Oxytocinergic projection from the hypothalamus to supramammillary nucleus drives recognition memory in mice

Junpei Takahashi; Daisuke Yamada; Wakana Nagano; Yoshitake Sano; Teiichi Furuichi; Akiyoshi Saitoh

doi:10.1371/journal.pone.0294113

. 2023 Nov 16;18(11):e0294113. doi: 10.1371/journal.pone.0294113

Oxytocinergic projection from the hypothalamus to supramammillary nucleus drives recognition memory in mice

Junpei Takahashi ¹, Daisuke Yamada ¹, Wakana Nagano ¹, Yoshitake Sano ², Teiichi Furuichi ², Akiyoshi Saitoh ^1,^*

Editor: Peng Zhong³

PMCID: PMC10653413 PMID: 37971993

Abstract

Oxytocin (OXT) neurons project to various brain regions and its receptor expression is widely distributed. Although it has been reported that OXT administration affects cognitive function, it is unclear how endogenous OXT plays roles in cognitive function. The present study examined the role of endogenous OXT in mice cognitive function. OXT neurons were specifically activated by OXT neuron-specific excitatory Designer Receptors Exclusively Activated by Designer Drug expression system and following administration of clozapine-N-oxide (CNO). Object recognition memory was assessed with the novel object recognition task (NORT). Moreover, we observed the expression of c-Fos via immunohistochemical staining to confirm neuronal activity. In NORT, the novel object exploration time percentage significantly increased in CNO-treated mice. CNO-treated mice showed a significant increase in the number of c-Fos-positive cells in the supramammillary nucleus (SuM). In addition, we found that the OXT-positive fibers from paraventricular hypothalamic nucleus (PVN) were identified in the SuM. Furthermore, mice injected locally with CNO into the SuM to activate OXTergic axons projecting from the PVN to the SuM showed significantly increased percentage time of novel object exploration. Taken together, we proposed that object recognition memory in mice could be modulated by OXT neurons in the PVN projecting to the SuM.

Introduction

Oxytocin (OXT) is a peptide hormone that is synthesized in the paraventricular hypothalamic nucleus (PVN) and the supraoptic nucleus (SON). Projections of OXT neurons are widely distributed in the regions responsible for cognitive function. For example, previous reports discovered that OXT neurons projects to the hippocampus (Hip), perirhinal cortex, entorhinal cortex (Ent), and supramammillary nucleus (SuM) [1, 2]. Additionally, OXT receptors are reported to be expressed in the Hip, Ent, and SuM [1, 2]. The OXT receptor, a 7-transmembrane G protein-coupled receptor capable of binding to either G_i/o or G_q/11 proteins, activates a set of signaling cascades, such as the MAPK, PKC, PLC, CaMK, and CREB pathways [1, 3, 4]. The OXT receptors in the hippocampal dentate gyrus (DG) are mainly expressed on GABAergic neurons [5], whereas the OXT receptors in the CA1, CA2, and CA3 are expressed on glutamatergic neurons [5, 6]. Moreover, OXT has been shown to regulate social memory. Mice lacking OXT or OXT receptor gene in the CA2 area exhibited aberrant social memory [5, 7]. Furthermore, OXT neuron-specific deficiency of Ca²⁺-dependent activator protein for secretion 2 (CAPS2), which regulates dense core vesicle exocytosis in OXT neurons, impairs social memory [8]. These reports suggest that endogenous OXT neurons play a pivotal role in regulating learning and memory functions, although the aspects regarding non-social memory are unclear.

Previous reports suggested that SuM, above the mammillary bodies, has an important role in learning and memory [9, 10]. Neurons in the SuM directly project to DG and CA2. SuM neurons regulate hippocampal theta rhythm [9, 11, 12]. The SuM-DG projections regulate new environmental memory, and the SuM-CA2 projections regulate social memory. Moreover, OXT receptors and OXTergic axons are localized in the SuM [12, 13]. In vitro electrophysiological experiments demonstrated that the application of OXT receptor agonist (Thr4,Gly7)-oxytocin increased the neuronal spikes of the SuM; these effects were antagonized when treated with the OXT receptor antagonist, (d(CH2)51,Tyr(Me)2,Thr4,Orn8,des-Gly-NH29)-Vasotocin [13]. It is unclear whether activation of endogenous OXTergic projections regulates OXT receptor-mediated neuronal activity in the SuM.

A previous study reported that intracerebroventricular administration of OXT enhanced long-term spatial and object memories [3, 14, 15]. Another study indicated that OXT perfusion in hippocampal slice enhanced the late phase of long-term potentiation (L-LTP) in electrophysical studies [3, 14]. Recently, we reported that OXT perfusion in hippocampal slice reversed Aβ-induced impairment of LTP [16]. Furthermore, intracerebroventricular and internal administration of OXT reversed Aβ-induced spatial memory impairment, as demonstrated by the undertaken Morris Water Maze (MWM) and Y-maze test [17]. With these results, we proposed that the administration of exogenous OXT in the central nervous system maintains or reinforces learning and memory. However, endogenous OXT’s role in cognitive function remains unclear.

Although exogenous OXT has been reported to regulate learning and memory performance, studies clarifying whether endogenous OXT regulates learning and memory are limited. Therefore, the present study employed a chemogenetic approach to activate OXTergic neurons in the PVN specifically. Here, the OXTergic neurons activation, projecting from the PVN to the SuM, enhanced mice object recognition memory.

Materials and methods

Animals

The Institutional Animal Care and Use Committee at Tokyo University of Science approved all the experimental protocols; these were conducted following the National Institute of Health and Japan Neuroscience Society guidelines and ARRIVE guidelines (approval no. Y22014). Total of 51 mice (3- to 4-months-old male Oxt-iCre knock-in mice, C57BL/6-Oxt^{tm1.1(Cre)Ksak}) [8] were used. All animals had free access to food and water in an animal room, with temperature (23°C ± 1°C) and relative humidity (45% ± 5%) and with a 12-h light-dark cycle (lights were automatically switched on at 8:00 am) and 3–5 mice per cage.

