GABA is synthesized by decarboxylation of glutamate by glutamic acid decarboxylase (GAD) (Figure 1) (2). Based on its molecular weight, it is possible to distinguish two isotypes, the 65 kDa isotype GAD65 and the 67 kDa isotype GAD67, which are involved in different aspects of GABAergic neurotransmission (3). GAD65 is responsible for rapid synthesis of GABA during periods of high synaptic demand; it is predominantly located on axon terminals and synaptic vesicle membranes and is thus primarily associated with packaging and release of GABA (4–7). GAD67 is responsible for basal GABA levels (4, 5) and the majority (80–90%) of GABA synthesis (8); it is located in the cytosol and is thus primarily associated with GABA synthesis and non-vesicular release (6, 7).

After synthesis in the presynaptic terminal, GABA is packaged into vesicles by the vesicular GABA transporter (VGAT), which is embedded in the vesicular membrane (9). A presynaptic action potential can induce a Ca²⁺-mediated fusion of the vesicle membrane and the presynaptic neuron membrane, which leads to release of GABA into the synaptic cleft. Alternatively, after strong depolarization or altered ion homeostasis, specific GABA transporters (GAT) may reverse their direction resulting in non-vesicular release of GABA (9, 10).

After release into the synaptic cleft, GABA exerts its inhibitory activity by binding to two types of receptors, such as GABA_A and GABA_B receptors. GABA_A receptors are ligand-gated Cl⁻ channels and produce most of the physiological actions of GABA (11). GABA_A receptors have a pentameric subunit structure derived from different gene families and include α, β, γ, δ, ε, π, and θ subunits. Some of these subunits have several isoforms (α1–6, β1–3, and γ1–3) (12). In most cases, the pentamers of subunits include a pair of α subunits and a pair of β subunits in combination with a fifth subunit (γ or δ) (13).

The synaptic activity of GABA is terminated when GABA is taken up by GAT that are embedded in the membranes of neurons and astrocytes (10). In humans, four types of GAT can be distinguished, GAT-1 to 3 and the betaine GABA transporter (BGT-1). GAT-1 is widely expressed in the brain, predominantly in presynaptic GABA neurons, and is thus primarily responsible for GABA reuptake (9, 10). GAT-3 is primarily responsible for GABA uptake into local astrocytes (14). In contrast to GAT-1 and GAT-3, GAT-2 and BGT-1 play a very limited role in GABAergic neurotransmission (10).

When GABA is taken up by neurons, it can either be repacked into vesicles or it can be degraded to succinic semialdehyde (SSA) by the enzyme GABA transaminase. After conversion of SSA to succinate, the latter enters the TCA cycle and is subsequently converted into glutamate (10, 15). The following conversion of glutamate to GABA by GAD65 or GAD67 completes the GABA cycle (Figure 1).

One of the most consistent postmortem findings in schizophrenia is a reduction of mRNA encoding for GAD67 in the dorsolateral prefrontal cortex (DLPFC) in layers 1 through 5 (3–5, 16–29), which results in a reduction of GAD67 protein levels although this has been less extensively studied (Table 1) (4, 30, 31). Since the majority of studies reported unaltered or increased neuronal density, it is unlikely that the reduction of GAD67 mRNA can be attributed to a decrease in the number of neurons in schizophrenia (16, 27, 32, 33). Rather, the density of neurons expressing a detectable level of GAD67 mRNA is decreased (27); expression of GAD67 mRNA is decreased below a detectable level in 25–35% of GABAergic neurons, while the remaining neurons have GAD67 mRNA levels similar to controls (27, 29). It has therefore been suggested that impaired GAD67 gene expression is limited to a certain subset of GABAergic neurons (27, 31). This subset could concern the chandelier, double bouquet, or wide-arbor neurons, which can be distinguished by the presence of specific calcium-binding proteins (Box 1) (1).

