Schizophrenia Research Forum - A Catalyst for Creative Thinking

Genetics, Expression Profiling Support GABA Deficits in Schizophrenia

25 June 2007. Several lines of study, in particular the assessment of gene expression in postmortem tissue, suggest that deficits in GABA neurotransmission may be intimately involved in the pathophysiology of schizophrenia. A trio of recent studies support this idea.

The most recent study, published in the June 12 issue of PNAS by Francine Benes at McLean Hospital, Belmont, Massachusetts, describes schizophrenia-specific, as well as bipolar disorder-specific, expression patterns of genes associated with the GABA synthesizing enzyme GAD67 in subregions and specific layers of hippocampus. The researchers suggest that such regional expression endophenotypes might help distinguish the roots of GABA-related dysfunction in schizophrenia from those of bipolar disorder.

In one of two earlier studies published online May 1 in Molecular Psychiatry, Richard Straub and colleagues at the NIMH also focused on GAD67. They report that inherited genetic polymorphisms in the gene for the enzyme are found more frequently in schizophrenia patients and their relatives. These results suggest that altered GABA levels may contribute to the disease, and they also hint that genetic variations affecting dopamine-based neurotransmission may exacerbate that pathology.

The second study in Molecular Psychiatry, led by David Lewis at the University of Pittsburgh, Pennsylvania, examines a wider range of genes that code for proteins related to GABA biology, specifically in dorsolateral prefrontal cortex (DLPFC). First author Takanori Hashimoto and colleagues report alterations in schizophrenia in genes involved in GABA synthesis, in genes for certain neuromodulators, and in genes that code for subunits of the GABAA receptor. This schizophrenia GABA "transcriptome" directs attention to specific subpopulations of DLPFC GABAergic neurons and raises the possibility of disease-related effects on both synaptic and extrasynaptic GABAergic signaling in DLPFC pyramidal neurons.

What's at the 5' End of Your GAD?
The evidence of GABAergic dysfunction in schizophrenia is multifaceted (as reviewed succinctly by Straub and colleagues in their introduction; see also Akbarian and Huang, 2006), and several studies have looked to genetic variations in the GAD67 gene, GAD1, to explain this, with mixed results to date (see entries in SchizophreniaGene. In their study, Straub and colleagues focused on genetic variations that are likely to affect GAD67 production, concentrating on single nucleotide polymorphisms (SNPs) near the promoter region, or on/off switch of the gene. They genotyped 19 polymorphisms in two independent data sets comprising parent-child trios. Data from the first, the NIMH’s Clinical Brain Disorders Branch’s sibling study database, which consists of samples of mixed ethnicity (though predominantly European American), revealed three SNPs that were significantly associated with schizophrenia, but only in female children. In contrast, data from the NIMH Genetics Initiative sample set (samples from only European American families) revealed six different SNPs that significantly associated with schizophrenia in females and yet another six SNPs that associate with the disease in males.

Because the genetic component of schizophrenia is predicted to be complex and dependent on variations in multiple genes, the researchers delved more deeply into the data, looking to see if any of the GAD1 variations are more common in individuals who have genetic variations in the catechol-O-methyltransferase (COMT) gene, which has also been linked to schizophrenia. One particular polymorphism in the COMT gene introduces either a methionine or valine at position 158 of the enzyme. The valine isoform is more active, and it has been suggested that this variant may increase susceptibility to schizophrenia because of more rapid degradation of dopamine in the brain. It is interesting, therefore, that when Straub and colleagues stratified the data by COMT genotype, they found additional GAD1 SNPs that associated with the disease—eight of the 19 GAD1 SNPs turned up a positive association with schizophrenia in families with the Val/Val genotype.

How might these GAD1 SNPs increase susceptibility for the disease? Given that they are located near the regulatory region of the gene, they might affect transcription and GAD67 levels. To test this, the researchers looked at GAD1 expression in postmortem brain samples. They found that one of the SNPs was associated with reduced levels of GAD1 mRNA in the DLPFC. How the other SNPs may affect the GAD1 gene or influence schizophrenia is unclear, but the authors found that genotype associated with cognitive performance. When they examined 15 different neurocognitive phenotypes, they found that 11 of them were associated with at least one of the 19 SNPs—there were both positive and negative associations. They also found that one of three SNPs tested associated with greater activation of the DLPFC, as judged by functional magnetic resonance imaging, during one of the cognitive tests.

The GABA-related Transcriptome
Hashimoto and colleagues looked more broadly at expression of GABA-related genes, using custom DNA arrays that detect 85 different GABA-related messenger RNAs, including those coding for GAD and other proteins involved in GABA synthesis, uptake, degradation, and binding. The researchers applied these microarrays to compare DLPFC gene expression profiles among 14 schizophrenia patient postmortem brain samples and age- and sex-matched control tissue.

