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Previous work has shown that chronic social defeat stress (CSDS) induces increased phasic firing of ventral tegmental area (VTA) dopamine neurons that project to the nucleus accumbens (NAc) selectively in mice that are susceptible to the deleterious effects of the stress. In addition, acute optogenetic phasic stimulation of these neurons promotes susceptibility in animals exposed to acute defeat stress. These findings are paradoxical since increased dopamine (DA) signaling in NAc normally promotes motivation and reward, and the influence of chronic phasic VTA firing in the face of chronic stress remains unknown.
We used CSDS with repeated optogenetic activation and pharmacological manipulations of the mesolimbic VTA-NAc pathway to examine the role of brain-derived neurotrophic factor (BDNF) and DA signaling in depressive-like behaviors. BDNF protein expression and DA release were measured in this model.
Pharmacological blockade of BDNF-TrkB signaling, but not DA signaling, in NAc prevented CSDS-induced behavioral abnormalities. Chronic optogenetic phasic stimulation of the VTA-NAc circuit during CSDS exacerbated the defeat-induced behavioral symptoms, and these aggravated symptoms were also normalized by BDNF-TrkB blockade in NAc. The aggravated behavioral deficits induced by phasic stimulation of the VTA-NAc pathway were also blocked by local knockdown of BDNF in VTA.
These findings show that BDNF-TrkB signaling, rather than DA signaling, in the VTA-NAc circuit is crucial for facilitating depressive-like outcomes after CSDS, and establish such BDNF-TrkB signaling as a pathological mechanism during periods of chronic stress.
Social stress is one of the most critical factors in the onset of depressive disorders in humans (1, 2). The effect of social stress on depressive-like behavioral abnormalities has been investigated with the chronic social defeat stress (CSDS) paradigm in mice (3–5), in which susceptible and resilient phenotypes are segregated after 10 days of the stress. Depressive-like behaviors in susceptible mice have been causally associated with molecular and physiological abnormalities in the mesolimbic dopamine (DA) pathway, which is comprised of the ventral tegmental area (VTA) and its projecting terminals to the nucleus accumbens (NAc) (3, 4, 6, 7). For example, phasic, but not tonic, firing of VTA DA neurons is increased in susceptible, not resilient, mice (4, 6).
Brain-derived neurotrophic factor (BDNF) in the mesolimbic DA pathway has been implicated in the susceptible phenotype after CSDS (3, 4). Elevated levels of BDNF protein expression in NAc are associated with depressive-like abnormalities induced by CSDS, and are not observed in resilient mice (4). Localized Bdnf gene deletion in VTA of adult mice reduces susceptibility to CSDS (3), suggesting that BDNF, transported from VTA to NAc, induces behavioral susceptibility. In addition, the combination of one day of defeat, plus acute optogenetic phasic stimulation of VTA-to-NAc DA neurons, induces social avoidance and other deficits, while either treatment alone does not induce behavioral abnormalities in normal mice (7). Phasic stimulation of this pathway increases BDNF protein levels in the NAc of one-day defeated mice, and blockade of BDNF-TrkB signaling in NAc prevents the ability of acute optogenetic stimulation to induce behavioral deficits in this acute stress paradigm (8).
Notably, phasic stimulation of the VTA-NAc pathway facilitates release of BDNF as well as DA from VTA DA terminals (9, 10). BDNF can also facilitate DA release from DA terminals (11). In addition, a subset of VTA DA neurons has been implicated in stress-elicited depressive-like abnormalities (12, 13). Thus, it is conceivable that both DA and BDNF signaling in NAc might promote depressive phenotypes. However, this view is contrary to the established role of DA in mediating reward. Indeed, DA deficiency in NAc has been postulated in depressed humans and animal models (14, 15). Several clinical studies have shown that depressed patients have attenuated concentrations of DA metabolites (16–18). Moreover, optogenetic activation of VTA DA neurons reverses chronic mild stress-induced depression-associated behaviors in mice, while inhibition of these neurons promoted thes e behaviors, suggesting an antidepressant-like role of DA signaling (19). Finally, some antidepressants increase DA transmission in NAc shell (20–22).
