %; n=3 animals; average n=8 c-Fos⁺ nuclei/animal) (New Figure 4—figure supplement 2A-C). To better identify which subtypes of GABAergic interneurons maybe involved in social interactions is indeed a very interesting question that would need, however, a complete new study and hence cannot be addressed in the present work.

4. The authors should consider determining whether MSDB Fos induction (Figure 3 suppl 2) is specific for social interaction. Does novel object and/or novel environment similarly increase Fos staining? Same could be asked regarding CA2 Fos.

We performed the requested experiments to exclude the possibility that c-Fos expression in the MSDB was induced by locomotor activity and environment exploration (a point raided also by Reviewer 2 in point 3). To this purpose mice (n = 4) were habituated to the three chamber arena (empty, EA) for 10 minutes (day 1) and again for 10 minutes after 24 hours (day 2). In this way, the animals underwent to the same handling and habituation phases as those exposed to social stimuli during the three chamber test (social interaction, SI). After one hour, we sacrified the animals. c-Fos immunolabeling revealed a certain degree of c-Fos expression after EA exploration as compared to HCC controls (New Figure 2, panels 2D-F). Nevertheless this was significantly lower than what observed in the SI condition (New Figure 2, panels 2G-H). Interestingly, we did not detect any ChAT⁺ neuron expressing c-Fos in the EA group, indicating that ChAT⁺ neurons selectively express c-Fos in response to social stimuli (New Figure 2, panels A-C, H).

Previous studies showed that a novel environment activates early gene transcription in the CA2 neurons (VanElzakker et al., 2008; Wintzer et al., 2014) and this activation is comparable to that induced by social stimuli (Alexander et al., 2016). In our case, the animals were habituated to the novel environment and the only discriminating factor between EA and SI conditions was the social stimuli. We performed c-Fos immunolabeling in EA group and we did not find a difference in the % of c-Fos⁺ nuclei in CA2 as compared to HCC (New Figure 4-supplementary 3).

5. The synaptic effects of MSDB axonal silencing and nicotine in vitro (Figure 6 and Figure 4 suppl) in CA2 are very interesting, but the results are lacking context in relation to the axonal coverage shown in Figure 1 (again, comparing CA1 and/or CA3). One would expect a dramatically smaller effect in CA1 and a similar effect in CA3. Additionally, the conclusions could be strengthened by experiments using excitatory DREADDs or optogenetic stimulation.

We collected new data regarding the effects of nicotine on synaptic transmission recorded from both CA3 and CA1 pyramidal cells. Contrary to what we previously observed in the CA2 region, where nicotine increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), we found a reduced frequency of sEPSCs in both CA3 and CA1 regions (New Figure 7—figure supplement 2). As already reported (Rosato-Siri et al., 2006; Tang et al., 2011; Hajós et al., 2005), the reduced frequency of sEPSCs in both CA3 and CA1 regions may be due to a nAChRs-dependent increase of GABAergic tone with consequential reduction of glutamatergic transmission.

Following Reviewer’s suggestion we injected the MSDB of ChAT-Cre mice with an AAV carrying the floxed sequence of hM3 excitatory DREADD. This allowed to the effects of endogenously released ACh on sEPSCs in CA3, CA2 and CA1 principal neurons before and during bath application of CNO. According to the effect observed in the presence of nicotine, CNO increased the frequency but not the amplitude of sEPSCs in CA2 principal neurons. In CA3, CNO mimicked the effect of nicotine by decreasing the frequency of sEPSCs. CNO also induced a significant decrease in sEPSCs amplitude, not observed during nicotine application. This discrepancy could be explained by different affinity and/or desensitization of nAChR subtypes when bound by ACh or nicotine (Albuquerque et al., 2009). In CA1, CNO did not change either the sESPCs frequency or the amplitude, possibly because of a reduced coverage of hM4-expressing fibers and hence an insufficient ACh release in response to hM3 activation (New Figure 8).

6. Likewise, the optogenetic activation of cholinergic neurons in MSDB increases the firing of CA2 principal cells in vivo is an exciting result, but data from CA3 and/or CA1 would reveal whether there is anything fundamentally different between the areas (such as what might be expected if the ACh receptor distribution is different, like chrm3, and perhaps chrna4. See http://mouse.brain-map.org/gene/show/12456)

Juxtacellular recordings combined with photo stimulation of ChAT⁺ neurons expressing ChR2 in the MSDB from both CA3 and CA1 regions were performed. No changes in the firing frequency or intraburst frequency from both CA3 and CA1 neurons could be observed during light stimulation. This may be related to the concomitant activation of mAChRs, which would probably counteract the depressing effect of nAChRs on glutamatergic transmission. These results were included in a new supplementary figure (Figure 9—figure supplement 2).

7. The conclusions based on the data in Figure 7 would be stronger if the authors would have included light-only controls, ideally in Chat-cre-negative- littermates. Although the light in MS is unlikely to be directly affecting CA2 neurons, heat generated in the MS but could be activating ChAT- neg neurons projecting to the hippocampus.

We agree with Reviewer that the experiment she/he suggested is an important control. Thus, juxtacellular recordings combined with light delivery in the MSDB were performed from the CA2 region of ChAT-Cre mice lacking ChR2 expression. No change in the frequency of spontaneous firing was observed in recorded neurons. These data were added to Figure 9—figure supplement 1.

Further comments:

1. In some places, the authors use 'impairs social novelty discrimination", which is a good description of what was actually measured. This should be used throughout the manuscript instead of 'memory', which is an interpretation (better brought up in the Discussion).

The text was edited accordingly (Lines 1, 35, 71, 75, 116, 164, 197, 209, 229, 343).

2. Methods: please expand on the methods used to count Fos+ nuclei (software for counting or manually counted? If manually, was the experimenter blinded to treatment?)

The method section used to count c-Fos was expanded as requested. The counting was done manually, and the experimenter was not blinded to treatment. A second experimenter who was blinded to treatment repeated the c-Fos-positive cell counting and the same results were obtained (Lines 657-659, 662).