Stereotaxic surgery

For surgery, Oxt-iCre mice were used. All mice were deeply anesthetized by intraperitoneal injection of a mixed solution of 0.75 mg/kg medetomidine (NIPPON ZENYAKU KOGYO CO., Ltd. Fukushima, Japan), 4.0 mg/kg midazolam (Maruishi Pharmaceutical Co. Ltd, Osaka, Japan), and 5.0 mg/kg butorphanol (Meiji Animal Health Co., Ltd, Kumamoto, Japan) dissolved in saline. Then, mice were placed in a stereotaxic apparatus. After the surgery, the mice were administered atipamezole (0.75 mg/kg, i.p.: Kyoritsu Seiyaku Co., Tokyo, Japan) to reverse the sedation. Then, the mice were monitored for ≥5 days for recovery (normal eating, drinking, and defecation).

Virus injection

OXT neurons were chemogenetically activated by Cre-dependent transfection of DREADD, hM3Dq. Male Oxt-iCre mice were injected with recombinant adeno-associated virus (hSyn-DIO-hM3D(Gq)-mCherry-AAV8; 1.25×10¹³ virus molecules/ml; Addgene) into the PVN (anterior–posterior: −0.8 mm, mediolateral: ±0.3 mm, dorsoventral: −5.2 mm). A Hamilton 10-μl syringe with a 30-gauge blunt-end needle was inserted into the brain for virus delivery. The virus was bilaterally injected at a speed of 100nl/min for 200nL per site using a pump (UMC4, Florida, USA). To activate hM3Dq, mice were administered clozapine N-oxide (CNO, 1 mg/kg, i.p.: ENZ Enzo Life Sciences, Inc. BML-NS105-0025). CNO was dissolved in saline.

Chemogenetic pathway-selective activation

A guide cannula (BRC bio research center, Ibaraki, Japan) was implanted in the SuM (anterior–posterior: −2.8 mm, mediolateral: ±0.3 mm, dorsoventral: −5.0 mm) for pathway-selective activation by hM3Dq. Guide cannulas were firmly fixed to the skull using dental cement. Finally, a dummy cannula (Plastics one, BRC bio research center, Ibaraki, Japan) was inserted in the guide cannula to avoid clogging. One week after recovery, 3 μM CNO (200 nL) was infused locally into the corresponding brain area at a speed of 300 nL/min through an internal cannula (Plastics one, BRC bio research center, Ibaraki, Japan) connected to the guide cannula. CNO concentration was determined based on previous studies [18–20].

Immunohistochemistry

All mice were anesthetized by intraperitoneal injection of a mixed solution of medetomidine (0.75 mg/kg), midazolam (4.0 mg/kg), and butorphanol (5.0 mg/kg) dissolved in saline. Mice were perfused transcardially with PBS followed by 4% paraformaldehyde (PFA, Nacalai tesque Inc, Kyoto, Japan). Brains were dissected, postfixed in 4% PFA overnight, and subsequently immersed in 30% sucrose in PBS for cryoprotection. Following the fixation and cryoprotection, tissues were embedded in O.C.T compound (catalog #4583, Sakura-Finetek, Tokyo, Japan) and frozen at −80°C. The brains were sectioned coronally and sagittally with a thickness of 50 μm. Brain sections were stored at −20°C in cryoprotection solution (30% ethylene glycol, 25% glycerol in 1× PBS).

In IHC, stored brains were washed using PBST (0.2% Triton X-100 in PBS) three times and subsequently deactivated with 0.3% hydrogen peroxide in PBST for 5 min. Subsequently, sections were washed using PBST three times and blocked with blocking solution [3% BSA (catalog #001-000-162, Lot; 90246 Jackson ImmunoResearch, Pennsylvania, USA) in PBST] for 1 h. Then, the sections were incubated with primary antibody diluted with blocking solution for two overnights. After washing with PBST, the sections were incubated with secondary antibody [biotinylated α-rabbit IgG (catalog #BA-1100, vector, California, USA)] diluted with blocking solution for 90 min. Furthermore, sections were washed using PBST three times and incubated with AB solution (catalog #PK6100, vector, California, USA) for 60 min. Subsequently, sections were washed using PBST three times and then incubated with DAB (catalog #SK4100, vector, California, USA) for six min. After washing with PBST three times, the sections were treated with hematoxylin (catalog# 8656, Sakura-finetek, Tokyo, Japan) for 5 min. Then, the sections were washed using PBST three times and attached to the glass slides. Finally, the sections were dipped in hexane and mounted with Eukitt (catalog# 6.00.01.0002.04.01.EN ORSAtec, Bobingen, Germany). NDP viewer (HAMAMATSU PHOTONICS, Shizuoka, Japan) was used to obtain the digital images.

For detecting c-Fos and mCherry, we used rabbit-c-Fos antibody (catalog # 226 003, Lot; 4–66, Synaptic Systems, Göttingen, Germany), rabbit-Ds Red antibody (catalog #632496, Clontech, Fitchburg, USA).

Analysis of c-Fos and mCherry

A digital file containing a brain atlas was superimposed over each photomicrograph to specify the brain regions. The number of c-Fos-positive neurons in the CA1, CA2, CA3, Perirhinal cortex (PRh), Ent, and Cingulate/Retrosprenial cortex (Cg/RS) was counted manually by an observer blinded to the treatment conditions. Five to six sections were used to analyze c-Fos positive cells in hippocampal regions (CA1, CA2, CA3, and DG). Two to three sections were used to analyze c-Fos positive cells in the other brain regions (PVN, SuM, Ent, PRh and Cg/RS). We analyzed the dorsal portion of the Hip, because dorsal, but not ventral Hip, is reportedly involved in spatial memory and object memory [21–24]. We examined c-Fos positive neurons following NORT in this study. To analyze c-Fos counting, we referred to several reports [25–27]. These reports analyzed c-Fos positive neurons in several brain regions by using unpaired t-test. The distribution of OXTergic projections was identified as follows. OXT-iCre mice were injected with AAV8-hSyn-DIO-hM3Dq-mCherry in the PVN to specifically express the mCherry on OXTergic neurons. Then, the distribution of mCherry-positive neurons or fibers was detected by immunohistochemical staining against mCherry.

Y-maze test

The spatial working memory was examined by measuring the spontaneous alternation behavior of the mice in the Y-maze test, as described previously with some modifications [17]. The maze was made of black acrylic board. Each arm was 40 cm long, 12 cm high, 3 cm wide at the bottom, 10 cm wide at the top, and converged at an equal angle. Each mouse was placed at the end of one arm and allowed to move freely through the maze during an 8 min session. The series of arm entries were recorded visually. Alternation was defined as successive entries into the three arms on overlapping triplet sets. The effect was calculated as the percent alternation following the formula: Alternation (%) = ((number of alternations)/(total number of arm entries– 2)) × 100 (%). The arms were wiped down with paper between sessions.

