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. Author manuscript; available in PMC: 2024 May 28.
Published in final edited form as: Mol Psychiatry. 2022 Nov 28;29(6):1671–1682. doi: 10.1038/s41380-022-01858-3

Ventral tegmental area glutamate neurons mediate nonassociative consequences of stress

Dillon J McGovern 1, Annie Ly 1, Koy L Ecton 1, David T Huynh 1, Emily D Prévost 1, Shamira C Gonzalez 1, Connor J McNulty 1, Andrew R Rau 2,4, Shane T Hentges 2,5, Tanya L Daigle 3, Bosiljka Tasic 3, Michael V Baratta 1,✉, David H Root 1,✉
PMCID: PMC10375863  NIHMSID: NIHMS1913420  PMID: 36437312

Abstract

Exposure to trauma is a risk factor for the development of a number of mood disorders, and may enhance vulnerability to future adverse life events. Recent data demonstrate that ventral tegmental area (VTA) neurons expressing the vesicular glutamate transporter 2 (VGluT2) signal and causally contribute to behaviors that involve aversive or threatening stimuli. However, it is unknown whether VTA VGluT2 neurons regulate transsituational outcomes of stress and whether these neurons are sensitive to stressor controllability. This work adapted an operant mouse paradigm to examine the impact of stressor controllability on VTA VGluT2 neuron function as well as the role of VTA VGluT2 neurons in mediating transsituational stressor outcomes. Uncontrollable (inescapable) stress, but not physically identical controllable (escapable) stress, produced social avoidance and exaggerated fear in male mice. Uncontrollable stress in females led to exploratory avoidance of a novel brightly lit environment. Both controllable and uncontrollable stressors increased VTA VGluT2 neuronal activity, and chemogenetic silencing of VTA VGluT2 neurons prevented the behavioral sequelae of uncontrollable stress in male and female mice. Further, we show that stress activates multiple genetically-distinct subtypes of VTA VGluT2 neurons, especially those that are VGluT2+VGaT+, as well as lateral habenula neurons receiving synaptic input from VTA VGluT2 neurons. Our results provide causal evidence that mice can be used for identifying stressor controllability circuitry and that VTA VGluT2 neurons contribute to transsituational stressor outcomes, such as social avoidance, exaggerated fear, or anxiety-like behavior that are observed within trauma-related disorders.

INTRODUCTION

Exposure to trauma has been implicated in a number of mood and anxiety disorders. Traumatic experiences can lead to a broad range of negative consequences that are supported by both associative (e.g., avoidance of or distress due to stimuli related to the trauma) and nonassociative (e.g., exaggerated transsituational responding to circumstances unrelated to the trauma) processes [1–3]. Although exposure-based therapies are considered an efficacious intervention for a variety of stress-linked disorders, a substantial number of individuals fail to achieve long-term symptom reduction [4–6]. Treatment resistance, in part, may be due to the fact that exposure therapy primarily targets the learned, associative components of the disorder, leaving the nonassociative aspects unaddressed [7].

Well known for its role in reward, the ventral tegmental area (VTA) also plays an important role in aversion [8, 9]. The VTA is a cellularly heterogeneous midbrain region. In addition to dopamine neurons, the VTA contains GABAergic and glutamatergic subpopulations [10, 11] that exhibit distinct synaptic architecture and connectivity [8, 9]. Subpopulations of dopamine, GABA, and glutamate neurons have been shown to participate in aversive processing [11–13]. Notably, VTA glutamate neurons, defined by expression of the vesicular glutamate transporter 2 (VGluT2), are also activated by defensive responses to perceived threat [14]. Even in the absence of an external threat, photostimulation of VTA VGluT2 projections to lateral habenula is sufficient to form conditioned place aversion [13]. Based on results showing the involvement of VTA VGluT2 neurons in aversive processing, we hypothesized that VTA VGluT2 neurons contribute to the nonassociative behavioral sequelae that follow stress.

In the present experiments, we adapted a stressor controllability paradigm from rats to mice to examine the role of VTA VGluT2 neurons in mediating the transsituational outcomes of stress. Prior work, largely in rats, has shown that exposure to uncontrollable stress produces behavioral sequelae (e.g. social avoidance, exaggerated fear) that do not occur with physically-identical controllable stress [15]. Further, uncontrollable stress alters subsequent behaviors in circumstances different from the original stress experience, demonstrating that outcomes dependent on the uncontrollability of the stressor are mediated by a nonassociative process [15–17]. We found in male mice that inescapable tailshock stress (IS) produced social exploration deficits, as well as exaggerated fear, that were absent if the tailshock was escapable (ES). Female mice showed increased anxiety-like behavior in the light/dark box following IS but did not show changes in sociability. Latency to exit the dark compartment of the light/dark box was sensitive to the dimension of controllability in females but overall time spent in the light chamber did not differ between ES and IS groups. Calcium recordings showed that tailshock robustly increased the activity of VTA VGluT2 neurons independent of shock controllability. Chemogenetic inhibition of VTA VGluT2 neurons during tailshock blocked IS-induced social exploration deficits and exaggerated footshock-elicited freezing responses in males as well as blocked IS-induced anxiety-like behavior in the light/dark box in females. Multiple subtypes of genetically-defined VTA VGluT2 neurons were activated by stress but the subpopulation of VGluT2 neurons that co-expresses vesicular GABA transporter (VGaT) showed the largest percentage of c-Fos activation. Lateral habenula neurons that receive projections from VTA VGluT2 neurons, mostly from VTA VGluT2+VGaT+ neurons [13, 18–20], were also activated by stress. Overall, we interpret these data to reflect that stress-induced activation of VTA VGluT2 neurons is required for the development of nonassociative behavioral consequences that follow stress in male and female mice. Because VTA VGluT2 neurons did not discriminate their activity between escapable and inescapable stress, other cell-types and circuits must play an important role in the discrimination of inescapable stress-induced behavior changes. Finally, these experiments establish a mouse stressor controllability paradigm that can be combined with other genetically diverse mouse driver lines for identifying the cell-types and circuits underlying the nonassociative aspects of traumatic stress exposure.