The subset that is affected in schizophrenia appears to include parvalbumin-containing GABAergic neurons. In schizophrenia, parvalbumin mRNA expression is reduced in prefrontal cortex (PFC) layers 3 and 4, but not layers 2, 5, or 6 (5, 27, 29, 51). The overall expression of parvalbumin mRNA is decreased whereas the density of neurons expressing detectable levels of parvalbumin is unaltered (5, 52, 53), implying that the reduction of parvalbumin mRNA is not accompanied by a loss of parvalbumin-containing neurons. The reduced parvalbumin mRNA expression is associated with the decreased density of GAD67 mRNA-positive GABAergic neurons. 50% of the parvalbumin-positive neurons lack detectable amounts of GAD67 mRNA (5), whereas calretinin mRNA (which is expressed by a different subset of neurons—see Box 1) expression and the density of calretinin-positive neurons remain unchanged in schizophrenia (5, 54). These findings imply that the reduced GAD67 mRNA expression may be selective for the parvalbumin-containing subgroup of GABA neurons in the PFC (5). Recent evidence suggests that GAD67 protein levels are unaltered in the chandelier neurons, suggesting that other parvalbumin-containing neurons, such as the basket cells, are involved (31).

The observed alterations regarding parvalbumin are not likely to be caused by exposure to antipsychotic medication. Long-term exposure to haloperidol and benzotropine did not lead to an altered expression of parvalbumin mRNA (5). Furthermore, transcript levels for parvalbumin were reduced to the same extent in the DLPFC of medication-naïve patients compared to patients receiving antipsychotic medication (20). Animal studies have shown that treatment with dopamine D2-receptor antagonists influences the expression of GAD67 mRNA in the basal ganglia (BG) (55–58) but not in the PFC; however, D2-receptor density in the PFC is much lower than in the BG (20, 27).

The transporter protein GAT-1 is present in the presynaptic neuron and is responsible for the synaptic reuptake of GABA (19, 59). It plays a role in both tonic and phasic GABA-mediated inhibition (60, 61). GAT-1 terminates the synaptic activity of GABA and regulates the duration and efficacy of synaptic GABAergic neurotransmission (62); therefore, reduced GAT-1 levels suggest increased availability of GABA in the synapse (63). Several studies found reduced mRNA levels encoding for the GAT-1 protein in schizophrenia. GAT-1 mRNA levels are decreased in GABAergic neurons in the DLPFC (Table 2) (20, 21, 59, 62, 64). Together with the diminished expression of GAD67 mRNA, it is unclear whether this results in a net increase or decrease of the inhibitory tone on pyramidal cells (63). Moreover, GAT-1 mRNA expression is reduced below detectable levels in a subset of GABAergic neurons and relatively unaltered in the majority of the GABAergic neurons (59). The affected subset appears to include parvalbumin-containing neurons (1, 59). The reduction of GAT-1 mRNA expression is limited to layers 2 through 5, the same layers in which parvalbumin-containing neurons are found (59, 65).

The subset of GABAergic neurons where reduced GAT-1 mRNA levels are detected is possibly the subset of chandelier neurons (see Box 1). A marker of chandelier neurons is their GAT-1 immunoreactivity; the density of GAT-1 immunoreactive cartridges is decreased in schizophrenia, while markers of other axon terminal populations remain unchanged (65, 66). The lower density of GAT-1 immunoreactive cartridges implies decreased GAT-1 protein, which is associated with decreased GAT-1 mRNA levels. Putting together these findings, reduced GAT-1 mRNA levels may therefore account for the decreased density of GAT-1 immunoreactive axon cartridges in chandelier neurons (59). The reduction of GAT-1 immunoreactive cartridges cannot be attributed to a reduction of chandelier neurons, since the density of GABAergic neurons [identified by parvalbumin (52, 53) and VGAT (67)] is unchanged. Thus concluding, the density of chandelier neurons containing GAT-1 protein in the DLPFC in patients with schizophrenia was reduced whereas the density of parvalbumin-containing neurons remains unaltered. This finding suggests that the reduced levels of GAT-1 mRNA are limited to the chandelier neurons (29, 65).

Long-term exposure to therapeutic blood levels of haloperidol in monkeys did not result in changes in the expression of GAT-1 mRNA or the expression of GAT-1 protein (65, 66, 68), nor did effects of alcohol abuse or benzodiazepine use explain the findings (20, 21).

GABA_A receptors are ligand-gated chloride ion channels and produce most of the physiological actions of GABA (11). GABA_A receptors have a pentameric subunit structure and the subunits are derived from different gene families encoding for different subunits including α1–6, β1–3, γ1–3, δ, ε, π, and θ (12). The pentamers of subunits include in most cases a pair of α subunits and a pair of β subunits in combination with a fifth subunit (γ or δ) (13). Early studies demonstrated increased binding of muscimol, a selective GABA_A receptor agonist, in pyramidal neuronal cell bodies in patients with schizophrenia (69–71); however, muscimol can bind to all types of GABA_A receptor subunits. Recent advancements in technology have enabled investigation of deficits of individual GABA_A receptor subunits (72).