3. Methods: please include a statement justifying use of male mice only.

A sentence in the method section was added to justify the use of male mice: “to reduce the variability due to female estrus cycle we restricted the analysis to male animals” (Line 419).

4. Results heading (line 72): "ACh released from cholinergic neurons in the MSDB is required for social novelty" should be '… social novelty discrimination'.

The text was modified accordingly (see point 1).

5. Legend in S2. (C and D) (and throughout) are not really a scatter plots.

“Scatter plots” were substituted with “aligned dot plots” accordingly.

6. Fos images are labeled with 'social behavior'. More accurate would be 'social interaction'.

The labels in c-Fos images were changed accordingly.

7. The axis labels for the Fos staining in Figure 3 suppl 2 (D) is unclear. In the methods, the authors state "for each biological sample, the percentage of CA2 Fos+ nuclei was calculated as a total from two hippocampal slices and normalized to that obtained in the HCC condition" but the axis is labeled "% of fos+ cholinergic neurons". Do the authors mean "% of cholinergic neurons that are Fos+"? Similarly for Figure 3 F "normalized % of Fos+ nuclei". Should be "% of nuclei that are Fos+ (normalized to HCC)".

In Figure 3—figure supplement 2D (New Figure 2D) we meant the % of cholinergic neurons that were cFos⁺. In the revised version of the manuscript we did not show normalized data to HCC in the CA2 region as well. We added a sentence in the method to explain that in MSDB, the % of c-Fos⁺ cholinergic nuclei was analyzed in HCC and social interaction conditions. The axis labeled in Figure 3 (F) (New Figure 4F) was changed to “% of cFos⁺ nuclei” as the Reviewer suggested.

8. The graphs in Supp 3-2 'D' and Figure 3F are low resolution.

The resolution of the graphs was increased (New Figure 2D and Figure 4F).

9. Please label CA1/CA2/CA3 on images.

CA1/CA2/CA3 labels were added to images.

10. Wording on lines 183-4: "In slices obtained from naive mice not expressing hM4, CNO did not affect the frequency and the amplitude of both sIPSCs (Figure 4-supplement 2C) and sEPSCs (Figure 4-supplement 2D)." should read: ".. CNO had no effect on either frequency or amplitude of sIPSCs or sEPSCs." Or something similar.

The sentence was changed accordingly: “In slices obtained from naïve mice not expressing hM4, CNO had no effect on either frequency or amplitude of sIPSCs or sEPSCs” (Lines 219-220).

11. Methods, line 494: spelling should be Xylazine, not Xilazine (unless brand name? please confirm).

The typing error was corrected.

12. Methods, line 432: please clarify or be consistent with usage of 'spontaneous' synaptic currents vs. 'miniature' currents. (stated: "Miniature GABAA-mediated inhibitory postsynaptic currents...", but abbreviate with sIPSCs)". If TTX was used in both cases, why the difference in nomenclature? (should be mEPSC and mIPSC in that case, no?).

The typing error was corrected.

13. Methods, line 498: "frost" should be "frozen".

The text was changed accordingly.

14. References: should cite Raam, et al., 2017, showing role for CA2 in social discrimination.

The reference was included in the Introduction (line 64) and in the Reference sections.

Reviewer #2:

In this manuscript, the authors investigate the role of acetylcholine in social memory and sociability. They combine pharmacological experiments with chemogenetics, in-vivo experiments and different behavior paradigms. In general, the proposed question of how a common neuromodulator such as ACh regulate social memory is of general interest. The authors find that social novelty recognition it is mediated by ACh release from Medial Septum, which specifically activates nicotinic receptors in local interneurons, what contributes to disinhibition of CA2 pyramidal neurons. The main caveat of the study is the lack of temporal resolution for most of the points regarding the mechanisms by which ACh controls different aspects of social memory. The nature of these experiments assumes social memory as a whole part, while it has been shown that social (and in general any type of memory) it is further subdivided in different processes (encoding, consolidation, recall…) which in turn involve different brain mechanisms. Yet, I think that in general the authors made a good effort combining different techniques to dissect the present mechanisms that could potentially relate ACh with social memory. Some specific points that could strengthen the main conclusions are detailed below.

1. In Supplemental Figure 1 (panel C), the authors show that after applying TeNT the frequency of spiking is not affected, however, there is a clear trend of a lower frequency with TeNT conditions, suggesting that there might be a masked effect due to low N.

We increased the number of MSDB ChAT⁺ neurons expressing eYFP (from 6 to 9, please note that one cell included in the previous version was excluded because it was an outlier, frequency = 14.9 Hz; ROUT method, Q=1%). Unfortunately we could not further increase the number of TeNT expressing neurons (n=8) because the viral injection of the AAV carrying TeNT did not lead to TeNTGFP expression. This was a consequence of the limited number of available animals due to the pandemic condition. In spite of the clear trend, the difference in the firing frequency did not reach the statistical significance by adding new eYFP cells.

2. In general, it could be nice to clarify in every part how long after infusion of different drugs were the animals tested, I couldn't find it for example for the TeNT experiments.

TeNT was durably expressed in ChAT-positive neurons in the MSDB starting at 3-4 weeks from viral infection. Both the behavioral and electrophysiological experiments were performed after 4 weeks from viral injection as described in the Methods section (Lines 442-443). Regarding the local drug infusions, behavioral experiments were performed 30 minutes after drug application (Lines 206, 234).

3. The cFos experiments are nice but lack the temporal resolution to correlate this activity dependent marker with any specifics of the task, i.e., social interactions/memory. It cannot be rule out that the cFos activity is free movement of the animal in any task (since there are no controls with any other behavioral paradigm). For this and in general for all group of experiments, it would be helpful to compare it with the responses obtained in the object recognition test (novel/familiar).

We agree with the Reviewer’s comment and, as requested also by Reviewers 1 and 3, we performed c-Fos analysis in animals exploring the three chamber arena in the absence of social stimuli (empty arena, EA). See point 4 of Reviewer 1.

