Bidirectional modulation of negative emotional states by parallel genetically-distinct basolateral amygdala pathways to ventral striatum subregions

Summary

Distinct basolateral amygdala (BLA) cell populations influence emotional responses in manners thought important for anxiety and anxiety disorders. The BLA contains numerous cell types which can broadcast information into structures that may elicit changes in emotional states and behaviors. BLA excitatory neurons can be divided into two main classes, one of which expresses Ppp1r1b (encoding protein phosphatase 1 regulatory inhibitor subunit 1B) which is downstream of the genes encoding the D1 and D2 dopamine receptors (drd1 and drd2 respectively). The role of drd1+ or drd2+ BLA neurons in learned and unlearned emotional responses is unknown. Here, we identified that the drd1+ and drd2+ BLA neuron populations form two parallel pathways for communication with the ventral striatum. These neurons arise from the basal nucleus of the BLA, innervate the entire space of the ventral striatum, and are capable of exciting ventral striatum neurons. Further, through three separate behavioral assays, we found that the drd1+ and drd2+ parallel pathways bidirectionally influence both learned and unlearned emotional states when they are activated or suppressed, and do so depending upon where they synapse in the ventral striatum – with unique contributions of drd1+ and drd2+ circuitry on negative emotional states. Overall, these results contribute to a model whereby parallel, genetically-distinct BLA to ventral striatum circuits inform emotional states in a projection-specific manner.

Introduction

The ability to evaluate a sensory stimulus and to correctly act upon it is paramount for survival. Abnormal associations of stimuli and/or abnormal actions towards stimuli are hallmark features of psychiatric disorders including anxiety disorders. The basolateral amygdala (BLA) has long been known to support emotional responses (Klüver and Bucy, 1939; Blanchard and Blanchard, 1972), including to both aversive (Weiskrantz, 1956; Cahill and McGaugh, 1990; LeDoux, 1992; Maren et al., 1996; Cousens and Otto, 1998) and appetitive stimuli (Hatfield et al., 1996; Setlow et al., 2002; Schoenbaum et al., 2003). Affording the BLA with this capacity are both its intrinsic plasticity (Rogan et al., 1997; Maren and Quirk, 2004) and its projections into ‘downstream’ structures which can directly influence decisions and behavioral outcomes. For instance, the BLA innervates the central nucleus of the amygdala and this input is necessary for the expression of learned avoidance (LeDoux, 2003). Photostimulating central amygdala projecting BLA neurons evokes avoidance, while photoinhibition of the same neurons reduces fear learning (Namburi et al., 2015). Other projections of the BLA can influence appetitive responses, including stimulation of BLA neurons that project to the ventral striatum’s nucleus accumbens (NAc) (Namburi et al., 2015). Thus, it is clear that regionally-separable downstream recipients of BLA input are sufficient to direct emotional responses.

There is also an interplay between BLA projection targets and the cell types which comprise those projections in how specific BLA outputs influence emotion. The genetic identity of BLA neurons is highly diverse and these different cell types appear to be uniquely be engaged following emotional responses (Hochgerner et al., 2023). A single genetically distinct neuronal population can drive opposing emotional responses if it were to project to two brain regions (Zhang et al., 2021) and likewise, two genetically distinct BLA outputs can drive opposing emotional responses if they each project to the same brain region (Kim et al., 2017). BLA excitatory neurons are divided into two main genetic classes, which are becoming increasingly understood to have diverse projection targets and functions. These include the Rspo2 expressing neurons (encoding R-spondin 2), which can drive aversive behaviors, and the Ppp1r1b expressing neurons (encoding protein phosphatase 1 regulatory inhibitor subunit 1B), which appear to support appetitive behaviors (Kim et al., 2016). BLA neurons distinguished by the expression of the transcription factor Rspo2, are also labeled by Fezf2 (encoding the transcription factor zinc-finger 2) (Zhang et al., 2021), and project to the NAc and also its neighboring ventral striatum subregion, the tubular striatum (TuS, also known as the olfactory tubercle) (Wesson, 2020). Activation of Fezf2 neurons innervating the NAc drives aversive states and contrastingly, activation of Fezf2 neurons innervating the TuS increases appetitive states. Together, these findings indicate that neither the downstream target nor the genetic identity alone sufficiently explain how the BLA broadcasts emotional information. Instead, where this information goes and who within the BLA sends it are both critical for regulating emotional states. Given that Rspo2/Fezf2+ BLA neurons support both appetitive and aversive states depending upon their downstream targeting, we sought to answer the question of whether the Ppp1r1b+ BLA neuron population also contribute to aversive states, and do so depending upon their regional innervation within the NAc and TuS.

Ppp1r1b (also known as Darpp-32, dopamine- and cAMP-regulated neuronal phosphoprotein) is a phosphoprotein regulated by both D1 and D2 dopamine receptors (Ouimet et al., 1984; Nishi et al., 1997; Svenningsson et al., 2004), which are encoded for by the drd1 and drd2 genes, respectively (Scibilia et al., 1992). Dopamine within the BLA is necessary for fear learning (Fadok et al., 2009). We know that drd1+ neurons in the BLA contribute to memory (Zhang et al., 2020a). While the role of drd2+ neurons in the BLA is not understood, prior pharmacological work has indicated a role for the D2 receptor in emotional responses (Guarraci et al., 2000; Berglind et al., 2006; de Oliveira et al., 2011). Overall, the respective contributions of drd1+ and drd2+ BLA neurons in regulating emotional states are unknown. Moreover, it is unknown if, like the Fezf2 neurons, the influence of drd1+ and drd2+ BLA neurons depends upon their projection targets. Here, using a combination of viral tracing, ex vivo brain slice recordings, chemo- and opto-genetics, and behavior, we identified that the drd1+ and drd2+ BLA neuron populations form two parallel pathways wherein each innervate both the NAc and the TuS for the modulation of negative emotional states depending upon which ventral striatum subregion they innervate. Overall, these results contribute to a model whereby parallel, genetically-distinct, BLA to ventral striatum circuits inform emotional states in a projection-specific manner and altogether expand our appreciation for how the BLA regulates emotions.

Results

drd1+ and drd2+ BLA neurons innervate the ventral striatum.

We first sought to determine if BLA drd1+ and drd2+ neurons form a circuit with ventral striatum neurons. We injected a Cre-dependent retrograde (rg) AAV expressing mCherry, rgAAV.hSyn.DIO.mCherry, into either the NAc or the TuS of drd1-Cre and drd2-Cre mice (Gong et al., 2007) (Fig 1A,B & 1E,F) and later inspected the BLA for mCherry+ neurons. mCherry+ cells in both groups of mice were found in the BLA in both drd1- and drd2-Cre mice (Fig 1C & G), indicating that these neurons indeed project to the ventral striatum. mCherry+ cells were found throughout the entire anterior-posterior extent of the BLA (Fig 1C & G). In contrast to the lateral amygdala (LA) which was largely void of mCherry+ cells, the basal nucleus of the amygdala (BA) displayed dense mCherry+ cells (Fig 1C & G). This organization was observed even when similarly injecting either the NAc or TuS of Ai9.TdTomato Cre reporter mice (Madisen et al., 2010) with rgAAV.hSyn.HI.eGFP-Cre.WPRE.SV40, suggesting the BA is a major conduit of ventral striatum input regardless of cell type (Supplementary Fig 1A–C & 1D–F). No reciprocal connection from ventral striatum to the BLA was found however (Supplementary Fig 1G–I).

