HDAC6 is enriched in serotonin neurons
To map distribution of HDAC6 we performed IHC and in situ hybridization (ISH) on serial mouse brain sections. For IHC we obtained robust cytoplasmic staining () with clear regional distribution in the mouse brain using a custom affinity-purified antibody raised against amino acids 991 to 1149 of murine HDAC6 (Gao et al., 2007
). Stereological counts of the number of labeled somas identified the midbrain raphe nuclei as the area with the densest populations of HDAC6-immunopositive cells (). Besides the raphe, numerous HDAC6 immunopositive cell bodies were also detected in a portion of the ventromedial forebrain comprising the subventricular zone and the lateral septum and in the dorsal hippocampus (). Dual labeling showed that 97% HDAC6+
DR neurons were positive for the neuronal marker NeuN (194/200) while none (0/174) were colabeled for the astroglial marker GFAP, a result indicating that the HDAC6-immunopositive cell population in this area is composed exclusively of neurons in adult mice ().
Distribution of HDAC6 immunopositive cells in the mouse brain
HDAC6 is enriched in serotonin neurons
Analysis of HDAC6 distribution by ISH (,) revealed a mRNA pattern consistent with that of HDAC6 protein. A search conducted using the Allen Brain Atlas (http://www.brain-map.org
) revealed a strong correlation between the distribution of HDAC6 mRNA and that of the serotonergic marker tryptophan hydroxylase 2 (TPH2) (Allen Brain Atlas, NeuroBlast score 0.70, see (Royball et al., 2008
)). No such overlap was observed for any other HDAC isoforms in the midbrain, including HDAC4 and HDAC10, the two isoforms most structurally related to HDAC6 (). Dual-label IHC revealed colocalized TPH signal in 94 % (283/300) of HDAC6 positive cells (). In contrast, HDAC6 immunolabeling was not observed in midbrain tyrosine hydroxylase (TH) positive cells in the ventral tegmental area and substantia nigra or locus coeruleus (not shown). To determine whether the enrichment of HDAC6 in 5-HT neurons is conserved across species, we examined the expression of HDAC6 in human postmortem DR and observed similar colocalization with TPH as seen in the mouse (not shown).
Downregulated HDAC6 expression in the dorsal raphe of resilient and imipramine-treated mice
Recent studies have reported the abnormal expression of several HDACs, including HDAC6, in patients with mood disorders (Covington et al., 2009
; Hobara et al., 2010
). To test whether the expression of HDAC6 in serotonin neurons is altered in a mouse model of stress-related disorders and treatment, we quantitated HDAC6 mRNA in the DR using qPCR. Tissues were collected using laser capture microdissection (LCM) in male ePet-YFP mice exposed to chronic social defeat for 10 days and subsequently treated for 28 days with the antidepressant imipramine (20 mg/kg) or vehicle. We have shown previously that this imipramine regimen restores social interaction to control levels in social defeat vulnerable mice, while it is devoid of behavioral effects in control animals (Berton et al., 2006
). Statistical analysis revealed a significant main effect of treatment condition (F3,25
= 6.55, P
= 0.0025). While there was no change in HDAC6 expression after social defeat in vulnerable mice treated with vehicle, we observed a significant 30 to 40% downregulation of HDAC6 in mice spontaneously expressing resilient phenotype as well as in vulnerable mice treated with imipramine (Fisher’s PLSD P
< 0.01, n=6-8). ().
Downregulation of HDAC6 in 5-HT neurons with imipramine and resilience
Conditional ablation of HDAC6 in raphe neurons
The enrichment of HDAC6 in 5-HT neurons positions it as a potential regulator of threat-related and socioaffective behaviors (Dayan and Huys, 2009
). To test this hypothesis we genetically depleted HDAC6 by taking advantage of a previously characterized floxed HDAC6 allele from which we derived two lines of mice lacking HDAC6, either selectively in 5-HT neurons (HDAC6Pet1Cre
KO) or in a pan-neuronal manner (HDAC6NestinCre
KO). In HDAC6Pet1Cre
KO mice, we observed a loss of HDAC6 mRNA signal by ISH () and a decrease of 85-90% in the total number of HDAC6 immunopositive neurons across the entire rostro-caudal extent of the raphe nucleus (). These results confirm genetically that the vast majority of HDAC6 immunopositive neurons in the raphe are serotonergic. We detected a small proportion of spared HDAC6 immunopositive neurons in the raphe nuclei of HDAC6Pet1Cre
KOs. These spared neurons were all TPH+
and represented 10 to 15% of the total number of TPH+
cells per slice (, inset). This observation is in good agreement with several reports indicating that a corresponding proportion of 5-HT neurons in raphe does not express the Pet1 gene (Braz et al., 2009
; Kiyasova et al., 2011
). Counts of HDAC6 immunoreactive somas throughout the brain indicated that the depletion HDAC6 in HDAC6Pet1Cre
KO mice was restricted to the raphe nuclei and did not extend to forebrain regions expressing HDAC6.