The researchers report that expression of 10 of the 85 genes was significantly different in patient tissue. For all 10, expression was higher in control samples. These genes code for three categories of protein: neuropeptides released by GABA neurons; GABA receptor subunits; and presynaptic regulators of GABA. The last included GAD67—its mRNA levels were about 33 percent lower in schizophrenia samples. The researchers report the biggest difference was in expression of the neuropeptide somatostatin (SST), which was 1.6-fold lower in schizophrenia samples. Expression of two other neuropeptides, cholecystokinin (CCK) and neuropeptide Y (NPY), was also lower in patient samples. The researchers validated the microarray data with real-time quantitative PCR and in situ hybridization studies in an extended cohort of 23 pairs.

Gene expression changes in GAD67 were closely tracked by SST, NPY, and CCK expression. Since GABAergic neurons expressing CCK form a separate subpopulation from one coexpressing SST and NPY, Hashimoto and colleagues propose that schizophrenia-related deficits in GABAergic transmission could affect two distinct neuronal populations. These two populations are also distinct from the parvalbumin-containing GABAergic cells that the Lewis lab has reported on previously (which express less parvalbumin and no detectable GAD67 in schizophrenia; for review, see Lewis et al., 2005).

The researchers also report that expression of the δ subunit of the GABAA receptor, which is only found in extrasynaptic receptors, as well as of subunits found in synaptic receptors, are lower in the patient sample; they thus surmise that schizophrenia DLPFC pathophysiology features alterations not just in synaptic GABA neurotransmission, but also in signaling via GABA receptors located outside synapses. This further suggests, the authors write, the presence in schizophrenia of both "decreased synaptic (phasic) and extrasynaptic (tonic) inhibition … in pyramidal neuron dendrites."

Two Paths to GABA Dysfunction?
In their PNAS paper, Benes and colleagues describe the continuation of their work on GABAergic dysfunction in limbic cortical structures such as hippocampus and anterior cingulate gyrus, particularly in the context of comparing GABAergic pathophysiology in schizophrenia and bipolar disorder (for review, see Benes and Berretta, 2001). In their current study, they apply a targeted approach to postmortem tissue analysis, isolating microscopic samples of hippocampal tissue with laser-capture microdissection. The researchers then used gene expression profiling to find changes in a network of proteins linked to GAD67, which were found to be downregulated in hippocampus in both disorders in previous studies. For sampling, the researchers used postmortem hippocampal tissue from schizophrenia, bipolar disorder, and normal control brains—seven samples in each case.

Their findings highlight the value of laser-capture microdissection (LCM). While their analysis suggests no difference between GAD67 expression in schizophrenia versus control when total hippocampus was examined, and only a 1.8-fold reduction in bipolar disorder, robust differences emerged when microscopic samples were analyzed. In regions CA2 and CA3, and specifically in the stratum oriens, the second most superficial hippocampal layer, which is home to GABAergic neurons, they found GAD67 was almost 10-fold lower in bipolar disorder compared to control. The difference in schizophrenia samples—nearly threefold lower—was not so dramatic in stratum oriens, and was similar to decreases found in deeper hippocampal layers (stratum radiatum and stratum pyramidale) in both disorders. In region CA1, only one difference was detected in GAD67 expression between schizophrenia or bipolar groups and control—a threefold decrease in schizophrenia versus control in the stratum oriens.

Focusing on the stratum oriens, which featured expression abnormalities in both disorders, Benes and colleagues found that 18 of 25 GAD67 network genes have altered expression patterns in schizophrenia or bipolar disorder. Some are involved in neurotransmission, namely, glutamate receptor subunits, but transcription factors, cytokines, and chromatin modifying proteins were also in the mix. Interestingly, the expression profiles were not the same in schizophrenia and bipolar disorder, and the authors draw attention to several of these discrepancies—transcripts for proteins involved in epigenetic modification of genes were upregulated in schizophrenia but not bipolar disorder, whereas transcripts for cell differentiation factors were altered only in bipolar disorder. This leads the authors to suggest that, "a common cellular phenotype in SZ and BD, the decreased expression of GAD67 in GABAergic interneurons, may involve different underlying molecular mechanisms that are in part related to susceptibility genes for the respective two disorders, as well as activity-dependent changes arising from specific afferent inputs to these interneurons."—Tom Fagan.