Figure 1. Ventral striatum projecting BLA neurons arise from the BA and are comprised of drd1+ and drd2+ neurons.

(A) Schematic of approach for identifying BLA drd1+ and drd2+ inputs to the NAc. (B) Example of an NAc injection in drd1-Cre (top) and drd2-Cre (bottom) mice (ac = anterior commissure, NAcC & NAcSh=nucleus accumbens core & shell, respectively), and (C) example images of NAc projecting drd1+ (top) or drd2+ (bottom) neurons along the anterior-posterior axis of the BLA. Scale bars=500µm. (Di) Anti-NeuN and anti-DsRed immunofluorescence images to identify the size of the NAc projecting BLA neural population. Scale bars=40µm. (Dii) Quantification of the drd1 (n=3) or drd2 (n=3) expressing BLA cells along the entire AP axis that project to the NAc (Two-way ANOVA, ROI main effect F(1,8)=26.65, p=0.001). (E) Schematic of approach for identifying BLA drd1+ and drd2+ inputs to the TuS. (F) Example of a TuS injection in drd1-Cre (top) and drd2-Cre (bottom) mice (TuS=tubular striatum), and (G) example images of TuS projecting drd1+ (top) or drd2+ (bottom) neurons along the anterior-posterior axis of the BLA. Scale bars=500µm. (Hi) Anti-NeuN and anti-DsRed immunofluorescence images to identify the size of the NAc projecting BLA neural population. Scale bars=40µm. (Hii) Quantification of the drd1 (n=3) or drd2 (n=3) expressing TuS projecting BLA cells along the entire AP axis (Two-way ANOVA, F(1,8)=295.3, p<0.001). Mean±SEM.

To quantify the spatial distribution and to identify the size of the drd1 and drd2 neural population innervating both the NAc and TuS, sections were immunolabeled for both NeuN and the red fluorescent protein DsRed (Fig 1Di & 1Hi). This revealed that indeed the vast majority of ventral striatum projecting drd1+ and drd2+ neurons arise from the BA (Fig 1Dii, NAc: drd1+ MLSD=|BA-LA|=18.07, p=0.002, drd2+ MLSD=|BA-LA|=11.95, p=0.019; Fig 1Hii, TuS: drd1+ MLSD=|BA-LA|=31.0, p<0.001, drd2+ MLSD=|BA-LA|=9.24, p<0.001; MLSD=mean least square difference). Further, more drd1+ BA neurons innervate the TuS than those that are drd2+ (Fig 1Hii, TuS: MLSD=|drd1-drd2|=21.6, p<0.001).

Where throughout the ventral striatum do BLA neurons innervate? To answer this, we injected drd1-Cre and adora2a (a2a)-Cre mice into the BLA with an AAV encoding a synaptophysin.mRuby fusion protein (AAV.hSyn.FLEx.mGFP-2A-Synaptophysin-mRuby) (Herman et al., 2016) (Fig 2A). A2a-Cre mice were chosen for this and later anterograde AAV-based experiments to attempt to achieve optimal presynaptic expression in drd2+ neurons. This resulted in mRuby+ puncta, indicative of BLA drd1+ or a2a+ neuronal synapses, throughout both the NAc and TuS (Fig 2B). As expected based upon the retrograde tracing results in Figure 1, drd1 mRuby+ puncta were highly visible in comparison to a2a+ (Fig 2B & C). The mRuby+ puncta spanned the entire medial-lateral and anterior-posterior extents of the TuS, and were especially prominent in layer 2 (Fig 2B) which is the densest cell layer. mRuby+ puncta were also observed throughout the medial-lateral and anterior-posterior extents of the NAc, with comparable amounts in both the NAc core and shell (Fig 2C, drd1+ MLSD=|NAcC-NAcSh|=0.035, p>0.999, a2a+ MLSD=|NAcC-NAcSh|=0.241, p>0.999). It is notable, given its roles in associative learning including fear learning (Li et al., 2008; Wilson and Sullivan, 2011), that the ventral striatum receives more BLA drd1+ and less a2a+ neuronal input than the neighboring piriform cortex (Fig 2C, drd1+: F(1,16)=30.8, p<0.001; a2a+: F(1,14)=10.6, p=0.006). Together these tracing results establish that both drd1+ and drd2+ neurons, largely form the BA, innervate the entire span of the ventral striatum.

Figure 2. BLA neurons expressing drd1 and drd2 innervate the entire span of the ventral striatum.

(A) Schematic of approach for identifying BLA drd1+ and drd2+ synaptic innervation of the ventral striatum. (B) Representative images showing direct innervation of BLA neurons into the ventral striatum in both drd1- and a2a-Cre mice. Scale bar=500µm. (C) Quantification of synaptophysin puncta in drd1-Cre (n=3) and a2a-Cre (n=3) mice. drd1+ BLA neurons densely innervate the ventral striatum, and relatively fewer drd2+ BLA neurons innervate the ventral striatum. Mean±SEM. PCX=piriform cortex, NAcC and NAcSh=nucleus accumbens core and shell, respectively.

drd1+ and drd2+ BLA neurons excite ventral striatum spiny projection neurons.

Next, we injected a Cre-dependent AAV expressing channelrhodopsin and EYFP (AAV.Ef1a.DIO.hChR2(E123T/T159C)-EYFP) or EYFP alone as a control (AAV.Ef1a.DIO.EYFP) into the BLA of drd1-Cre and a2a-Cre mice which were crossed with the Ai9 TdTomato Cre reporter line and later took coronal slices of the ventral striatum for ex vivo recordings to determine which ventral striatum spiny projection neurons (SPNs) the BLA neurons make synapses upon. TdTomato+ neurons were identified and used to identify drd1+ or drd2+ SPNs (those expressing TdTomato+ in either drd1-Cre or a2a-Cre mice respectively; Fig 3Ai & Aii). In the same slices we also patched onto TdTomato-SPNs to monitor activity of drd1Ø and drd2Ø (putative drd2+ or drd1+) SPNs, in either drd1-Cre or a2a-Cre mice respectively. During recordings, blue light pulses were delivered to excite ChR2-expressing BLA terminals in the ventral striatum. Importantly, we confirmed in drd1-RFP;drd2-GFP double transgenic mice (Shuen et al., 2008) that there is minimal co-expression of drd1 and drd2 in the same cells (Supplementary Fig 2A & B). Moreover, the BLA to ventral striatum projection is predominantly ipsilateral (Supplementary Fig 2C & D). Current injection into ventral striatum neurons confirmed their firing patterns are as expected for TuS SPNs (Supplementary Fig 3A & B) (White et al., 2019).

Figure 3. Synaptic properties of drd1 and drd2 expressing ventral striatum neurons receiving BLA neuronal projections.

(Ai) Schematic indicating Cre-dependent expression of ChR2 in drd1+ BLA neurons of drd1-Cre;Ai9 mice. In these mice, TdTomato+ neurons are presumably drd1+ and TdTomato-neurons are presumably drd1Ø. During whole-cell patch clamp recordings, ChR2 expressing BLA terminals were activated by 470nm light. (Aii) Schematic indicating Cre-dependent expression of ChR2 in drd2+ BLA neurons of A2a-Cre;Ai9 mice. TdTomato+ neurons are presumably drd2+, and TdTomato-neurons are presumably drd2Ø. (B) Example light-evoked monosynaptic EPSCs (top) and light-evoked polysynaptic EPSCs (bottom) from drd1+ TuS neurons under voltage clamp mode. (C) Neurons organized by response type upon stimulation of drd1+ TuS projecting BLA terminals. (D) Example evoked EPSCs from drd1Ø TuS neurons. (E) Neurons organized by response type upon stimulation of drd1Ø TuS projecting BLA terminals. (F) Example evoked EPSCs from drd2+ TuS neurons. (G) Neurons organized by response type upon stimulation of drd2+ Tus projecting BLA terminals. (H) Example evoked EPSCs from drd2Ø TuS neurons. (I) Neurons organized by response type upon stimulation of drd2+ Tus projecting BLA terminals. The holding potential was −70mV.

