We have found that the level of GAD-67, but not GAD-65, was significantly reduced in medication-free depressed subjects as compared to their matched controls. In contrast, depressed subjects medicated with antidepressants at the time of death had unchanged levels of both GAD isoforms as compared to corresponding control subjects. Together, these data raise the intriguing possibility that GAD-67 is involved in the pathophysiology of depression and antidepressants are able to normalize a GAD deficit in MDD.
GAD-67 is found abundantly in cell bodies and proximal dendrites, and GAD-65 is predominantly present in axon terminals (Kaufman et al., 1991
). Hence, a selective decrease in GAD-67 protein suggests that somata and dendrites of specific population of GABAergic neurons may be affected in depression. Low levels of GAD-67 protein immunoreactivity in depression may suggest either a loss of cell bodies and/or their dendritic processes or decreased rate of GAD-67 protein synthesis per unchanged number of neurons. In fact, we have previously reported a selective reduction in the density of calbindin-positive GABAergic interneurons in the dorsolateral PFC (Rajkowska et al., 2007
). Therefore, it is likely that reduced GAD-67 immunoreactivity in the dorsolateral PFC is due to a reduction in the density and size of calbindin-positive somata of GABAergic interneurons. Other post-mortem studies investigating GAD in depression found reductions in GAD-65/67 immunopositive structures in the dorsolateral PFC (Gos et al., 2008
) and in GAD protein level in the cerebellum (Fatemi et al., 2005
A reduction in GAD-67 in post-mortem dorsolateral PFC of medication-free MDD subjects reported herein supports a recent MRI spectroscopy observation of a reduced GABA level in the PFC in unmedicated living depressed subjects (Hasler et al., 2007
). This and other neuroimaging studies which consistently report reduced GABA levels in MDD (Sanacora et al., 1999
; Sanacora et al., 2004
) measure total amount of GABA, consisting of metabolic and neurotransmitter pools in cellular and extracellular compartments. Given that the present study demonstrates a reduction in the level of GAD-67 isoform which is predominantly involved in the synthesis of metabolic or cytosolic GABA, it could be suggested that reduced metabolic pool of GABA significantly contributes to the GABA deficits observed by neuroimaging studies in MDD.
Our findings of a reduction in GAD-67 levels in untreated depressed subjects and a lack of a reduction in depressed subjects treated with antidepressants raise the possibility that antidepressants may normalize GAD-67 level in depression. In the present study eight depressed subjects had antidepressants (sertraline, citalopram, paroxetine, venlafaxine, amitriptyline, and nortriptyline) present in their toxicology screenings (). Among these drugs are selective serotonin reuptake inhibitors (SSRIs), and inhibitors of reuptake of both serotonin and norepinephrine. Thus, the observed protective effect of antidepressants on the cortical GAD-67 level could be explained, to some extent, by the action of serotonin on GABAergic neurons.
More direct evidence for the influence of SSRIs on GABA system is provided by Sanacora et al (2002
demonstrating that chronic treatment with fluoxetine and citalopram as well as electroconvulsive therapy results in higher levels of GABA as compared to levels recorded before treatment. Moreover, acute administration of the SSRI citalopram increased total occipital GABA levels in healthy subjects (Bhagwagar et al., 2004
) with the magnitude similar to that observed after chronic (on average 8 weeks) treatment with SSRIs. The ability of serotoninergic antidepressant drugs to normalize GABA levels in depression and even increase it in healthy subjects indicates close interactions between these two systems and suggests that GABAergic mechanism may indirectly contribute to the mechanism of action of clinically active antidepressants.
The evidence exists that facilitation of GABAergic transmission produces antidepressant effects in humans (Nielsen et al, 1990
; Smith et al, 1998
; Petty et al, 1995
). Interestingly, agents acting at GABA-A or GABA-B receptors exhibit antidepressant-like activity in animal screening procedures (Lloyd et al., 1983
; Nowak et al., 2006
). If there is a deficit in GABA neurotransmission in depression, then adaptive changes in the density of GABA receptors might be predicted. Studies of GABA-A receptor binding in post-mortem tissues from depressed and suicide subjects have not demonstrated consistent alterations as increases, decreases or no change were found in the benzodiazepine binding sites (Pandey et al., 1997
; Cheetham et al., 1988
; Rochet et al., 1992
; Kugaya et al., 2003
; Zhu et al., 2006
). Moreover, gene expression studies report reduced (Merali et al, 2004
) or up-regulated (Choudary et al., 2005
) transcripts encoding specific subunits of the GABA-A receptor in depression and suicide. Collectively, these post-mortem studies of GABA-A receptors do not provide consistent evidence for GABA receptor dysregulation as a pathological marker of depression. Differences between experimental techniques and subject characteristics (e.g. medication exposure, post-mortem interval, brain tissue pH) are key factors that may be associated with discrepancies between post-mortem studies.
One of the shortcomings of this study is that GAD protein immunoreactivity was measured in post-mortem tissue homogenates as opposed to measuring GAD immunoreactivity localized to specific subpopulations of cortical GABA interneurons. However, we have previously reported that calbindin-positive GABAergic interneurons were selectively reduced in the same brain region in MDD (Rajkowska et al., 2007
); thus it was plausible to speculate that lower GAD-67 protein levels could reflect a reduction in interneurons expressing calbindin. However, among 21 MDD subjects examined in the present study, only 7 subjects who were antidepressant-free were the same as those analyzed in our study on reductions in GABA-calbindin immunoreactive neurons (Rajkowska et al., 2007
). There was no significant correlation between the amount of GAD-67 and the density or size of calbindin immunoreactive neurons. This discrepancy could be explained by the fact, that GAD-67 was measured in homogenates collected from the entire extend of available area 9 and across all six cortical layers. In contrast, calbindin immunoreactive neurons were counted only in a very narrow strip of cortical area 9 and only within one cortical layer (layer II).
Another possible weakness of our study is that we classify the MDD subjects as “medication-free” based on clean post-mortem toxicology screening. However, upon examination of the medical records we can determine that nine out of these 13 “medication-free MDD subjects did not have a prescription for antidepressants in the last month of life (). Therefore, it is likely that a majority of our “medication-free” MDD subjects were antidepressants naïve for at least 4 weeks before their death. Further studies are needed to test the influence of antidepressants on the GAD protein level in the brain.
Next limitation of the present study is a relatively small sample size (n=8) of “medicated” MDD subjects. Thus, our observation on unchanged level of GAD-67 and GAD-65 in these subjects needs to be tested on a larger number of subjects treated with antidepressants.
In summary, to our knowledge, this is the first observation of selective reduction in the GAD-67 immunoreactivity in the dorsolateral PFC of antidepressant-free MDD subjects and a lack of reduction in medicated MDD subjects as compared to matched controls. The present findings are consistent with studies using other approaches to implicate the GABA system in the pathophysiology of depression. Further indirect evidence is provided here of a regulatory effect of antidepressants on GABAergic system. Additional studies of the GABAergic markers will lead to a better understanding of the role of GABA in the pathology of depression and may lead to the development of more effective approaches to the treatment of depressive symptoms.