Social defeat stress-specific increase in c-Fos expression in the extended amygdala in mice: Involvement of dopamine D1 receptor in the medial prefrontal cortex

We recently reported that dopamine D1 receptor in the medial prefrontal cortex (mPFC) is activated by subthreshold social defeat stress and suppresses the induction of depressive-like behavior in mice. However, which mPFC projection(s) mediates this antidepressant-like effect remains poorly understood. Here we show that social defeat stress specifically increased c-Fos expression, a marker for neuronal activity, in distinct brain regions involved in emotional regulation, relative to novelty-induced exploration. Among these brain areas, D1 knockdown in the mPFC decreased social defeat stress-induced c-Fos expression in the interstitial nucleus of the posterior limb of the anterior commissure (IPAC), a subregion of the extended amygdala. Using retrograde adeno-associated virus vectors and transgenic mice expressing Cre recombinase under the D1 promoter, we also found that D1-expressing deep-layer pyramidal neurons in the mPFC send direct projections to the IPAC. These findings indicate that social defeat stress specifically activates neurons in distinct brain areas, among which the IPAC is regulated by dopamine D1 receptor in the mPFC perhaps through direct projections. Thus, this study provides hints toward identifying neural circuits that underlie antidepressant-like effects of stress-induced dopamine D1 receptor signaling in the mPFC.

Stress caused by aversive and demanding conditions induces different biological responses, depending on the stress conditions. It is thought that brief and moderate stress evokes adaptive responses, such as "fight-or-flight response", and promotes habituation and resilience to stress. By contrast, prolonged or excessive stress may induce cognitive and affective dysfunctions, and predisposes to mental disorders 1,2 . Previous studies using rodent stress models including restraint stress, chronic unpredictable stress and social defeat stress have revealed that stress alters structures and activities of neurons in several brain areas including the medial prefrontal cortex (mPFC), hippocampus, and nucleus accumbens (NAc) [3][4][5][6][7] .
It has been shown that repeated social defeat stress decreases firing rates of dopamine neurons in the ventral tegmental area (VTA) projecting to the mPFC 8 and stress-induced dopaminergic response in the mPFC 9 . These dopaminergic deficits have been shown to be crucial for repeated social defeat stress-induced social avoidance. On the other hand, we recently reported that subthreshold stress induced by single exposure to social defeat stress induces dopamine D1 receptor signaling in the mPFC 10 . Knockdown of dopamine D1 receptor in excitatory neurons in the mPFC facilitated the induction of social avoidance after single or repeated exposure to social defeat stress. Consistently, it was reported that dopamine D1 receptor in the mPFC is crucial for antidepressant-like effects of ketamine, and that infusion of a D1-like receptor agonist induces antidepressant-like effects 11 . These
To examine whether D1 activation is sufficient to induce c-Fos expression in the IPAC, we performed c-Fos immunohistochemistry in the IPAC as well as in the PL and the IL with or without mPFC infusion of SKF81297, a D1-like receptor agonist, at 400 mg/L (1.37 mM), a dose that is larger than the reported affinity (i.e. Ki~2.2 nM) Figure 1. Social defeat stress specifically increased c-Fos expression in distinct brain areas, relative to noveltyinduced exploration.(a) A behavioral schedule. Mice received social defeat stress for 10 min (the defeat stress group) or were allowed to explore a novel cage for 10 min (the exploration group), and were kept undisturbed in their homecages for 90 min before being sacrificed for immunohistochemistry. As the naïve group, mice were left undisturbed in their homecages until sacrifice. (b) Automatic detection and counting of c-Fos-positive cells (Fos + cells) visualized by immunohistochemistry (Fos IHC). For example, the prelimbic cortex (PL) and the infralimbic cortex (IL) adjacent to anterior forceps (fa) were defined in a fluorescent image of c-Fos immunoreactivity (magenta) and nuclear staining with Hoechst33342 (blue), based on corresponding brain areas in the Allen Mouse Brain Atlas, as shown in the left. A magnified image of c-Fos immunoreactivity (magenta) corresponding to the inset in the fluorescent image and binarized signals of automatically designated Fos + cells (yellow) are shown in the right. Note that c-Fos signals are well overlapped by the binarized Fos + cells. Scale bars: 500 μm (left) and 50 μm (right). (c-i) Quantification of the numbers of Fos + cells in the naïve group, the exploration group and the defeat stress group. The number of data points for each group is shown below each bar. Schematics were drawn based on the Allen Mouse Brain Atlas. PL; prelimbic cortex, IL; infralimbic cortex, NAc; nucleus accumbens, ACC; anterior cingulate cortex, LSv; lateral septal nucleus ventral part, BNST; bed nucleus of the stria terminalis, SI; substantia innominata, IPAC; interstitial nucleus of the posterior limb of the anterior commissure, MH; medial hypothalamus, ARH; arcuate nucleus, CeA; central amygdala, BLA; basolateral www.nature.com/scientificreports www.nature.com/scientificreports/ ( Fig. 3a,b). This stimulation of D1-like receptors in the mPFC did not alter the number of c-Fos-positive cells in the IPAC (Fig. 3c), suggesting that activation of D1-like receptors in the mPFC is not sufficient for social defeat stress-induced c-Fos expression in the IPAC. It should be noted, however, that this treatment did not increase the number of c-Fos-positive neurons in the PL or IL either (Fig. 3c), suggesting that this treatment did not increase the excitation of mPFC neurons.