We found that both BLA cell populations synapse upon both drd1+ and drd2+ SPNs, albeit with differing weights and strengths. The majority of drd1+ BLA neurons elicited large monosynaptic currents in drd1+ and drd1Ø SPNs (Fig 3B–D). While both ventral striatum cell types were excited by drd1+ BLA neurons, BLA drd1+ neurons send stronger input (viz., larger evoked excitatory postsynaptic currents [EPSCs]) and do so more predominantly upon drd1+ vs drd1Ø (putative drd2+) SPNs (drd1+ vs drd1Ø monosynaptic: X²(1, N=91)=5.45, p=0.02; drd1+ vs drd1Ø polysynaptic: X²(1, N=91)=0.096, p=.757; Fig 3C & E). In a subset of SPNs, monosynaptic glutamatergic connections were verified via pharmacological manipulations (Supplementary Fig 3C & D). Likewise, drd2+ BLA neurons also synapse upon drd2+ and drd2Ø SPNs, but compared to the drd1+ BLA input, input from drd2+ BLA neurons was both weaker and not as predominant (Fig 3F–I). A far larger percentage of SPNs displayed monosynaptic EPSCs upon drd1+ BLA neuron terminal stimulation than when stimulating drd2+ BLA terminals (X²(1, N=255)=33.947, p<0.001). Including polysynaptic EPSCs, 13.1 – 15.9% of SPNs (drd2+ and drd2Ø, respectively) displayed evoked potentials and these were notably modest in amplitude compared to what was observed when stimulating drd1+ BLA terminals (e.g., Fig 3B vs 3F). Together these results extend the anatomical circuitry (Figs 1 & 2) by showing that both drd1+ and drd2+ BLA neurons excite ventral striatum spiny projection neurons, albeit with differing likelihood of observing monosynaptic connections.

drd1+ BLA neurons innervating the NAc and drd2+ BLA neurons innervating the TuS both promote avoidance behavior.

Next, we sought to determine a functional role for BLA drd1+ and drd2+ input to the ventral striatum. In the first of three assays, we used an optogenetic approach to excite drd1+ and drd2+ BLA neuron terminals innervating either the NAc or TuS to determine if these pathways influence avoidance or approach behaviors. For this we unilaterally injected drd1-Cre and a2a-Cre mice with AAV.Ef1a.DIO.hChR2(E123T/T159C)-EYFP or AAV.Ef1a.DIO.EYFP as control into their BLA and later implanted optical fibers into the ipsilateral NAc or TuS (Fig 4A). Given the similar innervation of both the NAc core and shell (Fig 2C), we targeted both NAc subregions for stimulation. Four weeks post injection, we used a 3-chamber real time place preference/aversion assay wherein light was delivered to stimulate the drd1+ or drd2+ BLA neuron terminals (465nm, 15ms pulses, 40Hz) on only one side of the chamber, with no optical stimulus in either the center or the opposite chambers (Fig 4B). The location of the mice was tracked with infrared photobeams to trigger the optogenetic stimulation and video was captured for off-line quantification.

Figure 4. BLA drd1+ and drd2+ neurons innervating the ventral striatum promote aversive states depending upon projection target.

(A) Paradigm for optic activation of NAc or TuS projecting drd1+ or drd2+ BA neurons and (B) 3-chamber real time place preference/aversion assay where optic stimulation occurs in only one side of the chamber (chamber A, blue glow). (Ci) Optical stimulation of drd1+ BLA→NAc neurons resulted in less time spent in the light-paired chamber (One-way ANOVA, F(2,18)=5.04, p=0.018). (Cii) Stimulation of drd1+ BLA NAc neurons results in avoidance of the light-paired chamber (upper, t(6)=2.981, p=0.025), demonstrated by representative heat map of chamber preference from one mouse (lower). (Di) Optical stimulation of drd2+ BLA TuS neurons resulted in less time spent in the light-paired chamber compared to optical stimulation of EYFP controls (Welch’s ANOVA W(2.00,8.31)=6.02, p=0.024). (Dii) Stimulation of drd2+ BLA TuS neurons results in avoidance of the light-paired chamber (upper, t(5), p=2.916), demonstrated by representative heat map of chamber preference from one mouse (lower). Mean±SEM.

We found that optical stimulation of drd1+ BLA NAc neuron terminals resulted in less time spent in the light-paired chamber compared to optical stimulation of drd2+ BLA NAc neuron terminals and EYFP controls (Fig 4Ci & Cii) (One-way ANOVA F(2,18)=5.04, p=0.018). Indeed, compared to the non-stimulated side, mice spent 49.70±11.10 % (mean ± SEM) less time on the chamber paired with drd1+ BLA NAc neuron terminal stimulation (t(6)=2.981, p=0.025). Similarly, we found that optical stimulation of drd2+ BLA TuS neuron terminals resulted in less time spent in the light-paired chamber compared to optical stimulation of EYFP+ BLA TuS controls (Fig 4Di & Dii) (Welch’s ANOVA W(2.00,8.31)=6.02, p=0.024). Compared to the non-stimulated side, mice spent 31.42±6.39 % (mean ± SEM) less time on the chamber paired with drd2+ BLA TuS neuron terminal stimulation (t(5)=2.916, p=0.033). These results show that activation of drd1+ and drd2+ BLA input to the NAc and TuS respectively lead to avoidance behavior.

drd1+ BLA neurons innervating the NAc and drd2+ BLA neurons innervating the TuS support Pavlovian fear learning.

Next, we wanted to know the possible influence of these pathways on learned emotional behaviors. To do this we employed an odor-shock Pavlovian fear-learning paradigm (Best and Wilson, 2003; Jones et al., 2008; Hegoburu et al., 2011) wherein an otherwise neutral odor is paired with a mild foot shock (Supplementary Fig 4Ai). To quantify learning, we monitored both physical immobility and fear-associated respiratory power (4–6Hz) which increases in power when animals anticipate an aversively-paired stimulus (Hegoburu et al., 2011; Moberly et al., 2018). Mice were placed in a plethysmograph with a custom floor made out of metal connected to a shock stimulus generator. Also connected to the plethysmograph was a tube allowing delivery of clean air or an odor which were both controlled by an odor presentation machine. All behavioral measures and stimuli were controlled by the same computer allowing synchrony in measures and stimulus presentation events. In untreated C57BL/6J mice we validated that only odors paired with shock, were associated with elevations in physical immobility following the conditioning day (Supplementary Fig 4B). We also validated that fear-associated 4–6Hz respiratory power is similarly elevated as mice learn to associate an odor with a shock (Supplementary Fig 4C–H).