Depletion of HDAC6 in raphe nuclei leads to hyperacetylation of α-tubulin but not histone H3
To evaluate the influence of HDAC6 on protein lysine acetylation in the DR, we measured bulk levels of acetylated histone and non-histone proteins in this area. We first examined acetylation of α-tubulin at lysine 40, a well-characterized substrate of HDAC6 (Hubbert et al., 2002
; Matsuyama et al., 2002
; Haggarty et al., 2003
). Western blots from HDAC6NestinCre
KO brainstem homogenates revealed a two-fold increase in levels of acetylated α-tubulin at lysine 40 compared to WT (). This increase in tubulin acetylation occurred without concomitant changes in the level of acetylated histone H3 at lysine 14 () and at lysine 9 (WT = 1.32 ± 0.45, KO = 1.07 ± 0.44 n=3) nor of histone H4 (WT = 0.79 ± 0.37, KO = 1.48 ± 0.4; n=3).
Serotonin-selective depletion of HDAC6 does not result in major cellular abnormalities
Loss of function of certain HDAC isoforms is known to produce neurodevelopmental abnormalities in the mouse (Akhtar et al., 2009
; Montgomery et al., 2009
). To test if HDAC6 is critical to the development of DR 5-HT neurons, we evaluated the consequences of HDAC6 depletion on their number, morphology and intrinsic electrophysiological characteristics. Histological, morphological and electrophysiological evaluations conducted in naïve adult WT and HDAC6Pet1Cre
KO animals did not reveal any major abnormality (). Stereological counts of the numbers of 5-HT+
somas in the DR and stereological estimations of the density of 5-HT immunoreactive axon terminals in several serotonergic projection areas indicated that gross morphological features of 5-HT ascending pathways were preserved in the absence of HDAC6. Finer morphological analyses conducted in 5-HT neurons individually filled with biocytin and reconstructed using the Neurolucida software also revealed a lack of significant alterations in somatodendritic morphology and complexity of 5-HT neurons (). Evaluation of the levels of 5-HT and its major metabolite 5-HIAA in raphe tissues or major projection areas did not reveal any significant abnormalities in the ability of HDAC6 deficient neurons to synthesize and metabolize serotonin (). The sole significant cellular abnormality found in HDAC6Pet1Cre
KO compared to WT mice under baseline conditions was a 50% decrease in membrane resistance of serotonin neurons (effect of genotype F1,59
= 4.92, P
= 0.002, Fisher’s PLSD P
< 0.01) detected using whole cell recordings in acute DR slices. On the other hand, HDAC6Pet1Cre
KO mice had normal resting membrane potential, time constant (tau), action potential amplitude, threshold and width ().
