GABAB(1) receptor subunit isoforms differentially regulate stress resilience

Significance Stress can increase susceptibility to developing psychiatric disorders, including depression. Understanding the neurobiological mechanisms underlying stress resilience and susceptibility is key to identifying novel targets for the development of more effective treatments for stress-related psychiatric disorders. Here we show that specific isoforms of GABAB receptor subunits differentially regulate stress resilience. Specifically, GABAB(1a)−/− mice are more susceptible whereas GABAB(1b)−/− mice are more resilient to stress-induced anhedonia and psychosocial stress-induced social withdrawal, two features of depression. Furthermore, GABAB(1b)−/− mice were resilient to stress-induced decreases in the survival of newly born cells in the adult hippocampus, and hippocampal GABAB(1b) expression was increased in a genetic mouse model of depression. Taken together, GABAB receptor subunit isoforms may represent novel therapeutic targets for stress-related disorders. Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)−/− mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression.

Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABA B receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABA B (2) subunit with either a GABA B(1a) or a GABA B(1b) subunit. Here we show that mice lacking the GABA B(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABA B(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wildtype mice. In addition, increased hippocampal expression of the GABA B(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABA B(1b) −/− mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABA B(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression. neurogenesis | depression | anxiety | antidepressant A lthough chronic and/or severe stress is a risk factor for the development of several different psychiatric disorders including depression and anxiety, many individuals remain resilient to such negative effects of stress. The mechanisms underlying this resilience are not yet fully understood, although it is thought to involve a complex interplay between several environmental factors such as social support and biological and genetic risk factors (1,2). Currently, there is an impetus to determine the neural substrates underlying stress resilience and susceptibility, as these are poised to be key novel targets for the development of more effective treatments for depression and anxiety disorders.
Accumulating evidence suggests that GABA B receptors may be important therapeutic targets for the treatment of stress-related psychiatric disorders such as anxiety and depression (3)(4)(5). Functional GABA B receptors are composed of heterodimers of GABA B (1) and GABA B(2) subunits (6). Interestingly, the GABA B (1) subunit is expressed as different isoforms, and in the brain the predominant isoforms are GABA B(1a) and GABA B(1b) (6). Mice deficient in GABA B(1a) and GABA B(1b) exhibit differential cognitive and conditioned fear responses, indicating a potential role for these isoforms in psychiatric illness (7)(8)(9)(10). Recent postmortem brain studies report alterations in the expression of GABA B receptor subunits in depression (4,11), and clinical studies suggest that neurophysiology deficits in GABA B receptors may play a role in major depression (12) and the antidepressant response (13). In addition, mice lacking functional GABA B receptors exhibit an antidepressant-like phenotype and increased anxiety (14,15), and pharmacological blockade of these receptors induces antidepressant-like behavior (16)(17)(18). GABA B receptor antagonists have also recently been shown to increase cell proliferation in the adult hippocampus (16), which is an important regulator of stressand antidepressant-related neuroplasticity. However, the specific role of GABA B receptor isoforms in stress sensitivity is unclear. Therefore, we assessed the susceptibility and resilience to stress during either early life (maternal separation) or adulthood (psychosocial stress) in GABA B(1a) −/− and GABA B(1b) −/− mice. 0.018) and GABA B(1a) −/− mice (P = 0.004), but this effect was greater in GABA B(1a)

Results
−/− mice [SD stress GABA B(1a) −/− vs. SD stress WT, P = 0.006]. In contrast, GABA B(1b) −/− mice were resilient to SD stress-induced social avoidance. Interestingly, nonstressed GABA B(1a) −/− mice spent less time in the interaction zone in the presence of a social target than nonstressed WT and GABA B(1b) −/− mice (P = 0.036). There were no group differences in time spent in the interaction zone in the absence of a social target (Fig. S1A). Social avoidance was not due to altered locomotor activity (Fig. S1B). GABA B(1a) −/− mice were also more susceptible to MS-induced anhedonia than GABA B(1b) −/− mice. The effects of MS on anhedonia were measured using the saccharin preference test and the female urine sniffing test. Analysis of saccharin preference in female mice over 24 h (Fig. 1D) revealed an effect of genotype [F(2,50) = 8.107, P < 0.001]. Specifically, MS reduced saccharin preference in GABA B(1a) −/− mice compared with MS WT (P < 0.01) and MS GABA B(1b) −/− (P < 0.01) mice. Similar results were obtained when preference was measured over 36-and 48-h periods ( Fig. S2 A and B). The effect of MS in male mice on preference to sniff female urine is illustrated in Fig The level of maternal care that a pup receives can program stress sensitivity later in adulthood (19). To exclude the possibility that the resilient phenotype of GABA B(1b) −/− mice is due to increased maternal care, we measured high maternal care behaviors ( −/− mice. In females (Fig. S3D), there were no genotype differences. However, MS had no effect on this parameter in any genotype or sex.