We used a chemogenetic approach to suppress BLA ventral striatum input which included six separate groups of mice to establish the roles of each of the BLA NAc and BLA TuS pathways (Fig 5A). These included drd1+ and drd2+ mice injected with rgAAV.hSyn.DIO.hM4D(Gi)-mCherry or rgAAV.hSyn.DIO.mCherry as control. All mice were subsequently implanted with bilateral intracranial cannulae into the BLA for administration of either the DREADD ligand J60 (Bonaventura et al., 2019) or vehicle. J60 or vehicle were administered 30 minutes prior to the learning session following a single behavioral session on a prior day to acclimate the mice to the chamber.

Figure 5. BLA drd1+ and drd2+ neurons innervate the ventral striatum support Pavlovian fear learning depending upon projection target.

(A) Paradigm for DREADD induced silencing of NAc or TuS projecting drd1+ or drd2+ BLA cells. (B) Influence of DREADD agonist J60 (100nL, 10nM) on fear learning in all NAc injected mice, (C) left NAc injected mCherry controls (Two-way RM ANOVA, Trial main effect F(1,27)=86.2, p<0.001); middle drd1+ hM4D(Gi) mice (Two-way RM ANOVA, Trial main effect F(1,13)=48.9, p<0.001); and right drd2+ hM4D(Gi) mice (Two-way RM ANOVA, Trial main effect F(1,13)=301, p<0.001). (D) Influence of DREADD agonist J60 (100nL, 10nM) on fear learning in all TuS injected mice, (E) left mCherry TuS injected controls (Two-way RM ANOVA, Trial main effect F(1,29)=151, p<0.001); middle drd1+ hM4D(Gi) mice (Two-way RM ANOVA, Trial main effect F(1,13)=85.6, p<0.001); and right drd2+ hM4D(Gi) mice (Two-way RM ANOVA, F(1,12)=7.71, p=0.017). Mean±SEM.

Among both the BLA NAc and BLA TuS groups, all control groups displayed elevations in fear-associated respiration by the 10^th trial of odor-shock pairings (Fig 5B,C & F,G; NAc mCherry control: Two-way RM ANOVA, trial main effect F(1,27)=86.2, p<0.001; TuS mCherry control: Two-way RM ANOVA, trial main effect F(1,29)=151, p<0.001) indicating that they learned to associate an odor with an aversive outcome. As expected, similar elevations in physical immobility were also observed (Supplementary Fig 4). Importantly, there was no difference in learning between the vehicle and J60 infused groups supporting that there are no off-target effects of this DREADD ligand on odor-shock learning (Fig 5B,C & F,G; NAc mCherry controls Trial 10: MLSD=|Vehicle-J60|=−0.0443, p=0.601, TuS mCherry controls Trial 10: MLSD=|Vehicle-J60|=0.0118, p=0.888).

While neither inhibition of drd2+ BLA NAc and drd1+ BLA TuS pathways impacted fear-learning (Fig 5E & H, Two-way RM ANOVA, trial main effect: drd2+ BLA NAc: F(1,13)=301, p<0.001; drd1+ BLA TuS: F(1,13)=85.6, p<0.001), we found that inhibition of drd1+ BLA NAc and drd2+ BLA TuS pathways suppressed the magnitude of the learned association. Both drd1+ BLA NAc and drd2+ BLA TuS pathway inhibition resulted in less fear-related respiration by trial 10 in J60 infused mice compares to those infused with vehicle (Fig 5D & I; drd1+ BLA NAc Trial 10: MLSD=|Vehicle-J60|=0.319, p=0.049, drd2+ BLA TuS Trial 10: MLSD=|Vehicle-J60|=0.364, p<0.001). Fear-related physical immobility was likewise reduced upon drd2+ BLA TuS pathway inhibition, yet interestingly not upon drd1+ BLA NAc inhibition (Supplementary Fig 5). These results show that drd1+ and drd2+ BLA input to the NAc and TuS respectively are necessary for fear learning in addition to their role in real-time avoidance.

drd2+ BLA neurons innervating the TuS promote spontaneous avoidance of odors.

Finally, given the evidence that BLA to ventral striatum drd1+ and drd2+ neural pathways each influence both spontaneous / real-time aversive states and learned avoidance to odors, we examined if this circuitry might also influence spontaneous attraction or avoidance to odors. For this we used the same mice that completed the Pavlovian odor-shock fear learning (Fig 5), either a few days before or after the Pavlovian testing, and tested them in a spontaneous odor attraction/avoidance assay following intracranial infusion of either vehicle or J60. One side of the testing arena contained cotton laced with the aversive fox odor compound 2MT (2-Methyl-2-thiazoline), and the other side contained cotton laced with an attractive peanut oil odor (Fig 6A). Both stimuli were housed in clean perforated plastic tubes to prevent touching or tasting the stimulus yet still allowing release of volatiles. Video was collected for quantification of place preference. C57BL/6J mice, whether injected with J60 or vehicle, both spent more time on the peanut scented side of the apparatus than the fox odor side indicating that this approach can assay unlearned valence to odors (Fig 6B; vehicle: t(7)=3.71, p=0.004; J60: t(6)=2.24 p=0.033).

Figure 6. drd2+ BLA neurons innervating the TuS promote spontaneous avoidance to odors.

(A) Behavioral schematic of spontaneous odor attraction/avoidance task. (B-H) Influence of DREADD agonist J60 (100nL, 10nM) on spontaneous avoidance in (B) C57BL/6J mice (vehicle: t(7)=3.71, p=0.004; J60: t(6)=2.24, p=0.033), (C) BLA→NAc mCherry control mice (top J60: t(13)=3.13, p=0.004; bottom vehicle: t(14)=6.31, p<0.001, (D) top J60 drd1+: t(8)=2.30, p=0.025; bottom vehicle drd1+: t(9)=3.840, p=0.002, (E) top J60 drd2+: t(7)=4.4, p=0.002; bottom right trend of drd2+ vehicle: t(6)=1.7, p=0.073). (F) BLA→TuS mCherry control mice (top J60: t(15)=3.34, p=0.002; bottom vehicle: t(14)=2.89, p=0.012, (G) top J60 drd1+: t(6)=3.705, p=0.005; bottom vehicle drd1+: t(7)=2.014, p=0.042, (H) top J60 drd2+: t(6)=1.171, p=0.143; bottom vehicle drd2+: t(6)=5.02, p=0.001). Mean±SEM.

As anticipated, among both the BLA NAc and BLA TuS mCherry groups, all controls, regardless of J60 or vehicle treatment, spent more time on the peanut scented side of the apparatus compared to the fox odor side (Fig 6C & F) supporting that there are no off-target effects of this DREADD ligand on spontaneous approach or avoidance to odors (J60-inhibited BLA NAc: t(13)=3.13, p=0.004; vehicle-treated BLA NAc: t(14)=6.31, p<0.001; J60-inhibited BLA TuS: t(15)=3.34, p=0.002; vehicle-treated BLA TuS: t(14)=2.89, p=0.012). In line with our results from the Pavlovian odor-shock fear learning, we found that drd2+ BLA TuS pathway inhibition resulted in reduced approach and avoidance for peanut and fox, respectively (Fig 6H; J60-inhibited D2R+ BLA TuS: t(6)=1.171, p=0.143). Unlike in the Pavlovian odor-shock fear learning however, inhibition of drd1+ BLA NAc pathway did not influence spontaneous approach and avoidance (Fig 6D) (J60-inhibited drd1+ BLA NAc: t(8)=2.300, p=0.025). These results show that drd2+ BLA input to the TuS, but not drd1+ input to the NAc, contributes to unlearned odor avoidance in addition to its role in real-time avoidance and Pavlovian fear learning.

