CORTICAL INHIBITORY NEURONS AND SCHIZOPHRENIA

CORTICAL INHIBITORY NEURONS AND SCHIZOPHRENIA David A. Lewis*,Takanori Hashimoto* and David W. Volk* Abstract Impairments in certain cognitive functions, such as working memory, are core features of schizophrenia. Convergent findings indicate that a deficiency in signalling through the TrkB neurotrophin receptor leads to reduced GABA (γ-aminobutyric acid) synthesis in the parvalbumin-containing subpopulation of inhibitory GABA neurons in the dorsolateral prefrontal cortex of individuals with schizophrenia. Despite both pre- and postsynaptic compensatory responses, the resulting alteration in perisomatic inhibition of pyramidal neurons contributes to a diminished capacity for the gamma-frequency synchronized neuronal activity that is required for working memory function. These findings reveal specific targets for therapeutic interventions to improve cognitive function in individuals with schizophrenia. PSYCHOSIS This refers to distortions in inferential thinking, such as delusions (fixed, false beliefs that are firmly held in the face of contradictory evidence), and perceptual disturbances, such as hallucinations. Auditory hallucinations, usually experienced as voices distinct from one s own thoughts, are most common in schizophrenia. Departments of *Psychiatry and Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA. Correspondence to D.A.L. e-mail: lewisda@upmc.edu doi:1.138/nrn1648 Schizophrenia is a devastating illness that affects approximately 1% of the world s population 1.Affected individuals frequently come to clinical attention during late adolescence or early adulthood, about 1% will eventually commit suicide and most experience a lifetime of disability. As a result, schizophrenia is associated with a substantial emotional burden for the families of those affected and it incurs tremendous economic costs for society in terms of medical expenditure and lost productivity. The risk of developing schizophrenia is directly proportional to the degree of genetic relatedness to an affected individual 2, and several putative susceptibility genes for schizophrenia have been identified 3.However, the degree of risk conferred by each gene seems to be small, and, for most genes, the biological basis of the increased risk for illness remains unclear. Furthermore, the degree of concordance for schizophrenia among individuals with the same genetic make-up is only about 5% (REF. 2),which indicates that genetic liability alone might not be sufficient to cause the clinical features of the illness. Consistent with this interpretation, various environmental events across development, from advanced paternal age at the time of conception to frequent cannabis use during adolescence, seem to increase the likelihood of developing schizophrenia later in life 4. Therefore, the etiology of schizophrenia is thought to require an interaction between genetic susceptibility and environmental risk factors, which alters neurodevelopmental processes that occur before the onset of the diagnostic features of the illness. Although PSYCHOSIS is usually the presenting and most striking clinical aspect of schizophrenia, it is not diagnostic of the disorder. Schizophrenia is recognized by the presence of impairments in social and occupational functioning (BOX 1) that result from the confluence of a range of disturbances in brain function, including abnormalities in perception, inferential thinking, fluency and production of language, expression of emotion, capacity for pleasure, volition and attention 5. Of the many clinical features of schizophrenia, disturbances in certain cognitive processes, such as impairments in attention, memory and executive functions (that is, the ability to plan, initiate and regulate goaldirected behaviour), might represent the core features of the illness 6.Cognitive abnormalities have been found throughout the life span of individuals with schizophrenia, including during childhood and adolescence 7, and at the initial onset of psychosis 8.The unaffected relatives of individuals with schizophrenia also show similar, although milder, cognitive deficits 9,which indicates that the cognitive deficits are not caused by treatment with antipsychotic medications or the chronic nature of the illness. The cognitive disturbances of schizophrenia have 312 APRIL 25 VOLUME 6 www.nature.com/reviews/neuro

Box 1 Diagnostic criteria for schizophrenia At present, no findings from laboratory procedures, neuroimaging methods or psychological tests are diagnostic of schizophrenia. Schizophrenia is diagnosed on the basis of the clinical syndrome, that is, the clustered appearance of a characteristic set of signs and symptoms. For the purpose of diagnostic reliability, these features have been formulated into specific criteria 5.The first diagnostic criterion requires the presence of two or more of the following clinical features: delusions, hallucinations, disorganized thinking (as reflected by tangential or incoherent speech), grossly disorganized or catatonic behaviour, and negative symptoms. The latter include altered emotional expression (for example, flat affect), poverty of speech (for example, alogia) and avolition (for example, the inability to initiate and persist in goal-directed activities). The second diagnostic criterion requires evidence of social or occupational dysfunction, such as deterioration in interpersonal relationships, work habits or personal hygiene, from the level achieved prior to the appearance of the clinical features. Third, the signs of the disturbance must be continuously present for at least six months. Fourth, it must be clear that the clinical features are not attributable to another disorder, including substance abuse. Although establishing the validity of these criteria remains an ongoing area of investigation, the application of these criteria is very reliable, which results in a high degree of concordance across research clinicians in the diagnosis of schizophrenia. At present, available treatments for schizophrenia are primarily effective in suppressing the psychotic features of the illness; they have limited impact on negative symptoms, and do not appreciably improve the social or occupational outcome for most individuals. WORKING MEMORY The active maintenance of limited amounts of information for a short period of time to guide thought processes or sequences of behaviour. Working memory is typically assessed through delayed response tasks in which a stimulus cue is briefly presented and removed, a delay period ensues, and then a response is required based on information contained in the stimulus cue. Pathogenesis (reduced signalling through TrkB) a greater negative impact on daily activities than do the psychotic symptoms, and the degree of cognitive dysfunction might be the best predictor of long-term functional outcome for individuals with schizophrenia 1. Despite the