Anxiety Levels in Adulthood Are Unaffected by Early-Life Stress and GABA B(1) Receptor Subunit Isoforms. GABA B receptors have been reported to play a role in innate anxiety (14,15). However, the contribution of specific GABA B (1) isoforms to this effect is less obvious (20). Here, neither MS nor GABA B(1) receptor subunit isoform disruption nor MS coupled with GABA B(1) receptor , and GABA B(1b) −/− mice, with and without prior MS. Restraint stress was induced by placing mice in ventilated tubes for a period of 2 h, and animals were killed 2 h after cessation of restraint. The number of c-Fos-positive cells was measured in stress-related brain areas including the hippocampus, nucleus accumbens (NAcc), dorsal raphe nucleus (DRN), paraventricular nucleus (PVN), ventral tegmental area (VTA), medial prefrontal cortex, and amygdala. The PVN is known to be activated by restraint stress (21). Therefore, to evaluate the validity of our restraint stress paradigm, we first confirmed that it induced c-Fos activation in the PVN of the hypothalamus of WT mice that did not undergo early-life stress (Fig. S5F). All data including statistical analysis are summarized in Table S1.
One of the most striking observations was that in several areas of the hippocampus, the number of c-Fos-positive cells in response to acute stress was significantly increased in GABA B(1b) −/− mice compared with WT and GABA B(1a) −/− mice. Importantly, the hippocampus is a key brain area involved in regulation of the stress response (22,23). The enhanced stress-induced c-Fos activation was most apparent in the dorsal and ventral dentate gyrus and ventral CA3 (Fig. 2 and Table S1 Table S1), thus further supporting the hypothesis that GABA B receptors in the hippocampus might be important in the differential response to stress. A similar but weaker pattern of effects was also observed in the PVN and the DRN whereby GABA B(1b) −/− mice exhibited enhanced stress-induced neural activation irrespective of prior MS, although statistical differences were generally restricted to comparisons with GABA B(1a) −/− and not WT mice (Table S1). Of all the regions investigated, the NAcc was the only area where stress-induced c-Fos expression was differentially regulated in GABA B(1b) −/− mice by prior MS (Fig. 2 and Table S1). Specifically, although there were no statistically significant genotype  (Table S1).  (24), would exhibit alterations in hippocampal expression of this subunit. GABA B(1b) mRNA was increased in the dentate gyrus (t = 7.591, df = 10, P < 0.001), CA1 (t = 6.377, df = 10, P < 0.001), and CA3 (t = 6.674, df = 10, P < 0.001) of helpless H/Rouen (H) mice compared with nonhelpless (NH) controls ( Fig. 3 E-H). GABA B(1a) mRNA expression did not differ between the two groups ( Fig. 3 A-D). Adult hippocampal neurogenesis is thought to play important roles in the stress response, anhedonia, antidepressant action, and regulation of the stress response by antidepressants (25)(26)(27). We previously reported that pharmacological inhibition of the GABA B receptor increases hippocampal cell proliferation and induces antidepressant-like behavior (16). This finding along with the present study, which demonstrates increased expression of GABA B(1b) mRNA in the hippocampus of a mouse model of depression and enhanced stressinduced activation of the hippocampus in GABA B(1b) −/− mice, *Compared with the respective WT group; *P < 0.05; **P < 0.01. + Compared with the respective GABA B(1a) −/− group; + P < 0.05; ++ P < 0.01; +++ P < 0.001. # Compared with the respective NMS group; # P < 0.05. suggest that increased adult hippocampal neurogenesis may be a potential mechanism underlying the stress-resilient and antidepressant-like phenotype of GABA B(1b) −/− mice. Emerging evidence suggests that the effects of stress and antidepressant treatments on adult hippocampal neurogenesis might occur preferentially in the ventral (vHi) rather than the dorsal hippocampus (dHi) (28,29), and we previously reported that chronic treatment with a GABA B receptor antagonist increases cell proliferation in the vHi but not dHi (16). This is interesting in light of the preferential roles of the dHi in spatial learning and memory and the vHi in the regulation of the stress response and anxiety (30). Therefore, we determined whether the stressresilient and antidepressant-like phenotype of GABA B(1b) −/− mice was accompanied by increased proliferation and survival of newly born cells and whether such effects occurred preferentially in the vHi rather than in the dHi.