Reference:
Straub RE, Lipska BK, Egan MF, Goldberg TE, Callicott JH, Mayhew MB, Vakkalanka RK, Kolachana BS, Kleinman JE, Weinberger DR. Allelic variation in GAD1 (GAD67) is associated with schizophrenia and influences cortical function and gene expression. Molecular Psychiatry. 2007, May 1; online publication. Abstract

Hashimoto T, Arion D, Unger T, Maldonado-Aviles JG, Morris HM, Volk DW, Mirnics K, Lewis DA. Alterations in GABA-related transcriptome in the dorsolateral prefrontal cortex of subjects with schizophrenia. Molecular Psychiatry. 2007, May 1; online publication. Abstract

Benes FM, Lim B, Matzilevich D, Walsh JP, Subburaju S, Minns M. Regulation of the GABA cell phenotype in hippocampus of schizophrenics and bipolar. PNAS. 2007, June 4. Abstract

Comments on News and Primary Papers
Comment by:  Karoly Mirnics, SRF Advisor
Submitted 26 June 2007
Posted 26 June 2007

The evidence is becoming overwhelming that the GABA system disturbances are a critical hallmark of schizophrenia. The data indicate that these processes are present across different brain regions, albeit with some notable differences in the exact genes affected. Synthesizing the observations from the recent scientific reports strongly suggest that the observed GABA system disturbances arise as a result of complex genetic-epigenetic-environmental-adaptational events. While we currently do not understand the nature of these interactions, it is clear that this will become a major focus of translational neuroscience over the next several years, including dissecting the pathophysiology of these events using in vitro and in vivo experimental models.

View all comments by Karoly MirnicsComment by:  Schahram Akbarian
Submitted 26 June 2007
Posted 26 June 2007
  I recommend the Primary Papers

The three papers discussed in the above News article are the most recent to imply dysregulation of the cortical GABAergic system in schizophrenia and related disease. Each paper adds a new twist to the story—molecular changes in the hippocampus of schizophrenia and bipolar subjects show striking differences dependent on layer and subregion (Benes et al), and in prefrontal cortex, there is mounting evidence that changes in the "GABA-transcriptome" affect certain subtypes of inhibitory interneurons (Hashimoto et al). The polymorphisms in the GAD1/GAD67 (GABA synthesis) gene which Straub el al. identified as genetic modifiers for cognitive performance and as schizophrenia risk factors will undoubtedly spur further interest in the field; it will be interesting to find out in future studies whether these genetic variants determine the longitudinal course/outcome of the disease, treatment response etc etc.

View all comments by Schahram Akbarian

Comments on Related News


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Comment by:  David Yates
Submitted 18 April 2007
Posted 26 April 2007

Are these studies of relevance to the report from Israel that older men feed their mutations into the gene pool and this in part accounts for keeping the “schizophrenia gene” going despite poor fertility (Malaspina et al., 2002)? And might a comparison of the DNA of healthy siblings born before the mutations of an “older man” mutation with that of a sibling who got such a later mutation and developed schizophrenia reveal something of interest?

View all comments by David Yates

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Comment by:  John Krystal
Submitted 6 December 2007
Posted 9 December 2007

The paper by Behrens and colleagues provides exciting new data to suggest that NADPH oxidase plays an important role in the impact of the NMDA receptor antagonist, ketamine, upon parvalbumin-containing (PVC) fast-spiking GABA interneurons. The authors show that ketamine causes an activation of NADPH oxidase, resulting in increases in superoxide production. The elevation in free radicals, presumably toxic to these neurons, is associated with reduction in the expression of parvalbumin and GAD67. These effects of ketamine could be prevented by inhibition of NADPH oxidase.

These data were interpreted by the authors to help explain the schizophrenia-like effects of ketamine in healthy humans. I think that these data provide important insights into the impact of reductions in NMDA receptor function, and they may be relevant to schizophrenia. First, the data amplify the implications of the work of Kinney, Cunningham, and others who have shown that PVC interneurons express the NR2A subunit of the NMDA receptor and that deficits in NMDA receptor function may contribute to reduced GAD expression by these neurons. Since PVC deficits in GAD expression have been described in postmortem cortical tissue from people diagnosed with schizophrenia, the current data suggest that some of these findings may be attributable to activation of NADPH oxidase. It would be interesting to know whether there is an interaction between this consequence of deficits in NMDA receptor function, a feature associated with schizophrenia, and reductions in the cortical levels of glutathione, also associated with this disorder. Glutathione is a free radical scavenger. In other words, the emergence of GABA neuronal deficits may be an unfortunate consequence of the convergence of a disturbance in glutamatergic neurotransmission and a heritable abnormality in neural metabolism. These data highlight the potential importance of some very preliminary new data that suggest that N-acetyl-cysteine (NAC) may augment antipsychotic effects in treating schizophrenia. NAC raises intracellular glutathione and might be a treatment that targets the cellular process described by Behrens and colleagues.

The Behrens paper also highlights the importance of research studies exploring ketamine effects from a systems and cognitive neuroscience perspective. For example, it does not explain why ketamine effects produce symptoms and cognitive impairments associated with schizophrenia. It is likely that the work of scientists including H. Grunze, R. Greene, B. Moghaddam, R. Dingeldine, M. Cunningham, and others is important to consider. These investigators have shown that NMDA receptor antagonists reduce the recruitment of PVC interneurons in feed-forward inhibition pathways resulting in increased glutamatergic output. When NMDA receptors are blocked, the activity of these neurons produces dysfunctional effects, in that neural activity seems chaotic and the organized oscillatory activity of networks is disrupted. These disturbances in network function are paralleled by abnormal behaviors and cognitive impairments in animals and "schizophrenia-like" symptoms and cognitive deficits in humans. One potential solution to this problem would be to reduce glutamate release, a paradoxical suggestion for a disorder commonly thought of as "hypoglutamatergic" based on loss of cortical connectivity. Yet, in animals and humans, drugs that reduce glutamate release (lamotrigine, group II metabotropic glutamate receptor agonists) reduce the physiologic and behavioral effects of NMDA glutamate receptor antagonists. Further, there are now some encouraging clinical data that lamotrigine and, particularly, group II mGluR agonists, might have clinical efficacy in treating schizophrenia.