clinical importance of these cognitive abnormalities, there are no approved pharmacological treatments for these deficits. Furthermore, all medical treatments currently used for schizophrenia, which primarily suppress the psychotic features, were discovered by chance. The development of more effective treatments, and possibly of secondary prevention measures, requires greater knowledge of the underlying disease process (FIG. 1). Specifically, how do complex and variable combinations of genetic liabilities, environmental risk factors and developmental processes alter the structure and function of the brain and produce the characteristic clinical symptoms of schizophrenia? Typically, the starting point Pathophysiology (reduced gamma band power) Etiology Pathological entity Clinical syndrome (deficit in chandelier (impaired working cell-mediated inhibition) memory) Prevention Treatment ( α2 agonist) Figure 1 Components of the disease process of schizophrenia. The cluster of clinical signs and symptoms (clinical syndrome) characteristic of a brain disorder, such as schizophrenia, is the result of some cause or set of causes (etiology). The cause or causes induce the disease mechanisms (pathogenesis), which produce abnormalities in the brain (pathological entity) that alter its function (pathophysiology). The development of effective treatments or preventative measures requires the ability to interrupt or reverse the pathophysiological processes and pathogenetic mechanisms, respectively. For each component of the disease process of schizophrenia, the data reviewed in this article provide convergent evidence for the indicated abnormalities. TrkB, tyrosine kinase Trk (tropomyosin-related kinase) receptor B. for addressing this question is the clinico-pathological correlation the identification of a pathological entity that is associated with the clinical syndrome. An important challenge in schizophrenia research has been to identify a pathological entity that is associated with the core cognitive deficits of the illness. In this review, we illustrate one strategy for characterizing the components of the disease process of schizophrenia, beginning with the cognitive features of the clinical syndrome. Specifically, we review evidence that certain cognitive abnormalities in schizophrenia reflect pathology in specific cortical inhibitory circuits; that these pathological alterations might arise from specific pathogenetic mechanisms; and that these pathological alterations result in pathophysiological processes that have implications for new therapeutic interventions. As such, this article is not intended to provide a comprehensive review of the abnormalities found in inhibitory neurons in individuals with schizophrenia, which have been reviewed elsewhere 11,12. Working memory deficits in schizophrenia Certain crucial cognitive deficits in schizophrenia seem to reflect alterations in executive control, which includes context representation and maintenance functions, such as WORKING MEMORY,that depend on the DORSOLATERAL PREFRONTAL CORTEX (DLPFC) 13.Individuals with schizophrenia tend to perform poorly on working memory tasks and to show reduced DLPFC activation when attempting to carry out such tasks 14,15.In addition, under conditions in which individuals with schizophrenia perform normally on working memory tasks, activity in the DLPFC can be increased, which indicates that the DLPFC operates less efficiently 16. Several findings support the relevance of these alterations in DLPFC function to the disease process of schizophrenia. First, individuals with other psychotic disorders 17 or major depression 18 show normal activation of the DLPFC when carrying out working memory tasks, which indicates that the abnormalities observed in schizophrenia are, at least in part, specific to the clinical syndrome of schizophrenia. Second, the severity of deficits in activation of the DLPFC, but not of other cortical regions, during working memory tasks predict the severity of cognitive disorganization symptoms in individuals with schizophrenia 15.Third,it has been suggested that reduced working memory capacity might be rate limiting in the performance of other cognitive tasks in schizophrenia 19.Therefore, working memory deficits seem to be a central feature of schizophrenia, and identifying the pathological entity (or entities) in the DLPFC that produces these functional alterations is essential for understanding the underlying disease process. A pathological entity in schizophrenia Working memory and inhibition in the DLPFC. Although working memory is an emergent property of a neuronal network that is distributed across a number of brain regions 2, it depends on the coordinated and sustained firing of subsets of DLPFC PYRAMIDAL NEURONS between the temporary presentation of a stimulus cue NATURE REVIEWS NEUROSCIENCE VOLUME 6 APRIL 25 313

1 2 3 4 5 G 6 Grey matter Ch DORSOLATERAL PREFRONTAL CORTEX (DLPFC). Those regions on the dorsal surface of the primate frontal lobe that are located rostral to the motor and premotor regions, and which include Brodmann s areas 9 and 46. PYRAMIDAL NEURONS These constitute ~75% of cortical neurons, and are recognized by their triangular cell bodies, a single apical dendrite directed toward the cortical surface and an array of basilar dendrites. The dendrites of these neurons are studded with many spines, and their axons project into the white matter and provide excitatory projections to other cortical regions or subcortical structures. DB ais DB White matter Figure 2 Morphological and biochemical features of subpopulations of cortical GABA neurons in the dorsolateral prefrontal cortex. The diagram illustrates the calcium-binding proteins parvalbumin (blue), calbindin (red) and calretinin (yellow) and the locations of inhibitory synaptic inputs to a pyramidal neuron (green) by different morphological classes of cortical GABA (γ-aminobutyric acid) neurons. The chandelier (Ch) and wide arbor (WA) or basket neurons provide inhibitory input to the axon initial segment (ais) and the cell body proximal dendrites, respectively, of pyramidal neurons. By contrast, the calbindin-expressing double bouquet (red DB), neurogliaform (Ng) and Martinotti (M) neurons tend to provide inhibitory inputs to the distal dendrites of pyramidal neurons. Finally, calretinin-expressing (yellow) DB and Cajal Retzius cell (CRC) appear to target both pyramidal cell distal dendrites and other GABA (G) neurons. 