The present study was designed to address these paradoxical findings. Our data establish that BDNF, but not DA, mediates the ability of a hyperactive VTA-NAc pathway to promote depressive-like symptoms in the CSDS paradigm.
Male 7–12 week old C57BL/6J mice (25–30 g, Jackson), 7–15 week old floxed Bdnf mice (25–32 g, BL6/sv129 background) (3), 2–3 month old Drd2 (D2) GFP bacterial artificial chromosome (BAC) transgenic mice (25–32 g, C57BL/6J background, www.gensat.org) (23), and 4–6 month old CD1 retired breeders (35–45 g, Charles River) were used. Mice were fed ad libitum at 22~25°C on a 12-hr light/dark cycle. CD1 mice were singly housed except during social defeats. All other mice were group housed before social defeats and singly housed after social defeats. All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committees at Mount Sinai.
Stereotaxic surgeries were performed as described previously (8, 24). For repeated optical activation of the VTA-NAc pathway during the chronic social defeat stress (CSDS) paradigm, 0.5 µl of retrograde traveling adeno-associated virus (AAV2.5) vectors that express ChR2, fused with enhanced yellow fluorescent protein (EYFP) (i.e., AAV2.5-hsyn-ChR2-EYFP, purchased from University of Pennsylvania Vector Core) were bilaterally infused into the NAc (AP +1.5; ML ±1.5; DV −4.4 from bregma; 10° angle) at a rate of 0.1 µl/min. Three weeks later, optic fibers were bilaterally implanted into VTA (AP −3.2; ML ±1.0; DV −4.6; 7° angle). If necessary, a bilateral 26-gauge guide cannula (4 mm length from the cannula base) was implanted bilaterally into NAc (AP +1.5; ML +0.75; DV −3.9; 0° angle) for drug infusions. While these surgeries targeted to the NAc medial shell, the manipulations also affected the NAc core due to the small size of this brain region in the mouse.
For optical activation of the VTA-NAc pathway in an acute defeat stress paradigm, as described previously (8), a double floxed (DIO) Cre-dependent AAV vector expressing ChR2 fused with EYFP (i.e., AAV-DIO-ChR2–EYFP, purchased from University of North Carolina Vector Core) was bilaterally infused into VTA. Two weeks later, a replication-defective version of the retrograde traveling pseudorabies virus expressing Cre (i.e., PRV–Cre, obtained from Jeffrey M. Friedman, Rockefeller University) was bilaterally infused into NAc.
For localized Bdnf gene knockdown followed by repeated optogenetical activation of VTA-NAc pathway, AAV-Cre or AAV-GFP (purchased from University of North Carolina Vector Core) and AAV2.5-hsyn-ChR2-EYFP were infused into VTA and NAc of floxed Bdnf mice, respectively. Two weeks after the double surgery, optic fibers were bilaterally implanted into VTA, as described above.
Around 15 min before daily defeat stress for 10 days, animals received bilateral intra-NAc infusions of SCH 23390 (D1 receptor antagonist, 1 µg/0.5 µl/side) (24), eticlopride (D2 receptor antagonist, 1 µg/0.5 µl/side) (24, 25), or ANA-12 (TrkB inhibitor, 1 µg/0.5 µl/side) (8), at doses known to be behaviorally active (8, 24, 25), or vehicle as a control (sterile saline or 50% DMSO in ACSF) at a continuous rate of 0.1 µl/min via a micro-infusion pump (Harvard Apparatus). Injector needles remained in place for 5 min before being pulled out. Mice were allowed to sit undisturbed for ~5 min prior to the daily defeat stress. In the case of one-day defeat stress, mice received a bilateral intra-NAc infusion of SCH 23390, eticlopride, or vehicle 1 hr prior to the social interaction test.
Chronic and acute defeat stress were conducted as described previously. 24 hr after the last defeat, a social interaction test was performed (3, 4, 6–8). Based on social interaction ratios (time in interaction zone with social target/time in interaction zone without social target × 100%), mice were designated as susceptible or resilient: susceptible ratio<100; resilient ratio≥100. This measure of susceptibility vs. resilience has been shown to correlate with other defeatinduced behavioral abnormalities (4). In vivo phasic stimulation of VTA-NAc pathway was conducted (7) for 5 min, immediately after or during the daily defeat stress in 10-day chronic defeats. Acutely defeated mice received the phasic stimulation during the social interaction test when CD1 mice were presented for 2.5 min.