4. In line 358 the authors claim that "local release of ACh in the CA2 is sufficient for social memory's encoding". I think that what it can be proved with their experiments is that local release of ACh in the CA2 is necessary for social memory's encoding. In addition, the title of this part states "Local release of ACh in the CA2 hippocampal region is necessary for social novelty", shouldn't it be "for social novelty encoding/detection/recognition"?

We agree with the Reviewer’s comment, the text (Lines 196-197) and the paragraph title were changed accordingly: "Local release of ACh in the CA2 is necessary for social memory encoding" and “Local release of ACh in the CA2 hippocampal region is necessary for social novelty discrimination”.

5. In the main text, it is stated that n=14/n=13 animals were used for optogenetics groups, while in the figure 4 legend it is n=12/n=10. Were some animals excluded maybe due to virus or canula mistargeting?

The Reviewer’s comment is correct: the animals treated with CNO locally (in CA2) were n=13/n=14 (eYFP and hM4 respectively), but some were excluded due to canula mistargeting (1 eYFP and 4 hM4). We then performed an additional set of experiments and increased the number of animals in both groups (eYFP: n=15; hM4: n=14) as indicated in the figure legend (Figure 5). The text was changed accordingly.

6. Could the authors provide any insight with immunostaining (or even just speculate/discuss it), on which type of interneurons might be being involved in disinhibiting CA2 cells?

Looking at different GABAergic interneuron subtypes would be very interesting but too demanding at this stage of the work. It warrants a completely new study. As mentioned in the discussion (Lines 395-398) “A disinhibitory effect of ACh was observed also in the prelimbic area of mPFC, where α5 nAChR dependent activation of VIP⁺ interneurons inhibits downstream SOM⁺ cells, which in turn leads to the enhancement of principal neurons firing in the layer 2/3 (Koukouli et al., 2017)”. Thus, VIP⁺ and SOM⁺ interneurons could be candidates for nicotinic-mediated disinhibition in the CA2 region. PV⁺ neurons could also play a role (Pi et al., 2013). Nevertheless, our double-immunostaining experiments for c-Fos and PV allow us to exclude a scenario in which PV⁺ interneurons are involved. These hypotheses will certainly be pursued of future investigations.

Reviewer #3:

The current study by Pimpinella et al. investigates an important and timely question regarding the contribution of the neuromodulator acetylcholine to social memory. By conducting behavioral experiments with chemogenetic, pharmacological, and optogenetic manipulations of cholinergic neurons in the medial septum or cholinergic release sites in the hippocampal CA2 region in mice, the authors demonstrate that cholinergic signaling is critically important for social memory and also for social novelty-related neuronal activity in CA2. Cholinergic neuromodulation in the hippocampus has long been implicated in novelty encoding but the dependence of social memory on cholinergic modulation has not been shown so far. The authors further show that cholinergic modulation of social memory depends on nicotinic acetylcholine receptors as opposed to muscarinic receptors, an important step towards deciphering the mechanism of cholinergic action contributing to social memory encoding. The authors claim that they have identified the mechanism of cholinergic action underlying social memory formation as disinhibition of CA2 principal neurons via activation of nicotinic acetylcholine receptors on CA2 interneurons. However, this claim is currently only poorly supported by their experimental data and the authors do not rule out alternative hypotheses that may account for the observed effects. The authors further claim that the inhibition of cholinergic modulation in the CA2 region specifically affects social memory but no other types of memory such as spatial memory. However, they do not present convincing evidence that spatial memory is spared and that inhibition of cholinergic signaling is constrained to the CA2 region. In summary, the manuscript presents very interesting data on the importance of cholinergic signaling for social memory. However, the manuscript in its current form lacks critical evidence supporting the authors' claims regarding the proposed mechanism of cholinergic modulation of social memory.

1. With respect to the TeNT experiments: The authors convincingly demonstrate that cholinergic MSDB neurons are important for social memory. However, they then further claim in lines 102-107 that the memory effect is specific for social memory based on comparing the effect on social memory with effects on novel object recognition. I am not convinced that this is the correct control experiment because social memory is hippocampus-dependent, while novel object recognition is not (or at least there is no strong evidence for novel object recognition being hippocampus-dependent). It is odd that the authors chose the NOR test as opposed to a hippocampus-dependent test such as the novel object location task. If the authors want to make the point that cholinergic signaling is specifically important for social memory, they would need to show that other forms of hippocampal-dependent memory such as spatial memory is unaffected or less affected by the same manipulations. Given the current data, it seems that hippocampal acetylcholine release from MSDB cholinergic projection neurons is important for hippocampus-dependent novelty tasks including-but not necessarily specific-to CA2-dependent social memory.

Although there is indication that the NOR task is hippocampus-dependent (see review Cohen and Stackman, 2015), the issue is controversial. Thus, we agree with the Reviewer’s comment concerning the need to show how cholinergic disruption impacts on spatial memory using a hippocampus-dependent test such as the object location task (OLT). To this end, ChAT-Cre mice injected with the AAV carrying either eYFP or hM4 sequences were subjected to OLT. CNO was systemically delivered via i.p. injection 30 minutes before mice were exposed to the new location of the object. Although hM4 mice showed preference to novel location as the eYFP group, the time they spent in exploring the object in the new location was not significantly different from that in the familiar location. This result suggests that inhibition of ChAT⁺ neurons in the MSDB affects hippocampal-dependent spatial memory. These data were included in a new supplementary figure (Figure 4—figure supplement 5).

2. Lines 89-91, Figure 2—figure supplement 1C: "No changes in the (…) spontaneous firing frequency between eYFP and TeNT expressing neurons were observed." The authors only compared n = 5 TeNT cells with n = 6 eYFP cells using a non-parametric test that is underpowered at a sample size of 5. Nevertheless, they observe a more than threefold reduction in firing rate with a p-value of p = 0.08. This is very close to significance despite the low sample size and the test being underpowered. It is very likely that the authors would find a significant difference if they increase the sample size to n > 10. I am concerned that TeNT does not only affect synaptic release of vesicles but also basic firing properties of the cells. The authors may argue that, even if that is the case, the main conclusion of the paper may not change because the overall effect of TeNT expression is an inhibition of cholinergic activity. However, it should be discussed that TeNT may not be specifically affecting only synaptic acetylcholine release.

