Increased glutamic acid decarboxylase expression in the hypothalamic suprachiasmatic nucleus in depression

In mammals, the proper temporal organization of behavioral, physiological, and biochemical processes in synchronization with the environmental light/dark cycle is regulated by the central circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). In depression, these biological rhythms become disrupted. Fluctuating mood, such as early morning mood worsening, is a feature of major depressive disorder (MDD) (Morris et al. 2009), while in seasonal affective disorder (SAD), low mood onset and a relapse of depressive episodes begin when photoperiod shortens (Lewy et al. 2006). In addition, almost 90% of the MDD patients suffer from sleep disturbances (Neylan 1995), while light therapy, which acts via re-setting of the disrupted SCN, has proven to be a successful treatment not only for SAD patients (Martensson et al. 2015), but also for MDD patients (Yamada et al. 1995). Polymorphisms in SCN clock genes were also found to be a risk factor for bipolar disorder (BD) (Rybakowski et al. 2014). In our previous studies, we found a disturbance in the key output neuropeptide of the SCN, arginine vasopressin (AVP), in depression (Zhou et al. 2001).

A hyperactive hypothalamo–pituitary–adrenal (HPA) axis is often a feature of depression (Bao et al. 2008), and activation of the corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) is the central motor for HPA activity (for review, see Bao et al. 2008). Direct and indirect projections from the SCN to the PVN have been found in mouse and rat (Abrahamson et al. 2001) as well as in human (Dai et al. 1998). In nocturnal animals, such as rats, SCN AVP was found to inhibit corticosterone release (Kalsbeek et al. 1996). However, in a diurnal animal, the A. ansorgei, SCN AVP was found to stimulate corticosterone release (Kalsbeek et al. 2008). Most SCN neurons in rat and human are reported to be GABAergic (producing γ-aminobutyric acid as a neurotransmitter), and contain glutamic acid decarboxylase (GAD), which is the key enzyme for GABA production (Moore and Speh 1993; Buijs et al. 1995; Gao and Moore 1996). In rodents, the SCN consists of two parts: the ‘core’, which comprises vasoactive intestinal peptide (VIP) and gastrin-releasing peptide-producing neurons, and the ‘shell’, which mainly contains AVP-producing neurons (Moore et al. 2002). GABA is essential for interregional communication within the SCN network and can act both as an excitatory and inhibitory neurotransmitter in an adult rodent’s SCN (De Jeu and Pennartz 2002; Choi et al. 2008; Albus et al. 2005; Irwin and Allen 2009). This difference is probably due to the differential expression of the chloride cotransporters KCC1 and NKCC2 in the SCN (Choi et al. 2008; Belenky et al. 2010). It seems that GABAergic excitation relays the photic and phase information from ventral to dorsal SCN following a shifted light/dark cycle (Albus et al. 2005). Increased SCN GAD65-mRNA was observed in rats exposed to chronic intermittent stress (Bowers et al. 1998). These findings suggest that disordered GABA expression contributes to the disturbance of SCN functions in depression, in a way that may be distinct from rodents, which are nocturnal animals. To test this hypothesis, we used immunocytochemistry to measure the amount of GAD65/67-immunoreactivity (ir) and AVP-ir in the SCN, and used in situ hybridization to quantify the expression of SCN GAD67-mRNA, which is the dominant GAD-mRNA type in the SCN (Gao and Moore 1996), from a series of human hypothalamic tissue samples from individuals with a long-term history of depression as well as from well-matched controls. Since immunoreactive GAD fibers may arise from both inside and outside the SCN, special attention was paid to the quantification of the GAD mRNA. In addition, special attention was paid to potential sex differences, since depression is more prevalent in women than in men (Altemus 2006).