1 6, layers of dorsolateral prefrontal cortex. Modified, with permission, from REF. 41 (1994) John Wiley & Sons, Inc. Ng CRC WA and the later initiation of a behavioural response 21. Although other neurotransmitter systems are also involved, DLPFC neurons that use the inhibitory neurotransmitter GABA (γ-aminobutyric acid) seem to be crucial for such synchronization of pyramidal neuron activity during working memory processes. For example, fast-spiking GABA NEURONS in the monkey DLPFC are active during the delay period of working memory tasks 22, and are necessary for task-related neuronal firing and the spatial tuning of neuronal responses during working memory 23.In addition, the injection of GABA antagonists into the DLPFC disrupts working memory 24. Constantinidis and colleagues 25 suggested that during working memory tasks inhibition might serve both a spatial role (controlling which DLPFC pyramidal neurons are activated during working memory) and a temporal role (controlling when they are active during the different phases of working memory). These findings indicate that impairments in GABAmediated inhibition in the DLPFC could provide a M mechanism for the disturbances in working memory in individuals with schizophrenia. Consistent with this view, tissue concentrations of the mrna that encodes the 67 kilodalton isoform of glutamic acid decarboxylase (GAD67), an enzyme that synthesizes GABA, are reduced in the DLPFC of individuals with schizophrenia 26 31.Indeed, reduced GAD67 mrna expression in the DLPFC is one of the most consistent findings in postmortem studies of individuals with schizophrenia 32. The deficit in GAD67 mrna seems to be accompanied by a corresponding decrease in the cognate protein, although this has been less extensively studied 28.By contrast, mrna and protein levels of GAD65, another enzyme that synthesizes GABA, are not altered in the DLPFC of people with schizophrenia 28, and the density of GAD65-immunoreactive axon terminals also remains unchanged 33.Interestingly, elimination of the Gad65 gene in mice does not alter cortical levels of GABA 34, whereas reductions in GAD67 mrna are associated with marked decreases in cortical GAD activity and GABA content 35. Gene expression deficits in a subset of GABA neurons. Cortical GABA neurons are heterogeneous, with various subpopulations that can be distinguished by a combination of morphological, physiological and molecular attributes (FIG. 2).Distinct subpopulations of GABA neurons seem to have specialized functions in regulating pyramidal neuron activity 36,37.For example, the chandelier (or axo axonic) subpopulation of GABA neurons express the calcium-binding protein parvalbumin 38,have a fast-spiking, non-adapting firing pattern 39 and project a linear array of axon terminals (termed cartridges) that synapse exclusively on the axon initial segments of pyramidal neurons 4 (FIG. 3a,b). Wide arbor (basket) cells in the monkey DLPFC also express parvalbumin 41 and have fast-spiking electrophysiological features that are indistinguishable from those of chandelier neurons 42.However, the axons of wide arbor neurons have a much larger spread than those of chandelier cells, and their axon terminals mainly target the cell bodies and proximal dendrites of pyramidal neurons 38.The proximity of the perisomatic inhibitory synapses formed by parvalbumin-expressing chandelier and wide arbor neurons to the site of action potential generation in pyramidal neurons indicates that these GABA neurons might be specialized to regulate the output of pyramidal neurons. The calcium-binding protein calbindin is expressed in double bouquet, neurogliaform and Martinotti cells, which are not fast-spiking and provide axon terminals that synapse on the distal dendrites of pyramidal cells 43,44. Finally, ~5% of GABA neurons in the monkey DLPFC, many of which are double bouquet cells, express the calcium-binding protein calretinin 41,have a regularspiking, adaptive firing pattern 39,44 and produce axon terminals that target distal dendritic spines or shafts on pyramidal cells or other GABA neurons 45.So, the functional consequences of a deficit in GAD67 in schizophrenia depend on whether all or a specific subpopulation (or subpopulations) of GABA neurons are affected. 314 APRIL 25 VOLUME 6 www.nature.com/reviews/neuro

a b c GABA NEURONS These comprise ~25% of cortical neurons, have smooth or sparsely spiny dendrites and provide axons that project locally within the cortical grey matter. 1 µm Figure 3 Pre- and postsynaptic markers of chandelier neuron inputs to the axon initial segment of pyramidal neurons. a,b Immunoreactivity for GABA (γ-aminobutyric acid) transporter 1 (GAT1; a) and parvalbumin (b) clearly identifies vertical arrays of chandelier neuron axon terminals (cartridges) that are located below the cell bodies of unlabelled pyramidal neurons. c Immunoreactivity for the α2 subunit of the (GABA type A) receptor is localized postsynaptically, in the axon initial segment of pyramidal neurons. Modified, with permission, from REF. 116 (23) John Wiley & Sons, Inc. Studies of GAD67 mrna expression at the cellular level showed that, in individuals with schizophrenia, the density of neurons with detectable levels of GAD67 mrna was significantly decreased in DLPFC layers 3 5, but not in layer 6 (REFS 26,27).Furthermore, in neurons with detectable levels of GAD67 mrna, the expression level per neuron did not differ from control values 27. These observations indicate that in individuals with schizophrenia, most DLPFC GABA neurons express normal levels of GAD67mRNA. However, ~25 3% of GABA neurons do not express this transcript at a detectable level. In the same cohort of individuals, expression of the GABA membrane transporter 1 (GAT1) a protein responsible for reuptake of released GABA into nerve terminals was also decreased, with almost identical cellular patterns 46.Therefore, in schizophrenia, both the synthesis and reuptake of GABA seem to be greatly reduced in a subset of DLPFC inhibitory neurons. Recent evidence indicates that the affected GABA neurons include those that contain the calcium-binding protein parvalbumin, which is present in ~25% of GABA neurons in the primate DLPFC 41.In individuals with schizophrenia, the expression of parvalbumin mrna was shown to be significantly decreased in layers 3 and 4, but not in layers 2, 5 or 6, of the DLPFC 47 (FIG. 4). However, in contrast to the findings for GAD67 and GAT1 mrnas, the density of neurons with detectable levels of parvalbumin mrna was unchanged in individuals with schizophrenia, although the expression level per neuron was significantly decreased. In the same individuals, the parvalbumin mrna expression level per neuron correlated strongly with the change in density of GAD67 mrna-positive neurons. These findings indicate that GAD67 mrna expression was markedly reduced in parvalbumin-expressing neurons that also had reduced, but still detectable, levels of parvalbumin mrna. This was confirmed by dual label in situ hybridization studies, which revealed that ~5% of parvalbumin mrnapositive neurons lacked detectable levels of GAD67 mrna in individuals with schizophrenia 47.These findings are consistent with immunocytochemical studies that failed to find a reduction in the densities of parvalbumin-positive neurons in the DLPFC of individuals with schizophrenia 48,49.So, it seems that