Fast scan cyclic voltammetry was used (26) to characterize presynaptic DA release and uptake as well as the ability of the DA terminal to respond to phasic stimulation patterns in NAc shell. Animals were used for voltammetry experiments 18~22 hr after a social interaction test to identify susceptible mice after CSDS. To obtain baseline recordings from 400 µm thick coronal brain sections containing the NAc shell, DA release was evoked by 1 pulse stimulations (350 µA, 4 msec, monophasic) every 5 min. When a stable baseline was established (3 collections within 10% variability) phasic stimulation curves were run. Here we evaluated evoked DA release to single pulse and multiple pulses (5 stimulations at varying frequencies: 5 to 20 Hz).
An unbiased CPP paradigm was used (24, 27). Briefly, mice were placed in a three-chambered CPP box for 20 min to ensure no chamber bias. For the next two days of cocaine/light CPP, optic fibers were secured to the cannulae prior to saline or cocaine (10 mg/kg, ip) injections. Mice were conditioned to saline/no light and cocaine/blue light (473 nm, 20 Hz frequency, bursts of 5 light-pulses, 40 ms pulse duration, every 10 sec) for 30 min over two days. On the CPP test day, mice were allowed to freely explore all three chambers for 20 min. CPP scores represent time spent in the paired – time spent in the unpaired chamber.
Mice were anesthetized with a lethal dose of chloral hydrate and intracardially perfused with 0.1 M phosphate-buffered saline (PBS) and 4% (wt/vol) PBS-buffered paraformaldehyde 24 hr post social interaction test. Post-fixed brains were incubated overnight in 30% sucrose at room temperature before being sliced on a microtome at 35 µm on a microtome. Free-floating sections were washed with PBS and then blocked in 3% bovine serum albumin (BSA) and 0.3% Triton-X for 1 hr. For EYFP (ChR2)/TH double labeling, 1:4000 of mouse anti-TH (T1299, Sigma) was used for overnight incubation with 1:1000 of chicken anti-GFP (GFP-1020, Aves) at 4°C. The next day, 1:500 of donkey anti-mouse Cy3 for anti-TH was used in PBS together with 1:500 of donkey anti-chicken Cy2 for anti-GFP. For GFP/pERK double-labeling in D2 GFP mice, brain sections were incubated in 1:1000 of chicken anti-GFP (Aves) and 1:1000 mouse anti-pERK (4370S, Cell signaling) in block solution overnight at 4°C. The next day, sections were rinsed in PBS then incubated in 1:500 of donkey anti-chicken Cy2 (Immuno Research) and 1:500 of donkey anti-mouse Cy3 in PBS for 1 hr then subsequently rinsed in PBS. All sections were counterstained and mounted with antifade solution, including DAPI then subsequently imaged on a LSM 710 confocal microscope. GFP and pERK cell counting in the NAc was performed within a 200 µm × 200 µm square scale placed on the NAc shell or core. All immunopositive cells within the square scale were counted by an observer blind to experimental conditions.
Bilateral 14-gauge NAc punches were obtained 1 mm coronal NAc sections from mice 24 hr post social interaction test. Punches were then sonicated (Cole Parmer, Vernon Hills, Illinois, USA) in 30 µl of homogenization buffer containing 320 mM sucrose, 5 nM Hepes buffer, 1% SDS, phosphatase inhibitor cocktails I and II (Sigma, St. Louis, MO, USA), and protease inhibitors (Roche, Basel, Switzerland). The concentration of protein was determined using a DC protein assay (Bio-Rad, Hercules, CA) and 25 µg of total protein was loaded onto a 18% gradient Tris-HCl polyacrylamide gel for electrophoresis fractionation (Bio-Rad, Hercules, CA). Samples were then transferred onto a nitrocellulose membrane and then blocked in Odyssey® blocking buffer (Li-Cor, Lincoln, NE, USA) for 1 hr for Li-Cor analysis. After blocking, the same membrane was incubated in 4°C overnight with either antibodies against BDNF (1:500, Santa Cruz sc546), detecting truncated BDNF, or β-tubulin (1:10,000, cell signaling 2118) in Odyssey® blocking buffer. Following thorough washing with TBST, blots were incubated for 1 hr at room temperature with IRDye® secondary antibodies (1:10,000; Li-Cor, Lincoln, NE, USA) in Odyssey® blocking buffer. Blots were imaged with the Odyssey Infrared Imaging system (Li-Cor) and quantified by densitometry using NIH ImageJ (NIH, Bethesda, Maryland, USA). The amount of protein blotted onto each lane was normalized to levels of tubulin.