D1-expressing mPFC pyramidal neurons send direct projections to the IPAC. To examine
whether the IPAC receives direct projections from D1-expressing neurons in the mPFC, we injected retrograde AAV vectors (rAAV2retro) expressing EYFP only in the presence of Cre recombinase into the IPAC of transgenic mice expressing Cre recombinase under the promoter of dopamine D1 receptor (D1-cre) or wild-type mice without Cre expression (Fig. 4a). These AAV vectors would be retrogradely infected into and consequently induce EYFP expression in D1-expressing neurons in the mPFC, if these neurons would send direct projections to the IPAC. Neither the mPFC nor the IPAC showed EYFP expression in the wild-type mice (Fig. 4b,c), confirming the lack of leaky expression of EYFP in the absence of Cre recombinase. By contrast, in D1-cre mice, EYFP expression was predominantly detected in deep-layer pyramidal neurons in the mPFC ipsilateral to the IPAC with AAV infusion. The IL tended to show higher intensity of EYFP signals than the PL (P = 0.0738, t(5) = 2.255), suggesting that mPFC neurons projecting to the IPAC are more frequently distributed in the IL (Fig. 4d). EYFP-positive neurons were also detected in the agranular insular cortex (AI; Fig. 4c), though the number was much fewer. Note that EYFP-positive axonal processes were strongly detected in the IPAC, indicating that the infected neurons send projections to the IPAC (Fig. 4b). These findings indicate that D1-expressing deep-layer pyramidal neurons in the mPFC send direct projections to the IPAC.

Discussion
In the present study, we found that single exposure to social defeat stress induced c-Fos expression in multiple brain areas. Among these brain areas, in the NAc, LSv, BNST, IPAC, CeA, CoA and PA, social defeat stress specifically induced c-Fos expression relative to novelty-induced exploration. Furthermore, knockdown of dopamine D1 receptor in the mPFC decreased c-Fos expression in the IPAC. Using retrograde AAV vectors and transgenic mice expressing Cre recombinase under the D1 promoter, we found that D1-expressing neurons in the mPFC send direct projections to the IPAC. Thus, these findings identified distinct brain areas that are specifically activated by social defeat stress, and indicate that dopamine D1 receptor signaling in the mPFC regulates stress-induced activation of the IPAC, perhaps through direct projections.
Our findings revealed two types of brain areas with different stimulus selectivity: brain areas activated specifically by social defeat stress (stress-specific brain areas) and those activated by both social defeat stress and novelty-induced exploration (stress/exploration-responsive brain areas). The stress-specific brain areas include the extended amygdala composed of BNST, IPAC and CeA. The extended amygdala has been shown to be activated by aversive stimuli and involved in fear memory, anxiety-like behaviors and pain-induced aversion 15 . Given that social defeat stress is more aversive than novelty-induced exploration, the higher level of aversiveness could explain social defeat stress-specific activation of the extended amygdala.