Novel object recognition test (NORT)

The object memory was examined by measuring the exploring novel object behavior of the mice in the NORT, as described previously with some modifications [28]. NORT consists of three tasks: Habituation, training, and test. These tasks were performed in an open-field apparatus (30 × 30 × 30 cm). We used object A (cube) and object B (snowman); there were no differences in preference between the objects.

First, mice explored in open-field-apparatus for 5 min in habituation task. Twenty-four hours after the habituation task, mice explored in open-field-apparatus where objects A or B were placed for 10 min or until the total time spent exploring the objects to 20 s. We conducted counterbalance during training session. Half of the mice were presented with Object A and the other half with Object B. Then 72 h after the training task, mice explored in open-field-apparatus where object A and B were placed for 10 min. Object recognition memory was assessed by measuring the exploration time of novel objects during the test. We counted the object exploration time in a blinded fashion. Object exploration time was referred as the time the mouse touches an object. Exploration time of novel object (%) = (time of exploration time of object B) / (total time of exploration time of objects) × 100 (%). Data was manually analyzed.

Statistical analysis

Data were represented as mean ± SEM. The significance of differences in data was evaluated using unpaired t-test. Analyses were performed using Graphpad Prism (Graphpad Software Inc., San Diego, CA, USA); P values <0.05 were considered statistically significant.

Results

Projection of OXTergic neurons in PVN

We investigated the brain regions where receive projections of OXTergic neurons in the PVN. Mice injected with AAV8-hSyn-DIO-hM3Dq-mCherry in the PVN were perfused 4–6 weeks later and were analyzed expression of mCherry in OXTergic projection areas by immunohistochemical staining. mCherry-positive cells were identified in PVN (Fig 1A), and mCherry-positive fibers were identified in PVN (Fig 1A) and SuM (Fig 1B).

Fig 1 — Immunohistochemical staining of mCherry 4–6 weeks after injecting AAV8-hSyn-DIO-hM3Dq-mCherry into the PVN. Image of the PVN (A) and SuM (B).

The number of c-Fos positive cells in PVN after administration of CNO

We examined whether OXTergic neurons in Oxt-iCre mice with hM3Dq-AAV infection were activated by administering CNO. An overview of the experimental schedule is presented in (Fig 2A). We perfused mice 120 min after an administration of CNO (1mg/kg, i.p.) followed by neural activity analysis by immunostaining for c-Fos (Fig 2B and 2C). We counted the number of c-Fos positive cells in PVN. There were significantly more c-Fos positive cells in CNO treatment than saline treatment in PVN (Fig 2D; t(7) = 4.331, P = 0.0034, unpaired t-test, **P < 0.01).

Fig 2 — Immunohistochemical staining of c-Fos in the PVN 120 min after an intraperitoneal CNO administration (1.0 mg/kg). An overview of the experimental schedule is presented in (A). Image of PVN in saline-treated mice (B) and CNO-treated mice (C). The number of c-Fos positive cells in the saline and CNO (D). Data represents mean ± SEM (n = 4–5). Statistical analyzes were performed as unpaired t-tests. **P < 0.01.

Effects of activation of OXTergic neurons in PVN on the Y-maze test and NORT

We examined whether activating OXTergic neurons affects learning and memory. Fig 3A presents the experimental schedule in Y-maze. The administration of CNO (1 mg/kg, i.p.) did not affect spontaneous alternation (Fig 3B; t(13) = 0.1564, P = 0.8781, unpaired t-test, ns: not significant) and total arm entries (Fig 3C; t(13) = 1.533, P = 0.1493, unpaired t-test, ns: not significant) in Y-maze. Fig 3D presents the experimental schedule in NORT. The CNO (1 mg/kg, i.p.) -treated mice did not affect the exploration time of the object during the training session than saline-treated mice (Fig 3E; t(19) = 0.4889, P = 0.6305, unpaired t-test, ns: not significant). The CNO treatment mice had more exploration time of novel object (%) during the test session than saline-treated mice (Fig 3F; t(21) = 4.437, P = 0.0002, unpaired t-test, ***P < 0.001).

The number of c-Fos positive cells after NORT

We stained c-Fos 120 min after the NORT to examine which brain regions were involved in enhancing object recognition memory. An overview of the experimental schedule is presented in (Fig 4A). We counted the number of c-Fos positive cells in several brain regions. We found that the number of c-Fos positive cells was significantly increased in CNO-treated mice compared to saline-treated mice in DG (Fig 4B, 4C and 4F; t(12) = 2.645, P = 0.0214, unpaired t-test, *P < 0.05) and SuM (Fig 4D, 4E and 4G; t(12) = 2.265, P = 0.0428, unpaired t-test, *P < 0.05). However, there was no difference between the number of c-Fos positive cells in CNO-treated mice compared to saline-treated mice in CA1, CA2, CA3, PRh, Ent and Cg/RS (Table 1; CA1, t(5) = 0.4197, P = 0.6921, unpaired t-test, ns: not significant); (Table 1; CA2, t(5) = 2.068, P = 0.0935, unpaired t-test, ns: not significant); (Table 1; CA3, t(5) = 0.05772, P = 0.9562, unpaired t-test, ns: no-significant);. (Table 1; PRh, t(9) = 1.000, P = 0.3432, unpaired t-test, ns: not significant); (Table 1; Ent, t(9) = 0.5006, P = 0.6287, unpaired t-test, ns: not significant); (Table 1; Cg/RS, t(10) = 1.163, P = 0.2717, unpaired t-test, ns: not significant).

Fig 4 — Immunohistochemical staining of c-Fos 120 min after the NORT. An overview of the experimental schedule is presented in (A). Image of the DG in saline-treated mice (B). Image of the DG in CNO-treated mice (C). Image of the SuM in saline-treated mice (D). Image of the SuM in CNO-treated mice (E). The number of c-Fos positive cells in the DG (F) and SuM (G). Data were presented as the mean ± SEM (F; n = 7: E; n = 6–8). Statistical analyzes were performed as unpaired t-tests. *P < 0.05, ns: no-significant.

Table 1. Expressions of c-Fos in the various brain areas after NORT.

Immunohistochemical staining of c-Fos 120 min after the NORT. Table showed the number of c-Fos positive cells in the PRh, Ent, CA1, CA2, CA3 and Cg/RS. Data were presented as the mean ± SEM. Statistical analyzes were performed as unpaired t-tests.