The number of cells included in the analysis of TeNT group was 8 (circles shown in the figure overlap). We agree with the Reviewer’s comment on the possible effect of TeNT expression on neuronal firing activity, besides the one on acetylcholine release. We increased the sample of eYFP expressing neurons to 9 (see New Figure 3—figure supplement 1C). We could not increase the number of TeNT⁺ cells further (see answer to Reviewer 2-point 1). The additional data from eYFP group did not change the statistical outcome about the firing frequency (p=0.07, Mann-Whitney test). In addition, we performed a normality test (Shapiro-Wilk) in order to run a parametric test, of which the p value was 0.08.

We have two hypotheses regarding the TeNT-induced changes in intrinsic neuronal properties:

1. Diverse tropism of eYFP carrying AAV (serotype 2/9) vs TeNT carrying AAV (DJ serotype) may target different subtypes of cholinergic neurons in the MSDB.

2. TeNT could affect local (i.e. dendritic) ACh release with consequent effect on intrinsic properties modulated by cholinergic autoreceptors. This phenomenon was observed for neuromodulatory systems (i.e. dopaminergic neurons, see review article by Luwing et al., 2016).

3. With respect to systemic CNO experiments: It has been shown by Gomez et al. (2017) that CNO is very likely not the active component responsible for DREADD-effects but that CNO is instead converted to clozapine which then acts on DREADD receptors. The authors cite this paper and do the correct control experiments by comparing their results with CNO-injected eYFP mice. However, I am missing a more detailed discussion when introducing the method (line 120) and a short discussion about potential off-target effects that has been previously shown for the CNO concentrations used in the current study (10 µM for in vitro experiments and 100 µM or 3 mg/kg for in vivo experiments).

The discussion of the possible side-effects of CNO at the doses used in the current study was extended (Lines 150-152, 159-160, 202-204, 242-245).

4. Lines 145-147: "(…) social behavior elicited strong increase in c-Fos-positive cells (in the MSDB)". The authors do not provide evidence that c-Fos induction is caused specifically by social novelty as opposed to general novelty or exposure to the three-chamber test apparatus. The authors mention in the Discussion section that "(…) to rule out the contribution of c-Fos expression induced by a novel context, the animals were exposed to the three-chamber apparatus 24 hours before the social test" (lines 269-271). However, a single exposure to the test chamber one day before the test does very likely not reduce c-Fos expression on the next day. If the authors want to make that claim, they need to provide data showing that a single exposure to the same environment on the previous day significantly reduces c-Fos activation. As it stands, the current data do not show that social memory is related to c-Fos activation in the MSDB or hippocampus in addition to the c-Fos activation generally observed when taking mice out of their home cage and placing them in a test chamber.

We agree with the Reviewer that in the previous version of the manuscript we did not provide evidence that c-Fos activation in the MSDB and CA2 was caused by social novelty. As indicated in the answer to Reviewers 1 (point 4) and 2 (point 3), we performed c-Fos staining and quantification in the MSDB and in the CA2 hippocampal region of mice exposed to the three chamber empty arena (EA). We found a certain degree of activation in MSDB in animals exposed to EA (lower as compared to social stimuli). However, none of ChAT⁺ neurons analyzed were positive for c-Fos, indicating that ChAT⁺ neurons were selectively activated by social stimuli (New Figure 2). In the CA2 region of the hippocampus, we did not detect significant c-Fos activation in EA animals as compared to HCC group (Figure 4—figure supplement 3).

5. With respect to experiments using local injections of CNO or nicotine into the CA2 region, how can the authors be sure that CNO and nicotine (both injected at relatively high concentrations) do not diffuse to the surrounding areas CA1 and CA3? A caveat of those experiments is that the inhibition of cholinergic signaling may not be constrained to the CA2 region. Observed effects of cholinergic inhibition may therefore be completely or partially caused by blocking cholinergic action in surrounding areas CA1 and/or CA3. This point becomes even more important in the light of the results of the local CNO injection, which-contrary to what the authors argue in the manuscript-did not show a significant difference between eYFP and hM4 animals.

Related to this point, the authors claim that "local release of ACh in the CA2 is sufficient for social memory's encoding" (line 168). However, this claim is not supported by their data. The data shown in Figure 4C clearly show that there is NO significant difference in the difference score between eYFP mice and hM4 mice (right panel). It would be wrong to draw that conclusion simply because there is a significant difference between Familiar and Novel in the eYFP group but not the hM4 group (see Nieuwenhuis et al., 2011). The correct statistical comparison is the one shown in the right panel (as acknowledged by the authors themselves earlier in the manuscript when introducing the difference score).

The nicotinic antagonists DHßE delivered to the CA2 area caused an impairment of social novelty. We agree with the Reviewer’s comment regarding the absence of statistical significance in the score between eYFP and hM4 groups treated with CNO locally. We performed an additional set of experiments delivering CNO in the CA2 region of eYFP- and hM4-expressing mice. We increased the number of animals to 15 and 14 for eYFP and hM4 groups, respectively. The difference score is now significantly different between eYFP and hM4 mice (New Figure 5).

Our attempt at collecting data on the diffusion of drugs infused via canula by using a lipofilic dye (DiI) was unsuccessful, probably because of the different diffusion properties of the dye as compared to the drugs used in the experiments. In our experiments we used a small volume (150 nL) and we always checked the canula placement, to be sure that all drugs targeted CA2. However, we cannot exclude some diffusion to neighboring CA3 and CA1 regions.

A similar consideration applies to data presented in Figure 4 —figure supplement 2. Since CNO can have off-target effects, the authors should statistically compare effects of CNO in hM4 mice to effects of CNO in naïve mice.

We performed additional experiments in naïve mice to increase the sample of cells recorded (IPSCs n=10 cells; EPSCs n=9 cells). To evaluate possible off-target effects of CNO, we compared the % of CNO-induced changes between naïve and hM4 groups for frequency and amplitude of sIPSCs and sEPSCs. These results were included in Figure 5—figure supplement 2.