METHODS AND MATERIALS

Male and female C57BL/6J, VGluT2::Cre, or Ai193/VGluT2::Cre/VGaT::Flp mice were used. All animal procedures were approved by the University of Colorado Boulder Institutional Animal Care and Use Committee and conformed to National Institutes of Health guidelines. For manipulation of stressor controllability, mice were run in a triadic design consisting of escapable, yoked-inescapable, and no stress conditions. GCaMP6m recordings of VTA VGluT2 neuronal activity were performed as previously described [21]. Behavioral outcomes, conducted 24 h after stress, included social exploration, shock-elicited freezing, and exploration of the light/dark box. VTA VGluT2 subtype profiling was determined with immediate early gene immunolabeling and fluorescent microscopy. Detailed experimental procedures are provided in Supplementary Methods.

RESULTS

Stress-induced social avoidance depends on the uncontrollability of the stressor in male mice

We first determined if deficits in social behavior, a common feature of anxiety disorders [22, 23], would result from prior exposure to inescapable stress (IS). Male and female C57BL/6 J mice received IS or no stress (homecage control; HC) and twenty-four hours later sociability was evaluated with the three-chamber social preference test (Fig. 1A). The 10 min social preference test was conducted in a novel apparatus and in a novel procedure room so as to minimize the presence of common cues between the prior tailshock stress and subsequent behavioral testing environments. For male mice, a mixed ANOVA yielded a Group x Stimulus interaction, F(1,14) = 5.519, p = 0.034. Sidak-adjusted pairwise comparisons indicated that IS males spent significantly less time with the social stimulus than no stress HC males, p = 0.035 (Fig. 1B). In addition, no stress HC males demonstrated a significant preference for social stimulus interaction (p < 0.001) while IS males showed no difference in time spent between the social and nonsocial stimuli. In contrast to males, for female mice a mixed ANOVA yielded a main effect of social stimulus, F(1,10) = 8.790, p = 0.014. Sidak-adjusted pairwise comparisons indicated an overall preference for spending time with a novel conspecific versus exploring an empty chamber (p < 0.05) across both HC and IS females groups (Fig. 1C). There was no Group X Stimulus interaction, F(1,10) = 0.169, p > 0.05. These results establish that prior inescapable tailshock selectively induces social avoidance in male mice.

Fig. 1. Inescapable stress reduces social exploration in male, but not female, C57BL/6 J mice.

Fig. 1

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A Schematic of the experimental timeline. Mice first received either inescapable stress (IS; 100 tailshocks, 5 sec duration each) or home cage treatment (no tailshock) followed by testing in the three-chamber sociability test 24 h later. n = 8/group for males and n = 6/group for females. B Quantification of interaction behavior with a social (same-sex conspecific) or a nonsocial (empty chamber) stimulus for male and (C) female subjects. Data are mean ± SEM. *p < 0.05, ***p < 0.001; ANOVA with Sidak’s multiple comparison post hoc test.

We next tested whether the social-related consequence of tailshock was dependent on its uncontrollability. Here, pairs of male mice received physically identical tailshocks, with one member having control over shock termination (escapable stress; ES), and the other yoked to the ES subject but having no control (yoked-inescapable stress; Y-IS). For each trial, tailshock was terminated for both subjects when the ES mouse completed the wheel-turn instrumental response criterion. Thus, both ES and Y-IS mice received exactly the same duration, intensity, and temporal pattern of tailshocks during stress treatment (Fig. 2A). All ES mice acquired the wheel-turn controlling response, significantly increasing the number of required quarter wheel-turns across the session until reaching the maximum response criterion (12 quarter-wheel turns), Friedman’s test Χ2(9) = 30.941, p < 0.001 (Fig. 2A). Sidak-adjusted pairwise comparisons showed that the criterion quarter wheel-turns completed during the initial block of ten tailshock trials was significantly less than the last five blocks of ten tailshock trials, all p < 0.05. There was no significant change in latency to complete the criterion of quarter wheel-turns to terminate tailshocks over the training session, Χ2(9) = 11.69, p > 0.05. Termination latency for the last 10 trials (mean = 2.49, standard deviation = 1.23) was about half the duration of the non-yoked paradigm in the first experiment, which consisted of 5 sec programmed shocks for the IS condition.

Fig. 2. Stress-induced reduction in social exploration is dependent on the controllability of the stressor in male mice.