Area	Saline (n)	CNO(n)	P value
PRh (perirhinal cortex)	53±21　(5)	82±15　(5)	0.30
Ent (entorhinal cortex)	74±18　(5)	76±8.8　(5)	0.90
CA1	3.8±0.3　(3)	3.4±0.9　(4)	0.70
CA2	1.2±0.15　(3)	0.8±0.12　(4)	0.09
CA3	13±3.8　(3)	13±0.9　(4)	0.96
Cg/RS (cingulate/retrosplenial cortex)	100±24　(5)	143±25　(7)	0.27

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Effects of activation of OXTergic neurons in SuM by NORT

We implanted a guide cannula in SuM of mice to examine if OXTergic fibers in SuM were involved in enhancing object recognition memory. An overview of the experimental schedule is presented in (Fig 5A). Mice received a local injection of saline or CNO (3 μM, 200 nL) to SuM 20 min before the training session in NORT. The local injection of CNO did not affect exploration time of object during the training session compared to saline-treated mice (Fig 5B; t(8) = 0.8773, P = 0.4059, unpaired t-test, ns: not significant). CNO-treated mice exhibited a significant increase in exploration time of novel object (%) during the test session compared to saline-treated mice (Fig 5C; t(8) = 2.419, P = 0.0419, unpaired t-test, *P < 0.05).

Fig 5 — Mice received a local injection of saline or CNO (3μM, 200nL) to SuM 20 min before the training session in NORT. An overview of the experimental schedule is presented in (A). Exploration time of object (%) during the training session (B). Exploration time of novel object (%) during the test session (C). Data were presented as the mean ± SEM (B, C; n = 5). Statistical analyzes were performed as unpaired t-tests. *P < 0.05, ns: no-significant.

Discussion

For the first time, this study found that endogenous OXT participates in object recognition memory via SuM. To investigate the projections of OXTergic neurons in PVN to different brain regions, we analyzed mCherry expression with ABC staining methods. Results show that mCherry-positive cells observed in PVN and mCherry-positive fibers were observed in SuM. These results suggested that OXTergic neurons in PVN projected to SuM, similar to previous reports [1, 2]. However, mCherry-positive fibers were not observed in Hip (include in CA1, CA2, CA3 and DG). It was reported that alkaline phosphatase staining did not detect OXT neurons projection in the Hip by using Oxt ^cre/+; Z/AP mice [29]. It is possible that the areas where OXT neuron projections could be detected depend on the mouse type and staining methods used.

CNO treatment increased the number of c-Fos positive neurons in the PVN. To ascertain that the identified post-treatment c-Fos positive neurons were indeed OXTergic neurons, we detected c-Fos and OXT neurons by immunofluorescence staining. We found that the double-labeled neurons (c-Fos⁺ and OXT⁺ neuron) were detected in PVN of CNO-treated mice (S3 Fig). These results suggested that c-Fos positive neurons were OXTergic neurons.

There was no difference in spontaneous alternation in the Y-maze test between saline- and CNO-treated mice, suggesting that endogenous OXT does not affect short-term spatial memory. In NORT, naïve mice had approximately 70% of the exploration time to novel object after 6 h of the training task (S1 Fig). Therefore, naïve mice could maintain object recognition memory after 6 h of training task. However saline-treated mice exhibited about 50% of the exploration time to novel object after 72 h of the training task, indicating that normal mice could not maintain object recognition memory after 72 h of the training task. Interestingly, CNO-treated mice exhibited about 70% of the exploration time to novel object during NORT, suggesting that CNO-treated mice could distinguish a new object from an old object. Thus, we propose that endogenous OXT enhances long-term object recognition memory. Our results are compatible with previous study in which OXT improved hippocampal L-LTP, which is important to maintain memory, but not early-phase LTP [3, 14]. Moreover, it has been reported that OXT enhanced long-term, but not short-term, spatial memory in eight-arm radial maze task in rodents [3]. Thus, previous studies and our findings suggest that OXT regulates cognitive functions in long-term, but not short-term, memory.

Following NORT, CNO treatment upregulated the number of c-Fos positive neurons in the SuM and DG. These results suggested possibility that activating OXTergic neurons enhanced the maintenance of long-term memory in mice via neurons in DG and SuM. Several reports suggested that neurons in DG and SuM are involved in object recognition memory. Ex vivo electrophysiological experiment demonstrated that the ratio of membrane currents mediated by AMPA receptor to NMDA receptor (AMPA/NMDA ratio), an index of neuronal plasticity, was increased in DG granule cells after NORT [30]. Moreover, bilateral lesions of dorsal DG in rats showed impaired discrimination of the two objects compared to the sham-operated rats [31], suggesting that dorsal hippocampal DG neurons modulate long-term object recognition memory. Additionally, selective activation of axons of SuM projection neurons in the medial prefrontal cortex improved impairment of object recognition memory in 5×FAD mice [32], suggesting that SuM is also involved in object recognition memory. Thus, we propose that activating DG and SuM neurons enhance object recognition memory by activating OXT neurons.

Finally, chemogenetic pathway-selective activation of OXTergic axons in SuM increased the exploration time to novel object, suggesting that OXTergic axons in SuM projecting from PVN modulate object recognition memory. Neurons in the SuM directly project to DG and CA2. They are also involved in learning and memory [9, 12]. Thus, this study’s findings indicate that OXTergic axons in SuM modulate object recognition memory via DG. The SuM-DG pathway modulates new environmental memory [9, 10]. Chen et al. injected AAV-DIO-ChR2-eYEP or AAV-DIO-eNpHR-eYEP into the SuM in SuM-Cre mice to manipulate the selective SuM-DG pathway [10]. The SuM-DG pathway, activated or inhibited with optogenetics, showed altered exploration behavior in contextual novelty environment [10]. Li et al. injected AAV-DIO-hM3Dq-mCherry or AAV-DIO-hM4Di-mCherry into SuM in mice. This study reported that CNO treatment increased c-Fos positive neurons in the DG of hM3Dq expressing mice, while CNO treatment decreased c-Fos positive neurons in the DG of hM4Di expressing mice. In addition, Li et al. injected AAV-DIO-Ch2R-mcherry and AAV-shVglut2 (known as a marker of glutamatergic neurons) into SuM in mice. Activating DG with optogenetics decreased c-Fos positive neurons in the DG of deletion of Vglut2 mice. Therefore, these results suggested that glutamatergic neurons in the SuM activate DG. Moreover, Li et al. assessed cognitive behavior by novel place recognition test and NORT [9]. CNO-treated mice could distinguish new place from old place by novel place recognition test. However, they could not distinguish a new object from an old object by NORT. Thus, contrary to our results, these findings suggested that glutamatergic neurons in SuM modulate spatial memory but not recognition memory [9]. This difference in the findings could be due to the difference in the time course of experiments. Li et al. conducted the test task 1 h after the training task, whereas we conducted the test task 72 h after the training task in NORT. These differences in hours after training (72 hours (long-term memory) vs. 1 hour (short-term memory)) may suggest that SuM neurons modulate the type of memory and time-dependent memory. Taken together, we proposed that OXTergic neurons in SuM projecting from PVN could enhance object memory via the activating of DG projecting from glutamatergic neurons in SuM.