Effect of HDAC6 depletion on the distribution, morphology, serotonin synthesis/metabolism and intrinsic electrophysiological properties of serotonergic neurons
Serotonin-selective depletion of HDAC6 does not result in major behavioral abnormalities in naïve mice
To evaluate the behavioral impact of HDAC6 depletion in 5-HT neurons, male HDAC6Pet1Cre KO mice and their WT littermates were compared in a comprehensive phenotyping screen conducted in 8-10 week-old behaviorally naïve mice. This screen did not reveal any significant alteration of basic physiological or behavioral functions such as food and water intake, motor coordination, locomotor activity patterns, social interest, anxiety and startle responses ()
Effect of HDAC6 depletion on the behavioral phenotype of naïve mice
Serotonin-selective depletion of HDAC6 promotes an antidepressant-like phenotype in mice exposed to inescapable stress
The only two behavioral tests in which significant genotype-dependent differences were observed in previously unmanipulated mice were the tail suspension test (TST) and the forced swimming test (FST), where active escape behaviors are assessed in response to acute inescapable stressors (). Both of these measures are classically used as endpoints to evaluate antidepressant activity upon acute administration (Cryan et al., 2002
). In these two tests HDAC6Pet1Cre
KO mice displayed a modest antidepressant–like phenotype characterized by a reduction in the time spent immobile compared to their WT littermates (; for TST F1,16
= 5.29, P
= 0.04; for FST F1,22
= 4.79, P
We next evaluated whether depletion of HDAC6 in serotonin neurons influences resilience to chronic stress in the social defeat model () a paradigm that responds to chronic but not acute antidepressant administration. Social defeat induced a dramatic downward shift in the distribution of individual interaction ratios () and in the absolute time spent in the interaction zone () in WT mice but it had no significant effect on these variables in HDAC6Pet1Cre KO mice (Genotype × Defeat interaction on interaction ratios, F1,149 = 5.47; P<0.05, Fisher PLSD in WT group P < 0.001; Genotype × Defeat interaction on time in interaction zone, F1,149 = 5.78; P<0.05, Fisher PLSD in WT P<0.01). In WT animals, 31 % of the mice tested were categorized as resilient after social defeat, while 45 % displayed strong social avoidance (interaction ratio < 50). In contrast, a higher proportion of HDAC6Pet1Cre KO mice (50 %) was classified as resilient and only 23 % developed strong avoidance (Fisher exact test, P<0.01). Importantly, there was no difference between undefeated WT and HDAC6Pet1Cre KO regarding interaction ratios (WT 177.86 % ± 19.34 and KO 142.25% ± 17.79; NS) or absolute time spent in the interaction zone (WT = 61.94 ± 6.29 sec and KO = 50.90 ±4.82 sec; NS), indicating that serotonergic depletion of HDAC6 does not interfere with baseline expression of social investigation behaviors. Rather, the finding that HDAC6Pet1Cre KO mice fail to display the typical social avoidance response after social defeat suggests that HDAC6 in serotonin neurons may play a role in the experience-dependent modulation of socioaffective responses.
Depletion of HDAC6 prevents social defeat-induced hypoexcitability of serotonin neurons
Because the behavioral phenotype of HDAC6Pet1Cre KO mice emerges primarily after exposure to inescapable stressors, we reasoned that the neurobiological influence of HDAC6 may also become more readily observable under these conditions. We thus extended our previous electrophysiological analyses in HDAC6Pet1Cre KO and WT mice by incorporating slices from animals exposed to 10 days of social defeat. Raphe brain slices were collected for whole cell recordings 24h after the last exposure to social defeat or control conditions. To correlate resiliency status with cell properties, control and defeat-exposed mice were tested for social avoidance immediately prior to tissue collection. Recorded neurons were sampled exclusively from the ventromedial subfield of the DR and their 5-HT phenotype was confirmed post-recording using TPH immunohistochemistry (not shown). In WT mice, social defeat had a profound impact on physiological properties of 5-HT neurons. First, we observed a robust defeat-induced hypoexcitability in 5-HT cells, as revealed by frequency-intensity plots (). When exposed to the same input current, 5-HT neurons of defeated WT mice consistently fired at frequencies 50% lower than those of controls. This difference was significant for input current steps ranging from 40 to 80 pA (, social defeat × genotype interaction F1,59 = 7.3, P = 0.009; Fisher’s PLSD P < 0.05). Second, we found that both the AHP amplitude () and activation gap (the difference between the RMP and AP threshold) were significantly increased by defeat in WT mice (control = 29.00 ± 2.90 mV and defeat = 41.00 ± 2.40 mV, defeat × genotype interaction F1,59 = 6.29, P = 0.01, Fisher’s PLSD P < 0.01). Finally, we evaluated the effect of social defeat on the functional coupling of the 5-HT1A autoreceptor. 