the number of parvalbumin-expressing GABA neurons are not reduced in the DLPFC of individuals with schizophrenia, but that they have decreased expression of several important genes and might, therefore, be functionally impaired. Alterations in chandelier neurons. In addition to the unique role of parvalbumin-expressing GABA neurons in regulating pyramidal cell activity, their distinctive developmental trajectories in the monkey DLFPC, especially during adolescence (BOX 2), indicate that alterations in these neurons might contribute to working memory dysfunction in schizophrenia. Consistent with this hypothesis, the density of chandelier neuron axon cartridges that were immunoreactive for GAT1 was significantly reduced in the DLPFC of individuals with schizophrenia 5 (FIG. 5),with the most extensive reduction seen in the middle cortical layers 51.By contrast, immunoreactivity of GAT1 in other axon terminal populations was unchanged 5.Together with the gene expression studies reviewed above, these findings indicate that chandelier neurons in the DLPFC of individuals with schizophrenia express decreased levels of parvalbumin mrna and undetectable levels of GAD67 and GAT1 mrnas, the latter of which results in reduced GAT1 protein in their axon cartridges. Despite the convergence of these findings for a common GABA-containing cell type, it is unclear how GABA neurotransmission at the synapse between the chandelier neuron and the pyramidal cell axon initial segment is altered in schizophrenia. These findings could reflect deficient inhibition, resulting from a primary reduction in GABA synthesis, or excessive inhibition, secondary to a reduction in GABA reuptake. To distinguish between these alternatives, it might be useful to analyse (GABA type A) receptors on the postsynaptic targets of chandelier neuron axons. receptors are pentameric hetero-oligomers that are composed of subunits from seven different classes, several of which have more than one member. For example, most receptors in the cortex contain one of six α subunits that have different subcellular distributions and physiological properties 52.Although present in only ~15% of cortical receptors 53,the α2 subunit is found at >95% of inhibitory synapses onto pyramidal neuron axon initial segments 54 (FIG. 3c), especially in the superficial layers of the human cerebral cortex 55.In addition, receptors that have the α2 NATURE REVIEWS NEUROSCIENCE VOLUME 6 APRIL 25 315

a PV mrna 1 2 b 12 1 8 Control Schizophrenia * 3 4 5/6 6 4 White matter Low density High density 2 1 2 3 4 5/6 Cortical Layer CR mrna 12 1 Control Schizophrenia 1 2 8 3 4 5/6 White matter 1 mm 6 4 2 1 2 3 4 5/6 Cortical Layer Figure 4 Expression of parvalbumin and calretinin mrnas in the dorsolateral prefrontal cortex. a Pseudocoloured autoradiograms showing the different laminar distribution patterns of the GABA (γ-aminobutyric acid) neurons that express parvalbumin (PV) or calretinin (CR) mrnas in the dorsolateral prefrontal cortex of human control subjects. b The bar graphs show the mean (SD) expression level of each transcript, by cortical layer, in individuals with schizophrenia and matched control subjects. The expression of parvalbumin mrna is significantly decreased in layers 3 4 of individuals with schizophrenia, whereas the expression of calretinin mrna is unchanged across all layers., nanocuries/gram; 1 6, layers of dorsolateral prefrontal cortex. *P =.12, P =.5. Modified, with permission, from REF. 47 (23) Society for Neuroscience. subunit seem to have a higher affinity for GABA, which results in faster activation and slower deactivation times than receptors that have the more common α1 subunit 56,57.So, receptors that have the α2 subunit seem to be anatomically located and functionally specialized to mediate a potent inhibitory influence on the output of pyramidal neurons. In the DLPFC of individuals with schizophrenia, the density of pyramidal neuron axon initial segments that are immunoreactive for the α2 subunit is increased by >1% compared with control subjects 58 (FIG. 5). This seems to reflect higher levels of α2 subunits at the axon initial segment rather than an increase in the density of pyramidal neurons 59 or of their axon initial segments 6.Furthermore, the density of α2-immunoreactive axon initial segments was inversely correlated with the density of GAT1-immunoreactive cartridges in the same individuals with schizophrenia (FIG. 5). So, in the DLPFC of individuals with schizophrenia, receptors seem to be upregulated at pyramidal neuron axon initial segments in response to deficient GABA release from chandelier axon terminals. Furthermore, as changes in GAD are not seen in mice that lack GAT1 (REF 61),both the increase in postsynaptic receptors and the decrease in presynaptic GAT1 are likely to be compensatory responses to a more primary deficit in GAD67 mrna expression. Although they are more difficult to assess, similar preand postsynaptic changes might also occur in the inputs of parvalbumin-expressing wide arbor neurons to the perisomatic region of pyramidal neurons. For example, the density of parvalbumin-immunoreactive puncta possibly the axon terminals of wide arbor neurons 62 is reduced in the middle but not the superficial layers of the DLPFC in individuals with schizophrenia 63,which parallels the laminar pattern of decreased expression of parvalbumin mrna in schizophrenia (FIG. 4). Furthermore, the increased density of receptors in the DLPFC of individuals with schizophrenia in ligand binding studies 64,65 was most prominent at pyramidal neuron cell bodies 65.These data indicate that, in schizophrenia, receptors at the soma and axon initial segments of pyramidal neurons might be locally upregulated in response to a reduction in perisomatic inhibitory input from chandelier and wide arbor neurons (FIG. 6). Given the restricted locations of these synaptic alterations, and the fact that inhibitory inputs to the cell bodies and axon initial segments of pyramidal neurons represent <1% of the total complement of inhibitory synapses on the cell, it is not surprising that changes in the mrna levels of the main postsynaptic receptor subunits have not been detected in the DLPFC of individuals with schizophrenia 66.However, some studies have reported increases in the mrna for the α1 subunit 67,68. 316 APRIL 25 VOLUME 6 www.nature.com/reviews/neuro