The effects of MS on cell proliferation in the subgranular zone (SGZ) and granule cell layer (GL) are illustrated in Fig. 4 B and C, respectively (representative photographs are in Fig. S6; the experimental design is in Fig. 4A) , P < 0.001) but that this effect was prevented by MS. Cell proliferation in the SGZ of the dHi did not differ between any of the groups. In the GL, neither genotype, stress, nor stress x genotype affected cell proliferation. However, upon segregation of the GL, there was an effect of genotype [F(1,24) = 7.726, P < 0.05] in the ventral GL. Specifically, NMS but not MS GABA B(1b) −/− mice displayed an increased number of Ki67-positive cells (P < 0.05) compared with their corresponding WT counterparts. Cell proliferation in the GL of the dHi was not different between any of the groups. Surviving newly born neurons generated in the SGZ migrate to the GL, where they mature to become integrated into the neural circuitry of the hippocampus. Therefore, we also determined the number of surviving newly born cells in the GL of these mice ( Hippocampal Neurogenesis. Experiments using doublecortin (DCX) immunohistochemistry in female mice confirmed that increased neurogenesis is restricted to GABA B(1b) −/− mice (P < 0.05) and that GABA B(1a) −/− mice were not different from WT mice (Fig. S8, SI Materials and Methods, and SI Results). Although a similar pattern of effects was observed in the dHi and vHi of NMS and MS mice, statistically significant effects were observed only in the dHi. Variability in these data might be a function of the limited sample number available for analysis (n = 6 vs. n = 10 in the cytogenesis study), estrus cycle regulation of neurogenesis in females (males were used in the cytogenesis study), and variation in the age or maturation state of individual DCX-positive cells, which future studies could investigate using appropriate immunohistochemical analyses.

Discussion
Understanding the molecular mechanisms underlying stress susceptibility and resilience is a key step toward identifying novel targets that could be exploited in the development of new, more effective treatments for stress-related psychiatric disorders, including depression. Here we show that the GABA B receptor may be one such target. Specifically, mice lacking the GABA B(1b) subunit are resilient to both stress-induced anhedonia and psychosocial stress-induced social withdrawal and exhibit antidepressant-like behavior, whereas GABA B(1a) −/− mice are more susceptible to stress-induced anhedonia and psychosocial stressinduced social withdrawal. These findings have important implications for the pathophysiology and treatment of depression. Furthermore, they support the contention that GABA B receptor subunit isoforms play differential roles in mediating behavioral responses (7)(8)(9).
GABA B(1a) −/− mice have previously been shown to exhibit sleep disturbances (6), cognitive impairments (7)(8)(9), and a reduced threshold for fear generalization (10). Here we show that GABA B(1a) −/− mice are more susceptible to stress-induced anhedonia. We cannot rule out that the increased maternal care behavior provided by GABA B(1a) −/− dams may have contributed to this phenotype. Future studies examining the phenotype of GABA B(1a) −/− mice following cross-fostering with WT mice would give further insight. Interestingly, a recent small postmortem study reported that GABA B(1a) mRNA expression is decreased in the dentate gyrus of depressed individuals (4), whereas antidepressant-induced increases in GABA B(1a) mRNA expression have been reported in the rat hippocampus (31). Taken together, this suggests that the GABA B(1a) receptor subunit may play a role in depression and antidepressant action.
On the other hand, data from GABA B(1b) −/− mice point to a novel role for this isoform in stress resilience. Previously, these mice have been shown to exhibit impaired fear conditioning (10) and cognitive deficits, including impairments in spatial working memory and extinction of aversive taste memory (7,8). Here we show that GABA B(1b) −/− mice are stress-resilient and that the hippocampus is a key area that is differentially activated in GABA B(1b) −/− and GABA B(1a) −/− mice in response to stress. Moreover, in another model of depression, helpless H/Rouen mice, hippocampal GABA B(1b) mRNA expression was increased compared with nonhelpless counterparts. This suggests that increased hippocampal GABA B(1b) mRNA expression is associated with a depression-like phenotype and that reducing its expression may have antidepressant-like effects. Indeed, we observed that compared with both WT and GABA B(1a) −/− mice, GABA B(1b) −/− mice were resilient to psychosocial stress-induced social avoidance and the anhedonic effects of early-life stress in the female urine sniffing test. We also observed that GABA B(1b) −/− mice exhibited antidepressant-like behavior in the forced swim test, although this effect should be interpreted with caution given their hyperactive phenotype. Importantly, GABA B(1a) and GABA B(1b) proteins are up-regulated 29% and 15% in CA1 of GABA B(1b) −/− and GABA B(1a) −/− mice, respectively (9), and thus it cannot be ruled out that their differential phenotypes are due to compensatory up-regulation of the other subunit.