Recent study reported that OXTergic axons in SuM modulate social memory via CA2 [33]. Keerthi et al. reported that the selective inhibition pathway of OXTergic axons in SuM decreased the investigation time to the novel rodents in social memory test [33]. Additionally, they found that the microinjection of OXT receptor antagonist in SuM decreased the investigation time to the novel rodents. Moreover, this report exhibited that 61% of vglut2 positive neurons in the SuM co-expressed OXT receptors with in situ fluorescent hybridization techniques. This indicates that OXTergic axons projecting to SuM from PVN modulates social memory via OXT receptors, which are predominantly expressed in the glutamatergic neuron in the SuM. Moreover, Keerthi et al. proposed that the PVN-SuM-CA2 pathway may be important for social memory, although they reported that it is unclear whether glutamatergic neurons with OXT receptors in SuM project to the hippocampal CA2 region [33]. Based on these reports, OXTergic axons in SuM modulate the SuM neurons projecting to DG; this neuronal participation enhances object recognition memory in NORT.

There are several reports demonstrating the modulatory effect of OXT on cognitive function. For example, we recently reported that OXT recovered impairment of spatial memory in Alzheimer’s disease (AD) model mice established by intracerebroventricular injection of β-amyloid protein (Aβ), commonly used to mimick the pathogenesis of AD [17]. Furthermore, OXT recovered Aβ_25–35-induced impairment of hippocampal long-term potentiation [16]. Other researchers also reported the therapeutic effects of OXT in AD-like symptoms in animal studies. Additionally, intranasal OXT attenuated aluminum chloride (AL)-induced impairment of cognitive function, decreased AL-induced elevation of Aβ_1–42, and Tau levels [34]. Interestingly, intravenous injection of OXT nanogel reversed spatial memory deficits of APP/PS1 mice in MWM [35]. Moreover, AD patients demonstrated a decrease in plasma OXT concentration compared to healthy individuals [36]. Although we cannot examine the direct relationship between AD and OXTergic neuron activity in this study, our result suggested that OXTergic neuron activity may be involved in each stage of AD pathological progression.

Female mice may exhibit differences since the distribution of OXT receptors and OXT projection areas differ between female and male mice [2]. However, we did not use female Oxt-iCre mice to avoid the possibility of recombination in all cells in a Cre expressing fertilized egg. Moreover, DAT-Cre KI mice show sex-dependent changes in amphetamine-induced locomotor activity [37]. This report suggested that the phenotypes of Cre-KI mice were sex-dependent. Thus, we used male Oxt-iCre mice in this study.

Conclusion

This study found that OXTergic projection from PVN to SuM drives recognition memory in mice for the first time. This study could be beneficial for future investigation of the role of physiological OXT in AD.

Supporting information

S1 Fig. The exploration preference to object in our laboratory conditions in NORT.

Figure showed the exploration time of object (%) during the training session and the test session. Data was presented as the mean ± SEM. (n = 8). Statistical analyzes was performed as unpaired t-tests. *P < 0.05.

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S2 Fig. The difference exploration time of object during the training session and the test session in NORT.

Figure showed the difference exploration time of object (s) during the training session and the test session. Test session was conducted 72 h after the training session Data was presented as the mean ± SEM. (n = 12). Statistical analyzes was performed as unpaired t-tests. **P < 0.001.

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S3 Fig. The immunohistochemical staining of c-Fos and OXT neurons.

Immunohistochemical staining of c-Fos and OXT neurons 120 min after administration of CNO. Magnification of 20x image of the PVN in saline-treated mice (A) and CNO-treated mice (B). Magnification of a 40X image of the PVN in saline-treated mice (C) and CNO-treated mice (D). Arrow indicate the double-labeled neurons (c-Fos + and OXT+).

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S1 Raw data

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S2 Raw data

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S3 Raw data

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S4 Raw data

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Acknowledgments

We would like to thank Enago (www.enago.jp) for the English language review.

Data Availability

Data are all contained within the Supporting Information files.

Funding Statement

This study was supported by a Grant-in-Aid for JSPS Fellows (Grant number JP 21J20036 to J.T.). https://www.jsps.go.jp/english/e-fellow/ YES -The funders had role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0294113.r001

Decision Letter 0

Peng Zhong

25 Aug 2023

PONE-D-23-22233Oxytocinergic Projection from the Hypothalamus to Supramammillary Nucleus Drives Recognition Memory in MicePLOS ONE

Dear Dr. Saitoh,

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Additional Editor Comments:

The findings are interesting. However, please provide the more convincing image demonstrating the projections of SuM. The reviewers' comments are needed to be addressed carefully, especially ones from Reviewer 1.

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Reviewer #2: Yes

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Reviewer #1: This manuscript describes results of experiments testing the influence of oxytocin projection neurons from periventricular nucleus (PVN) to the supramammillary nucleus (SuM) on long-term memory for objects. The excitatory DREADDs receptor, hM3Di was expressed in oxytocin neurons of the PVN, mice were subsequently administered systemic CNO and tested for the influence on c-Fos expression in SuM, spatial working in a Y-maze task, and object recognition memory. Systemic CNO enhanced the preference of the mice to explore the novel object during a test session conducted 72 h after the initial training session. A follow-up study demonstrated a similar enhancing effect after mice received local infusion of CNO into the SuM. The authors conclude that oxytocin projection neurons of the PVN play a significant role in object recognition memory. There is compelling evidence from prior reports that oxytocin influences social memory, and converging evidence has defined circuits supporting this effect. The present manuscript expands on this literature by demonstrating the involvement of oxytocin in a different form of recognition memory. The results are interesting and the breadth of the approach here is commendable. There are some issues outlined below that should be addressed.