Box 2 Postnatal development of chandelier neuron inputs The axon terminals of chandelier neurons are vertical arrays of boutons (cartridges) that are immunoreactive for parvalbumin (PV) or GABA membrane transporter 1 (GAT1), and outline the axon initial segment of pyramidal neurons (FIG. 3a,b). Although the developmental time course differs somewhat for these two markers, as illustrated in the diagram, the density of labelled cartridges is low in the DLPFC of newborn monkeys, increases to reach a peak before the onset of puberty, and then declines markedly during adolescence (shaded area between 15 and 42 months of age) to adult levels 116. These density changes in parvalbumin- and GATimmunoreactive cartridges seem to reflect developmental shifts in the concentration of these proteins (and so in the detectability of cartridges) as cartridges are readily visualized with the Golgi technique over this same Cartridge and ais number per mm 2 35 3 25 2 15 1 5 Birth PV cartridges GAT1 cartridges.1.1 1 1 1 1, time period 117.Interestingly, the peak and subsequent decline in the density of labelled cartridges occurs prior to the age at which the peak density of parvalbumin-immunoreactive varicosities putative axon terminals from the wide arbor class of parvalbumin-expressing GABA (γ-aminobutyric acid) neurons (FIG. 2) is achieved 62.Postsynaptically, the detectability of the α2 subunit of the (GABA type A) receptor in pyramidal neuron axon initial segment is high at birth, and then markedly declines during adolescence before stable adult levels are achieved. The marked developmental changes in these pre- and postsynaptic markers of inhibition at the inputs from chandelier and wide arbor cells to the perisomatic region of pyramidal neurons indicate that the capacity to synchronize pyramidal neuron output in the DLPFC might be in substantial flux until adulthood. Consequently, the protracted developmental time course of improvements in performance on working memory tasks 118 might depend on both refinements in the number of excitatory connections among pyramidal neurons and changes in their proximal inhibitory inputs (for a review, see REF. 119). These developmental changes during adolescence might contribute to unmasking the consequences of inherited abnormalities in the regulation of GABA mediated neurotransmission and might help to explain why certain life experiences during adolescence (for example, stress or cannabis exposure) seem to increase the risk for schizophrenia 4. Panel adapted, with permission, from REF. 116 (23) John Wiley & Sons, Inc. P PV varicosities α2 ais 3 m 15 m 42 m Log age (months) Adult 2 15 1 5 PV varicosity number per 4 µm 2 Specificity to the disease process of schizophrenia. Whether these findings represent a clinically relevant pathological entity in schizophrenia depends, in part, on the extent to which they are specific to the disease process of schizophrenia. In this regard, neither parallel studies in individuals with other psychiatric disorders (FIG. 5),nor studies in monkeys that were chronically exposed to antipsychotic medications in a fashion that mimics the clinical treatment of schizophrenia, replicated the alterations in markers of GABA neurotransmission that are described above 27,46,47,51,58. Furthermore, these deficits seem to be relatively specific to parvalbumin neurons and are not found in many other GABA neurons in the DLPFC in individuals with schizophrenia. For example, most GABA neurons in the DLPFC express normal levels of GAD67 and GAT1 mrnas 27,46.Moreover, the expression of calretinin mrna, which is found in about half of the GABA neurons in the primate DLPFC 41, is also unchanged in individuals with schizophrenia 47 (FIG. 4), as is the density of calretinin-immunoreactive puncta, which presumably represent a substantial proportion of GABA axon terminals 48.However, abnormalities in parvalbumin neurons alone might not completely account for the deficits in expression of GAD67 and GAT1 mrnas, as such changes were also observed in cortical layers 1 and 2, where relatively few parvalbumin-expressing GABA neurons are located 41, and where no changes in the expression of parvalbumin mrna were found 47 (FIG. 4). Regional specificity. Are these abnormalities in GABA neurotransmission restricted to the DLPFC, or do they represent a disturbance that is distributed across other cortical regions that might contribute to other aspects of the clinical syndrome of schizophrenia? Initial studies of the hippocampus reported a decrease in the density of non-pyramidal, putative GABA neurons 69 and an increase in receptor binding 69 in schizophrenia. However, a recent study from the same research group did not detect a difference in the expression of either GAD67 or GAD65 mrnas in the hippocampus of individuals with schizophrenia, although both NATURE REVIEWS NEUROSCIENCE VOLUME 6 APRIL 25 317

REELIN Reelin is a large protein that is secreted into the extracellular matrix by Cajal Retzius cells. It regulates the migration of cortical neurons during development. The absence of reelin in the reeler mouse results in a distinctive alteration of the normal cellular architecture of the cortex. Change from matched control group (%) 15 1 5 5 1 Schizophrenia Major depression GAT1 cartridge α2-ais Figure 5 Changes in pre- and postsynaptic markers of GABA input to the axon initial segment of pyramidal neurons in the dorsolateral prefrontal cortex of individuals with schizophrenia. The bar graph shows the mean percentage change in the densities of GABA (γ-aminobutyric acid) transporter 1 (GAT1)-immunoreactive cartridges and (GABA type A) receptor α2 subunit immunoreactive axon initial segments (ais) in the dorsolateral prefrontal cortex of individuals with schizophrenia or major depressive disorder compared with matched control subjects. Note that the changes of these markers in schizophrenia are reciprocal and seem to be disease-specific. Modified, with permission, from REF. 58 (22) Oxford University Press. transcripts were decreased in individuals with bipolar disorder 7.In the anterior cingulate cortex of individuals with schizophrenia, the densities of non-pyramidal neurons 69 and of neurons that were immunoreactive for the calcium-binding protein calbindin 71 were reported to be reduced in layer 2, as was the density of GAD67 mrna-positive neurons 72.In the superior temporal gyrus, the level of GAD67 mrna was reduced 67, as was the density of GAT1-immunoreactive axon cartridges, although to a lesser extent than in the DLPFC of the same individuals with schizophrenia 73.So, although the current knowledge base is not sufficient to provide a definitive answer, the available data indicate that changes in GABA neurotransmission in schizophrenia might be a common feature of neocortical regions, but not of the hippocampus 7, and that the magnitude of these changes might be larger in the DLPFC than in other neocortical regions. Uncovering a pathogenetic mechanism The importance of the changes in parvalbumin-expressing GABA neurons as a pathological entity in schizophrenia would be strengthened by showing that they co-occur in the illness with abnormalities that represent potential pathogenetic mechanisms, and by confirming in animal models that these correlations represent cause and effect. In this section, we review the evidence for several pathogenetic mechanisms that could produce such changes in GABA neurons in the DLPFC. Reduced levels of reelin. Reductions in mrna and