Subunits of the α-type can be characterized by their subcellular localization within the central nervous system. Over 95% of the GABAergic synapses on the axon initial segment (AIS) of pyramidal neurons contain the α2 subunit, while only 15% of cortical GABA receptors contain the α2 subunit (73, 74). It appears that this subunit is characterized by high affinity, fast activation, and slow deactivation (75). Given its anatomical position and functional features, the GABA_A α2 subunit serves as a major source for inhibitory tone on pyramidal neurons (46). Parvalbumin-containing neurons, which appear to exhibit a reduced expression of GAT-1 and GAD67 mRNA in schizophrenia, target the AIS of pyramidal neurons. Indeed, it has been demonstrated that in schizophrenia, the GABA_A α2-receptor subunit is upregulated in the AIS of pyramidal neurons (46, 69, 76). This increase in α2 subunit density may occur in response to reduced extracellular GABA concentrations due to diminished GABA synthesis (1, 46). Furthermore, GAT-1 immunoreactive cartridges and the density of α2 subunits at the postsynapse of pyramidal neurons demonstrate an inverse correlation, which implies that GABA_A α2 subunits are upregulated at the AIS of pyramidal neurons and GAT-1 is downregulated to provide a synergetic compensation for the diminished GABAergic activity (46). In contrast to GAD67 mRNA and GAT-1 mRNA, mRNA expression levels of postsynaptic GABA_A α2-receptor subunits seem to be unaltered (16, 17). Reductions of α2-receptor subunits are exclusively found at the AIS synapses; the lack of upregulation of α2 subunit mRNA might be explained by the fact that inhibitory synapses at the AIS of pyramidal neurons make up less than 10% of the total number of inhibitory synapses of the pyramidal neuron (16, 77).

mRNA levels of the GABA_A α1, γ2, α4, α5, and δ receptor subunits are suggested to be downregulated in the DLPFC of patients with schizophrenia (Table 3) (20, 21, 47, 78–80). However, two studies reported an increase of α1 subunit mRNA expression (34, 64), one study revealed an increase of α5 subunit mRNA (34), one study observed an increase of the GABA_A receptor α1 subunit protein (72), and one study demonstrated no change of the α4 receptor subunit (80). In contrast to the a2 subunit localized at the AIS of pyramidal neurons, GABA_A receptors containing the α1, α5, γ2, and δ (often co-expressed by α4) subunits are predominantly localized in the dendrites of pyramidal neurons (73, 81, 82). The observed alterations in the postsynaptic GABA_A receptors do not seem to be a consequence of an increased number of neurons, because the majority of studies have reported no change or an increase in neuron density (27, 32, 33, 51).

Animal studies in which rats were exposed to benzodiazepines did not reveal changes in the expression level of α2 subunit mRNA or protein levels and long-term exposure to haloperidol or olanzapine did not result in altered α1, α2, α5, β2, or δ subunit mRNA levels (20, 78, 84). Postmortem studies show that α1 and δ subunits are reduced to the same extent in the DLPFC of patients who were not taking antipsychotic medication at the time of death, which is unlikely to be driven by the effects of alcohol abuse or benzodiazepine use (20, 21). (For an overview of pre- and postsynaptic GABAergic alterations, see Figure 2.)

There is sufficient histological–pathological evidence to link impairments in GABAergic neurotransmission in other cortical regions than the DLPFC to pathologies and cognitive dysfunctions observed in schizophrenia (63).

Similar to the DLPFC, the anterior cingulate cortex (ACC), primary visual cortex (VC), and primary motor cortex are characterized by the same deficits in GABAergic gene expression as seen in the DLPFC, including selective involvement of parvalbumin-containing subsets of GABA neurons. The largest declines were reported for the levels of mRNA encoding for parvalbumin (21). These brain areas also exhibit a decrease of GAD67 mRNA, GAD65 mRNA, GAT-1 mRNA, and GABA_A receptor α1 and δ subunits (1, 21, 28). Calretinin levels remained unchanged (21). GABA-related transcript expression is suggested to be decreased to the same extent in all aforementioned brain regions, so there possibly is no preferential involvement of the DLPFC (21). The reduced expression of GABA_A receptor α1 and δ subunits in these cortical areas also imply that reduced phasic and tonic inhibition, respectively, might be a feature shared by multiple cortical regions.