Fig. 2

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A Illustration of the stressor controllability procedure. Male escapable (ES) with a functional wheel and yoked-inescapable stress (IS) mice with a locked wheel received 100 identical tailshocks (average intertrial interval 60 sec) that were terminated by the ES mouse. Subpanel: Average number of wheel quarter-turns per 10 block trials. B Quantification of interaction behavior with a social (same-sex conspecific) or a nonsocial (empty chamber) stimulus. ES and home cage (HC) subjects spent more time with the social stimulus than the nonsocial stimulus. IS mice spent equal time with both stimulus types. C Preference for the social stimulus compared to the nonsocial stimulus (social score) was significantly different between ES and IS groups, as well as HC and IS groups, but not ES and HC groups. Social scores were quantified by the following formula: (social interaction time – nonsocial interaction time) / (social interaction time+nonsocial interaction time). n = 10 ES, 9 Y-IS, 10 HC. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ANOVA with Sidak multiple comparison post hoc test.

Twenty-four hours following stress treatment, ES, Y-IS, and no stress HC mice were tested in the three-chamber sociability test. The mixed ANOVA yielded a Group x Stimulus interaction, F(2,26) = 3.56, p < 0.05. Sidak-adjusted pairwise comparisons indicated that both ES and HC mice spent significantly more time with the social stimulus than the nonsocial stimulus (ES: p < 0.001; HC: p < 0.01), however preference for the conspecific was absent in Y-IS mice (p > 0.05) (Fig. 2B). Specifically, ES mice had a significantly higher social score than Y-IS mice, main effect of Group: F(2,26) = 6.27, p < 0.01, Sidak-adjusted pairwise comparison p < 0.01 (Fig. 2C). The social score of Y-IS mice was near 0, indicating a complete lack of preference between social and nonsocial stimuli. Total social interaction time was significantly less in IS mice when compared to home cage controls using an unpaired t-test, t(17) = 2.457, p = 0.0342, reconfirming that inescapable stress is sufficient to impair social exploration. These results establish that stressor-induced reduction in social exploration with a novel conspecific is controllability-dependent, with Y-IS reducing social exploration, but not physically identical ES, or nonstressed HC controls.

VTA VGluT2 neurons respond to both uncontrollable and controllable stressors

In addition to dopamine and GABA neurons, the VTA contains glutamate-releasing VGluT2-expressing neurons [10]. VTA VGluT2 neurons show increased neuronal activity following a wide range of aversive events [24], including shock [18], and can produce place aversion or reward following their photostimulation in a projection-defined manner [13, 25, 26]. To understand whether controllability of the stressor modifies the activation of VTA VGluT2 neurons, we virally expressed the Cre-dependent Ca2+ sensor GCaMP6m in the VTA of VGluT2::Cre male mice and implanted an optical fiber into the VTA to record changes in VGluT2 neuron GCaMP6m activity during the stressor controllability paradigm (Fig. 3A, B; Supplementary Fig. 2). We found that GCaMP6m Ca2+ activity of VTA VGluT2 neurons significantly increased immediately following tailshock when compared to activity immediately prior to tailshock in ES and IS groups during both the early phase acquisition of the wheel-turn response (initial 20 trials) and the late phase (final 20 trials) of the session. Wilcoxon signed ranked tests indicate that Ca2+ activity was significantly higher at shock than baseline during the ES early phase Z = 60.712, p < 0.05, the IS early phase Z = 49.848, p < 0.05 (Fig. 3C, D), the ES late phase Z = 23.728, p < 0.05, and the IS late phase Z = 8.524, p < 0.05 (Fig. 3E, F). Ca2+ activity was not significantly different between ES and IS conditions at shock onset for the early stage t(10) = 0.4439, p = 0.666, or for the late stage of the session, t(10) = 0.6476, p = 0.5318. Thus, the strong activation of VTA VGluT2 neurons to tailshock was not sensitive to its controllability across the tailshock session.

Fig. 3. VTA VGluT2 neuron signaling is enhanced by both inescapable and escapable stress.

Fig. 3

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A Schematic of viral delivery of Cre-dependent GCaMP6m and optical fiber implantation in VTA of VGluT2::Cre mice. B Integration of fiber photometry with the stressor controllability paradigm. Light path for fluorescence excitation and emission is through a single 400 µm optical fiber patch cord to the VTA fiber implant. C Mean trial-by-trial time course of average GCaMP6m z-scores event-locked to tailshock delivery and average peak activity over the first twenty trials. Dark red/green lines show mean activity, while lighter shaded lines indicate standard error. D Individual normalized z-scored data during the baseline (BL) period prior to and following tailshock (shock) during the first twenty trials. Average activity significantly increased compared to BL for both escapable (ES) and inescapable (IS) mice, *p < 0.05. ES and IS did not differ during the first twenty trials. E Mean trial-by-trial time course of average GCaMP6m z-scores event-locked to tailshock delivery and average peak activity over the final twenty trials. Dark red/green lines show mean activity, while lighter shaded lines indicate standard error. F Average activity significantly increased during tailshock compared to BL for both ES and IS, *p < 0.05, but they did not differ between groups during the last twenty trials. n = 6/group. Data are mean ± SEM.