protein levels for REELIN, in association with deficits in the expression of GAD67 mrna, have been reported in the DLPFC and several other brain regions in individuals with schizophrenia 67.However,in contrast to control subjects, the levels of reelin and GAD67 mrnas were not correlated in individuals with schizophrenia 28.Reelin is expressed in ~5% of GABA neurons in the adult rodent cortex 74, and heterozygote reeler mice (expressing 5% of the normal levels of reelin) show decreased GAD67 mrna expression 75.However, reelin is apparently not expressed in parvalbumin-expressing cortical GABA neurons 76 ;therefore, the relationship between decreased reelin expression and the preferential reduction of GAD67 mrna expression in parvalbumin-positive neurons in individuals with schizophrenia requires further study. Reduced excitatory drive. It has been suggested that the deficit in GAD67 mrna expression represents an activity-dependent change in response to reduced activity of excitatory circuits in the DLPFC 26. One source of such excitatory inputs is the mediodorsal nucleus of the thalamus, which is the principal source of thalamic projections to the DLPFC. Interestingly, initial studies reported that the number of neurons in this nucleus was reduced in individuals with schizophrenia; however, recent studies have not confirmed these observations (for a review, see REF. 77). Furthermore, experimental reductions in neuron number in the mediodorsal thalamus of rodents did not change the expression of GAD67 mrna in the prefrontal cortex 78.However, other changes, such as a decreased density of dendritic spines (a marker of excitatory synaptic inputs to pyramidal neurons) in the DLPFC of individuals with schizophrenia 79,8,are consistent with reduced excitatory drive in DLPFC circuits, although the source of these altered inputs has yet to be determined. In addition, convergent lines of evidence indicate that schizophrenia might be associated with reduced neurotransmission through NMDA (N-methyl-D-aspartate) glutamate receptors 81 and, interestingly, the administration of NMDA receptor antagonists to rodents reduces parvalbumin levels in cortical GABA neurons 82. One potential source of such altered excitatory inputs is the hippocampus. Various structural and functional abnormalities have been observed in the hippocampi of individuals with schizophrenia, and some of these seem to be correlated with alterations in the DLPFC. For example, Weinberger and colleagues observed that tissue levels of N-acetyl-aspartate, a putative marker of neuronal integrity, were decreased in the hippocampus and DLPFC, but not in other brain regions, of individuals with schizophrenia 83.To explore this association, they used a rodent model in which lesions of the ventral hippocampus are created neonatally 84.In adulthood, these animals, in addition to mimicking a number of other phenotypic features of schizophrenia, show deficits in GAD67 expression in the prefrontal cortex 85.However, whether the deficits in GAD67 mrna expression show the cell type specificity, and are accompanied by the other changes in GABA markers that are present in schizophrenia, has not yet been investigated. 318 APRIL 25 VOLUME 6 www.nature.com/reviews/neuro

1 2 GAD67 GAT1 CR mrnas 3 4 GAT1 IR α2 IR 5 6 GAD67 GAT1 PV mrnas Grey matter White matter Figure 6 Schematic summary of alterations in GABA circuitry in the dorsolateral prefrontal cortex of individuals with schizophrenia. Reduced levels of gene expression in chandelier neurons (blue) are associated with a decrease in immunoreactivity (IR) for GABA (γ-aminobutyric acid) transporter 1 (GAT1) in the axon cartridges of these neurons and an upregulation of (GABA type A) receptor α2 subunit immunoreactivity in the postsynaptic axon initial segment of pyramidal neurons (green). Gene expression in calretinin (CR)-expressing subpopulations of GABA neurons does not seem to be altered (yellow). GAD67, 67 kd isoform of glutamic acid decarboxylase; PV, parvalbumin; 1 6, layers of dorsolateral prefrontal cortex. Variants in the GAD67 gene. Recently, allelic variants in the GAD67 gene have been reported to be associated with an increased risk for schizophrenia 86.Whether and how these variants would result in cell-type specific changes in GAD67 mrna expression are unknown. Reduced neurotrophin signalling. Signalling by brainderived neurotrophic factor (BDNF) through its receptor, the tyrosine kinase Trk (tropomyosin-related kinase) B receptor (TrkB), promotes the development of GABA neurons and induces the expression of GABA-related proteins, including GAD67, GAT1 and parvalbumin 87,88. These effects might be selective for parvalbumin-expressing cortical GABA neurons, as TrkB is predominantly expressed by GABA neurons that express parvalbumin, but not by those that express calretinin 89.So,reduced BDNF TrkB signalling might contribute to the altered expression of GABA-related genes in the DLPFC of individuals with schizophrenia. Consistent with this hypothesis, the mrna and protein levels for both BDNF and TrkB have been found to be reduced in the DLPFC of individuals with schizophrenia 31,9 (FIG. 7), and the expression of these mrnas seems to decrease early in the course of the illness. Comparisons with the results of other studies indicate that, in individuals with schizophrenia, the decreased tissue levels of these mrnas represent changes in gene expression rather than a loss of DLPFC neurons 91.In contrast to BDNF and TrkB, the mrna expression of TrkC the receptor tyrosine kinase for neurotrophin 3 is unchanged 31. The changes in the expression levels of TrkB and GAD67 mrna were strongly correlated (r =.74, P <.1) in the same individuals, and this correlation was significantly stronger than that between BDNF and GAD67 mrnas, indicating that changes in TrkB expression might be a pathogenetic mechanism that results in a reduction in GABA-related gene expression in individuals with schizophrenia. However, these correlations in human studies do not show a causal relationship. Consequently, as a proof of concept test that reduced signalling through TrkB receptors could cause changes in expression of GABA-related genes in schizophrenia, we used TrkB hypomorphic mice in which the insertion of floxed TrkB cdna (fbz) resulted in decreased TrkB expression 92. Compared with wild-type mice, expression levels of TrkB mrna in the prefrontal cortex were significantly NATURE REVIEWS NEUROSCIENCE VOLUME 6 APRIL 25 319