The present study identified the IPAC as brain areas in which dopamine D1 receptor in the mPFC affects social defeat stress-induced c-Fos expression. Previous studies showed that systemic administration of antidepressants or antipsychotics induces Fos-like immunoreactivity in the IPAC 16,17 . Rodent studies using in vivo microdialysis have shown that conventional antidepressants which inhibit serotonin and/or noradrenaline reuptakes increase the extracellular level of dopamine in the mPFC 18,19 . Thus, dopamine D1 receptor in the mPFC could also be involved in antidepressant-induced Fos expression in the IPAC. However, pharmacological stimulation of D1-like receptors in the mPFC did not induce c-Fos expression in the IPAC. Since this stimulation did not induce c-Fos expression in the mPFC either, stimulation of D1 receptor alone may not be sufficient to increase the excitation of mPFC neurons. It has been reported that NMDA receptor is involved in dopamine-induced c-Fos expression in striatal neurons 20 . Thus, concurrent stimulation of dopamine receptors with NMDA receptor could be necessary to increase the excitation of mPFC neurons.
Using retrograde AAV vectors and D1-cre mice, we found that the IPAC receives direct projections from D1-expressing mPFC neurons. Given that mPFC neurons projecting to the IPAC are glutamatergic, these direct projections could facilitate social defeat stress-induced excitation of IPAC neurons. Whether direct projections from the mPFC to the IPAC are involved in antidepressant-like actions of dopamine D1 receptor in the mPFC warrants to be investigated. However, the possibility that some indirect projection mediates the effect of mPFC D1 receptor on the IPAC cannot be excluded, until the expression of dopamine D1 receptor would be manipulated selectively in mPFC neurons directly projecting to the IPAC. Since D1-expressing neurons also exist in the IPAC, the dopaminergic system may also directly affect IPAC neurons 21 .
In conclusion, the present study identified the IPAC as a brain area in which social defeat stress-induced c-Fos expression is affected by dopamine D1 receptor in the mPFC. To examine the roles of this brain area in antidepressant-like effects of dopamine D1 receptor in the mPFC may provide hints toward identifying neuronal circuits that underlie adaptive stress responses. amygdala, CoA; cortical amygdala, VTA; ventral tegmental area, SUM; supramammillary nucleus, PA; posterior amygdala, PAG; periaqueductal gray, DRN; dorsal raphe nucleus, LC; locus coeruleus, PBN; parabrachial nucleus. Values are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 for multiple comparison tests with Tukey-Kramer correction. Social defeat stress. Male C57BL/6 N mice (9 to 10-week old in Fig. 1 and 14-week old in Fig. 2) were subjected to single exposure of social defeat stress, as previously described with minor modifications 9,10,22,23 . Prior to the stress, ICR mice were screened as aggressor mice based on their aggression against novel C57BL/6 N mice for 3 min daily for 3 consecutive days. The aggression was evaluated by the latency to the first attack and the frequency of the attacks during the 3-min exposure. On the day of the social defeat stress, a male C57BL/6 N mouse was transferred to the home cage of one of the aggressor mice and was attacked for 10 min. The defeated mice were sacrificed at 90 min after the stress for histological analyses. www.nature.com/scientificreports www.nature.com/scientificreports/ immunohistochemistry. For c-Fos immunohistochemistry, we used 9 mice for the naïve group, 12 mice for the exploration group and 13 mice for the defeat stress group in Fig. 1, 5 mice for the control miRNA group and 6 mice for the D1 miRNA group in Fig. 2 (see the definitions of respective groups in the Results section), and 4 mice each for D1-like receptor agonist infusion and for saline infusion in Fig. 3. We did not exclude any of them from our analyses, unless a brain slice or its part was damaged during the immunohistochemistry procedure.

Scientific RepoRtS
Immunohistochemistry was performed as previously described with minor modifications 10,23 . Briefly, mice were deeply anesthetized with intraperitoneal injection of sodium pentobarbital (100 mg/kg, Nacalai Tesque, Kyoto, Japan) and transcardially perfused with a flush of saline followed by 0.1 M sodium phosphate buffer containing 4% paraformaldehyde. Brains were obtained from the mice and postfixed in the same fixative at 4 °C overnight. For c-Fos immunohistochemistry, frozen sections were used. After cryoprotection in Dulbecco's modified phosphate-buffered saline (D-PBS) containing 30% sucrose two overnights, the brains were embedded and frozen in OCT compound (Sakura Finetek, Tokyo, Japan) and cut into 30-μm sections using a cryostat (CM1860, Leica Biosystems, Wetzlar, Germany). For immunohistochemistry for anatomical tracing, the brains were cut into 50-μm sections using a vibratome (NLS-AT, Dosaka EM, Kyoto, Japan). The tissue sections were kept in D-PBS containing 25% glycerol and 25% propylene glycol at −30 °C until use. For immunohistochemistry, the sections were incubated in blocking solution (D-PBS containing 1% normal donkey serum (017-000-121, Jackson ImmunoResearch Laboratories, Inc, West Grove, PA, USA) and 0.3% Triton X-100) for 1 h at room temperature (RT), followed by incubation in the blocking solution containing primary antibodies against c-Fos derived from rabbits To analyze c-Fos expression, we defined brain areas to be analyzed based on the Allen Mouse Brain Atlas and a mouse brain atlas 24 , as previously described 25 . We applied the Transfluor application module of the Metamorph software (Molecular Devices Corporation, PA, USA) to detect and count objects that are within the range of 9-30 μm in diameter and brighter than a threshold determined by adjacent background signals in each brain area in each hemisphere. These objects were defined as c-Fos-positive nuclei. We averaged the numbers of c-Fos-positive cells from each hemisphere.