Discussion

It is well established that BLA outputs to specific brain regions influence emotional responses (e.g., (Cardinal et al., 2002; Paré et al., 2004; Ambroggi et al., 2008; Stuber et al., 2011; Janak and Tye, 2015; Namburi et al., 2015; Beyeler et al., 2016)). More recently, several lines of evidence have uncovered divergent valence responding through genetically-distinct neurons within the BLA, including by means of Pppr1r1b and Rspo2 neurons (Kim et al., 2016, 2017; Zhang et al., 2021). Together, both the genetic identity and downstream targets of BLA neurons are necessary to incorporate when understanding the role of BLA cell types in orchestrating the many functions of the BLA.

In the present study we focused on defining the contributions of drd1+ and drd2+ BLA neurons to emotional responding. The drd1 and drd2 genes encode for the D1 and D2 receptors, respectively (Scibilia et al., 1992), which regulate Pppr1r1b – a marker for one of the two main classes of BLA excitatory neurons. It has been long known that D1 and D2 receptors are in the BLA (e.g., (Meador-Woodruff et al., 1991)). We established that both drd1+ and drd2+ BLA neurons innervate the NAc and TuS, and we subsequently focused upon these two pathways (BLA NAc and BLA TuS) given the recent evidence of regulation of emotional responses through BLA output into these regions (Zhang et al., 2021). Our findings extend the work of (Zhang et al., 2021) by showing that in addition to the Rspo2/Fezf2 BLA neuron class, both drd1+ and drd2+ BLA neurons in the Ppp1r1b neuron class also each innervate the NAc and TuS. Far more drd1+ neurons comprise this circuit than drd2+ neurons, with more drd1+ neurons innervating both the TuS and NAc. These neurons originate from nearly the entire anterior-posterior extent of the BLA, and specifically the vast majority from within the BA (Fig 1). Further, our synaptophysin tracing suggests that they innervate nearly all of TuS and NAc space (all layers of TuS and both the NAc core and shell; Fig 2). While the spatial innervation of drd1+ and drd2+ BLA neurons into the ventral striatum is not unlike that reported by (Zhang et al., 2021), it is important to emphasize that Fezf2+ BLA neurons do not co-express Pppr1r1b (Zhang et al., 2021) which suggests these three neuron types connecting the BLA with the ventral striatum are distinct.

While the synaptophysin tracing suggests synaptic innervation of the ventral striatum by drd1+ and drd2+ BLA neurons, we used brain slice recordings to quantify this. This is interesting given that the primary cell type in the ventral striatum are spiny projection neurons which also express drd1+ and drd2+. We focused our recordings on TuS spiny projection neurons given the comparable innervation of both structures (NAc and Tus) by drd1+ and drd2+ BLA neurons which allowed us to also perform recordings to identify if there is logic by which ventral striatum neurons these BLA neurons synapse upon. We found that both BLA cell types excite drd1+ and drd2+ (identified in this experiment by expression of A2a) TuS neurons in manners which appeared to be largely glutamatergic, with especially drd1+ BLA neurons sending a large amount of monosynaptic currents (Supplementary Fig 3). Further, drd1+ BLA neurons monosynaptically excited predominately drd1+ TuS neurons, and drd2+ BLA neurons non-preferentially excited a small population of both drd1+ and drd2+ TuS neurons. While these results were initially surprising given that the drd1+ BLA TuS pathway was dispensable for the fear and avoidance behaviors explored in this work, this may indicate a potential role for this pathway in other behaviors, such as those involved in reward. Thus, BLA input to the TuS, and therefore possibly also the NAc, has an organization which allows for recruitment of specific postsynaptic neurons in the TuS which could therefore allow differential output from the ventral striatum in manners supporting specific outputs into the basal ganglia and other brain networks important for behavioral responses.

We found within this circuitry that the parallel pathways generated by the drd1+ and drd2+ BLA neuron populations modulates negative emotional states depending upon their ventral striatum projection target (Fig 7). To show this, we used three distinct behavioral paradigms in combination with either projection specific chemo- or optogenetic manipulations. In all behavioral paradigms, we were able to uncover a role for either drd1+ and/or drd2+ neurons, yet, in not all cases did each cell population impact behavior. Instead, the impact on behavior was in most cases also projection target specific. For instance, drd1+ BLA neurons innervating the NAc increased negative valence states in the real-time place preference/aversion paradigm, whereas the same cell population projecting to the TuS did not (Fig 4). Likewise, drd2+ BLA neurons innervating the TuS increased negative valence states in the real-time place preference/aversion paradigm, whereas the same cell population projecting to the NAc did not. Similar differences in how these genetically-distinct BLA cell populations influenced Pavlovian fear learning and spontaneous valence behaviors were also observed to be cell-type and projection target specific. As mentioned, not in all cases did each pathway impact one of the three behaviors assayed, possibly hinting towards a role for these pathways in other behaviors. These findings lay the foundation for future work to systematically target drd1+ or drd2+ BLA inputs into specific regions within the NAc (core vs shell (West and Carelli, 2016)) or TuS (medial vs lateral (Murata et al., 2015; Zhang et al., 2017)) which may provide even more specific behavioral outcomes. This work extends a role for drd1+ BLA neuron output to the central amygdala which was found to influence extinction memory (Zhang et al., 2020b), into two ventral striatum subregions which are important for valence-based behavioral responses, and by allowing for comparison with the influence of the neighboring drd2+ neurons.

Figure 7. Overview of findings illustrating the behavioral consequences of activating (left) or suppressing (right) drd1+ or drd2+ BLA neuron inputs to the NAc and TuS.

Dotted lines annotate pathways that are dispensable for regulating the behaviors tested.

Interestingly, when comparing changes in fear-associated respiration (well known to be influenced by sympathetic state (Stevenson and Ripley, 1952; Boiten, 1998; Homma and Masaoka, 2008; Hegoburu et al., 2011; Moberly et al., 2018)) and fear-induced immobility, we saw that manipulation of NAc projecting drd1+ BLA neurons did not similarly influence both of those fear-associated behaviors (Supplementary Fig 5). This may be due to either distinct inputs or outputs (collaterals) of the drd1+ BLA neurons which might differentially guide changes in respiratory behavior versus motor behavior. For instance, differential innervation of the periaqueductal grey might allow for one cell-type to influence respiration over another given the periaqueductal grey’s influence on breathing (Walker and Carrive, 2003; Subramanian and Holstege, 2013). While we did not identify the differential pathway, it is interesting to identify instances wherein fear-related behaviors are not simultaneously displayed. Further manipulation of this pathway while similarly taking multiple measures of fear-related behaviors, including even heart rate, skin conductance, and ultrasonic vocalizations for instance, will help refine our understanding of circuitry which specifically supports each to be displayed during emotional contexts.