394 21 143 17 82 61 25 741 59 397 336 21 129 68 3,47 154 112 81 55 32 11 2,8 1,2 86 62 39 21 9 Control BDNF mrna TrkB mrna GAD67 mrna PV mrna 1 mm Schizophrenia 1 mm BDNF mrna % control TrkB mrna % control GAD67 mrna % control PV mrna %control 14 12 1 8 6 4 2 14 12 1 8 6 4 2 14 12 1 8 6 4 2 14 12 1 8 6 4 2 C C C C F 1,23 = 18.8 P <.1 S F 1,24 = 22.4 P <.1 S F 1,24 = 19.4 P <.1 S F 1,12 = 1.8 P =.6 Figure 7 Reduced neurotrophin and GABA-related gene expression in the dorsolateral prefrontal cortex of individuals with schizophrenia. The autoradiograms show the laminar distribution of brain-derived neurotrophic factor (BDNF), tyrosine kinase Trk (tropomyosin-related kinase) receptor B (TrkB), 67 kd isoform of glutamic acid decarboxylase, (GAD67) and parvalbumin (PV) mrnas in the dorsolateral prefrontal cortex of a control individual and the reduced levels of these transcripts in a matched individual with schizophrenia. The densities of hybridization signals are presented in pseudocolours according to the calibration scales for each mrna. Solid and broken lines indicate the pial surface and the border between the grey and white matter, respectively. The bar graphs show the mean (SD) expression of each transcript in individuals with schizophrenia (S) and matched control subjects (C). F, Fisher s F distribution resulting from the analyses of variance; the subscript numbers indicate the corresponding degrees of freedom., nanocuries/gram. Modifed, with permission, from REF. 31 (25) Society for Neuroscience. S decreased by 42% and 75% in fbz/+ and fbz/fbz mice, respectively; and in fbz/fbz mice, the expression levels of GAD67 and parvalbumin mrnas in the prefrontal cortex were significantly decreased by 25% and 4%, respectively 31 (FIG. 8).In addition, in the fbz/+ mice, the expression levels of GAD67 and parvalbumin mrnas were intermediate between the wild-type and fbz/fbz mice. The cellular pattern of reduced GAD67 mrna expression in these mice also precisely paralleled that seen in individuals with schizophrenia 27.That is, the density of neurons with detectable levels of GAD67 mrna was significantly reduced, but the expression level of GAD67 mrna per neuron was unchanged 31.Furthermore, consistent with the selective vulnerability of a GABA neuron subpopulation in schizophrenia, the TrkB genotype did not affect the expression of calretinin mrna. So, the changes in the expression of GABA-related genes in TrkB hypomorphic mice replicate those found in individuals with schizophrenia at both the tissue and cellular levels. By contrast, although the expression of BDNF mrna was decreased by 8% in the prefrontal cortex of mice with a neuron-specific inducible knockout of Bdnf 93, there were no changes in the expression levels of GAD67 or parvalbumin mrnas in these animals, whether the Bdnf knockout was induced during embryogenesis or in adulthood 31.These findings indicate that changes in TrkB but not BDNF expression regulate the expression of GABA-related genes in the prefrontal cortex. 32 APRIL 25 VOLUME 6 www.nature.com/reviews/neuro

6,84 2,84 1,4 85 55 Wild Type 26 TrkB mrna 4,89 2,56 1,54 97 59 27 GAD67 mrna 3,7 1,58 98 65 41 19 PV mrna fbz/+ fbz/fbz 1 mm TrkB mrna % wild type GAD67 mrna % wild type PV mrna % wild type 14 12 1 8 6 4 2 14 12 1 8 6 4 2 14 12 1 8 6 4 2 F 2,6 = 51.1, P <.1 a b c F 2,6 = 11.1, P =.1 a b b F 2,6 = 9.92, P =.13 a ab b WT fbz/+ fbz/fbz Figure 8 Changes in gene expression in the prefrontal cortex of the TrkB hypomorphic mice are similar to those in individuals with schizophrenia. Representative pseudocoloured autoradiograms show the decreased expression of tyrosine kinase Trk (tropomyosin-related kinase) receptor B (TrkB), 67 kd isoform of glutamic acid decarboxylase (GAD67) and parvalbumin (PV) mrnas in mice that are heterozygous or homozygous for the floxed TrkB (fbz) locus. In each graph, bars that do not share the same letter (a, b or c) are significantly different (p<.5). The mean (SD) expression level of each transcript is reduced in a gene-dose dependent fashion. F, Fisher s F distribution resulting from the analyses of variance; the subscript numbers indicate the corresponding degrees of freedom., nanocuries/gram; WT, wild type. Modifed, with permission, from REF. 31 (25) Society for Neuroscience. Investigating the pathophysiological process The findings reviewed above provide evidence for both a pathological entity (deficient chandelier cell-mediated inhibition in pyramidal neurons) and a pathogenetic mechanism (reduced neurotrophin signalling through the TrkB receptor) in the DLPFC, the dysfunctional activation of which is associated with a core component (working memory deficits) of the clinical syndrome of schizophrenia. However, the explanatory strength of these findings, and their relevance to the development of new treatment interventions, requires the demonstration of a pathophysiological process by which this pathological entity could give rise to this component of the clinical syndrome. In this regard, it is of interest that gamma band oscillations, which reflect the synchronized firing of neuronal networks at 3 8 Hz, are induced and sustained in the DLPFC during the delay period of working memory tasks 94.In addition, the amplitude, or power, of gamma band oscillations in the DLPFC seems to increase in proportion to working memory load 95.In the frontal cortex of individuals with schizophrenia, phase-locking of gamma activity in response to the stimulus onset is impaired 96, and the power of gamma band oscillations in the DLPFC is reduced during the delay period of a working memory task 97. Interestingly, different subpopulations of GABA neurons seem to be specialized to selectively regulate the output of pyramidal cells, giving rise to oscillatory activity in different frequency bands 98 1.For example, networks of parvalbumin-expressing, fast spiking GABA neurons in the middle cortical layers, formed by both chemical and electrical synapses, give rise to oscillatory activity in the gamma band range, whereas multipolar bursting GABA neurons in the superficial layers that express both parvalbumin and calbindin give rise to theta frequency (4 7 Hz) oscillations 1.The coordinated oscillatory context that is provided by these networks of inhibitory neurons is thought to create the discrete temporal structure that is necessary for ensembles of pyramidal neurons to perform specific functions, such as those involved in working memory. So, a deficit in the synchronization of pyramidal cells, which results from impaired perisomatic inhibition by parvalbumin-expressing GABA neurons, might contribute to the reported deficits in gamma band oscillations, and, consequently, to working memory dysfunction, in individuals with schizophrenia. Several features of parvalbumin-expressing neurons, and of their changes in schizophrenia, could explain how this might occur. First, the axonal arborizations of individual chandelier neurons are highly divergent and target the axon initial segments of a large number of pyramidal neurons. A single chandelier cell can provide inputs to >2 pyramidal neurons within a 1 15 µm radius 11 and can thereby regulate the firing of local groups of pyramidal neurons 98.Second, in the monkey DLPFC, parvalbumin-expressing GABA neurons and pyramidal cells share certain excitatory inputs, including projections from neighbouring pyramidal neurons and from the mediodorsal thalamus 12 14. Excitatory input from these sources stimulates both parvalbumin-expressing and pyramidal neurons NATURE REVIEWS NEUROSCIENCE VOLUME 6 APRIL 25 321