Overall, we seem to be working in a period where a wide variety of data from many sources is rapidly converging to capitalize on the insight that NMDA receptor antagonists, when administered to healthy people, transiently produce effects that resemble schizophrenia.

View all comments by John Krystal

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Comment by:  Steven Siegel (Disclosure)
Submitted 6 December 2007
Posted 9 December 2007

The article by Behrens and colleagues provides evidence for a mechanistic link between NADPH oxidase and disruption of normal protein expression in some interneurons following the drug ketamine. Data presented demonstrate that addition of an NADPH oxidase inhibitor, given in the animal’s drinking water, blocked the effects of ketamine on a specific class of interneurons that contains parvalbumin. Several lines of research suggest that this population of cells is disrupted in schizophrenia, and that reductions of NMDA-type glutamate receptor activity may lead to that impairment. The important iterative advance in the current study links the reduction in NMDA receptor-mediated glutamate transmission to a specific intracellular mechanism and molecular pathway. Furthermore, the authors demonstrate that they can effectively block the cellular changes by inhibiting that pathway, suggesting a novel therapeutic target.

This leads to two major questions: 1) Could NADPH oxidase inhibitors, or similar mechanisms be used to avert the onset of schizophrenia if administered during a prodromal period? 2) Is the process of reduced parvalbumin expression reversible? Some studies have shown that drugs like ketamine, which reduce activity at NMDA receptors, actually lead to cell death, suggesting that only prevention would be possible. Alternatively, there is evidence that the parvalbumin-containing cells in schizophrenia may not be dead and gone, but rather have impaired function and loss of this particular protein. In this latter scenario, it is possible that the effects of the illness could be reversible. Given that ketamine also causes a variety of functional changes in animals, including electrical brain activity and behavior, the current work lays the groundwork for future studies to determine if co-administration of NADPH oxidase inhibitors can block the functional consequences of ketamine and, by extension, reduce NMDA receptor activity in general.

View all comments by Steven Siegel

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Comment by:  Dan Javitt, SRF Advisor
Submitted 7 December 2007
Posted 10 December 2007

The study by Behrens and colleagues is an excellent illustration of how breaking with traditional paradigms can lead to identification of novel potential targets for intervention in schizophrenia. As detailed on the pages of Schizophrenia Research Forum (e.g., Interview with D. Lewis) and the cited articles from F. Benes, one of the most consistent findings in schizophrenia is the downregulation of PV and GAD67 expression in PV+ GABAergic interneurons. Dysfunction of these neurons, in turn, may be responsible for frontal neurocognitive and dopaminergic deficits. The underlying cause of the GABAergic interneuron changes, however, has only intermittently been investigated.

One of the leading potential mechanisms underlying reduced PV and GAD67 expression in brain in schizophrenia has always been NMDA dysfunction, given the strong expression of NMDA receptors on GABA interneurons, as described by Behrens and colleagues, and the well-known ability of NMDA antagonists to induce both symptoms and neurocognitive deficits closely resembling those of schizophrenia. Last year, Kinney and colleagues demonstrated that exposure to the NMDA antagonist ketamine reduced PV and GAD67 expression in GABAergic interneurons in vitro (Kinney et al., 2006). The present study builds upon this finding and demonstrates a similar phenomenon in vivo. Moreover, it builds upon this finding to demonstrate that these changes can be reversed by antagonists of NADPH oxidase, suggesting a potential target for intervention.

This study thus adds reduced GAD67 and PV expression in PV+ GABAergic interneurons to the long list of findings in schizophrenia that can be viewed as “downstream” of a more proximal deficit in NMDA-mediated neurotransmission, and supports interventions aimed specifically at frontal GABAergic interneurons, as well as more generally at reduced NMDA activity throughout brain. This preparation, moreover, may be appropriate to the testing of novel glutamatergic agents.

Behrens and colleagues' article, however, also leaves many questions unanswered. For example, loss of PV and GAD67 in schizophrenia is not confined to prefrontal cortex. It would be of interest to know, therefore, whether histological changes induced by ketamine are or are not confined to this region. As with all proposed new drug targets, it will also be important to know what other processes NADPH oxidase is involved with both inside and outside brain before proposing it too seriously as a drug target. It is one thing to reverse a specific deficit in a short-term treatment model, another to contemplate long-term treatment. At first glance, NADPH oxidase would seem to be a very general enzyme, which is being targeted to treat a very specific condition. Nevertheless, if NADPH oxidase activity can safely be blocked throughout the body long term, the present findings may point the way for new treatments for schizophrenia.