Chemogenetic inactivation of VTA VGluT2 neurons prevents the transsituational outcomes of stress

The sensitivity of VTA VGluT2 neurons to repeated tailshock experience led us to hypothesize that VTA VGluT2 neurons may participate in the enduring consequences of stress. To begin testing this, we first validated a chemogenetic approach for reducing VTA VGluT2 activity using the inhibitory Gi-coupled designer receptor, hM4Di, and its actuator compound, low-dose clozapine [27]. VGluT2::Cre mice were injected with an adeno-associated viral vector encoding either Cre-dependent mCherry (VGluT2 control mice) or Cre-dependent hM4Di tethered to mCherry (VGluT2 hM4Di mice) (Fig. 4A, B). Three weeks later, mice were injected with clozapine (0.1 mg/kg) ten minutes prior to testing and received five brief (0.5 sec) footshocks, a stimulus that rapidly activates VTA VGluT2 neurons via increased glutamate input [21]. Activation of VTA VGluT2 neurons was assessed by c-Fos expression within mCherry-labeled neurons (Fig. 4C–E). On average, 22.55% ± 1.89% of mCherry neurons co-expressed c-Fos in VGluT2 control mice, which was significantly greater than VGluT2 hM4Di mice of 6.00% ± 0.43%, t(11) = −9.191, p < 0.001. In addition, VTA mCherry-labeled neurons from VGluT2 hM4Di mice were whole-cell recorded in the presence or absence of clozapine (Fig. 4F). Electrophysiological properties of VTA VGluT2 neurons were similar to prior reports [28, 29] (Supplementary Fig. 3). Current-evoked action potentials prior to clozapine wash (10 µM) were normalized and compared to post-clozapine current-evoked firing (Fig. 4G, H). Clozapine activation of the hM4Di receptor on VTA VGluT2 neurons significantly reduced the percent of current-evoked action potentials compared to normalized baseline evoked action potentials, t(6) = 2.553, p = 0.043. Together, results are consistent with hM4Di activation reducing VTA VGluT2 neuronal activity.

Fig. 4. hM4Di-receptor activation reduces VTA VGluT2 neuronal activity.

Fig. 4

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A VGluT2::Cre male mice were injected in VTA with Cre-dependent mCherry (VGluT2 control, CTL) or B hM4Di-mCherry (VGluT2 hM4Di). Mice were exposed to brief footshocks and VTA mCherry-labeled neurons were examined for co-expression of c-Fos immunoreactivity C, D. E mCherry was co-expressed with c-Fos significantly more often in VGluT2 CTL mice compared with VGluT2 hM4Di mice. n = 7 hM4Di-mCherry and 6 mCherry mice. F Schematic of targeting Cre-dependent viral vector encoding the inhibitory designer receptor hM4D(Gi) to VTA in VGluT2::Cre mice and subsequent recording. Representative traces G and summarized data H from whole-cell recordings show that the designer receptor actuator clozapine (10 µM) significantly decreased the number of current-evoked action potentials in hM4D(Gi)-mCherry VTA VGluT2 neurons. Red line indicates normalized baseline firing.

Next, to test the role of VTA VGluT2 neurons in mediating the behavioral outcomes of IS, VGluT2::Cre mice were injected in VTA with a Cre-dependent adeno-associated viral vector encoding hM4Di-mCherry (VGluT2 hM4Di mice) or a Cre-dependent control vector encoding either GFP or mCherry (VGluT2 control mice or HC no stress mice) (Fig. 5A). Uninjected VGluT2::Cre mice were assigned to ES in order to provide a temporal pattern and duration of tailshock that was comparable to the fiber photometry (ES condition) experiment (Fig. 4D, E). The ES mouse simultaneously controlled the duration, intensity, and temporal pattern of tailshocks for both yoked inescapable stress hM4Di mice and yoked inescapable stress VGluT2 control mice. Ten minutes prior to stress treatment, VGluT2 hM4Di and IS VGluT2 control mice were injected with a behaviorally subthreshold dose of clozapine (0.1 mg/kg) [27]. A separate group of VGluT2 control mice were designated as no stress HC and were injected with the same dose of clozapine before being returned to the colony. ES mice acquired the instrumental controlling response, as demonstrated by a significant increase in the number of criterion quarter wheel-turns to terminate tailshocks over the shock session, Friedman’s test Χ2(9) = 17.03, p < 0.05. Sidak-adjusted pairwise comparisons showed that the criterion quarter wheel-turns completed during the initial block of ten tailshock trials was significantly less than the last block of ten tailshock trials, p < 0.01.

Fig. 5. Chemogenetic suppression of VTA VGluT2 neuron activity blocks nonassociative behavioral consequences of inescapable stress.

Fig. 5

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A Illustration of the experimental setup in which VGluT2 hM4Di or VGluT2 control (CTL) mice received yoked-inescapable shock (IS; 100 tail shocks, average intertrial interval 60 sec). Ten minutes prior to IS or home cage treatment, VGluT2 hM4Di, VGluT2 CTL, and no stress home cage controls (HC) received clozapine (0.1 mg/kg; i.p.). B Twenty-four hours following tailshock stress, mice were tested for sex-specific outcomes of IS. Males were tested for sociability and fear; females were tested for exploratory behavior in the light/dark box. n = 13 IS VGluT2 CTL, 8 IS VGluT2 hM4Di, 9 HC, 14 ES for males and 9 HC, 6 IS, 8 IS VGluT2 CTL, 9 IS VGluT2 hM4Di, 10 ES for females. C Quantification of interaction behavior with a social (same-sex conspecific) or a nonsocial (empty chamber) stimulus 24 h following stress treatment. HC, IS VGluT2 hM4Di, and ES mice spent significantly more time with the social stimulus compared to the nonsocial stimulus. IS VGluT2 CTL mice spent equal time with both social and nonsocial stimulus types. D Preference for the social stimulus compared to the nonsocial stimulus (social score) significantly differed between HC, ES, IS VGluT2 hM4Di, and IS VGluT2 CTL groups. IS VGluT2 hM4Di, HC, and ES groups did not differ from one another. E Mean time spent freezing in response to the delivery of two footshocks in a novel context. IS VGluT2 CTL mice showed enhanced freezing which was absent in IS VGluT2 hM4Di, unstressed HC mice, and ES mice. F–G In wild-type female mice, IS led to increased latency to exit the dark compartment compared to HC and ES female mice (F) and decreased time spent in the light compartment (G) of the light/dark box compared to HC female mice. H–I Following IS, VGluT2 CTL mice, but not VGluT2 hM4Di mice show increased latency to exit the dark compartment (H) and decreased time spent in the light compartment (I) of the light/dark box. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ANOVA with Sidak multiple comparison post hoc test.