Top-down glutamatergic inputs to the BLA profoundly influence associative learning and behaviors. Given the fact that these BLA ventral striatum neurons express dopamine receptors, it is tempting to speculate how this pathway may be modulated by dopamine. Dopamine within the BLA is necessary for fear learning (Fadok et al., 2009). Local antagonism of both D1Rs and D2Rs within the BLA blocks the expression of fear during a potentiated startle paradigm (Lamont and Kokkinidis, 1998; Greba et al., 2001). Antagonism of BLA D1Rs also perturbs the timing of fear behavior (Shionoya et al., 2013), and antagonism of BLA D2Rs attenuates freezing during Pavlovian fear conditioning (Guarraci et al., 2000; de Oliveira et al., 2011; de Souza Caetano et al., 2013). The role of dopamine receptors is similarly mixed in appetitive behaviors, where antagonizing both D1Rs and D2Rs within the BLA attenuates conditioned reward seeking and taking (See et al., 2001; Berglind et al., 2006; Kim and Lattal, 2019). Indeed, local application of D1 agonists increases intrinsic excitability and the evoked firing of BLA neurons (Kröner et al., 2005). D1 receptors have a lower affinity for dopamine than D2 receptors (Richfield et al., 1989; Schultz, 2007). Further, when dopamine levels are low, D2 receptors are agonized, but when DA levels are elevated, like when receiving an emotionally salient stimulus, both D1 and D2 receptors become agonized (Guarraci et al., 1999; Horvitz, 2000; Bristol et al., 2004). It is possible these differential roles of D1 and D2 receptors in the BLA might explain our finding that drd2+ neurons vs drd1+ neurons contributed differently to the regulation of emotional states.

Overall, this work has uncovered that drd1+ and drd2+ neurons within the Ppp1r1b BLA neuron class forms parallel pathways which bidirectionally influence emotional states when they are activated or suppressed and do so depending upon where they synapse – with unique contributions of drd1+ and drd2+ BA NAc vs BA→TuS circuitry on negative valence states. Overall, our results contribute to a model whereby parallel, genetically-distinct BLA to ventral striatum circuits inform emotional states in a projection-specific manner. This work adds to our understanding of the complex interplay between projection cell types and their projection targets, in how the BLA helps orchestrate emotions.

Materials and Methods

Animals

Adult male and female mice, 2–5 months of age, were housed in a temperature-controlled vivarium on a 12:12 hour (hr) light/dark cycle with ad libitum access to food and water, except during behavioral testing. All behavioral testing occurred during the light cycle. Mice that only underwent viral injections were group housed (≤5 mice/cage) and mice with chronic implants were single housed following surgery. All experimental procedures were conducted within the AALAC animal research program of the University of Florida in accordance with the guidelines from the National Institute of Health, and were approved by the University of Florida Institutional Animal Care and Use Committee.

Mouse lines included the following transgenic lines which were maintained on a C57BL/6J background (strain #000664; RRID:IMSR_JAX:000664, The Jackson Laboratory) and were bred in house within a University of Florida vivarium. drd1-Cre (B6.FVB(Cg)-Tg(Drd1-cre)EY262Gsat/Mmucd, RRID:MMRRC_030989-UCD), drd2-Cre (B6.FVB(Cg)-Tg(Drd2-cre)ER44Gsat/Mmucd, RRID:MMRRC_032108-UCD), and a2a-Cre (B6.FVB(Cg)-Tg(Adora2a-cre)KG139Gsat/Mmucd, RRID:MMRRC_036158-UCD) mice were obtained from the UC Davis Mutant Mouse Regional Resource Center. Ai9 TdTomato Cre reporter mice (B6.Cg-Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}/J; RRID:IMSR_JAX:007909, (Madisen et al., 2010)) were obtained from the Jackson Laboratory.

Viral vectors

rgAAV.hSyn.HI.eGFP-Cre.WPRE.SV40 (Addgene #105540-AAVrg, 7×10¹² vg/ml), Ef1α.DIO.Synaptophysin-mRuby and Ef1α.FLEX.Synaptophysin.GFP (both generous gifts from Dr Marc Fuccillo, University of Pennsylvania) (Herman et al., 2016), and AAV.hSyn.FLEx.mGFP-2A-Synaptophysin.mRuby (Addgene #71760-AAV1, 9.8×10¹² vg/mL) were used for tracing. AAV.Ef1a.DIO.hChR2(E123T/T159C)-EYFP (Addgene #35509-AAV5, 1×10¹² vg/ml vg/ml) was used for patch-clamp recording and for optogenetic stimulation during the optogenetic real time place preference/avoidance task. AAV.Ef1a.DIO.EYFP (Addgene #27056-AAV5, 1×10¹² vg/ml) was used as a control virus for the optogenetic real time place preference task. rgAAV.hSyn.DIO.hM4D(Gi)-mCherry (Addgene #44362-AAVrg, 1.2×10¹³ vg/ml) and rgAAV.hSyn.DIO.mCherry (50459-AAVrg, 1.8×10¹³ vg/ml) were used for chemogenetic inhibition.

Surgical procedures

For all surgical procedures, mice were anesthetized with 2%–4% isoflurane (IsoFlo, Patterson Veterinary, Greeley, CO) in 1 L/min O₂, and head fixed in a stereotaxic apparatus while their body temperature was maintained using a 38°C water bath heating pad. The scalp was shaved and cleaned with betadine and 70% ethanol. Following subcutaneous (s.c.) administration of Meloxicam (20 mg/kg) analgesia and local administration of the anesthetic lidocaine (lidocaine, 3 mg/kg, s.c., Patterson Veterinary) to the scalp, a small midline cranial incision was made.

For viral injections, craniotomies were made above the target regions. A pulled glass micropipette containing the AAV was slowly inserted for injection. For TuS injections, 50nl of viral solution was injected bilaterally at the following coordinates: anteroposterior (AP) +1.4mm bregma, mediolateral (ML) ±1.2mm lateral midline, dorsoventral (DV) −4.85mm from the brain surface. For NAc injections, 100nl of viral solution was delivered bilaterally (AP 1.5mm, ML ±1.0mm, DV −3.75mm). For BLA injections, 100nl of viral solution was delivered either unilaterally into the right hemisphere (AP −1.6mm, ML +3.25mm, DV −4.25mm) for Opto-RTPP/A and brain slice electrophysiology experiments, or bilaterally (AP −1.6mm, ML ±3.25mm, DV −4.25mm) for tracing experiments. All injections were performed at a rate of 2nl/second (s), with 20–40s intervals using a Nanoject III (Drummond Scientific). Following injection, at least 5min went by before slowly withdrawing the pipette from the brain. Craniotomies were then sealed with dental wax and the incision was closed with wound clips.

For cannula implantation, the skull was scrubbed with 3% H₂O₂ and covered with a thin layer of cyanoacrylate glue (Vetbond, 3M). Bilateral craniotomies were drilled over the BLA and 26-gauge(G) guide cannulae (#C315GMN/SPC, P1 Technologies) extending 3.5mm below pedestal were implanted at the coordinates AP −1.3mm, ML ±3.2mm. Cannulae were then lowered into the brain and secured to the skull with a small amount of Vetbond followed by dental cement, and dust caps with a 3.5mm projection wire (C315DCMN/SPC, P1 Technologies) were inserted.

For optical fiber implantation, following skull preparation for implantation as above, a craniotomy was made and drilled above the ventral striatum on the right hemisphere. Fibers (300µM core diameter, 0.39NA, 6.0mm length) for optogenetic stimulation were lowered into the NAc (AP 1.4mm, ML 1.0mm, DV-3.85mm) or the TuS (AP 1.5mm, ML 1.2mm, DV −4.9mm). The fiber was secured with Vetbond followed by dental cement as described for the cannulae implantation.

Following surgery, mice were allowed to recover on a heating pad until ambulatory, and were given immediate ad libitum access to food and water. Meloxicam analgesic (20mg/kg, s.c.) was administered for at least 3 days following surgery. Mice will indwelling cranial implants were single housed and given 7 to 14 days after surgery to recover before the being acclimated to behavioral procedures.