Data were analyzed with SigmaPlot 13.0 (Systat) and Prism 6.0 (GraphPad). Student’s t-tests were used for the analysis of experiments with two experimental groups. One-way ANOVAs were used for analysis of three or more groups, followed by Fisher's PLSD post-hoc tests, when appropriate. For social interaction data that were generated from the phasic stimulation experiment without pharmacological infusions (Figure 2G), two-way ANOVAs were used followed by Fisher's PLSD post-hoc tests. For all analysis of voltammetry data, Demon Voltammetry and Analysis software was used (28). To evaluate DA kinetics, evoked levels of DA were modeled using Michaelis-Menten kinetics. Burst frequency response curves, were subjected to a two-way repeated measures ANOVA with burst frequency as the within subjects factor and experimental group as the between subjects factor. All p values of < 0.05 were considered to be statistically significant. All data are expressed as mean ± SEM.
We first assessed the role of DA signaling in NAc using a one-day defeat paradigm and an optogenetic method to activate the VTA-NAc pathway (Figure S1A,B). Previous work showed that intra-NAc infusion of the TrkB inhibitor, ANA-12, blocked social avoidance elicited by this acute stress paradigm (8). We found that intra-NAc infusion of a D1 (SCH23390) but not D2 (eticlopride) receptor antagonist blocked the ability of acute optogenetic stimulation of the VTA-NAc pathway to induce social avoidance in this one-day stress procedure (Figure S1C). These data demonstrate that both BDNF and DA signaling in NAc mediate the ability of acute optogenetic stimulation to induce social avoidance during acute social stress.
However, while the one-day defeat paradigm is useful to reveal pro-susceptibility phenotypes (4, 29), it has the major limitation of involving acute, not chronic, stress. Therefore, we used the standard CSDS (10 day) paradigm to determine the underlying mechanism in the VTA-NAc pathway responsible for CSDS-induced social avoidance. We infused bilaterally SCH23390, eticlopride, or ANA-12 into NAc, at a dose known to be behaviorally effective (8, 24), 15 min prior to each daily defeat during the 10 day protocol (Figure 1A–C). As seen for the one-day defeat paradigm, TrkB inhibitor pretreatment counteracted the social avoidance induced by CSDS (Figure 1D). Surprisingly, however, neither D1 nor D2 antagonist pretreatment blunted the social avoidance induced under these chronic stress conditions (Figure 1D).
To determine the behavioral consequences of phasic stimulation of the VTA-NAc pathway during chronic stress, compared to effects in the one-day paradigm (7), we first injected retrograding AAV2.5-hsyn-channelrhodopsin-2 (ChR2)-eYFP into NAc bilaterally and 3 weeks later implanted optic fibers in VTA (Figure 2A–C). At four weeks ChR2 (EYFP+, green) was well expressed in VTA DA neurons (TH+, red) (Figure 2D). We then optically stimulated the VTA daily during CSDS using two stimulation protocols: stimulation during the defeat episodes (Figure 2E) vs. immediately after each defeat (Figure 2F). The phasic stimulation during defeat had no additional effect on the social avoidance produced by CSDS. In contrast, the phasic stimulation post-defeat exacerbated the effect of CSDS (Figure 2G). Phasic activation of the VTA-NAc pathway by itself (i.e., without defeat stress) had no effect on social interaction (Figure S2).