Twenty-four hours later, male mice were first assessed in the social exploration test (Fig. 5B). A mixed ANOVA yielded a Group x Stimulus interaction, F(3,28) = 4.253, p < 0.05. Sidak-adjusted pairwise comparisons showed that VGluT2 hM4Di (p < 0.001), HC (p < 0.001), and ES (p < 0.001) groups spent significantly more time with the social stimulus than the nonsocial stimulus, but this social preference was absent in IS VGluT2 control mice (p > 0.05) (Fig. 5C). Social score was significantly lower in IS VGluT2 control mice compared to IS VGluT2 hM4Di, HC groups, and ES groups (F(3,32) = 9.238, p < 0.01; all Sidak-adjusted pairwise comparisons p < 0.05) (Fig. 5D). Thus, chemogenetic inhibition of VTA VGluT2 neurons during uncontrollable stress exposure blocked the social deficits that resulted from IS.

We then examined another transsituational outcome that follows uncontrollable stress in rats—enhanced fear [30] via shock-elicited freezing in a novel context and procedure room (Fig. 5B). During the two minute baseline exploration period, subjects showed minimal to no freezing in the novel chamber, suggesting that subjects did not generalize fear across the tailshock and footshock contexts, F(2,21) = 2.229, p > 0.05 (Supplementary Fig. 1D). Total freezing was calculated over the two minutes following the first footshock. The univariate ANOVA yielded a significant main effect of Group, F(3,39) = 9.592, p < 0.0001. Sidak-adjusted pairwise comparisons showed that IS VGluT2 hM4Di, HC, and ES mice spent significantly less time freezing than IS VGluT2 control mice (all p < 0.01) indicating that silencing VTA VGluT2 neuronal activity during inescapable stress prevented subsequent IS-induced enhanced fear (Fig. 5E). IS VGluT2 hM4Di mice and HC groups did not differ from one another in total time spent freezing, p = 0.983.

We next returned to female mice, which previously showed no change in social exploration behavior following inescapable stress. We reasoned that because sex differences exist in mouse social behavior [31–34] that female mice might differentially display stress-induced changes in behavior in a nonsocial context. We hypothesized that females would show a general exploratory deficit when faced with an ambiguous but mildly threatening environment without inducing freezing behavior. Thus, we tested for exploratory behavior in the light/dark box in which performance is comparable between males and females [35] and reliably shows sensitivity to anxiolytics [36]. We found that female mice that received yoked IS had significantly longer latency to exit the dark compartment when compared to home cage no stress females (HC) and ES female mice, F(2,22) = 5.245, p < 0.01, all Sidak-adjusted pairwise comparisons of IS versus ES or HC p < 0.05 (Fig. 5F). Total time in the light side of the compartment was also recorded for HC, IS, and ES female mice. Data were significantly different across experimental conditions, F(2,22) = 9.907, p < 0.001. However, pairwise comparisons were only significant between HC and IS conditions (p < 0.001) and there was no significant difference between ES and IS conditions (p > 0.05) (Fig. 5G).

Following the identification of stress-modulated exploratory behavior in the light/dark box of female mice, we next chemogenetically inhibited VTA VGluT2 neurons during IS in females and examined the same behaviors in the light/dark box (Fig. 5A, B). Chemogenetic inhibition of VTA VGluT2 neurons in female VGluT2 hM4Di mice blocked the increased latency to exit the dark compartment of the light/dark box observed in female VGluT2 control mice expressing mCherry, t(15) = 3.004, p = 0.0089 (Fig. 5H). In addition, the reduced total time in the light compartment observed in female VGluT2 control mice was rescued in female VGluT2 hM4Di mice, t(15) = 2.190, p < 0.05 (Fig. 5I). Together, both male and female mice show changes in behavior following inescapable stress that are dependent on VTA VGluT2 neuronal activity.