5-HT1A receptor responses were triggered by bath application of the 5-HT1A receptor agonist 5-carboxyamodotryptamine maleate (5-CT, 100 nM) and the amplitude of the resulting outward current was measured. We found that, in WT mice, social defeat potentiated 5-HT1A receptor mediated responses () while it had no effect on 5-HT1A receptor mediated responses in raphe slices from HDAC6Pet1Cre KO mice () (defeat × genotype interaction F1,50 = 3.55, P = 0.05, Fisher’s PLSD control vs Defeat P = 0.05 in WT). These results indicate HDAC6 in 5-HT neurons mediates the neurophysiological consequences of social defeat stress on 5-HT neurons
Defeat-induced hypoexcitability of serotonin neurons is prevented by HDAC6 depletion
Depletion of HDAC6 prevents social defeat-induced hypertrophy of 5-HT neurons
In conjunction with the electrophysiological changes identified above in 5-HT neurons, certain morphological neuroadaptations may contribute to alter the net functional output of raphe circuits after defeat. To date, however, evidence for such structural changes in raphe neurons after stress or stress hormone exposure has remained relatively indirect (Azmitia et al., 1993
; Azmitia and Liao, 1994
). Thus, we took advantage of the fact that 5-HT neurons from WT and HDAC6Pet1Cre
KO mice exposed to social defeat or control conditions were individually filled with biocytin during whole cell recordings to test whether changes in the somatodendritic morphology of 5-HT neurons predicted resilience in the social defeat paradigm. Biocytin was processed using fluorescently conjugated streptavidin to visualize neurons and confocal stacks were collected to generate 3D neuronal reconstructions that were subsequently analyzed using Neurolucida software ().
Defeat-induced hypertrophy of serotonin neurons is prevented by HDAC6 depletion
As summarized by representative drawings in , social defeat induced increases in soma size and dendritic complexity of DR 5-HT neurons in WT mice. Significant correlations were observed in defeat-exposed WT mice between the time spent in the interaction zone (target present) and three morphological variables, namely the mean cell body surface (; R2 = 0.45, P < 0.001), the cumulative number of Sholl intersections, an index of dendritic complexity (; R2 = 0.29, P < 0.01), and the mean dendrite length (; R2 = 0.3,P < 0.01). In contrast, no correlations were observed between these variables in controls from both genotypes (not shown) or in defeat-exposed HDAC6Pet1Cre KO mice, in which expression of social avoidance was reduced, in agreement with our previous observation. When defeat-exposed mice were stratified into vulnerable and resilient subgroups on the basis of their interaction ratios, the rightward shift in the distribution of interaction scores in HDAC6Pet1Cre KOs led to an incomplete factorial design (i.e., lack of KO/vulnerable mice) that impeded the use of parametric statistics. Kruskall-Wallis non-parametric tests revealed significant increases in mean cell body surface (; H4,45 11.26, P < 0.05), in the mean dendrite length (; H4,45 = 10.99, P < 0.05) and in dendritic complexity (; H4,45 = 9.58, P < 0.05) in WT vulnerable but not resilient mice. Sholl analysis conducted along the entire extent of the dendritic tree up to 380 um from the soma revealed that the increase in cell complexity after social defeat was specific to proximal dendrites found within a 140 um distance from soma (not shown; two-way ANOVA, soma distance × social defeat interaction F1,19 = 1.88, P < 0.05).
In summary, our results indicate that social defeat leads to an increased cell body and increased dendritic length and complexity of 5-HT neurons that constitutes a robust morphological biomarker of vulnerability to defeat. Like behavioral and electrophysiological effects of social defeat, these morphological consequences were prevented by serotonergic depletion of HDAC6.
HDAC6 depletion leads to Hsp90 hyperacetylation and impaired GR chaperoning
Having demonstrated that 5-HTergic depletion of HDAC6 opposes the behavioral and certain neuroplastic consequences of social defeat in raphe circuits, we next examined intracellular mechanisms mediating the pro-resilient effects of HDAC6 depletion. We focused on the GR chaperone protein Hsp90, a well characterized substrate of HDAC6. Hyperacetylation of Hsp90 following pharmacologic or genetic inactivation of HDAC6 has a major inhibitory impact on intracellular responses to glucocorticoids (Kovacs et al., 2005
; Murphy et al., 2005
; Zhang et al., 2008
). Because GR is abundantly expressed in raphe nuclei by both serotonergic and non-serotonergic cells (), we hypothesized that the pro-resilient effects of HDAC6 depletion may result, at least in part, from an inhibition of GR responses in 5-HT circuits.