View all comments by Dan Javitt

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Comment by:  Julie MarkhamJames I. Koenig
Submitted 10 December 2007
Posted 10 December 2007

The role of reactive oxygen species in the pathogenesis of schizophrenia is currently unclear. Several lines of evidence support a greater production of these reactive molecules in schizophrenia because of reduced levels of important buffers for superoxides, such as glutathione. Other research, however, suggests that antipsychotic drugs themselves increase the production of oxygen radicals. In this week’s issue of Science, Behrens and colleagues present data supporting the involvement of reactive oxygen species in the pathophysiology of schizophrenia. The authors have previously shown that administration of an NMDA receptor antagonist to primary cultures of cortical neurons results in the loss of GAD67 and parvalbumin (PV; a calcium-binding protein) from PV positive GABAergic interneurons (Kinney et al., 2006), similar to what has been observed in studies using postmortem tissue from patients with schizophrenia (e.g., Volk et al., 2000; Hashimoto et al., 2003). In this study, administration of the NMDA receptor antagonist ketamine was found to increase production of reactive molecules both in vitro (following bath application of the drug to cultured neurons) and in vivo (following two injections of the drug to mice). Moreover, inhibition of the enzyme NADPH oxidase prevented the reduction of both PV and GAD67 expression. The authors suggest that inhibition of NADPH oxidase may represent a novel treatment for both ketamine-induced psychosis and schizophrenia.

While the authors’ findings are undoubtedly exciting, some limitations of their approach need to be addressed before over-enthusiasm regarding NADPH oxidase inhibition as a treatment for schizophrenia is generated. Although the title advertises a “loss of phenotype of fast-spiking interneurons,” the reduction in PV and GAD67 expression from neurons that remain PV positive does not represent a loss of phenotype, and the ketamine-induced increase in superoxide production was not specific to interneurons (only 5-10 percent of primary cortical neuron cultures are PV positive, yet the effect was observed throughout sampled cells). Also, although their findings bear similarity to those observed in schizophrenia, there are notable differences. For instance, whereas the level of PV expression per cell is reduced in schizophrenia (Hashimoto et al., 2003), the level of GAD67 mRNA expression per cell does not differ between individuals with schizophrenia and controls; rather, it appears to be a reduction in the density of neurons that express the transcript. In contrast, Behrens and colleagues report a reduction in the expression per cell for both PV and GAD67. While this difference may simply be due to the fact that Behrens and colleagues examined levels of the proteins, the potential discrepancy should be recognized.

Perhaps the most important limitation to the work is the absence of a functional measure to determine whether the reduction in PV and GAD67 in cortical interneurons observed following ketamine administration results in any of the schizophrenia-associated endophenotypes which can be modeled in rodents. Animal models of schizophrenia employing developmental strategies have been very successful in this regard (reviewed in Carpenter and Koenig, in press), and it is unclear how functional outcomes from the acute pharmacological challenge in mature animals used in the present study might compare. Although the data as they stand are promising, they would be much more compelling if a functional deficit as a result of the treatment was observed and the authors could demonstrate that inhibition of NADPH oxidase prevented this deficit. Unfortunately, such a deficit is unlikely to be found following such a limited ketamine exposure. This is actually quite fortunate since ketamine is a popular general anesthetic in both human and veterinary medicine. Additionally, countless biomedical investigators routinely use ketamine as an anesthetic for survival surgeries; even in cases where the experimental design calls for multiple anesthetizations over the course of the study, no major functional disturbances in experimental animals have been reported. Our conclusion is that, while exposure to ketamine may induce features of neuropathology that bear some similarity to those observed in schizophrenia, the excitement about a treatment for ketamine-induced superoxide production should be tempered until it can be demonstrated that the treatment reverses a functional deficit that is relevant to schizophrenia.

References:

Carpenter WT, Koenig JI. The evolution of drug development in schizophrenia: past issues and future opportunities. Neuropsychopharmacology. (In press, 2007)

Hashimoto T, Volk DW, Eggan SM, Mirnics K, Pierri JN, Sun Z, Sampson AR, Lewis DA. Gene expression deficits in a subclass of GABA neurons in the prefrontal cortex of subjects with schizophrenia. J Neurosci. 2003 Jul 16;23(15):6315-26. Abstract

Kinney JW, Davis CN, Tabarean I, Conti B, Bartfai T, Behrens MM. A specific role for NR2A-containing NMDA receptors in the maintenance of parvalbumin and GAD67 immunoreactivity in cultured interneurons. J Neurosci. 2006 Feb 1;26(5):1604-15. Abstract