Subsets of VTA VGluT2 neurons release glutamate alone, release both dopamine and glutamate, or release both glutamate and GABA [8, 9, 18, 19, 29, 37]. Given the lack of discrimination within VTA VGluT2 GCaMP signaling in response to escapable and inescapable stress, we aimed to identify which VTA VGluT2 neuronal subtypes are activated by stress in general. We crossed Ai193 reporter mice [38] with double transgenic VGluT2::Cre/VGaT::FlpO mice [18], generating triple transgenic Ai193/VGluT2::Cre/VGaT::FlpO mice (Fig. 6A). Ai193 mice express GFP in the presence of Cre and express tdTomato in the presence of Flp. Therefore, Ai193/VGluT2::Cre/VGaT::FlpO mice express GFP in VGluT2-expressing glutamate neurons and tdTomato in FlpO-expressing GABA neurons (Fig. 6B). We then exposed these mice to IS (n = 3 male and 2 female mice) using tailshock onset/offset patterns from previously recorded ES mice, or delivered no stress (n = 2 female mice). Together with immunohistochemistry for tyrosine hydroxylase (TH) to label dopamine neurons, and c-Fos immunohistochemistry to identify stress-activated neurons, we counted neurons expressing GFP alone (VGluT2 + neurons), co-expressing GFP and TH (VGluT2 + TH + neurons), and co-expressing GFP and tdTomato (VGluT2 + VGaT+ neurons) with and without c-Fos co-expression (Fig. 6C–E). In HC mice, c-Fos co-expression in any cell-type was minimal, on average less than 5% for each. However, after IS all VTA VGluT2 neuron subtypes expressed large percentages of c-Fos co-expression, with the largest co-expression within VGluT2+VGaT+ neurons (41.4% ± 5.73%), followed by VGluT2+ neurons (27.11% ± 4.96%), and VGluT2+TH + neurons (24.03% ± 8.78%) (Fig. 6F). The percent of c-Fos expressing VGluT2+VGaT+ neurons was significantly greater than c-Fos expressing VGluT2+ and VGluT2+TH + neurons (Overall Friedman test: X2(2) = 8.4, p = 0.015; posthoc Wilcoxon tests: VGluT2+VGaT+ vs VGluT2 + z = −2.023, p = 0.043; VGluT2+VGaT+ vs VGluT2+TH + z = −2.023, p = 0.043) and c-Fos co-expression did not differ between VGluT2+ and VGluT2+TH + neurons, z = −1.753, p = 0.08. These results support prior work indicating that multiple types of VTA VGluT2 neurons are sensitive to aversive stimuli [18, 21, 24] but highlight VTA VGluT2+VGaT+ neurons as particularly sensitive to aversive outcomes.

Fig. 6. VTA VGluT2 cell-types and pathways activated by inescapable stress.

Fig. 6

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A Breeding strategy to generate triple transgenic Ai193/VGluT2::Cre/VGaT::FlpO mice that were then exposed to inescapable shock. B VTA section from a Ai193/VGluT2::Cre/VGaT::FlpO mouse and example c-Fos expressing VGluT2+ neuron C VGluT2+ TH + neuron D, and VGluT2+ VGaT+ neuron E (gray arrows). F Percentage of VTA VGluT2 neuron subtypes co-expressing c-Fos following inescapable stress. G Viral strategy to label postsynaptic neurons of VTA VGluT2 neurons. n = 5 mice. H Section from a HSV-tdTomato expressing VGluT2::Cre mouse at the level of the lateral habenula/hypothalamus and example c-Fos expressing lateral habenula neurons I or lateral hypothalamus neurons J that receive synapses from VTA VGluT2 neurons (tdTomato, gray arrows show co-expression). K Percent of c-Fos co-expressing HSV-tdTomato LHb or lateral hypothalamus neurons following inescapable tailshock stress. n = 25 sections from ten mice. Data are average ± SEM. Scale bars: 100 µm B, 25 µm C–E, 500 µm H, 25 µm I–J. TH tyrosine hydroxylase, LHb lateral habenula, LH lateral hypothalamus.

Based on the result that VTA VGluT2+VGaT+ neurons expressed c-Fos more often than other VGluT2 cell-types following stress, and that VTA VGluT2+VGaT+ neurons predominantly target the lateral habenula [18–20], we hypothesized that lateral habenula neurons receiving synapses from VTA VGluT2 neurons are activated by stress. To label neurons postsynaptic to VTA VGluT2 neurons, VGluT2::Cre mice were injected in VTA with a Cre-dependent adeno-associated viral vector encoding thymidine kinase (TK) that is required for anterograde transsynaptic spread of herpes simplex virus [39, 40]. After three weeks these mice were injected in VTA with the transsynaptic anterograde TK-deleted herpes simplex vector encoding tdTomato, H129-ΔTK-tdTomato (Fig. 6G). After one week, mice were exposed to inescapable stress where tailshock onset and offset patterns from previously ran ES mice were played back. TdTomato neurons were counted with and without c-Fos co-expression in lateral habenula (LHb) and lateral hypothalamus (LH) neurons within the same sections. We found that approximately one-third of lateral habenula (LHb) neurons receiving input from VTA VGluT2 neurons co-expressed c-Fos following stress (32.63% ± 10.4%) (Fig. 6H–J). The c-Fos activation of lateral habenula neurons was significantly greater than lateral hypothalamic neurons also receiving synaptic inputs from VTA VGluT2 neurons, t(18) = 10.99, p < 0.0001 (Fig. 6K).

DISCUSSION

Traumatic experiences can lead to a broad range of negative consequences that are supported by both associative (avoidance of stimuli related to the trauma) and nonassociative (exaggerated transsituational responding to circumstances unrelated to the trauma) processes [1, 2, 7, 41]. Here, we adapted a stressor controllability paradigm from rats to mice to test the hypothesis that VTA VGluT2 neurons play a role in the nonassociative consequences of stress.