To test whether resilience to social defeat, which is associated with HDAC6 downregulation in the DR, also correlates with hyperacetylation of Hsp90, we measured Hsp90 acetylation by IHC. Several acetylation sites have been previously identified in Hsp90 (Yang et al., 2008
; Choudhary et al., 2009
). We examined a specific site (lysine 294) that is directly involved in the ability of Hsp90 to recruit clients and cochaperones (Scroggins et al., 2007
) and to promote folding of steroid hormone receptors (Ai et al., 2009
; Kekatpure et al., 2009
), using an antibody that specifically recognizes the acetylated form of Hsp90 at K294. Cell counting in fields of view sampled across the entire rostrocaudal axis of the raphe revealed a strong trend toward an increase in the average number of raphe cells immunopositive for acHSP90 after social defeat in resilient compared to vulnerable mice (resilient = 38 ± 7, vulnerable = 20 ± 2 cells, P = 0.06, n=3).
We then asked whether the role of HDAC6 as a modulator of GR responses, previously established in cultured fibroblasts (Kovacs et al., 2005
; Murphy et al., 2005
; Zhang et al., 2008
), and in the liver in vivo (Winkler et al., 2011
), extends to serotonin neurons. To accomplish this, we used an immortalized rat raphe neuronal precursor cell line (RN46A) that expresses steroid receptors (White et al., 1994
; Bethea et al., 2003
). In this cell line we compared the ability of three HDAC inhibitors, with varying affinity for HDAC6, to antagonize GR translocation following hormonal stimulation. To induce GR translocation, cells were treated with 1 μM dexamethsone (DEX) for 1 hour and were subsequently fixed and immunostained for GR. Relative distribution of GR fluorescence across the nucleus and cytoplasm of individual cells was quantified. To examine the modulation of GR responses by HDAC inhibitors, cells were pretreated with vehicle or Trichostatin A (TSA, 5 μM), sodium butyrate (NaBu, 1 mM), or tubacin (10 μM), for 4 hours prior to GR stimulation. DEX led to an increase in relative nuclear GR signal that reflects the movement of GR from the cytoplasm to the nucleus (F1,178
= 30.0, P
< 0.0001). This effect was significantly attenuated by pretreatment with TSA (F1,107
= 5.20, P
= 0.02) and tubacin (F1,69
= 8.87, P
= 0.004), two inhibitors with nM affinity for HDAC6 (Bradner et al., 2010
), but not by the selective class I inhibitor NaBu (F1,46
= 1.89, P
= 0.18; ). Treatment with HDAC inhibitors alone had no effect on GR translocation in the absence of DEX.
Having confirmed the influence of HDAC6 on GR signaling in 5-HT neurons under tissue culture conditions, we next evaluated whether a similar function of HDAC6 in the DR can be evidenced in vivo in adult mice exposed to a psychosocial stressor. We did so by quantitating defeat-induced nuclear translocation of GR in the DR of WT and HDAC6 KO mice. In WT mice, social defeat exposure induced a robust increase in nuclear GR in the DR that presumably reflected GR activation by endogenous glucocorticoids released during social confrontation (). In striking contrast, there was no significant change in nuclear GR levels after social defeat in mice with pan-neuronal HDAC6 depletion (genotype × treatment interaction F1,18 = 4.95, P = 0.04; WT Fisher PLSD P = 0.002). GR signal was normalized to levels of histone H3 as a control for loading and fractionation procedures. No differences were observed between genotype and treatment groups in levels of H3. Importantly, plasma corticosterone levels were not different between WT and HDAC6 KOs at the same time point after social defeat (WT = 124.7 ± 9.0 ng/ml, KO = 139.7 ± 17.4 ng/ml, n = 4), a result suggesting that HDAC6 depletion affects stress resilience via a GR-regulated mechanism autonomous to raphe circuits and independent of HPA axis regulatory feed-back loops.