Given the focus on the role of adult hippocampal neurogenesis in stress-related disorders (32) and the fact that we recently showed that chronic treatment with a GABA B receptor antagonist increases hippocampal cell proliferation (16), we thus assessed whether the stress-resilient phenotype of GABA B(1b) −/− mice is accompanied by changes in adult hippocampal cytogenesis and neurogenesis. Indeed, GABA B(1b) −/− mice exhibited increased proliferation and survival of newly born cells in the adult hippocampus, and these effects occurred predominantly in the ventral hippocampus, particularly under stress conditions. The vHi is thought to play a preferential role in the modulation of the stress response and anxiety, whereas the dHi is thought to play a preferential role in spatial learning and memory (30). Intriguingly, emerging evidence suggests that stress and antidepressant-like treatments may exert their effects on adult hippocampal neurogenesis preferentially in the vHi rather than in the dHi (28,29). Moreover, analogous findings have been reported in humans, with antidepressants increasing neurogenesis in the anterior hippocampus (33). In addition to increases in the survival of newly born cells, GABA B(1b) −/− mice were also resistant to stress-induced decreases in the survival of newly born cells. The regulation of adult hippocampal neurogenesis by GABA B receptors has remained largely unexplored, but we previously reported that chronic treatment with a GABA B receptor antagonist that has antidepressant-like behavioral effects increases cell proliferation specifically in the vHi (16). Similarly, it was recently reported that the GABA B receptor is expressed on neural stem cells, and that mice lacking both GABA B isoforms exhibit increased proliferation and accelerated neuronal differentiation (34). Taken together, it is clear that GABA B receptors modulate adult hippocampal neurogenesis and that these effects are coupled with alterations in antidepressant-like behavior and stress resilience, with specific GABA B(1) receptor subunit isoforms playing differential roles in these behavioral effects. Future studies investigating neurogenesis-dependent behaviors such as pattern separation, or antidepressant-induced decreases in anxiety in the novelty-suppressed feeding test (27), will give further insight into the role of the GABA B(1b) -neurogenesis relationship in behavior, although it is also important to note that differences in innate anxiety were not observed in other tests (20). Although our mechanistic studies focused on the hippocampus, it is important to note that GABA B(1b) −/− mice also exhibited enhanced stress-induced activation of some other brain regions, although these effects were less robust. Interestingly, the nucleus accumbens was the only region that was differentially affected in NMS and MS GABA B(1b) −/− mice by restraint stress. GABA B interactions at the level of the NAcc are well-described in the context of drug addiction (35) and may play a role in stress-induced anhedonia (18). Previous studies have reported serotonin-GABA B receptor interactions (17,36), and here we show that stress-induced c-Fos activation was increased in the dorsal raphe nucleus in both NMS and MS GABA B(1b) −/− mice. This is interesting in light of recent data showing that the activity of GABAergic neurons in the DRN regulates resilience to SD stress (37) and that the antidepressant fluoxetine suppresses GABA B receptor activity in the DRN (38).
The molecular mechanisms underlying phenotypic differences between GABA B(1a) −/− and GABA B(1b) −/− mice are not yet known, but differences in cellular localization, physiological properties, and ontogenic expression of these subunits have been reported (6). Unlike GABA B(1b) , GABA B(1a) can localize to axons via Sushi domains, which also increase surface stability of GABA B(1a,2) receptors (6,39). In dendrites, GABA B(1a) localizes to glutamatergic terminals for heteroceptor function whereas GABA B(1b) localizes to spines opposing glutamate release sites, thus affecting pre-or postsynaptic inhibition (6). Interestingly, GABA B(1a) and GABA B(1b) are preferentially expressed in the developing and adult brain, respectively (3), and thus GABA B(1b) levels are relatively lower during the plastic period of brain development. In parallel, the present data suggest that inhibiting GABA B(1b) expression facilitates plasticity in the form of neurogenesis. Future studies characterizing the roles of these subunits will give further insight into the mechanisms underlying the differential phenotypes observed here.
In conclusion, GABA B(1) receptor subunit isoforms differentially regulate resilience to stress-induced anhedonia, with reductions in GABA B(1b) receptors associated with resilience whereas reductions in GABA B(1a) receptors are associated with increased susceptibility. These effects were coupled with alterations in stressinduced neural activity of reward pathways and adult hippocampal neurogenesis, and are further supported by alterations in the expression of GABA B(1) subunit isoforms in a genetic mouse model of depression and in response to antidepressant treatment (31) as well as recent postmortem findings in the human hippocampus (4). Taken together, these data further support the concept that the GABA B receptor may play a crucial role in the pathophysiology and treatment of stress-related disorders.