Arginine vasopressin-stained neurons as well as nerve fibers were found to be widely distributed, not only in the SCN but also in the PVN, SON, and the accessory nuclei (Supplement Figure 1). Figure 3 shows that the majority of the SCN neurons were GABAergic, i.e., that the SCN of these patients contains more GAD-mRNA positive neurons than AVP-staining neurons. GAD65/67-staining and GAD67-mRNA were observed to be widely distributed in the hypothalamus, including in the SCN (Fig. 2). Almost all SCN neurons were surrounded by GAD65/67-ir containing beads, probably terminals, while only few GAD65/67-stained neurons were visible in the SCN (Fig. 2f). In the rostral part of the SCN, a relatively clear boundary of GAD-ir was observed, which was similar to that stained by AVP, while more caudally, the possibility of delineating the SCN by GAD65/67-staining gradually disappeared as the GAD65/67-staining became widely spread throughout the hypothalamus, except for the optic tract (Fig. 2c, d). This was why we used AVP-staining in alternating sections to delineate the GAD65/67-staining in the SCN. The IODs of SCN GAD65/67-ir and GAD67-mRNA were significantly (58 and 169%) higher, respectively, in the depressive samples [21.63 (13.33–27.46) and 2.23 (1.40–4.70), respectively, n = 13] compared with control subjects [13.65 (8.29–18.47) and 0.83 (0.27–2.56), respectively, n = 13, P = 0.044, and P = 0.029, respectively, Fig. 4]. In addition, there were a highly significant increase of AVP-ir by 253% in the female depression patients [5.39 (4.63–6.37), n = 5] compared with female control subjects [1.53 (0.76–4.14), n = 5, P = 0.008, Fig. 5a–e], while there was no significant difference between male controls [3.25 (2.36–3.84), n = 8] and male depression patients [2.96 (1.66–3.70), n = 8, P = 0.574, Fig. 5a–e]. Multiple linear regression analysis revealed that there was indeed a clear trend for an association between AVP-ir and sex (β = 0.423, P = 0.057). Moreover, since the possible sex difference in the expression of SCN AVP-ir in depression was not investigated in one of our group’s previous studies (Zhou et al. 2001), we re-analyzed those data and confirmed the presence of a significantly increased number of AVP-expressing neurons only in females [female control 3422 (1945–4298); female depression 7947 (6362–11,070), P = 0.029], while only a trend of increase was present in the male depression group [male control 3534 (2467–6098); male depression 6178 (3832–7276), P = 0.073]. Furthermore, in that cohort, we observed a significantly higher ratio of AVP-ir/AVP-mRNA in the depression group [0.78 (0.51–3.24)] compared with the control group (0.30 (0.23–0.51), P = 0.002), although the ratio did not show a sex difference. There was a significant negative correlation between AVP-ir and age in the control group (ρ = −0.745, P = 0.003, n = 13), which was also present in the male depression group (ρ = −0.766, P = 0.027, n = 8, Fig. 5f, g) but not in the female depression group (ρ = −0.014, P = 0.964, n = 5). Such a negative correlation between AVP-ir and age was not present in male (P = 0.123, R = −0.590, n = 8) or female (P = 0.188, R = −0.700, n = 5) controls, but it should be noted that the sample sizes of these subgroups were small. SCN GAD65/67-ir or SCN GAD67-mRNA did not show a significant correlation with age, either in the control or in the depression group (all P ≥ 0.291).

There were no significant differences in the levels of AVP-ir, GAD65/67-ir, and GAD67-mRNA among the MDD, BD, and control groups (P = 0.192, P = 0.127, and P = 0.074, respectively). Please note that the sample sizes of these subgroups were small. No significant correlation was observed between depression duration and GAD65/67-ir (ρ = −0.127, P = 0.680), GAD67-mRNA (ρ = 0.242, P = 0.426), or AVP-ir (ρ = 0.030, P = 0.922).

In the present study, we found, for the first time, significantly increased GAD67-mRNA and GAD65/67-ir in the SCN, which indicates the presence of increased SCN GABA neurotransmission in depression. In addition, increased SCN AVP-ir was observed only in female patients, not in male patients. This significant sex difference was confirmed in a different cohort after re-analysis of our previously published data (Zhou et al. 2001).

It has been shown in rodents that both SCN GABA and AVP take part in generating circadian activities by interacting with other SCN neuropeptides (Irwin and Allen 2010). A major and novel finding of our present study is that both GAD65/67-ir and GAD67-mRNA were up-regulated in the SCN of depression patients. Based upon the evidence that (1) most human SCN neurons seem to express GABA (Gao and Moore 1996), as was also confirmed by the present study, especially in depressed patients, (2) all of these neurons were surrounded by GAD65/67-ir containing beads, probably terminals, (3) in rodents, GABA may up- or down-regulate SCN function, depending on the photoperiod (Farajnia et al. 2014; Myung et al. 2015), time of the day, and the sub-area of the SCN (DeWoskin et al. 2015; Irwin and Allen 2009; De Jeu and Pennartz 2002; Choi et al. 2008; Albus et al. 2005), (4) tonic release of GABA shifts the molecular clock and affects SCN synchrony (DeWoskin et al. 2015), and (5) SCN GABA may also serve as an efferent signal that conveys timing information to other brain areas and/or regulates the responsiveness of SCN neurons to afferent signals (Trachsel et al. 1996; Buijs et al. 1994), the strong SCN GABA alterations we observed can make a clear contribution to the disrupted biological rhythms in depression. It should be noted that previous studies have found a deficit in cortical GABA levels (Hasler et al. 2007): GABA_A receptors (Klumpers et al. 2010) and decreased cortical GABAergic neurons (Rajkowska et al. 2007) in depression. In addition, a significant reduction of GAD-mRNA and GAD-protein was observed in BD (Fatemi et al. 2005; Heckers et al. 2002) and MDD patients (Fatemi et al. 2005; Perry et al. 1977). Several studies have shown that plasma GABA levels remain low after the depression symptoms improved (Petty et al. 1995) or following anti-depressant intake (Petty et al. 1993). However, Bielau et al. (2007) have found increased density of GAD neurons in the orbitofrontal cortex, and in the present study, we have found increased GAD65/67-ir and GAD67-mRNA in the SCN of depression patients. These data indicate, therefore, a presence of brain area-dependent changes of GABAergic systems in depression, which may explain, at least partly, the inconsistent curative effect of using GABAergic agonists as antidepressants (Brambilla et al. 2003).