6. Regarding experiments on nicotinic control of neuronal activity in CA2: The authors claim that the mechanism of nicotinic receptor-dependent social memory is disinhibition of principal neurons in area CA2 via nicotinic activation of interneuron-selective interneurons. While this is an intriguing model consistent with their data, the authors do not provide convincing evidence for such a model or mechanism. In particular, recordings of interneurons are missing to support the author's conclusion and alternative hypotheses have not been addressed.

We agree with the Reviewer that recordings from interneurons would greatly increase the understanding of the mechanism of nicotinic modulation. It is known that different types of interneurons are modulated by nicotinic receptors. To dissect out those involved in nicotinic mediated control of CA2 circuit would require a significant effort and imply the use of several mouse strains. Hence, we feel that this important endeavour is out of the scope of this work but will be surely be a matter for future investigations.

7. Previous studies (Malezieux et al., 2020 and Dannenberg et al., 2015) have shown that brain states associated with high cholinergic activity and theta oscillations result in reduced firing of CA3 principal neurons via activation of interneurons. If the net effect of acetylcholine release in CA2 is activation of principal neurons via disinhibition as opposed to inhibition of principal neurons as shown in CA3, that would be a very interesting finding.

As suggested by Reviewer 1, we obtained a new data set from slice recordings showing that nicotinic activation (both using nicotine or hM3-CNO strategy) decreases the frequency of spontaneous glutamatergic transmission in the CA3 region (Figure 7—figure supplement 2 and Figure 8). This suggests that indeed, in contrast to CA2, the CA3 region is inhibited by nicotinic activation. in vivo experiments using juxtacellular recordings combined with photo stimulation of ChAT⁺ neurons in the MSDB were performed to assess the effect of ACh release on the CA3 output. Photo stimulation did not change the frequency of spontaneous firing in CA3. We included these results in a new supplementary figure (Figure 9—figure supplement 2). This may be related to the concomitant activation of mAChRs, which would probably counteract the depressing effect of nAChRs on glutamatergic transmission.

However, the authors should discuss the effect of tiletamine/xylazine anesthesia in their experiments.

To address this point, in a subset of experiments we used a different mix of anesthetics (ketamine-xylazine). Photo stimulation of MSDB ChAT⁺ neurons under these conditions significantly increased the firing of CA2 bursting neurons, mimicking the results obtained in the presence of previously used anaesthetics (zoletyl-xylazine). These results were included in a new supplementary figure (Figure 9—figure supplement 1).

Moreover, the authors could address alternative explanations. For example, cholinergic stimulation could result in a general increase of network activity resulting in higher firing rates in both interneurons and principal neurons. Data on interneurons would help distinguish between those hypotheses.

Data from interneurons will be important, but we think it would be too demanding since we expect different interneuron behavior in response to photo stimulation of MSDB cholinergic inputs (i.e. different classes of interneurons should be identified by a post hoc morphological analysis).

8. I couldn't find details on optogenetics experiments in the Methods section (e.g., light power, wavelength, viral construct).

The information regarding the light power and wavelength was added in the Method section (Lines 599-603), the information pertaining to the viral construct that was used was already described (former line 329, now lines 425-432).

References:

The suggested references were added to the Reference list

Further comments:

1. Title and elsewhere (e.g., line 69): "Septal cholinergic input (…) controls social memory (…)." The wording "controls social memory" is very vague. Can the authors describe the major finding of the manuscript more precisely?

The title was changed accordingly :“Septal cholinergic input to CA2 hippocampal region controls social novelty discrimination via nicotinic receptor-mediated disinhibition”.

2. Lines 29-30 and elsewhere: Patch clamp recordings in hippocampal slices are usually referred to as "in vitro" experiments as opposed to "ex vivo". Is there any specific reason why the authors have chosen "ex vivo" as opposed to "in vitro"?

We chose “ex vivo” since we think it is more appropriate for acute slices. “in vitro” definition is usually referred to cell culture or organotypic slices.

3. Lines 59: The authors could cite more recent reviews of the cholinergic system in addition to the reference to Teles-Grilo Ruivo and Mellor (2013), for example Dannenberg et al. (2017) and Haam and Yakel (2017).

The suggested Reviews were included in the manuscript (Lines 59-60).

4. Figure 2: I find the way the symbols and colors are used confusing. For example, the circle symbol is used for Animal, Object, Novel, and Familiar. It is further confusing that in E, orange and blue colors are used to indicate Novel and Familiar, but in B, D, and F, orange and blue colors are used to indicate eYFP and TeNT.

The colours of the objects in the schematic drawings were changed.

5. Line 173: "isolated pharmacologically". How?

The sentence “pharmacologically isolated spontaneous inhibitory and excitatory postsynaptic currents, were recorded before and after CNO (10 M) application in the presence of CNQX (10 µM), gabazine (10 µM) and physostigmine (3 M) to block AMPA, GABA-A receptors and acetylcholinesterase respectively” was added (Lines 215218).

[Editors’ note: what follows is the authors’ response to the second round of review.]

Reviewer #1:

This study is an important contribution to the field and is likely of interest to a broad readership of neuroscientists. The revised manuscript is much improved from the first submission and presents some exciting new data. Although the authors addressed most of the reviewer concerns, there are still a number of issues that should be addressed in the text before it is acceptable for publication. These, along with some minor issues, are listed below.

1. The authors now include data from Novel Object Recognition and Object Location tests (Figure 3E, F), however they still did not show whether investigation of a Novel Object induces Fos in a way similar to that induced by the social stimulus. The data now included showing Fos in response to exploration of an empty arena and home cage controls are important additions, but the possibility that an increase in Fos staining in CA2 may not be selective for a social stimulus should be clearly acknowledged and discussed since a similar response to a novel object has not been ruled out. This is an important point given that place field remapping occurs with novel objects to a similar degree as that occurring with a social stimulus (Alexander, 2016).