Brain infusion of adeno-associated virus vectors.
For knockdown of dopamine D1 receptor, adeno-associated virus (AAV) vectors expressing either artificial microRNA (miRNA) targeting dopamine D1 receptor (D1 miRNA) or negative control miRNA were produced, as previously described 10 . Briefly, these AAV vectors encode artificial microRNA targeting either dopamine D1 receptor (D1 miRNA) or control sequence (Ctrl miRNA), and emerald green fluorescent protein (EmGFP), both of which were placed in double-floxed inverse open reading frames (DIOs), thereby allowing expression of microRNAs along with EmGFP only in the presence of Cre recombinase. These AAV vectors (AAV-DIO-EmGFP-D1miRNA or AAV-DIO-EmGFP-CtrlmiRNA) were used with another AAV vector constitutively expressing Cre recombinase under the CMV promoter (AAV-Cre). For retrograde tracing experiments, an rAAV2retro vector, a recently evolved variant of AAV that infects neurons via a retrograde access, encoding enhanced yellow fluorescent protein (EYFP) inserted into a DIO cassette (rAAV2retro-DIO-EYFP) was produced, as previously described 26 .
For AAV infusion, stereotaxic surgeries were performed, as previously described with minor modifications 10 . For D1 knockdown experiments, after mice were anesthetized with isoflurane, their skulls were fixed on a stereotaxic apparatus (Stoelting, Wood Dale, IL, U.S.A). Then we vertically injected 500 nL of artificial cerebrospinal fluid (124 mM NaCl, 3 mM KCl, 26 mM NaHCO 3 , 2 mM CaCl 2 , 1 mM MgSO 4 , 1.25 mM KH 2 PO 4 , 10 mM D-Glucose) containing 5.0 × 10 8 copies of each AAV vector into the mPFC (1.8 mm anterior, 0.4 mm lateral and 2.8 mm ventral from the bregma) of both hemispheres. Stereotaxic coordinates were determined based on a mouse brain atlas 24 . Social defeat stress was applied after 4-week recovery. For retrograde tracing experiments, after mice were anesthetized with isoflurane, their skulls were fixed on a stereotaxic apparatus. Then we vertically injected the same volume and concentration of rAAV2-retro-EYFP unilaterally into the IPAC (0.0 mm anterior, 2.2 mm lateral and 4.9 mm ventral from the bregma) of D1-cre mice or wild-type mice. Immunohistochemistry was performed after 2-or 4-week recovery.
Infusion of a D1-like receptor agonist to the mPFC. Infusion of SKF81297, a D1-like receptor agonist, to the mPFC was performed, as described previously with minor modifications 11 . Briefly, after mice were anesthetized with isoflurane, their skulls were fixed on a stereotaxic apparatus. Then guide cannulas of 0.5-mm thickness were vertically implanted into the mPFC (1.8 mm anterior, 0.4 mm lateral and 2.8 mm ventral from the bregma) of both hemispheres and fixed with cement on the skull. The stereotaxic coordinate was determined based on a mouse brain atlas 24 . After 1-week recovery, 500 nL of SKF81297 dissolved in saline at 400 mg/L, or saline as a vehicle control was infused bilaterally into the mPFC at 100 nL/min. After 2 h, mice were deeply anesthetized with intraperitoneal injection of sodium pentobarbital and transcardially perfused with 0.1 M sodium phosphate buffer containing 4% paraformaldehyde for c-Fos immunohistochemistry.