Histology

Immunohistochemistry

Mice were anesthetized with Fatalplus (150mg/kg; Vortech Pharmaceutical Ltd, Dearborn, MI) and transcardially perfused with cold 0.9% NaCl (Physiological Saline), followed by cold 10% phosphate buffered formalin (#SF100-4, Thermo Fisher Scientific) for fixation. Brains were collected and further fixed and cryoprotected in a 30% sucrose/10% formalin solution for 72hr at 4 °C. Serial 40μm thick coronal sections were collected using a sliding microtome (Leica) and were stored at 4 °C in a solution of Tris-buffered saline (TBS) with 0.03% sodium azide.

Sections from drd1- or drd2-Cre mice injected with Cre-dependent retrograde mCherry AAV underwent antigen retrieval in citrate buffer (pH 6.0) for 30mins at 80°C. After being rinsed with tris buffered saline (TBS; 0.242% Tris base, 2.924% sodium chloride, pH=7.4 ± 0.2) and diluting buffer (2% bovine serum albumin (Sigma Aldrich), 0.9% sodium chloride (Sigma Aldrich), 0.4% Triton-X 100 (Sigma Aldrich), and 1% normal goat serum (Sigma Aldrich) in TBS), samples were blocked in 20% normal donkey serum solution, then incubated in the primary antibody overnight at 4°C. Sections were then incubated in the secondary antibody at room temperature and washed with TBS prior to slide-mounting with DAPI Fluoromount-G^® mounting medium (SouthernBiotech, catalog #0100-20). Primary antibodies included rabbit anti-DsRed (Takara Bio, catalog #632496, 1:1000) and chicken anti-NeuN/FOX3 (EnCor, catalog #CPCA-FOX3, 1:1000). Secondary antibodies included anti-chicken Alexa Fluor 488, anti-rabbit Alexa Fluor 680 (both from Invitrogen, 1:1000 dilution).

Imaging and quantification

Brain regions were identified using the mouse brain atlas (Paxinos and Franklin, 2000). Images were acquired using a Nikon Eclipse Ti2e fluorescent microscope. For quantification of the number of drd1+ and drd2+ TuS and NAc projecting BLA neurons, at least three BLA sections from three mice of each genotype and injection site were acquired spanning from −1.10mm to −2.10mm posterior to Bregma. Images were acquired at 20x magnification across both hemispheres and Z-stacked every 4µm. For quantification, regions of interest (ROIs) were drawn around the areas of interest (LA, BA). Images were preprocessed to remove background and to enhance local contrast, a rolling ball algorithm was applied to remove background, and images underwent Gaussian smoothing and Laplace sharpening. A semi-automated thresholding and counting algorithm created within NIS elements (Nikon) software was used to identify cells within selected ROIs, allowing for unbiased estimation of cell numbers. Cells were identified based on fluorescence intensity (via threshold) and diameter.

For quantification of drd1+ and a2a+ BLA to ventral striatum synaptophysin puncta within the ventral striatum, at least three sections from three mice of each genotype were acquired spanning from 1.7mm to 0.6mm anterior to Bregma. Images were acquired at 20x magnification for the hemisphere ipsilateral to the injection site, and Z-stacked every 0.9µm. For quantification, ROIs were drawn around the areas of interest (TuS, NAcC, NAcSh, PCX). Images were preprocessed to remove the average background. A semi-automated thresholding and counting algorithm created within NIS elements software was used to identify fluorescent puncta within selected ROIs, allowing for unbiased estimation of the number of fluorescent puncta. Puncta were identified based on fluorescence intensity (via threshold) and diameter.

Brain slice electrophysiology

Whole-cell patch-clamp recordings were performed in ex vivo brain slices from drd1-Cre;Ai9 or a2a-Cre:Ai9 mice, in which tdTomato expression was directed within cells expressing either drd1 or drd2, respectively. A Cre dependent AAV encoding for ChR2 (AAV-Ef1a-DIO hChR2(E123T/T159C)-EYFP) was injected bilaterally into the BLA of drd1-Cre:Ai9 or a2a-Cre:Ai9 mice, 2–3 months of age. After waiting a minimum of one month to allow for ample AAV expression, acute brain slices were prepared as follows.

Mice were deeply anesthetized with intraperitoneal injection of ketamine-xylazine (200–15 mg/kg body weight) and decapitated. The cranium was dissected and the brain was immediately removed and placed in ice-cold HEPES buffered cutting solution containing (in mM): 92 N-methyl-d-glucamine, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 5 sodium l-ascorbate, 2 thiourea, 3 sodium pyruvate, 10 MgSO4 and 0.5 CaCl2 (osmolality ~300 mOsm and pH ~7.4, bubbled with 95% O2 and 5% CO2). Coronal brain slice (180–200µM) containing the OT were cut using a Leica VT 1200S vibratome. Brain slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.2 MgSO4, 1.4 NaH2PO4, 11 glucose, 25 NaHCO3 and 0.6 sodium L-ascorbate (osmolality ~300mOsm and pH ~7.4, bubbled with 95% O2 and 5% CO2) for 1hr at 31°C and at room temperature thereafter. Slices were transferred to the recording chamber for whole-cell patch-clamp recordings and continuously perfused with oxygenated ACSF. 4-Aminopyridine (4-AP; 200µM) was added to enhance optically evoked synaptic release in ChR2+ axonal terminals. Fluorescent D1-/A2A-tdTomato+ cells in OT were visualized with a 40 X water-immersion objective under an Olympus BX51WI upright microscope equipped with epifluorescence. Electrophysiological recordings were controlled by an EPC-10 amplifier combined with Pulse Software (HEKA Electronic) and analyzed using pulse and Clampfit (Axon instruments). Whole-cell patch-clamp recordings were made in both current and voltage-clamp mode. Patch pipettes were pulled from thin-wall borosilicate glass-capillary tubing (WPI, Sarasota, FL, USA) on a Flaming/Brown puller (P-97; Sutter Instruments Co., Novato, CA, USA). The tip resistance of the electrode was 5–8MΩ. The pipette solution contained the following (in mM): 120 K-gluconate, 10 NaCl, 1 CaCl2, 10 EGTA, 10 HEPES, 5 Mg-ATP, 0.5 Na-GTP, and 10 phosphocreatine.

To activate ChR2 in the OT slices, blue light (pE-300ultra, CoolLED, ~25mW) was delivered through the same 40X objective. Pharmacological reagents including tetrodotoxin (TTX) citrate (Abcam), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), d,l-2-amino-5-phosphonopentanoic acid (AP5), and 4-Aminopyridine (4-AP) (Sigma-Aldrich) were bath perfused during recording.

in vivo DREADD-based chemogenetic inhibition

For DREADD-based chemogenetic inhibition of Gi coupled inhibitory DREADD receptor (hM4Di) expressing neurons, drd1+ and drd2+ mice were injected with rgAAV.hSyn.DIO.hM4D(Gi)-mCherry (1.2×10¹³vg/ml, 100nl/hemisphere in NAc, 50nl/hemisphere in TuS, catalog #44362-AAVrg, Addgene) or rgAAV.hSyn.DIO.mCherry (1.8×10¹³vg/ml, 100nl/hemisphere in NAc, 50nl/hemisphere in TuS, catalog #50459-AAVrg, Addgene) as control. All mice were implanted 1–2 weeks later with bilateral intracranial guide cannulae (Protech International, Inc, catalog #8IC315GMNSPC, 26ga) extending 3.5mm beyond the pedestal, for direct administration of either the DREADD ligand J60 (Bonaventura et al., 2019) or vehicle into the BLA. Dust caps without a projection wire (Protech International, Inc, catalog #8IC315DCMNSP) were inserted immediately following surgery, and mice were given 1–2 weeks to recover.