We agree with the Reviewer that our experiments do not rule out the possibility that, in the presence of a new object, the same degree of c-Fos activation would occur in CA2. We have now discussed this issue in the Discussion section “However, we cannot state that the increase in c-Fos staining in CA2 is selective for social stimuli since a similar response to a novel object has not been assessed (Alexander et al., 2016)”. Line 358

2. The authors should address the locations of the cannulas, several of which look like they were in CA1. In this case, they should acknowledge in the discussion the limitations of the method.

We think that the limitation of the method is stated in the sentence “Hence this finding warrants further support that ideally would be based on another experimental approach” already present in the Discussion section. Line 364. We added in the method section the maximal distance from CA2 we considered as an acceptable correct placement (within 350 µm from the CA2 border to CA1). Line 635

3. It was interesting that most of the Fos+ neurons in CA2 were likely interneurons, and so it is somewhat surprising that the authors still do not show recordings from interneurons. The findings do not appear to be overstated ( "…provides insight into the mechanism" in the abstract), but some discussion stating the limitations of the study is warranted. These recordings would be critical for supporting the authors model of cholinergic regulation of CA2 output, and so the authors are encouraged to submit those recordings as an eLife "Research Advance".

We agree with the Reviewer about the need to record from interneurons and we added a sentence in the discussion “Recordings from GABAergic interneurons will be critical to identify how different subtypes contribute to social memory via nAChR-mediated disinhibition”. Line 409

4. The graphic is confusing in light of (1) the new data showing the increase in Fos in the interneurons, (2) no recordings from interneurons, and (3) there is some discrepancy with the observation of increased firing of CA2 pyramidal cells in response to opto-stimulation. I suggest deleting the graphic unless it can be revised to better reflect the findings.

We agree with the Reviewer and we removed the graphic (Figure 10) from the manuscript.

Reviewer #2:

In the revised version of the manuscript, the authors have added new experimental support to their previous point, which substantially helped to strengthen the points made. Increasing the n numbers for several measures also helped to make sure the conclusions are solid. I am more convinced now about the mechanism that they proposed, stating that ACh, via nicotinic receptors, is necessary for encoding social novelty. However, although is interesting that they provide several possible models at the end, I still regret the lack of more specific data in relation to the type of interneurons. Overall, I think the authors made a decent job tackling most of the points raised in the previous review round and the information provided in this manuscript will be altogether of interest for the community.

Figure 1

– Units in figure 1 for axon density? Should it be "fluorescence density (AU, X20)"?

The axis label refers to the density of fluorescent axons counted in bins of 50x50 µm from images acquired with a 20x objective as described in the Method section. Lines 669-678

– 676 line reads: "stacks of 7 focal planes with a distance of 2?m" should it be "stacks of 7 focal planes with a distance of 2mm".

The unit has been corrected as µm. Line 671.

– No background subtraction was used I assume?

The Reviewer is correct, no background subtraction was used.

Figure 2

– This figure, still compares "stimulus" versus "non-stimulus" condition, without assessing whether the responses cFos+ are exclusive for an animal (social) or could be anything, object, spatial trajectory, foraging…

Experiments shown in figure 2 were performed to exclude c-Fos activation due to the new context, as discussed in point 1 raised by Reviewer 1.

Figure 7

– Scales missing in A and C traces (or are they all the same one?), please clarify.

Scales in A and C are the same of E.

Figure 8

– Scales missing in A and C traces.

Scales in A and C are the same of E.

Figure 9

– Duration of the bursts would also be informative.

The duration of bursts was analyzed and no significant differences between control and light were detected (Control: 26.4 ± 6.9, Light: 25 ± 5.5 ms; p=0.62, Wilcoxon test).

– Number of bursts would also be informative.

The burst number was analyzed and no significant differences between control and light were detected (Control: 24 ± 4.7, Light: 29 ± 6.3; p=0.41, Wilcoxon test).

Figure 5—figure supplement 2

– Temporal scale missing in traces showed in A.

Temporal scale in A and C is the same.

– Scales missing in traces showed in E.

Scales in E and G are the same.

– Y labels in I and J are confusing (shown in "%"), sIPSCs frequency is shown in Hz (in the above plots), so perhaps the authors meant difference between conditions or ratio compare to control in these plots?

In I and J we plotted the % of changes between pre- and post- CNO application to compare the difference among conditions (i.e. CNO effect in naïve vs hM4 expressing mice) as requested by Reviewer 3 (second question point 5) in the previous round of revision.

Figure 7—figure supplement 1

– Temporal scale missing in trace showed in A.

Temporal scale in A and C is the same.

Figure 7—figure supplement 2

– Temporal scale missing in trace showed in A.

Temporal scale in A and C is the same.

Figure 9—figure supplement 1

– Temporal scale missing in trace showed in B.

Temporal scale in B and F is the same.

Figure 9—figure supplement 2

– It is interesting that the spiking frequency of CA3 neurons is not affected at all by optical stimulation of MSDB, despite the frequency of sEPSCs being affected by nicotine.

We agree with Reviewer 2 that this is an interesting point. Our results may suggest that sEPSCs modulated by nicotine could be subthreshold events.

– Temporal scale missing in trace showed in B.

Temporal scale in B and F is the same.

Reviewer #3:

The revised manuscript entitled "Septal cholinergic input to CA2 hippocampal region controls social novelty discrimination via nicotinic receptor-mediated disinhibition" by Pimpinella et al. now presents additional experimental data and analyses that address previous concerns raised by reviewers. In particular, the authors now demonstrate that local delivery of CNO to the CA2 region of hM4-expressing mice impairs social novelty detection. Furthermore, the authors added in vitro experimental data that demonstrate an increase in spontaneous EPSCs in the presence of nicotine in CA2 but not in CA1 and CA3. In fact, a decrease in spontaneous EPSCs is observed in CA3. These new data provide evidence that nicotinic cholinergic receptors differentially affect network activity in hippocampal subregions and support the authors' hypothesis that cholinergic disinhibition of CA2 via nicotinic receptors is important for social novelty discrimination. Furthermore, the authors now report experimental results that demonstrate that social interaction increases cFos expression in cholinergic neurons in the medial septal complex further highlighting a significant activation and contribution of the septal cholinergic system during social interaction. I have few remaining comments, mostly addressing statistical analyses and the clarity of data presentation.