To investigate a role of BDNF in mediating the aggravated social avoidance induced by phasic stimulation of the VTA-NAc pathway during CSDS, we infused ANA-12 into NAc 15 min prior to each daily defeat for 10 days and optogenetically stimulated the pathway immediately after each defeat (Figure 3A–C; ‘stimulation post-defeat’). Social interaction testing on day 6 revealed no changes in social behavior (Figure 3D). However, after 10-days of CSDS, phasic activation of the VTA-NAc pathway worsened social avoidance, and this effect was blocked by intra-NAc ANA-12 infusions (Figure 3E).
Consistent with these behavioral data, CSDS increased BDNF protein levels in NAc 24 hr after the social interaction test (Figure 3F). Post-defeat phasic stimulation of the VTA-NAc pathway during CSDS (‘CSDS Veh+ChR2’) further increased BDNF levels compared to the non-stimulated defeated mice (‘CSDS Veh+EYFP’) (Figure 3F). ANA-12 treatment had no effect on BDNF protein levels (Figure 3F).
To complement the pharmacological approach, we knocked down BDNF in the VTA of floxed Bdnf mice by local infusion of AAV-Cre. Control mice received intra-VTA injections of AAV-GFP. We found that, whereas phasic stimulation of the VTA-NAc pathway during CSDS aggravated social avoidance in GFP control mice, this effect was lost in mice with a local VTA BDNF knockdown (Figure 3G–I). These data establish that BDNF expressed in VTA is required for the ability of repeated phasic stimulation of the VTA-NAc pathway to exacerbate social avoidance induced by chronic social stress.
We employed ex vivo fast-scan cyclic voltammetry to investigate the effects of CSDS and optogenetic stimulation of the VTA-NAc pathway on DA release in NAc shell. We found that CSDS, which by itself produced social avoidance behavior (Figure 4A), did not alter electrically-evoked DA release in NAc slices over broad frequencies (5–20 Hz) of stimulation (Figure 4B–D). Moreover, while phasic stimulation of the VTA-NAc pathway during CSDS worsened avoidance behavior (Figure 4A), it did not alter electrically-evoked DA release (Figure 4B–D).
Next, to assess the cell-type specificity of the effect of CSDS on BDNF-TrkB signaling in NAc, we measured levels of phosphorylated (active) ERK (pERK), which is downstream of TrkB, after CSDS. We used D2-GFP mice, which contain a bacterial artificial chromosome expressing GFP selectively in D2-type medium spiny neurons (MSNs) (23, 30) (Figure 5A–C). We found that 10 days of CSDS increased the number of pERK+/D2− cells in NAc shell of susceptible mice, with no effect seen in resilient mice (Figure 5D,E). In contrast, CSDS had no effect on pERK immunoreactivity in D2+ cells (Figure 5F) or on total pERK+ cell counts (one-way ANOVA, F2,9 = 3.021, p = n.s.). This effect was specific to NAc shell, as no effect of CSDS was found for pERK immunoreactivity in either D2− or D2+ core cells in susceptible or resilient mice (Figure S3).
In the present study, we demonstrate that BDNF, but not DA, signaling in the mesolimbic DA circuit is necessary for the susceptible phenotype produced by chronic social stress. BDNF-TrkB blockade in NAc prevented CSDS-induced social avoidance behavior, whereas DA receptor antagonism did not. Repeated optogenetic phasic stimulation of the VTA-NAc circuit, which approximates enhanced burst firing of the pathway that occurs uniquely in susceptible mice (4, 6, 7), increased BDNF levels in NAc and aggravated CSDS-induced social avoidance behavior. This exaggerated susceptible phenotype too was prevented by BDNF-TrkB blockade in NAc and by localized BDNF knockdown in VTA. These data agree with previous studies showing that NAc BDNF, transported from VTA, is critical for the susceptible phenotype after CSDS (3, 4, 8) and establish BDNF signaling by VTA DA neurons as an abnormal, pathological mechanism that arises during a period of chronic stress.