1. The details of how the object recognition task was conducted are missing. (A) For example, page 7 (lines 174-175) indicates that object A and B were used, and line 177 indicates that two object A's were presented during the training session. The object recognition task is based on spontaneous responses of the mice, and one must be careful to counterbalance the objects presented. For example, it would be important for the training session to present two object A's to half of the mice, and two object B's to the other half of the mice. The authors state that there were no differences in preference between the objects; however, counterbalancing objects presented during training and testing brings rigor to the approach.

(B) The measure used to assess memory was preference ratio, which suffers from a limited range: chance performance in 50%, and substantial preference for the novel object is rarely more than 75%. A better measure of test session memory performance is the Discrimination Ratio, calculated by taking the difference score (time exploring object B - time exploring object A) and dividing the result by the total time spent exploring both objects. Discrimination ratio scores better account for overall differences in total object exploration between individual mice, and the data tend to have a wider range of scores than that of preference ratio. The discrimination ratio is a well accepted and more rigorous measure of object memory than preference ratio. The authors are encouraged to present their test session object recognition data as discrimination ratio scores.

(C) Page 7, line 182 states that the "Data was manually analyzed." The authors should provide more detail as to what this means. How was object exploration scored, what constituted object exploration, and who scored the data? How was scientific rigor upheld while manual analysis was occurring?

2. Details are missing as to how the c-Fos counts were conducted and analyzed. How many sections from each mouse were counted for each of the regions (CA1, CA2, CA3, ... etc.)? Were the counts conducted blind? c-Fos counts were normalized by the area of the respective region. However, how was this determined given the need to control for the thickness of tissue and the differences in the size of the respective structures across sections? Also, background varies across a piece of tissue and across slides, so what approach was taken to achieve consistency across slides?

3. The c-Fos data were analyzed by multiple t-tests - that is, an unpaired t-test (saline- vs. CNO-treated) for normalized c-Fos counts in each region. The authors should consider that conducting multiple t-tests carries the risk of yielding a significant outcome by chance. Given that the data were normalized (though see comment #2 above), why not instead conduct a two-way RMANOVA (treatment group as between-subjects variable, and region of interest as the repeated measures/within-subjects variable)?

4. The manuscript refers to Table 1, but this information was not provided.

Minor issues

a) page 6, line 153: the statement that "...dorsal, but not ventral Hipp[ocampus] is involved in cognitive function..." is not well supported by the literature. For example see: Jin & Maren (2015) Scientific Reports; Okuyama et al. (2016) Science; Contreras et al. (2018) Hippocampus; Rao et al, (2019) Cell Reports; Jimenez et al. (2020) Nature Communications; to name a few example references.

b) the manuscript contains an overwhelming number of acronyms (OXT, ORM, SuM, PVN, ABC, CNO, NORT, AD, etc.), which makes some of the sentences challenging to understand. This reviewer suggests limiting the acronyms to a minimal number of terms that are used throughout.

c) page 10, line 249-252: This sentence is quite difficult to understand. Please revise.

d) page 11, line 267: "competed" should be revised to "compared"

e) page 13, line 314-315: The statement "...our result suggested that OXTergic neuron activity may be involved in each stage of AD pathological progression." is not supported by the results described in the current manuscript.

Reviewer #2: 1. As stated by the author in the introduction, "Projections of oxytocin (OXT) neurons exhibit extensive distribution across regions associated with cognitive functions. For instance, prior studies have revealed that OXT neurons project to the hippocampus (Hip), perirhinal cortex, entorhinal cortex (Ent), and supramammillary nucleus (SuM)." However, the findings from immunohistochemical staining (Fig. 1) merely illustrate the fibers within the paraventricular nucleus (PVN) and supramammillary nucleus (SuM). It would be advantageous to incorporate outcomes from other cerebral regions, notably the hippocampus (DG).

2. Regarding c-Fos staining and quantification subsequent to CNO treatment, it is advisable for the authors to undertake additional verification to ascertain that the identified c-Fos positive neurons post-treatment indeed are OXTergic neurons.

3. Does the localized administration of CNO to the SuM have an impact on the DG? While the results section does not explicitly address the SuM-DG pathway, it extensively delves into this topic in the discussion. It would be better for the authors to substantiate their conceptual arguments in the discussion by presenting relevant empirical findings.

4. In the discussion section (page 11, line 270), the authors put forth the assertion that “Thus, we propose that activating DG and SuM neurons enhance ORM by activating OXT neurons.” It would be judicious for the authors to complement this proposal by providing evidential support. Specifically, they could present data that demonstrate the comparable effects observed in “Effects of activation of OXTergic neurons in SuM by NORT”, where local activation of oxytocinergic fibers in the DG produced similar outcomes.

5. In this study, only male mice were used as the subjects. Could potential distinctions arise in female mice?

**********

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Reviewer #1: Yes: Robert W. Stackman Jr.

Reviewer #2: No

**********

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PLoS One. 2023 Nov 16;18(11):e0294113. doi: 10.1371/journal.pone.0294113.r002

Author response to Decision Letter 0

26 Sep 2023

Responses to the comments raised by Reviewer #1

Reviewer #1 comment

This manuscript describes results of experiments testing the influence of oxytocin projection neurons from periventricular nucleus (PVN) to the supramammillary nucleus (SuM) on long-term memory for objects. The excitatory DREADDs receptor, hM3Di was expressed in oxytocin neurons of the PVN, mice were subsequently administered systemic CNO and tested for the influence on c-Fos expression in SuM, spatial working in a Y-maze task, and object recognition memory. Systemic CNO enhanced the preference of the mice to explore the novel object during a test session conducted 72 h after the initial training session. A follow-up study demonstrated a similar enhancing effect after mice received local infusion of CNO into the SuM. The authors conclude that oxytocin projection neurons of the PVN play a significant role in object recognition memory. There is compelling evidence from prior reports that oxytocin influences social memory, and converging evidence has defined circuits supporting this effect. The present manuscript expands on this literature by demonstrating the involvement of oxytocin in a different form of recognition memory. The results are interesting and the breadth of the approach here is commendable. There are some issues outlined below that should be addressed.