Volk DW, Austin MC, Pierri JN, Sampson AR, Lewis DA. Decreased glutamic acid decarboxylase67 messenger RNA expression in a subset of prefrontal cortical gamma-aminobutyric acid neurons in subjects with schizophrenia. Arch Gen Psychiatry. 2000 Mar;57(3):237-45. Abstract

View all comments by Julie Markham
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Comment by:  Gavin Reynolds
Submitted 10 December 2007
Posted 10 December 2007

For two decades, following the work by Benes and her colleagues, it has been increasingly apparent that there is a deficit in cortical GABAergic neurons in schizophrenia. Ten years ago we found that the parvalbumin (PV)-containing, but not calretinin-containing, subgroup of these neurons was selectively affected, and recently this specific deficit has been seen in animal models of the disease. Repeated administration of non-competitive NMDA receptor antagonists such as PCP, MK801, and ketamine can induce in rats some behaviors reminiscent of schizophrenia, as well as enduring deficits in PV expression.

Behrens and colleagues have identified some of the molecular mechanisms underlying this specific neurotoxicity of ketamine and, probably, other NMDA antagonists. That the effects of ketamine involve generation of reactive oxygen species (ROS) is not surprising, given the ubiquity of oxidative free radical production in neurotoxic processes. However, identifying the role of NADPH oxidase in producing ROS in response to ketamine, and demonstrating that this process determines the consequent toxic effects of ketamine on PV-containing and other neurons, are potentially important developments.

The importance of these findings to schizophrenia relies on the assumption that repeated administration of ketamine and, presumably, other NMDA antagonists not only models (some of) the pathophysiology of schizophrenia, it also mimics the process leading to this neuronal pathology. This is far from proven, although the NMDA receptor hypofunction hypothesis of Olney and Farber provides a useful model mechanism for this pathogenesis.

A useful proof of concept would be to move away from pharmacological approaches to other animal models of the disease. One such is the isolation rearing paradigm; in this model, induction of abnormal “schizophrenia-like” behaviors is also paralleled by a deficit in PV-containing neurons (Harte et al., 2007). A simple but very informative study here would be to determine whether inhibition of NADPH oxidase might protect against the development of these deficits. Of course, how the NMDA receptor-mediated deficits relate temporally to the natural history of schizophrenia is unclear; we do not know when the PV deficits occur in schizophrenia. There may be some hope for targeted treatment with, e.g., NADPH oxidase inhibitors if the neuronal pathology parallels a neurotoxic process that underlies the progressive cognitive disturbances as implied by Olney and Farber, but not if the PV deficits relate to an early and primary pathology of the disease.

References:

Harte M, Powell S, Swerdlow N, Geyer M, Reynolds GP (2007) Deficits in Parvalbumin and Calbindin Immunoreactive cells in the Hippocampus of Isolation Reared Rats. J Neural Transm 114, 893-898. Abstract

View all comments by Gavin Reynolds

Related News: Does Oxidative Stress Link NMDA and GABA Hypotheses of Schizophrenia?

Comment by:  Kenneth Johnson
Submitted 18 December 2007
Posted 18 December 2007

The recent study by Behrens and colleagues provides in vitro evidence that blockade of NMDA receptors by ketamine leads to a selective reduction in PV and GAD67 that appears to be due to the toxic effects of superoxide anion arising subsequent to the activation of NADPH oxidase. Blockade of the sublethal, toxic effects of ketamine in neuronal culture is consistent with our report demonstrating that the apoptotic effect of phencyclidine (PCP) on cortical neurons in vivo also could be prevented by the superoxide dismutase mimetic, M40403 (Wang et al., 2003). Though seemingly non-specific, superoxide dismutase mimetics may prove to be useful in the treatment of ketamine or PCP-induced psychosis because of the relative sparseness of critical life-promoting processes that require superoxide anion. Perhaps more importantly, a better understanding of the mechanisms underlying ketamine-induced loss of PV/GAD67 may lead to novel treatment modalities for schizophrenia.

While the primary focus of the report by Behrens and colleagues is on PV-expressing GABAergic interneurons, Fig. 1 clearly demonstrates that ketamine also affects a large population of non-PV neurons. This is consistent with our recent in vivo experiments in developing rats demonstrating that PCP administration on PN7 induces apoptosis of cortical PV-containing interneurons as well as principal neurons in layers II-IV of the cortex (Wang et al., 2007). Early postnatal administration of PCP also results in neuronal apoptosis in the hippocampus, striatum, and thalamus (Wang and Johnson, 2007. Thus, it may be premature to focus solely on this population of interneurons.