We next tested whether depletion of HDAC6 induces hyperacetylation of Hsp90 and alters GR-Hsp90 protein-protein interactions in the brain in vivo. Hsp90 was immunoprecipitated from WT and HDAC6 KO brainstem lysates and resulting pulldowns were blotted for AcHsp90 or GR (). Values were normalized to the total amount of Hsp90 pulled down. Results from these experiments show that, while total levels of Hsp90 were not altered following neuronal depletion of HDAC6, Hsp90 became hyperacetylated at lysine 294 (optical density WT = 0.33 ± 0.03, KO = 0.67± 0.03, F1,6 = 68.95, P = 0.0002, n = 4 replicates) and the association between the Hsp90 chaperone and GR was dramatically diminished under these conditions (WT = 0.71 ± 0.09, KO = 0.29± 0.09, F1,4 = 10.85, P = 0.03, n=3 replicates).
HDAC6 depletion prevents transcriptional and electrophysiological effects of glucocorticoids in 5-HT neurons
Previous studies relying on the use of reporter systems in non-neuronal cells have shown that HDAC6 inactivation blunts GR-mediated transcriptional responses (Kovacs et al., 2005
; Zhang et al., 2008
). In order to assess whether HDAC6 KO has a similar functional impact on downstream GR signaling in 5-HT neurons, we evaluated hormone-induced regulation of two well-characterized transcriptional targets of GR in this neuronal population, namely the TPH2 and 5-HT1A
genes (Ou et al., 2001
; Albert and Lemonde, 2004
; Clark et al., 2008
). To pharmacologically mimic the pulsatile pattern of GR activation with SD exposure, WT and HDAC6Pet1Cre
KO mice were treated subchronically with DEX (1 mg/kg i.p.; daily for 4 days) and TPH2 and 5HT1A
mRNA levels were evaluated in DR tissues using qPCR. In line with previous literature (Ou et al., 2001
; Clark et al., 2008
), we observed a repression (35 and 30% decrease respectively) of TPH2 and 5HT1A
gene expression in the DR of WT mice treated with DEX (). In contrast, the same DEX regimen produced no significant effect in HDAC6Pet1Cre
KO on the expression of TPH2 (one-way ANOVA, main effect of genotype on Dex-induced change for TPH2 F1,14
= 5.53 P = 0.03 and 5HT1A
= 6.61 P
= 0.02). These results indicate that HDAC6 is a required mediator of the transcriptional effects of glucocorticoid hormones on these two serotonergic genes. As expected based on cell-type selectivity of HDAC6Pet1Cre
KO, the genotype-dependence of DEX effects did not extend to canonical GR targets genes such as SGK (serum glucocorticoid regulated kinase, ) or FKBP5 (not shown), which are ubiquitously expressed by both 5-HT and non-5-HT cells. The latter results further support a cell-autonomous mechanism for the pro-resilient effect of HDAC6 depletion in 5-HT circuits.
HDAC6 depletion blunts the transcriptional and electrophysiological effects of GR activation in 5-HT neurons
To determine whether diverging transcriptional responses to subchronic GR activation in WT and HDAC6Pet1Cre KO brains coincide with differential neurophysiological adaptations of 5-HT neurons, whole cell recordings of DR 5-HT neurons were conducted under the same DEX treatment regimen. DR brain slices were collected 1 hour after the last administration of VEH or DEX and recordings were conducted either in the absence or presence of bath applied DEX (100 μM). While acute bath application of DEX to tissues from VEH treated mice was devoid of electrophysiological effect (not shown), subchronic DEX treatment fully recapitulated the effects of social defeat on the excitability of 5-HT neurons. Thus, like social defeat (), subchronic DEX inhibited the ability of WT 5-HT neurons to fire action potentials in response to graded current inputs, but was devoid of effect in HDAC6Pet1Cre KOs (; three-way ANOVA, genotype × treatment × input current interaction, F4,104 = 3.09, P = 0.01). Together, these results identify repeated GR activation as a likely mechanism promoting the hypoexcitability of 5-HT neurons during social defeat and confirm the critical dependence of this mechanism on HDAC6 activity.