1. Regarding major point 2 (TeNT experiments): Do the authors now interpret their findings as an effect of TeNT on intrinsic firing properties? To more clearly distinguish between the alternative and null hypotheses that TeNT has or has not an effect on intrinsic firing properties, I suggest a statistical analysis using Bayes factor (Keysers, Christian, Valeria Gazzola, and Eric-Jan Wagenmakers. "Using Bayes Factor Hypothesis Testing in Neuroscience to Establish Evidence of Absence." Nature Neuroscience 23, no. 7 (July 2020): 788-99. https://doi.org/10.1038/s41593-020-0660-4.; Dienes, Zoltan. "Using Bayes to Get the Most out of Non-Significant Results." Frontiers in Psychology 5 (2014): 781. https://doi.org/10.3389/fpsyg.2014.00781). The Bayes factor will indicate whether (a) the data support the null hypothesis or (b) the data support the alternative hypothesis, or (c) the sample size is too low to draw a conclusion.

We performed a statistical analysis using Bayes factor with the aim to distinguish between the alternative and null hypotheses. The Bayes factor was calculated from http://pcl.missouri.edu/bftwo-sample. The Value obtained was 1.2, hence based on Keysers et al., 2020 we cannot draw a conclusion in favour of null or alternative hypothesis meaning that we cannot claim that TeNT had effect on intrinsic firing properties.

2. Why does the eYFP virus has a different serotype (2/9) than the TeNT carrying AAV (DJ serotype)? The possibility that diverse tropism of eYFP carrying AAV vs TeNT carrying AAV may target different subtypes of cholinergic neurons – as stated by the authors in the rebuttal letter – should also be discussed in the manuscript itself.

An AAV with DJ serotype carrying the eYFP reporter was not available at the time we performed the experiments.

The possibility that diverse tropism of eYFP carrying AAV vs TeNT carrying AAV may target different subtypes of cholinergic neurons – as stated by the authors in the rebuttal letter – should also be discussed in the manuscript itself.

Based on the statistical analysis suggested by the Reviewer we cannot draw any conclusion regarding the effect of TeNT on firing properties of MSDB cholinergic neurons. Thus, we think that discussing the issue of viral tropism is not relevant for the study. The properties of the viruses used in this study have been described in the Method section. Lines 426-434

3. To Figure 3C and Figure 4C: It is not immediately clear from looking at the figure if Novel and Familiar refers to an object or to an animal. I suggest to replace the circles with symbols that make this immediately clear to the reader (e.g., a drawing of a mouse in Figure 3C and drawing of an object in Figure 3A).

The circles have been replaced with drawings of mice in the main Figure 3 and 4.

4. Figure 4 —figure supplement 5: In my opinion, this figure should be part of main Figure 4 because it is an important control experiment and shows that chemogenetic inactivation of cholinergic neurons in the medial septal complex also affects spatial memory in addition to social memory, though the effect size appears smaller for effects on spatial memory compared to the larger effects on social memory. But I leave the final decision to the authors.

We decided to keep the figure concerning the OLT as supplementary because we think that adding the OLT experiments in the main Figure 4 would make it too crowded.

5. Line 211: "necessary" instead of "sufficient"?

The text has been changed accordingly. Line 209

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure 2—source data 1. Raw data of c-Fos quantification in the MSDB.

elife-65580-fig2-data1.xlsx^{(20.3KB, xlsx)}

Figure 3—source data 1. Interaction times and scores for three-chamber and novel object recognition tests.

elife-65580-fig3-data1.xlsx^{(12.3KB, xlsx)}

Figure 4—source data 1. Interaction times and scores for three-chamber and c-Fos quantification in CA2.

elife-65580-fig4-data1.xlsx^{(18.6KB, xlsx)}

Figure 5—source data 1. Interaction times and scores for three-chamber test.

elife-65580-fig5-data1.xlsx^{(13.6KB, xlsx)}

Figure 6—source data 1. Interaction times and scores for three-chamber test.

elife-65580-fig6-data1.xlsx^{(12.4KB, xlsx)}

Figure 7—source data 1. Frequency and amplitude of spontaneous excitatory postsynaptic currents .

elife-65580-fig7-data1.xlsx^{(12.8KB, xlsx)}

Figure 8—source data 1. Frequency and amplitude of spontaneous excitatory postsynaptic currents.

elife-65580-fig8-data1.xlsx^{(12.1KB, xlsx)}

Figure 9—source data 1. Spike analysis.

elife-65580-fig9-data1.xlsx^{(9.8KB, xlsx)}

Transparent reporting form

elife-65580-transrepform1.pdf^{(231KB, pdf)}

Data Availability Statement

Data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2–9.

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PERMALINK

Septal cholinergic input to CA2 hippocampal region controls social novelty discrimination via nicotinic receptor-mediated disinhibition

Domenico Pimpinella

Valentina Mastrorilli

Corinna Giorgi

Silke Coemans

Salvatore Lecca

Arnaud L Lalive

Hannah Ostermann

Elke C Fuchs

Hannah Monyer

Andrea Mele

Enrico Cherubini

Marilena Griguoli

Roles

Abstract

Introduction

Results

ACh released from cholinergic neurons of the MSDB is required for social novelty discrimination

Figure 1. Distribution of ChAT+ neurons in the medial septum/diagonal band of Broca (MSDB) and their axon fibers in the hippocampus.

Figure 2. c-Fos detection in a subset of medial septum/diagonal band of Broca (MSDB) ChAT+ neurons following social behavior and environment exposure.

Figure 3. Inhibition of acetylcholine (ACh) release from medial septum/diagonal band of Broca (MSDB) ChAT+ neurons impairs social novelty but not object recognition.

Figure 3—figure supplement 1. Intrinsic properties of TeNT-expressing ChAT+ neurons in the medial septum/diagonal band of Broca (MSDB).