It is of particular interest that CSDS-induced social avoidance was blocked by neither D1 nor D2 antagonism, even though repeated, severe stress exposure facilitates DA release in NAc shell (31–33). Our observations here suggest that increased DA transmission in NAc during severe stress is not associated with depressive-like phenotypes. Moreover, we show that CSDS has no effect on electrically-evoked DA release in NAc ex vivo compared to stress naïve animals. Previous clinical and animal studies have shown a negative correlation between concentrations of DA metabolites and depressive symptoms, but a positive correlation between antidepressant effects and DA transmission in NAc shell (see Introduction). However, in contrast to the lack of involvement of DA signaling in NAc after CSDS, D1 but not D2 antagonism in NAc—like acute BDNF-TrkB antagonism (8)—blocked the ability of acute optogenetic activation of the VTA-NAc pathway to worsen the effects of acute stress. These data suggest that very different mechanisms are at play during responses to initial stress compared with more pathological changes that occur with chronic stress. Repeated, excessive stress may promote greater release of BDNF, but not of DA, from VTA nerve terminals, resulting in depressive-like pathologies (34).
We provide evidence that D1 MSNs are the site of action of BDNF in NAc after CSDS. We show that pERK levels are increased solely in D2-negative cells in NAc shell of susceptible mice, with no change in resilient mice. Prior work has established that GFP-negative cells in D2-GFP mice provide a highly reliable measure of D1-type MSNs (35, 36). This finding suggests that BDNF signaling in D1 MSNs contributes to the susceptible phenotype after CSDS. Interestingly, enhanced ERK phosphorylation has been associated with a reduction in neuronal activity of D1 MSNs (27), and reducing neuronal activity of D1 MSNs renders resilient mice more susceptible (37). Moreover, excitatory synaptic input to D1 MSNs is reduced in susceptible mice after CSDS (37) and in mice subjected to repeated restraint stress (38). Together, these results support a scheme wherein increased BDNF signaling in NAc contributes to CSDS-induced behavioral susceptibility by inhibiting the activity of D1 MSNs. An important caveat is that ERK is downstream of several signaling pathways in addition to BDNF, therefore, further work is needed to directly test this and alternative hypotheses.
BDNF signaling in NAc has also been implicated in drug reward, particularly for cocaine (27, 39). We observed here that blockade of BDNF signaling in NAc inhibits cocaine reward (Fig. S4) in addition to CSDS-induced social avoidance, and that both of these behaviors are enhanced by optogenetic activation of the VTA-NAc pathway. However, we have demonstrated previously that enhancement of cocaine reward by BDNF in NAc is mediated by D2 MSNs (27).This is in contrast to the present results where we provide evidence that NAc BDNF-TrkB enhancement of stress susceptibility is mediated by D1 MSNs, thus establishing very different mechanisms for the ability of BDNF acting in NAc to promote drug reward vs. behavioral susceptibility to chronic stress.
In conclusion, the present study demonstrates a required role of mesolimbic BDNF signaling, rather than DA signaling, in NAc in mediating social avoidance induced by CSDS. Our data suggest that NAc BDNF, which originates from VTA (3), mediates social avoidance through activation of TrkB on D1 MSNs, as evidenced by exclusive induction of ERK phosphorylation in D1 MSNs of susceptible mice. Our findings in chronically stressed and chronically optogenetically stimulated animals demonstrate clear differences from findings with acute stress and acute stimulation paradigms, since D1 dopamine function in NAc is involved in acute responses, but not chronic responses. Thus, our findings address the paradox of why increased firing of VTA dopamine neurons, which might otherwise be expected to be associated with increased reward and decreased depression-like behavior, is causally linked to susceptibility to CSDS. The sustained activation of these neurons, in the context of chronic stress, promotes increased release of BDNF which produces pathological effects within the mesolimbic DA circuit. This pro-depressant role of mesolimbic BDNF signaling is in direct contrast to the antidepressant-like actions of BDNF in hippocampus, which emphasizes the circuit-specific nature of molecular mechanisms involved in brain disease (40).
This work was supported by the National Institute of Mental Health (R01MH051399 and P50MH096890 to E.J.N. and R01MH092306 to M.H.H.) and National Institute on Drug Abuse (R01DA014133 to E.J.N.), the Hope for Depression Research Foundation, IMHRO/Johnson & Johnson Rising Star Translational Research Award (M.H.H.), and National Research Service Award (F31AA022862 to B.J.).
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All authors report no biomedical financial interests or potential conflicts of interest.