Serotonergic depletion of HDAC6 alters the socioaffective effects of glucocorticoid hormones
Results presented above suggest that HDAC6 depletion modulates neuroadaptive responses of 5-HT circuits under chronic stress, in part by blocking some of the slow “genomic” actions of adrenal steroids released during repeated social confrontations. However, adrenal steroids, such as corticosterone (Cort) and cortisol, also exert rapid behavioral and cognitive effects in animals and humans, detectable a few minutes after exogenous hormone administration and thus presumably independent of gene transcription or de novo protein synthesis(Gasser et al., 2009
). Because certain rapid “non-genomic” effects of steroid hormones have been attributed to the signaling activity of certain components of the chaperone complex, released from their interaction with Hsp90 upon hormone stimulation (Han et al., 2009
; Groeneweg et al., 2011
), we asked whether the rapid socioaffective effects of exogenous glucocorticoids also differ between WT and HDAC6 KO mice.
We first examined the ability of acute exogenous Cort to alter the expression of the resilient or vulnerable behavioral phenotype in a social avoidance test. Mice were exposed to repeated social defeat and were subsequently tested twice for social avoidance, one week apart with or without Cort administered in a counter balanced order. Results from the tests conducted under vehicle conditions were used to stratify individual mice as resilient or vulnerable. When administered as a single injection 20 min prior to the social interaction task, a low dose of Cort (0.5 mg/kg, i.p) did not influence social approach in control mice (interaction ratio WT Veh = 146.5 ±18.2, WT Cort = 160.3 ±14.5, KO Veh = 142.3 ± 11.3, KO Cort = 139.5 ±16.6, n=13-22/group) and was devoid of effect on the expression of social avoidance in vulnerable mice, in both genotypes (). On the other hand, the same single Cort injection was sufficient to trigger the expression of social avoidance in otherwise non-avoiding defeat-exposed WT mice, thus switching their behavioral profile from defeat-resilient (average interaction ratio 138.50 ± 10.80) to defeat-vulnerable (average interaction ratio 85.24 ± 8.51; F1,14 = 10.05, P = 0.007). These results indicate that experience-dependent expression of social avoidance in WT mice is facilitated by an acute surge in adrenal steroids and GR activation at the time of testing. In contrast, acute corticosterone administration was devoid of effect in resilient HDAC6Pet1Cre KO mice. Thus, the rapid facilitating effect of adrenal steroids on social avoidance after defeat, which likely reflects non-genomic action of glucocorticoids, also requires intact HDAC6 in 5-HT neurons. Modulation of the rapid behavioral effect of Cort by HDAC6 was not restricted to mice exposed to social defeat, as differential behavioral responses to Cort were also detected in a cohort of naïve WT and HDAC6Pet1Cre KO mice examined in two anxiety tasks, the elevated plus maze (EPM) and open-field (OF). In these tests acute Cort induced robust anxiogenic responses in WT mice (i.e, decreased time spent in the center of the OF and open arms of the EPM) while it produced no significant effects, or anxiolytic-like responses in HDAC6Pet1Cre KO mice (; OF test, genotype × treatment interaction F1,65 = 19.8, P = 0.00004 , Fisher PLSD P = 0.001 vs vehicle in WT and P = 0.05 vs vehicle in KO; EPM test, genotype × treatment interaction F1,34 = 14.7, P = 0.0005, Fisher PLSD P = 0.001 vs vehicle in WT).
Serotonin-selective depletion of HDAC6 alters socioaffective responses to exogenous corticosterone
Because Pet1 expression was recently reported in the pancreas, (Ohta et al., 2011
) where HDAC6 could also regulate GR signaling, it is possible that Pet1-driven recombination may influence glucose homeostasis. We verified that differential behavioral responses to acute corticosteroids in WT and HDAC6Pet1Cre
KO mice do not reflect differential plasma glucose regulation under baseline conditions or after corticosterone administration. Despite a significant enhancement of plasma glucose with cort (WT baseline = 110 ± 6.5 mg/dl, WT cort = 161.2 ± 17.6 mg/dl, main effect of Cort, F(1,16) = 19.5, P < 0.001, Fisher PLSD P < 0.01) there was no significant effect of genotype or interaction between cort and genotype on glucose levels (KO baseline = 113.8 ± 7.8 mg/dl, KO cort = 160.8 ± 9.0 mg/dl). Thus, despite a lack of significant baseline phenotypic differences between WT and HDAC6Pet1Cre
KO mice in socioaffective behaviors, an acute rise in adrenal steroid hormones unmasks such differences in a context and experience dependent manner, through a 5-HT and HDAC6 mediated mechanism.