Prior to behavior, mice underwent 2 days of handling in which the dummy cannulae were removed and replaced. On the habituation behavior day, mice received a “mock” infusion, wherein the internal cannulae (Protech International, Inc, catalog #8IC315MNSPC, 5.75mm projection, 33ga) connected to tubing from a 1µL Hamilton Syringe (Hamilton, catalog #86211) were inserted into the guide cannulae, and the Harvard Apparatus 22 Syringe Pump (catalog #PY2 55-2222) was turned on for 2 min to simulate the noise of the infusion. This mock infusion occurred 30 min prior to being placed in the plethysmograph for the Pavlovian fear learning behavioral paradigm, and occurred on a separate day from the spontaneous odor attraction/avoidance assay. On the learning day (Day 2) of the Pavlovian fear learning paradigm, and on the day of the spontaneous odor attraction/avoidance assay, mice were once again tethered to the Hamilton syringe, but this time received an infusion of 100nL of either 10nM J60 or vehicle at a rate of 50nl/min, 30 min prior to the start of the behavioral task.

Behavioral Tasks

Odor-shock Pavlovian fear learning

We used a whole-body plethysmography chamber (Data Sciences International, St. Paul, MN) that was adapted for the infusion of a neutral odor and the administration of a mild foot shock for an odor-shock Pavlovian fear learning test, as originally developed for use in rats (Hegoburu et al., 2011). We constructed an air-dilution olfactometer (Gadziola et al., 2015; Johnson et al., 2020) and used custom code in Synapse (Tucker Davis Technologies) to control the delivery of an otherwise neutral odor, isopentyl acetate (1 torr in liquid state; Sigma Aldrich), at a flow rate of 1 L/min (20s) which co-terminated with the presentation of a mild foot shock (0.5mA for 1s). Respiratory transients were detected using a Data Sciences pressure transducer, gain amplified 100 X (Cygnus Technology Inc), and digitized (0.1–20Hz) at 300Hz in Synapse. Positive pressure of clean room air was continuously applied to the chamber using a stable-output air pump (Tetra Whisper). Following each stimulus trial, odor-vaporized air was exhausted from the plethysmograph through an outlet at the chamber’s ceiling.

Mice were acclimated to handling in the behavioral room for two days prior to entering the plethysmograph. Mice were then acclimated to the plethysmograph by undergoing a session in which no odors or shock were delivered, but the associated sounds were present (Supplementary Fig 4). Twenty-four hr later on the acquisition day, mice were allowed to acclimate to the plethysmograph for a 4-minute (min) period and were then presented with 10 trials of 20s odor delivery co-terminating with an odor-paired 1s foot shock (0.5mA) with an inter-trial interval (ITI) of 180s. For the unpaired fear conditioning task, the foot shock was presented pseudorandomly in the ITI (90s after the foot shock). For the odor only control mice, the 10 trials consisted of only 20s odor delivery without the administration of the foot shock. The shocked mice received no odor delivery during the trials, but received a foot shock either at the end of the trial (trial shock group) or pseudorandomly in the ITI (ITI-shock group). Mice were then returned to their home cage. Twenty-four hr later on the retrieval day, the odor was presented for 10 trials without the foot shock for all groups receiving odor (paired, unpaired, and odor only groups). Mice who did not previously receive the odor underwent the 10 trials without odor delivery or foot shock. Mobility behavior was recorded throughout the entire fear conditioning task using two digital cameras (Microsoft, 10Hz frame rate), and was scored in 0.4s bins during the 19s of odor presentation prior to shock using ezTrack (Pennington et al., 2019) to identify periods of physical immobility. Respiration digitized from the pressure transducer was imported into MATLAB and a MATLAB script was used to calculate fast-fourier transform (FFT) power spectra of the respiratory signal during odor (excluding the 1s when the shock co-occurred) as compared to pre-odor (see Supplementary Fig 4).

Spontaneous odor attraction or avoidance

To test the spontaneous attraction or avoidance towards odors, a 30.48 × 30.48 × 30.48 cm (length × width × height) dark acrylic chamber was divided into two equal sides by a transparent acrylic plate with a tunnel in the bottom center to allow mice to pass through (Fig 6). An infrared video camera was placed above the chamber to record activity of the mouse in each chamber (12Hz frame rate).

Cotton swabs laced with peanut oil (diluted in mineral oil, 1:12.5), an appetitive odor, were placed in a perforated microcentrifuge tubes to prevent touching or tasting the stimulus while still allowing the release of volatiles. Tubes containing this appetitive odor were placed on one side of the chamber, while perforated microcentrifuge tubes containing cotton swabs laced with 2-Methyl-2-thiazoline (2MT, 97%, Fisher Scientific, diluted in mineral oil, 1:50), a component of fox feces, were placed on the opposite side. These dilutions were selected to achieve a comparable intensity of odor from each tube.

Mice were handled for two days prior to the behavioral assay to acclimate the mice to experimenter handling, and mice received a mock infusion to acclimate the mice to the sound of the infusion pump. On the day of the behavioral assay, mice received an infusion of either J60 or vehicle 30min prior to being placed in the center of the chamber, with the odors arranged on opposite sides. Mice were allowed to explore the appetitive peanut oil and aversive fox urine sides for 10min. All testing was performed in a dark room with a single dim light to illuminate subjects, and infrared video recordings were used to assess the amount of time spent in each side, after subtracting out the middle third of the apparatus – i.e. location of the tunnel. Analyses were performed in ezTrack (Pennington et al., 2019).

Optogenetic real time place preference or aversion test (Opto-RTPP/A)

Mice were gently handled and acclimated to the behavior room the day prior to the opto-RTPP/A test. Prior to starting the opto-RTPP/A test, mice were gently scruffed, the dust cap was removed, and the mice were tethered to a 400µm, 0.57NA fiber (Thorlabs, catalog #M58L01) and placed in a 15.24 × 40.64 × 27.94 cm (length × width × height) apparatus divided into three chambers. This fiber was connected to an LED (Doric, 465nm) through a rotary joint connected to a 400µm, 0.39NA patch cable. Mice were placed in the center of a three-chamber apparatus and allowed to explore for 30min. An infrared video camera was placed above the chamber to record activity of the mouse in each chamber (12Hz frame rate). When mice entered into one of the three chambers, and subsequently broke the infrared beam path, light stimulation (465nm, 15ms pulse width, 40Hz) was initiated and continuously delivered until mice left the chamber and ceased breaking the infrared beams (controlled by an Arduino). At the end of the 30min, mice were gently restrained and the tether was removed, following which the mice were returned to their home cage. The mice were euthanized and perfused the same day, and brains were collected for histological verification of virus injection and optic fiber placement. Analyses were performed in ezTrack (Pennington et al., 2019) to quantify the time spent in each chamber and to generate maps of physical space for illustration purposes.

Data analysis

Data were analyzed for statistical significance in GraphPad Prism. All data are reported as mean±SEM unless otherwise noted. Specific tests used can be found in the Results sections or the figure legends. All t-tests were paired. When possible, experimenters handling the data were blinded to treatment conditions.