Furthermore, in addition to the ACC, primary VC, and primary motor cortex which demonstrated similar GABAergic expression deficits as the DLPFC, the orbital frontal cortex (OFC), superior temporal gyrus (STG), striatum, and thalamus show a diminished GAD67 mRNA expression as well (3). In addition, the STG and auditory gyri demonstrated reduced GAT-1 protein levels (34). Reduction in GABAergic activity in the OFC could lead to disturbances related to emotional and cognitive functioning and may therefore underlie symptoms regarding social withdrawal and apathetic behavior (85). In addition, abnormalities in the STG could contribute to deficit auditory processing and auditory hallucinations (3). These findings imply that the aberrations seen in the DLPFC may not be due to alterations in DLPFC circuitry only, but that the altered transcript levels appear to be the consequence of a common upstream mechanism that operates across multiple cortical areas.

A possible integrative model for the alterations in GABA neurotransmission is that a subset of prefrontal GABA neurons is affected in schizophrenia. In contrast to the reduced GAD67 and the consequent attenuation of inhibitory GABAergic neurotransmission, the reduction of GAT-1 mRNA expression tends to increase the synaptic activity of GABA (63). In addition, GABA_A receptors are upregulated in postsynaptic pyramidal neurons, which suggests a compensatory increase in response to the decreased extracellular GABA concentrations (46, 70, 76). However, based on postmortem studies, it is not possible to identify the initial deficit in the pathological chain and, therefore, two scenarios are possible (see Figure 2).

The most likely scenario is an overall reduced GABAergic activity in schizophrenia. This implies that the initial step in this specific pathologic process is the presynaptic reduction of GABA synthesis, followed by a secondary, compensatory reduction of reuptake by means of GAT-1 and by compensatory upregulation of postsynaptic GABA receptors (1, 18, 86). This synergetic attempt, to improve the GABAergic neurotransmission at the synapse of the pyramidal neuron AIS, serves to compensate for the initial deficit in synthesis of GABA. Consistent with the theory that the reduction of synthesis is the first step in the pathological chain, mice lacking the GAT-1 gene do not develop diminished levels of GAD67 mRNA. This indicates that the reduction of GAD67 is the initial event (87). Furthermore, GABA hypofunction due to decreased synthesis reflected by the diminished levels of GAD67 mRNA was imitated in rats by means of pharmacological blockade of prefrontal GABA_A receptors. This resulted in impaired working memory performance, a cognitive function characteristically disturbed in patients with schizophrenia (88, 89). However, it is still controversial whether the compensatory mechanisms are sufficient to overcome the decreased GABA synthesis. In other words, it is unknown if the net effect of the diminished presynaptic synthesis on the one hand and the decreased reuptake increased postsynaptic reception on the other hand result in an increase or decrease of the inhibitory tone on pyramidal cells by GABAergic neurons (63). In conclusion, the most likely scenario is that reduced presynaptic GABA production results in a reduced reuptake of GABA and in upregulated postsynaptic GABA receptors in schizophrenia.

Alternatively, an excessive increase of GABAergic activity due to both primary diminished reuptake and upregulated postsynaptic receptors may also be an initial step in the pathological process followed by secondary compensatory downregulation of GAD67 mRNA in chandelier neurons due to the excessive GABAergic activity. Furthermore, the effects of pharmaceuticals involved in GABAergic neurotransmission seem to be in line with the hypothesis of excess GABAergic activity. For example, lorazepam, a positive allosteric modulator of GABAergic neurotransmission, results in a deterioration of working memory aberrations while flumazenil, a partial inverse agonist, leads to improvement of the working memory deficits (63). Thus, according to this scenario, excessive GABAergic activity could be the result of an initial postsynaptic upregulation of the GABA_A receptor and downregulation of the presynaptic GABA reuptake transporters as a first step in the pathological chain (63).

Finally, the aberrations seen in the DLPFC may not be due to alterations in DLPFC circuitry, but instead reflect transcript levels that are a consequence of a common upstream mechanism that operates across multiple cortical areas in schizophrenia.

In conclusion, the most likely scenario involves reduced GABA concentrations due to a compromised production of GABA reflected by the diminished concentration of GAD67 mRNA. However, due to the observation that presynaptic GAT-1 is reduced and postsynaptic receptors are upregulated, postmortem studies do not provide a conclusive answer about the net GABAergic concentrations and activity. Therefore, in vivo studies could provide additional insights into GABA levels in clinical states contributing to a more definitive formulation about the pathological cascade and GABAergic alterations in schizophrenia.