Reviewer #1 comment 1

The details of how the object recognition task was conducted are missing. (A) For example, page 7 (lines 174-175) indicates that object A and B were used, and line 177 indicates that two object A's were presented during the training session. The object recognition task is based on spontaneous responses of the mice, and one must be careful to counterbalance the objects presented. For example, it would be important for the training session to present two object A's to half of the mice, and two object B's to the other half of the mice. The authors state that there were no differences in preference between the objects; however, counterbalancing objects presented during training and testing brings rigor to the approach.

Response to comment 1

Thank you for your helpful suggestions. (A) We conducted counterbalance during the training session. Half of the mice were presented with Object A and the other half with Object B. We have now added this information to the Materials & Methods section (line 187). (B) We analyzed the difference score (time exploring new object – time exploring old object). These results were presented in supplement figure 2. (C) We counted the object exploration time in a blinded fashion. Object exploration time is referred to the time during which the mouse touches an object. We added this information to the Materials & Methods (line 190).

Reviewer #1 comment 2

Details are missing as to how the c-Fos counts were conducted and analyzed. How many sections from each mouse were counted for each of the regions (CA1, CA2, CA3, ... etc.)? Were the counts conducted blind? c-Fos counts were normalized by the area of the respective region. However, how was this determined given the need to control for the thickness of tissue and the differences in the size of the respective structures across sections? Also, background varies across a piece of tissue and across slides, so what approach was taken to achieve consistency across slides?

Response to comment 2

Thank you for your helpful suggestions. We conducted c-Fos counts blindly. Five to six sections were used to analyze c-Fos positive cells in hippocampal regions (CA1, CA2, CA3 and DG). Two to three sections were used to analyze c-Fos positive cells in other brain regions (PVN, SuM, Ent, PRh, and Cg/RS). We added relevant text responding to these comments in the Materials & Methods section (line 156). Furthermore, slices were cut using a cryostat, which can perfectly regulate section thickness. We calculated the area of the respective regions by NDP views. We think that size variation can be minimized by measuring several slices. Finally, we used the same reaction time for antibody incubation, AB reaction, and DAB staining to achieve a consistent background level.

Reviewer #1 comment 3

The c-Fos data were analyzed by multiple t-tests - that is, an unpaired t-test (saline- vs. CNO-treated) for normalized c-Fos counts in each region. The authors should consider that conducting multiple t-tests carries the risk of yielding a significant outcome by chance. Given that the data were normalized (though see comment #2 above), why not instead conduct a two-way RMANOVA (treatment group as between-subjects variable, and region of interest as the repeated measures/within-subjects variable)?

Response to comment 3

Thank you for your helpful suggestions. To analyze c-Fos counting, we referred to several reports (Ref. 25-27), which analyzed c-Fos positive neurons in several brain regions using unpaired t-test but not two-way ANOVA. We think it reasonable to use unpaired t-test in our study because we wanted to compare the average difference between saline treated mice and CNO treated mice. Moreover, we could not apply a two-way repeated ANOVA because the sample size and samples differed among brain regions. We added the description on the method used to analyze c-Fos counting to the Materials & Methods section (line 157).

Reviewer #1 comment 4

The manuscript refers to Table 1, but this information was not provided.

Response to comment 4

We apologize for this miss. Table 1 is inserted on page 40 and can be downloaded.

Reviewer #1 comment Minor issues

a) page 6, line 153: the statement that "...dorsal, but not ventral Hippocampus is involved in cognitive function..." is not well supported by the literature. For example see: Jin & Maren (2015) Scientific Reports; Okuyama et al. (2016) Science; Contreras et al. (2018) Hippocampus; Rao et al, (2019) Cell Reports; Jimenez et al. (2020) Nature Communications; to name a few example references.

c) page 10, line 249-252: This sentence is quite difficult to understand. Please revise.

d) page 11, line 267: "competed" should be revised to "compared"

Response to comment Minor issues

(a) Thank you for recommending the given references. Accordingly, we added some new references to the manuscript. We would like to count c-Fos positive neurons following NORT. Therefore, we investigated the interaction between the hippocampus and object recognition memory. Several reports showed that the dorsal hippocampus was involved in object recognition memory (Ref. 21-24). We added relevant text to the Materials & Methods (line 160)

(b) ORM was removed.

(d) We revised this word.

(e) Thank you for your helpful suggestions. Indeed, in this study we did not perform any experiments related to Alzheimer’s disease. However, the present results show that the activity of OXTergic neurons regulates cognitive function. Further studies are required showing that OXTergic neurons are involved in the pathological progression of Alzheimer’s disease. We consider that this possibility is one of the future impacts of this study.

Responses to the comments raised by Reviewer #2

Reviewer #2 comment 1

As stated by the author in the introduction, "Projections of oxytocin (OXT) neurons exhibit extensive distribution across regions associated with cognitive functions. For instance, prior studies have revealed that OXT neurons project to the hippocampus (Hip), perirhinal cortex, entorhinal cortex (Ent), and supramammillary nucleus (SuM)." However, the findings from immunohistochemical staining (Fig. 1) merely illustrate the fibers within the paraventricular nucleus (PVN) and supramammillary nucleus (SuM). It would be advantageous to incorporate outcomes from other cerebral regions, notably the hippocampus (DG).

Response to comment 1

Thank you for your helpful suggestions. We have now added the missing information on OXT neurons projecting to the hippocampus. However, we could not detect the mCherry positive fiber in the hippocampus, including hippocampal DG, in this study. In a previous study, alkaline phosphatase staining did not detect OXT neurons projection in the hippocampus of Oxt cre/+; Z/AP mice [29], which might be because of the projection areas of OXT neurons detected depend on mice and staining methods. We have added relevant text to the Discussion section (line 304).

Reviewer #2 comment 2

Regarding c-Fos staining and quantification subsequent to CNO treatment, it is advisable for the authors to undertake additional verification to ascertain that the identified c-Fos positive neurons post-treatment indeed are OXTergic neurons.

Response to comment 2

Thank you for your pertinent suggestions. To ascertain that the identified c-Fos positive neurons post-treatment were indeed OXTergic neurons, we detected c-Fos and OXT by immunofluorescence staining. We detected double-labeled neurons (c-Fos+ and OXT+) in the PVN of CNO-treated mice. These results suggested that the c-Fos positive neurons observed post-treatment are OXTergic neurons. Therefore, CNO administration can activate OXT neurons. These results were added to supplement figure 3 and commented on in the Discussion section (line 309).

Reviewer #2 comment 3

Does the localized administration of CNO to the SuM have an impact on the DG? While the results section does not explicitly address the SuM-DG pathway, it extensively delves into this topic in the discussion. It would be better for the authors to substantiate their conceptual arguments in the discussion by presenting relevant empirical findings.