BENZODIAZEPINE Benzodiazepines bind to receptors, where they increase the frequency of opening of Cl channels in the presence of GABA, but do not directly open channels in the absence of GABA. At present, the available benzodiazepines are not selective for receptors that contain the α2 subunit, and so produce a range of effects, such as the sedation mediated by the α1 subunit, which can impair cognitive processes. simultaneously, which results in a secondary, temporally delayed perisomatic inhibitory input to pyramidal neurons. This disynaptic inhibitory input seems to limit the window of time, and thereby increases the temporal precision, for the summation of the excitatory inputs that are needed to evoke pyramidal neuron firing 15.Consequently, a deficiency in inhibitory perisomatic input to pyramidal neurons would be expected to reduce the magnitude of pyramidal cell synchrony, and, therefore, the power of gamma band oscillation, in the DLPFC. This interpretation predicts that some changes in parvalbumin-expressing GABA neurons in schizophrenia represent compensatory, but inadequate, responses to the deficit in GABA synthesis in these neurons. For example, by buffering presynaptic Ca 2+ transients, parvalbumin is thought to reduce the Ca 2+ -dependent facilitation of GABA release 16.During periods of repetitive firing, parvalbumin binds Ca 2+ molecules, thereby reducing the level of residual intraterminal Ca 2+,which results in decreased GABA release. Consistent with this interpretation, in parvalbumin-knockout mice, GABA release from fast-spiking neurons is facilitated and is associated with an increase in the power of gamma band oscillations 16. Similarly, although the level of GAT1 does not seem to affect single inhibitory postsynaptic currents (IPSCs), the blockade of GABA reuptake prolongs the duration of IPSCs when synapses that are close to each other are synchronously activated 17.The prolongation of IPSCs, in turn, leads to an increased probability of IPSC summation and, therefore, enhanced efficacy of IPSC trains. This observation is of particular relevance to the inputs of chandelier neurons to pyramidal cell axon initial segments, where the GABA synapses are close together and the presynaptic action potential reaches them almost simultaneously. So, a combined reduction in parvalbumin and GAT1 proteins in the chandelier axon cartridges of individuals with schizophrenia could function synergistically to increase the efficacy of GABA neurotransmission at pyramidal cell axon initial segments during the types of repetitive activity that are associated with working memory. However, these compensatory mechanisms and the upregulation of postsynaptic receptors are not adequate to overcome the effects of decreased GABA synthesis in schizophrenia. Treatment implications The real value of understanding the pathophysiology of schizophrenia rests in the prediction of novel targets for pharmacological intervention. The observations reviewed above indicate that drugs with selective agonist activity at α2 subunit-containing receptors could provide an effective approach for enhancing chandelier neuron inhibition of DLPFC pyramidal neurons in schizophrenia, by increasing the synchronization of pyramidal cell firing at gamma frequencies, and consequently improving working memory function 18.The α2 subunit of the receptor represents a selective target for enhancing inhibition at the axon initial segment of pyramidal neurons because it is predominantly restricted to this location 54.In addition, the increased density of α2 containing receptors at pyramidal neuron axon initial segments in individuals with schizophrenia 58 indicates that it might be possible to devise dosing strategies that would activate these receptors without affecting synaptic sites in other brain regions that have normal levels of the receptor α2 subunit. Such differential activation could reduce potential side effects. Drugs that directly activate α2-containing receptors independently of the presence of GABA, or those that generally increase the firing rate of chandelier cells, might disrupt the timing of disynaptic inhibition of pyramidal neurons. Furthermore, agents that inhibit GABA reuptake non-selectively could function too broadly, and, in individuals with schizophrenia, the decrease in the level of GAT1 expression in chandelier neurons indicates that the brain has already used this compensatory approach. By contrast, drugs that augment the release of GABA by chandelier cells during normal firing, or those that enhance the postsynaptic response to the release of GABA from chandelier cell axons, such as a α2-selective BENZODIAZEPINE, might be expected to improve disynaptic inhibition. So, treatment with an α2-selective benzodiazepine would be predicted to augment the postsynaptic inhibitory response at pyramidal neuron axon initial segments in a manner that incorporates the crucial timing of chandelier neuron firing, which is essential for synchronizing pyramidal neuron activity. Furthermore, as the anxiolytic effects of benzodiazepines seem to be mediated by α2 subunit-containing receptors 19, α2-specific agents might both improve cognitive function and reduce the stress responses that have been linked to the exacerbation of psychotic symptoms in individuals with schizophrenia 11. Understanding schizophrenia As summarized in FIG. 1, the findings reviewed in this article converge on the idea that a deficiency in TrkB signalling is a pathogenetic mechanism that produces reduced GAD67 mrna expression and GABA synthesis in the parvalbumin-expressing subpopulation of GABA neurons in the DLPFC of individuals with schizophrenia. Despite the apparent compensatory responses, which include a decrease in the levels of presynaptic GAT1 and parvalbumin, and upregulation of postsynaptic receptors, the resulting pathophysiological process changes in the perisomatic inhibitory regulation of pyramidal neurons that are required for gamma frequency oscillations contributes to the impairments in working memory function that represent a core feature of the clinical syndrome of schizophrenia. However, these abnormalities in GABA neurons are unlikely to be the only contributors to working memory dysfunction in this disorder. For example, changes in dopamine and glutamate neurotransmission in the DLPFC also seem to be involved 111,112.Interestingly, in the monkey DLPFC, dopamine terminals provide synaptic inputs to parvalbumin-expressing, but not calretinin-expressing, GABA neurons 113, and parvalbumin-positive cortical neurons express a combination of glutamatergic receptor subunits that differs from those in other populations of GABA neurons 114. 322 APRIL 25 VOLUME 6 www.nature.com/reviews/neuro