We first tested whether uncontrollable tailshock results in social deficits, a common feature of anxiety disorders [22, 23]. Tailshock was explicitly chosen as an aversive stimulus over footshock because tailshock ensures that all subjects receive equivalent shock (duration, temporal pattern, intensity) that cannot be mitigated by postural adjustments or other behaviors such as jumping or locomotion [42]. In male mice we found that inescapable stress results in reduced social exploration compared with unstressed mice. Having identified that repeated tailshock stress results in social deficits in male mice, we next tested whether these social deficits were dependent on its uncontrollability. Male mice that were able to terminate tailshock (escapable stress) showed no social deficits, similar to mice that did not receive stress. In contrast, mice that received identical tailshock stress, but in an inescapable manner, showed a social deficit involving a lack of discrimination between social and nonsocial stimuli. We also found that in males, inescapable stress resulted in enhanced freezing behavior following mild footshock, compared with mice that experienced escapable stress or no stress. We interpret these results as demonstrating that behavioral control over stress confers transsituational resilience against an inescapable stress-induced reduction in social exploration and increase in fear-related behavior. These results establish the stressor controllability paradigm in mice that has largely been performed in rats [15, 42, 43].

We next examined the role of VTA VGluT2 neurons in stressor controllability. Glutamate neurons within the VTA, defined by the expression of VGluT2 [44, 45], contribute to reward, aversion, and threat [14, 24, 26, 46]. Optogenetic stimulation of the VTA VGluT2 pathway to lateral habenula results in conditioned place aversion [13]. Therefore, we performed two examinations: one investigating the extent to which VTA VGluT2 neuron activity is sensitive to stressor controllability and whether VTA VGluT2 neuron activity is required for the consequences of uncontrollable stress. VTA VGluT2 neurons are highly sensitive to aversive stimuli [18, 21, 24]. We hypothesized that when ES mice learned to terminate tailshocks by wheel-turning, this element of behavioral control over stress would attenuate aversion-related VTA VGluT2 neuronal activity. Instead, we found that both ES and IS tailshocks similarly increased VGluT2 neuronal activity. Although there was no population-level difference in VTA VGluT2 neuronal signaling between ES and IS groups, it is possible that distinct VTA VGluT2 neurons differentially signal or contribute to stressor control or loss of control. VTA VGluT2 neurons show heterogeneous electrophysiological dynamics for rewarding and aversive stimuli [24] and receive distinct reward or aversion-related glutamatergic inputs [21]. Further, genetically-defined subtypes of VTA VGluT2 neurons show different signaling patterns related to reward, aversion, and learned cues that predict these events [18]. To begin examining whether genetically distinct subtypes of VTA VGluT2 neurons show differences in stress activity in general, we combined fluorescent genetic reporting of VGluT2 and VGaT VTA neurons with immunolabeling of VTA dopamine neurons, as well as c-Fos following stress. We found that VGluT2+, VGluT2+TH+, and VGluT2+VGaT+ neurons all showed increased c-Fos expression following stress compared with unstressed mice. Because VGluT2+VGaT+ neurons were the highest c-Fos co-expressing cell-type following stress, and VGluT2+VGaT+ neurons predominantly target LHb [18, 19] we investigated whether LHb neurons receiving synapses from VTA VGluT2 neurons were activated by stress. We found that about one-third of LHb neurons receiving synapses from VTA VGluT2 neurons co-expressed c-Fos following stress, which was significantly more than lateral hypothalamus neurons receiving synapses from VTA VGluT2 neurons. Additional research will be needed to further test the involvement of each genetically-distinct VTA VGluT2 neuron subtype and circuits in their signaling and role in the consequences of stress.

Before determining the role of VTA VGluT2 neurons in the consequences of inescapable stress, we further investigated the behavioral effects of inescapable stress in female mice. Our original investigation showed that inescapable stress results in a social exploration deficit in male but not female mice. We also examined exaggerated fear that can follow trauma [47, 48]. We found that inescapable stress enhances footshock-induced freezing in males but preliminary experiments resulted in variable rates of freezing in females, perhaps due to darting behavior that is more often observed in female rodents [49]. While previous research has demonstrated differences in stress responses between sexes [50], we reasoned that our protocol was not yet optimized in female mice, rather than demonstrating a sex difference in stress responses. We hypothesized that female mice would show a general exploratory behavior deficit when challenged with an ambiguously threatening environment that does not induce freezing behavior. Thus, we tested for exploratory behavior in the light/dark box in female mice because this test shows comparable behavior between sexes [35] and is reliably sensitive to anxiolytics [36]. We found that inescapable stress resulted in “anxiety-like behavior” in female mice consisting of significantly increased latency to exit the dark compartment into the light compartment, as well as a reduced amount of time spent in the light compartment, compared with unstressed female mice. Control over stress prevented the increased latency to exit the dark compartment that was observed in IS females; however, time in the light compartment did not differ between these two conditions. This indicates a global stress effect for time in light, which is not mediated by controllability, suggesting a complex effect of stress in female mice. Specifically, while latency to exit the dark compartment was sensitive to controllability, females did not benefit from controllability training as assessed by total time in the light compartment of the light/dark box. The latter result is consistent with female rats that similarly do not benefit from escapable stress training assessed with juvenile social exploration [50–52]. We interpret differences between sexes in our experiments as the following: Male and female mice are both affected by inescapable stress but display their behavioral consequences differently. Following inescapable stress, male mice show a controllability-dependent reduction in sociability as well as enhanced fear. Following inescapable stress in females, a reduced exploratory behavior in the light/dark box was observed that reflects both controllability-dependent and independent mechanisms. Additional research will be necessary to identify if other measures show benefits of controllable stress training in female rats and mice.