Response to comment 3

According to the reviewers’ suggestions, we have added the required description to the Discussion section (line 351).

“Li et al. injected AAV-DIO-hM3Dq-mCherry or AAV-DIO-hM4Di-mCherry into the SuM in mice. CNO treatment increased c-Fos positive neurons in the DG of hM3Dq expressing mice but decreased c-Fos positive neurons in the DG of hM4Di expressing mice. In addition, Li et al. injected AAV-DIO-Ch2R-mCherry and AAV-shVglut2 (a marker of glutamatergic neurons) into the SuM in mice. Activating DG with optogenetics decreased c-Fos positive neurons in the DG of Vglut2 deletion mice. Accordingly, these results suggested that glutamatergic neurons in the SuM activate the DG (Ref. 9)”.

Reviewer #2 comment 4

In the discussion section (page 11, line 270), the authors put forth the assertion that “Thus, we propose that activating DG and SuM neurons enhance ORM by activating OXT neurons.” It would be judicious for the authors to complement this proposal by providing evidential support. Specifically, they could present data that demonstrate the comparable effects observed in “Effects of activation of OXTergic neurons in SuM by NORT”, where local activation of oxytocinergic fibers in the DG produced similar outcomes.

Response to comment 4

We appreciate your attempt to understand our discussion. However, we consider that it cannot activate OXTergic neurons in the DG because we could not detect OXT neuron projections in the hippocampus. Therefore, we proposed that OXTergic neurons in SuM projecting from PVN could enhance ORM via the activating of gluterergic neurons in DG projecting from SuM. We have added these comments to the Discussion section (line 367).

Reviewer #2 comment 5

In this study, only male mice were used as the subjects. Could potential distinctions arise in female mice?

Response to comment 5

Thank you for your helpful suggestions. Female mice may exhibit differences since the distribution of OXT receptors and OXT projection areas differ between female and male mice (Ref. 2). However, we did not use female Oxt-iCre mice to avoid the possibility of recombination in all cells in a Cre expressing fertilized egg. Moreover, DAT-Cre KI mice show sex-dependent changes in amphetamine-induced locomotor activity (Ref. 37). This report suggested that the phenotypes of Cre-KI mice were sex-dependent. Thus, we used male Oxt-iCre mice in this study. We have added these comments to the Discussion section (line 396).

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PLoS One. doi: 10.1371/journal.pone.0294113.r003

Decision Letter 1

Peng Zhong

25 Oct 2023

Oxytocinergic Projection from the Hypothalamus to Supramammillary Nucleus Drives Recognition Memory in Mice

PONE-D-23-22233R1

Dear Dr. Saitoh,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewer #1: Yes

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6. Review Comments to the Author

Reviewer #1: The revised manuscript is improved with the changes that the authors have made. A few issues remain to be addressed.

1. Measures of effect size should be included for each significant statistical value.

2. In response to the critique that the NORT results would be better analyzed as measures of discrimination ratio (difference score divided by total object exploration) instead of preference ratio. The authors seem to have disregarded this message and have now included difference score measures in the supplementary material. Both preference ratio and difference score measures suffer from the issue of high variability between groups and between individual mice. That is the reason for the original message that discrimination ratio measures represent a more rigorous measure of object memory in the NORT task. The discrimination ratio scores have a wider range of possible scores and generally have a reduced variability. Thus, analyses of NORT discrimination ratios tends to lead to fewer false interpretations of results.

3. The c-Fos counts indicate no differences in any of the examined areas after NORT between mice receiving saline or CNO. This result is a bit surprising given several reports of increased c-Fos in hippocampal cell layers and related regions 90 min after the training or test session of the NORT task (see Beer et al.; Mendez et al 2015a, 2015b; Cinalli et al. 2020; Bernstein et al., 2019; Barbosa et al., 2013, etc.). Given those reports, one might have expected differential counts between cingulate and CA1 and CA3, for example. As well differences might have been expected in the CNO treated mice if the drug was expected to have indirectly increased activity of hippocampal neurons. The authors should provide more interpretative information regarding these issues.

Reviewer #2: (No Response)

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Reviewer #1: Yes: Robert Stackman Jr.

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PLoS One. doi: 10.1371/journal.pone.0294113.r004

Acceptance letter

Peng Zhong

7 Nov 2023

PONE-D-23-22233R1

Oxytocinergic Projection from the Hypothalamus to Supramammillary Nucleus Drives Recognition Memory in Mice

Dear Dr. Saitoh:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

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

Supplementary Materials

S1 Fig. The exploration preference to object in our laboratory conditions in NORT.

(TIF)

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S2 Fig. The difference exploration time of object during the training session and the test session in NORT.

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S3 Fig. The immunohistochemical staining of c-Fos and OXT neurons.

(TIF)

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S1 Raw data

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S2 Raw data

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S3 Raw data

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S4 Raw data

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Attachment

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Data Availability Statement

Data are all contained within the Supporting Information files.

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PERMALINK

Oxytocinergic projection from the hypothalamus to supramammillary nucleus drives recognition memory in mice

Junpei Takahashi

Daisuke Yamada

Wakana Nagano

Yoshitake Sano

Teiichi Furuichi

Akiyoshi Saitoh

Roles

Abstract

Introduction

Materials and methods

Animals

Stereotaxic surgery

Virus injection

Chemogenetic pathway-selective activation

Immunohistochemistry

Analysis of c-Fos and mCherry

Y-maze test

Novel object recognition test (NORT)

Statistical analysis

Results

Projection of OXTergic neurons in PVN

Fig 1. Projection of OXTergic neurons in the PVN.

The number of c-Fos positive cells in PVN after administration of CNO

Fig 2. The number of c-Fos positive cells in PVN after administration of CNO.

Effects of activation of OXTergic neurons in PVN on the Y-maze test and NORT

Fig 3. Effects of activation of OXTergic neurons in PVN on the Y-maze test and NORT.

The number of c-Fos positive cells after NORT

Fig 4. The number of c-Fos positive cells after NORT.

Table 1. Expressions of c-Fos in the various brain areas after NORT.

Effects of activation of OXTergic neurons in SuM by NORT

Fig 5. Effects of activation of OXTergic neurons in SuM during the NORT.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Peng Zhong

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Author response to Decision Letter 0

Decision Letter 1

Peng Zhong

Roles

Acceptance letter

Peng Zhong

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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