Figure 3—figure supplement 2. TeNT expression in ChAT+ neurons in the medial septum/diagonal band of Broca (MSDB) does not affect locomotor activity.

Figure 4. Silencing of hM4-expressing ChAT+ neurons via systemic delivery of clozapine N-oxide (CNO) inhibits social novelty and c-Fos expression in the CA2 region.

Figure 4—figure supplement 1. Clozapine N-oxide (CNO) application inhibits the spontaneous firing of hM4-expressing ChAT+ neurons in the medial septum/diagonal band of Broca (MSDB).

Figure 4—figure supplement 2. c-Fos activation of PCP4− neurons after social interaction.

Figure 4—figure supplement 3. c-Fos activation in CA2 after empty arena exploration.

Figure 4—figure supplement 4. The inhibition of medial septum/diagonal band of Broca (MSDB) ChAT+ neurons does not affect c-Fos expression in the CA3 and CA1 regions.

Figure 4—figure supplement 5. Silencing of hM4-expressing ChAT+ neurons via systemic delivery of clozapine N-oxide (CNO) affects spatial memory.

Local release of ACh in the CA2 hippocampal region is necessary for social novelty discrimination

Figure 5. The inhibition of acetylcholine (ACh) release in the hippocampus impairs social novelty.

Figure 5—figure supplement 1. CA2 principal neuron properties.

Figure 5—figure supplement 2. Clozapine N-oxide (CNO) affects both inhibitory and excitatory neurotransmission in CA2 from ChAT-Cre mice-expressing hM4 in medial septum/diagonal band of Broca (MSDB) neurons but not from naive mice.

ACh controls social novelty discrimination via nAChR activation in CA2 neurons

Figure 6. Local application of nAChRs, but not mAChRs antagonists in CA2 affects social novelty.

Figure 6—figure supplement 1. Clozapine N-oxide (CNO) or cholinergic receptor antagonists did not affect locomotor activity within the social context (three chamber).

nAChR-mediated increase of glutamatergic transmission in CA2 principal cells via disinhibition

Figure 7. nAChR-mediated modulation of synaptic transmission in CA2.

Figure 7—figure supplement 1. nAChR-mediated modulation of miniature synaptic events in CA2.

Figure 7—figure supplement 2. nAChR-mediated modulation of synaptic transmission in CA3 and CA1 regions.

Figure 8. Activation of hM3-expressing cholinergic axon fibers modulates synaptic transmission in CA2 and CA3 but not CA1.

Figure 8—figure supplement 1. Clozapine N-oxide (CNO) application enhances the spontaneous firing of hM3-expressing ChAT+ neurons in the medial septum/diagonal band of Broca (MSDB).

Optogenetic activation of ChAT+ neurons in the MSDB increases the firing of CA2 neurons in vivo

Figure 9. Photoactivation of ChAT+ neurons in medial septum/diagonal band of Broca (MSDB) controls CA2 output.

Figure 9—figure supplement 1. Light delivery in MSDB combined with CA2 recordings in not injected or ChR2-expressing mice under ketamine–xylazine anesthesia.

Figure 9—figure supplement 2. Photoactivation of ChAT+ neurons in MSDB does not affect CA3 and CA1 output.

Discussion

Materials and methods

Animals

Viruses

In vivo stereotactic injections

Cannula implantation for local drug delivery

Drug and injection procedure

Behavioral experiments

Three-chamber test

Open-field exploration test

NOR test

Object location test

Slice preparation

Electrophysiological recordings in slices

Data analysis

Drugs

Electrophysiological recordings in vivo

Tissue preparation for immunohistochemistry

Histological verification of the cannula placements

Immunohistochemistry

Image acquisition

Analysis of axon density

Statistical analysis

Acknowledgements

Funding Statement

Contributor Information

Funding Information

Additional information

Competing interests

Author contributions

Ethics

Additional files

Data availability

Figure 1. Distribution of ChAT⁺ neurons in the medial septum/diagonal band of Broca (MSDB) and their axon fibers in the hippocampus.

Figure 2. c-Fos detection in a subset of medial septum/diagonal band of Broca (MSDB) ChAT⁺ neurons following social behavior and environment exposure.

Figure 3. Inhibition of acetylcholine (ACh) release from medial septum/diagonal band of Broca (MSDB) ChAT⁺ neurons impairs social novelty but not object recognition.

Figure 3—figure supplement 1. Intrinsic properties of TeNT-expressing ChAT⁺ neurons in the medial septum/diagonal band of Broca (MSDB).

Figure 3—figure supplement 2. TeNT expression in ChAT⁺ neurons in the medial septum/diagonal band of Broca (MSDB) does not affect locomotor activity.

Figure 4. Silencing of hM4-expressing ChAT⁺ neurons via systemic delivery of clozapine N-oxide (CNO) inhibits social novelty and c-Fos expression in the CA2 region.

Figure 4—figure supplement 1. Clozapine N-oxide (CNO) application inhibits the spontaneous firing of hM4-expressing ChAT⁺ neurons in the medial septum/diagonal band of Broca (MSDB).

Figure 4—figure supplement 2. c-Fos activation of PCP4⁻ neurons after social interaction.

Figure 4—figure supplement 4. The inhibition of medial septum/diagonal band of Broca (MSDB) ChAT⁺ neurons does not affect c-Fos expression in the CA3 and CA1 regions.

Figure 4—figure supplement 5. Silencing of hM4-expressing ChAT⁺ neurons via systemic delivery of clozapine N-oxide (CNO) affects spatial memory.

Figure 8—figure supplement 1. Clozapine N-oxide (CNO) application enhances the spontaneous firing of hM3-expressing ChAT⁺ neurons in the medial septum/diagonal band of Broca (MSDB).

Optogenetic activation of ChAT⁺ neurons in the MSDB increases the firing of CA2 neurons in vivo

Figure 9. Photoactivation of ChAT⁺ neurons in medial septum/diagonal band of Broca (MSDB) controls CA2 output.

Figure 9—figure supplement 2. Photoactivation of ChAT⁺ neurons in MSDB does not affect CA3 and CA1 output.