In thinking about the mechanism underlying the selective loss of PV interneurons following PCP, it is important to note that PV is not yet expressed on PN7, which is when PCP was administered in our paradigm (Wang et al., 2007). (The loss of PV-containing interneurons was measured at PN56, well after the time of PV expression on about PN10.) Interestingly, interneurons expressing calretinin and calbindin at the time of PCP administration were spared. These neurons showed no colocalization with cellular markers of apoptosis (terminal dUTP nick-end labeling [TUNEL] of broken DNA or cleaved caspase-3), indicating that calretinin- and calbindin-containing neurons were protected from the toxic effect of PCP and survived into adulthood (Wang et al., 2007). The mechanism underlying this selectivity for cortical PV-containing interneurons is unknown, but as Behrens and colleagues suggest, it could be because these neurons are dependent on a relatively large glutamatergic input for survival. It is also possible that the differing calcium buffering capacities of these interneurons play a role in the selective neurotoxic effect of NMDA receptor blockade. That is, since calcium binding proteins could also act to buffer decreases in intracellular Ca2+ levels caused by ketamine-induced blockade of NMDA receptors, it is possible that the lack of PV in these vulnerable interneurons reduces the ability of these cells to adequately buffer the ketamine-induced decrease in intracellular calcium. This is consistent with the lack of effect on other interneurons that express the calcium binding proteins calretinin and calbindin at the time of PCP administration. This suggests NMDA receptor blockade could cause the deletion of PV neurons because of a specific effect at a critical stage of development. However, cleaved caspase-3 (a hallmark of apoptosis) showed no colocalization with BrdU, a specific marker of S-phase proliferation (Wang et al., 2007). These data suggest that the loss of PV-containing neurons in this paradigm was not due to an effect of PCP on proliferating neurons, but rather an effect on postmitotic neurons.

We have reported recently that PCP in cortical neuronal culture causes neuronal apoptosis by interfering with the Akt-GSK-3β cascade, which is necessary for neuronal survival during development (Lei et al., 2007). Moreover, increasing synaptic strength by various means such as increasing calcium current via activation of L-type calcium channels completely blocks PCP-induced cell death by increasing Akt phosphorylation. It would be of great interest to determine whether PV-containing interneurons respond in a similar fashion.

In order to fully appreciate the significance of ketamine-induced loss of PV-containing neurons, it will be necessary to carefully compare the in vivo dose-related effects of ketamine or PCP that are truly selective for PV/GAD67-containing interneurons to those cortically mediated behaviors that have relevance to schizophrenia.

References:

Wang C, McInnis J, West JB, Bao J, Anastasio N, Guidry JA, Ye Y, Salvemini D, Johnson KM. Blockade of phencyclidine-induced cortical apoptosis and deficits in prepulse inhibition by M40403, a superoxide dismutase mimetic. J Pharmacol Exp Ther. 2003 Jan 1;304(1):266-71. Abstract

Wang, C.Z., Yang, S.F., Xia, Y. and Johnson, K.M. Induction of a selective cortical deficit of parvalbumin-containing interneurons by phencyclidine administration during postnatal brain development. Neuropsychopharmacology (In press, 2007).

Wang CZ, Johnson KM. The role of caspase-3 activation in phencyclidine-induced neuronal death in postnatal rats. Neuropsychopharmacology. 2007 May 1;32(5):1178-94. Abstract

Lei, G., Xia, Y. and Johnson, K.M. The role of Akt-GSK-3β signaling and synaptic strength in phencyclidine-induced neurodegeneration. Neuropsychopharmacology (In press, 2007).

View all comments by Kenneth Johnson

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Comment by:  Patricia Estani
Submitted 11 January 2008
Posted 13 January 2008
  I recommend the Primary Papers

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Comment by:  Keri Martinowich
Submitted 30 April 2008
Posted 1 May 2008

The recent paper by Vetencourt et al., showing that fluoxetine can restore neuronal plasticity in the adult visual system, has quite obvious and exciting potential applications for the treatment of amblyopia. However, beyond this potential unexpected use for fluoxetine in the clinical treatment of eye disorders, lie implications and new insight into antidepressant mechanisms of actions in mood disorders. It has been speculated for some time that the need for chronic treatment with antidepressants to achieve a therapeutic effect is dependent on changes in neuronal and synaptic plasticity. Time and again, regulation of BDNF has emerged as a candidate underlying various depressive and/or anxiety-like phenotypes, as well as being a possible mediator of the effect of antidepressant/mood-stabilizer drugs. It is now well accepted that beyond its role as a trophic factor during development, BDNF plays a key role in regulating neuronal plasticity in the adult central nervous system.

Clinicians, patients and clinical trials alike attest that antidepressants have strong effects, but the understanding of how exactly they exert their function has remained a mystery despite intense speculation and study. Mechanistic understanding of antidepressant function may provide valuable clues about the functional effects of these drugs, which will likely facilitate our ability to develop treatments with faster therapeutic onset and fewer or less severe side effects by allowing us to precisely target the necessary sites of action.