In rats, SCN AVP was found to increase the firing rates of neurons and the circadian amplitude and to cause phase shifts (Murphy et al. 1998). The significantly increased SCN AVP-ir in female but not male patients (the present study and the paper by Zhou et al. 2001) indicates a stronger functional SCN alteration in female than in male depression patients, which is in agreement with the finding that young moderately depressed women showed higher homeostatic sleep pressure than depressed men (Frey et al. 2012). In addition, the significant negative correlation between SCN-AVP-ir and age in control subjects is in accordance with our previous findings (Swaab et al. 1985). It is a novel finding that this correlation was present in the male but not in the female depression group. These data fit well with the findings that females are more vulnerable to depression than males (Altemus 2006). The SCN molecular clock mechanism is based upon elegant interactive transcription–translation feedback loops for clock genes. AVP is required for the circuit-level organization of the expression of the SCN clock genes (Edwards et al. 2016). Indeed, our group has observed lower levels of AVP-mRNA together with higher levels of AVP-ir in depression in a previous study (Zhou et al. 2001). There seems to be a disbalance in the transcription–translation relationship for SCN AVP in depression, which was confirmed by the present findings following re-analysis of the published data, i.e., there were indeed a significant increase in the ratio of AVP-ir to AVP-mRNA in the depression group compared with controls in that study (Zhou et al. 2001). An important question is whether the alterations in the SCN may not only be the root of circadian changes, but could also be contributing factors to the increased activity of the HPA-axis and so to the symptoms of depression. In nocturnal animals, such as rats, SCN-AVP was found to inhibit hypothalamic HPA activity in the PVN (Kalsbeek et al. 1996), which may imply that a disorder in the SCN in rodents may cause disinhibition of the HPA-axis activity and thus contribute to HPA-axis hyperactivity, leading to depression-like symptoms (Holsboer 2001). However, in a diurnal animal, i.e., the Arvicanthis ansorgei, AVP from the SCN was found to have a stimulating effect on the hypothalamic PVN and thus on HPA activity (Kalsbeek et al. 2008). Whether the increased SCN AVP-ir found in the female depression group also leads to a stimulating effect on the HPA activity and thus to more depressive symptoms should be a topic for further studies, since in our previous study (Zhou et al. 2001), we found decreased SCN AVP-mRNA in depression, which prompted our proposal of a reduced production and release of AVP from the SCN. In this respect, it merits attention that recent studies have found that AVP plays a crucial role in the generation of overt circadian rhythms and that diminished AVP signaling in the SCN may cause a loss of coherent circadian rhythms (Li et al. 2009; Yamaguchi et al. 2013; Mieda et al. 2015; Loh et al. 2015). It should also be noted that the hyperactivity of the HPA axis in depression may contribute to the disturbance of the SCN, since we observed that glucocorticoids show an inhibitory effect on AVP mRNA expression in the human SCN (Liu et al. 2006). To the best of our knowledge, there are no data on the effects of CRH or corticosteroids on GABA expression in the SCN, which remains an intriguing topic for future studies.

A strongly increased SCN GABA was found that may be central to the disrupted clock function in depression. In addition, the functional consequences of the significantly increased SCN AVP-ir in female depression patients for SCN function and its output may be related to the higher vulnerability for depression in women. Taken together, our findings indicate a reduced stimulatory output from the SCN, i.e., there were more inhibitory GABAergic signaling and less excitatory AVP signaling from the SCN to the projection areas in depression, especially in female patients.