Following the identification of stress-induced behavioral changes in both sexes we determined whether VTA VGluT2 activity is necessary for the consequences of stress in male and female mice. We expressed the inhibitory hM4Di designer receptor in VTA VGluT2 neurons and administered a behaviorally-subthreshold dose of clozapine [27] to reduce neuronal activity within VGluT2 neurons during inescapable stress. Whereas male VGluT2 control mice showed social deficits following inescapable stress, chemogenetic silencing of VTA VGluT2 neurons prevented the development of sociability deficits that result from inescapable stress. We also measured footshock-elicited freezing in a novel context. Both unstressed and hM4Di mice showed low levels of freezing compared with the elevated levels of freezing observed in VGluT2 control mice. Thus, chemogenetic silencing of VTA VGluT2 neurons during inescapable stress blocked the development of exaggerated fear responses in male mice. There was no difference in freezing behavior prior to the first footshock between groups, highlighting the nonassociative aspects of the task by which associative fear from the wheel-turn box did not transfer to the footshock apparatus. Finally, chemogenetic silencing of VTA VGluT2 neurons in female mice prevented exploratory behavior deficits in the light/dark box following inescapable stress. These results indicate that neuronal activity of VTA VGluT2 neurons is required for the consequences of stress in male and female mice. Given that VTA VGluT2 neuron inhibition blocked male stress measures that depend on controllability, as well as female stress measures that do and do not depend on controllability, these results indicate that VTA VGluT2 neurons mediate the development of nonassociative consequences of stress in general. While critical for stress-induced behavior changes, due to their lack of discrimination between ES and IS, we hypothesize that there are other brain regions or cell-types involved in the specific discrimination of inescapability that also facilitate IS-induced behavior changes.

Understanding which neural circuits and cell-types contribute to both associative and nonassociative consequences following stress is a critical step toward developing targeted therapeutics to ameliorate mental health disorders that can result from trauma. These experiments establish a mouse model of stressor controllability that can be leveraged with neuronal strategies such as optogenetics to identify stress-related neural circuitries. Additionally, this model provides a means to interrogate nonassociative consequences following stress, such as social exploration, enhanced fear, and anxiety-like behavior in contexts independent from the original traumatic experience. Using this model, we established that VTA VGluT2 neurons are activated by both escapable and inescapable stress and that silencing these neurons during inescapable stress prevents transsituational stress-induced changes in behavior in male and female mice. Additional research will be necessary to determine if VTA VGluT2 neurons also participate in escapable stress as this was untested in the present study. Further, because VTA VGluT2 neurons can signal learned cues predictive of aversive events [21] and associative processes were not investigated in the present study, it is possible that VTA VGluT2 neurons have a role in both associative and nonassociative consequences of inescapable stress. Moreover, we found that multiple subtypes of VTA VGluT2 neurons were activated by stress, as well as LHb neurons that receive synapses from VTA VGluT2 neurons. Additional research will be needed to determine which VTA VGluT2 neuron subtypes and circuits are causally involved in the consequences of stress. Nevertheless, VTA VGluT2 neurons are embedded within stress-related circuits, including the nucleus accumbens shell, lateral habenula, dorsal raphe, and lateral hypothalamus [13, 14, 25, 53–56]. This work sets a foundation for mechanistically exploring how VTA VGluT2 neurons and related neural circuitry confer stress susceptibility or resilience. However, other VTA cell-types also play important roles in regulating stress. Inhibition of VTA GABA neurons in general promotes anxiolytic, reward-promoting effects [57–59]. Further, the role of dopamine appears to be dependent on stressor experience and behavioral task following stress. For example, VTA dopamine neurons may causally contribute to anxiety-like or depression-like behavior [60–63] or relieve these behaviors [64]. Together, the VTA is an important contributor to a variety of stress-related behaviors and this work extends the contributions of VTA VGluT2 cell-types in stress-related behavior changes in male and female mice.

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ACKNOWLEDGEMENTS

This research was supported by the Webb-Waring Biomedical Research Award from the Boettcher Foundation (DHR), R01 DA047443 (DHR), F31 MH125569 (DJM), a 2020 NARSAD Young Investigator grant from the Brain and Behavior Research Foundation (DHR), R01 MH050479 (MVB), R21 MH116353 (MVB), American Australian Association Fellowship (MVB), DK078749 (STH), and The University of Colorado Boulder. Ai193 mice were generously provided by the Allen Institute and generated with funds from 1U19MH114830–01. The imaging work was performed at the BioFrontiers Institute Advanced Light Microscopy Core (RRID: SCR_018302). Laser scanning confocal microscopy was performed on a Nikon A1R microscope supported by NIST-CU Cooperative Agreement award number 70NANB15H226. The PerkinElmer Opera Phenix is supported by NIH grant 1S10OD025072. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Prism and Biorender were used to make figures and schematics.

Footnotes

COMPETING INTERESTS

The authors declare no competing interests.

ADDITIONAL INFORMATION

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41380-022-01858-3.

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