Reports like the current one are refreshing to the field, reminding us that sometimes, the answers to our questions might be best answered by looking in a different direction rather than focusing and refocusing on the current studies. The sites of action of antidepressant drugs and mood stabilizing therapies are likely to be not just in one place, but in several or even many, interacting circuits within the limbic system of the brain. Teasing apart these circuits and visualizing the effects that antidepressants have on these functional circuits is of course, technically challenging. The comparative simplicity and better understanding of the visual system, makes it an attractive system for the present type of molecular studies. Clearly, biology has made use of redundancy with systems that work well. This biological redundancy may afford us the use of more manageable systems that allow us to observe physiological effects of antidepressants and mood-stabilizing drugs that can be extrapolated to areas of the brain important for regulation of mood and behavior.

View all comments by Keri Martinowich

Related News: Down With Inhibition: Modulating GABAergic Neocortical Control

Comment by:  Miles Whittington
Submitted 24 July 2008
Posted 24 July 2008

This paper by Kruglikov and Rudy examines in detail the profile of neuromodulatory influences on GABA release from fast-spiking (FS), parvalbumin-containing interneurons in sensory neocortex. The work elegantly demonstrates that this interneuron subtype is exquisitely sensitive to a diverse range of neuromodulatory chemicals including those acting on muscarinic, purinergic, serotonergic, and GABAB receptors. Agonists at each of these receptors produced a strong inhibition of GABA release from electrically stimulated FS synaptic terminals and, as a result, reduced inhibitory influence both locally in cortex and on ascending thalamocortical projections. These interneurons are of particular interest currently in schizophrenia research as functional markers for them are found to be robustly reduced in postmortem brain samples from schizophrenic patients. They are also one of the key interneuron subtypes involved in the generation of certain EEG rhythms—in particular, those in the gamma (30-80 Hz) band—involved in primary sensory processing, short-term memory, and cortico-cortical communication. The pattern of generation of gamma rhythms is disrupted in people with schizophrenia.

However, it is hard to see direct implications for our understanding of processes underlying cortical dysfunction in schizophrenia from this work. This is mainly due to the fact that only a subset of parvalbumin-containing FS cells appear to be affected in schizophrenia and related animal models. It is not yet known what makes this subset of interneurons so labile in psychiatric illness, but there is much exciting work ongoing which is highly suggestive of a role for NMDA receptor-mediated excitation and changes in redox state. The paper does not subclassify the FS interneurons studied directly, but there are a couple of issues which may be of interest to the field:

1. Firstly, the importance of neuromodulation for generating gamma rhythms can clearly be seen in Figure 6. Forty Hz artificial stimulation—matching the modal frequency of activation of these neurons during network gamma rhythms—terminates pyramidal cell action potential generation in the absence of neuromodulatory influences. This suggests that inhibition-based gamma oscillations in cortex would be useless as an information coding strategy. However, the reduced inhibitory post-synaptic potential size (and thus effective duration) under muscarinic neuromodulation permits sparse, but precisely timed pyramidal cell action potentials—exactly the signature for principal cell spiking during gamma rhythms in vivo. Loss of parvalbumin from FS cells causes a large increase in GABA release, and thus increased inhibitory post-synaptic potential size and duration. It is therefore interesting to consider what the reduced parvalbumin levels in schizophrenic cortex can tell us: is it a primary cause of the observed decrease in gamma rhythm generation, owing to its enhancement of GABA release and thus perhaps termination of pyramidal cell spiking? Or is it a compensatory mechanism, as previously proposed, for an underlying deficit in FS cell excitation?

2. Secondly, it is interesting to note in this paper that cannabinoid receptor activation did not change evoked GABA release from FS cells at all. There is a growing corpus of work linking cannabis use to increase risk of psychotic episodes and exacerbate symptoms of schizophrenia. Given the proposed critical role of FS cells in cortical dysfunction in schizophrenia, it is perhaps surprising that no effect was seen. However, the study used artificial, electrical stimulation to evoke GABA release from these cells. In active networks, cannabinoid agonists can act on CCK-containing interneurons, which may indirectly change excitability of parvalbumin-containing FS interneurons, changing GABA release patterns via altered rates of action potential generation. In addition, cannabinoid receptors have been shown to directly reduce excitatory post-synaptic potential size, something that might be expected to directly reduce FS cell recruitment during network activity.

View all comments by Miles Whittington

Related News: GABA Receptor Drug for Schizophrenia Is Put Through Its Paces

Comment by:  Robert McCarley
Submitted 7 November 2008
Posted 7 November 2008

This paper is further evidence of an important and laudable new trend in schizophrenia psychopharmacology: namely the development and test of compounds on the basis of their relationship to circuit abnormalities, evidence derived from postmortem, genetic, and animal model studies. The authors based their choice of MK-0777 for test in schizophrenia on evidence for decreased cortical GABA neurotransmission onto pyramidal neurons at receptors having the α2 subunit, and other evidence pointing to the GABA-pyramidal neuron interaction as important in cognition and in generation of γ band oscillations. In this add-on, double-blind placebo study, the Ns were underpowered and more subjects need to be studied to be certain about clinical effects. However, one test, the Preparing to Overcome Prepotency Test (POP), had significant improvements in response latency and showed concomitant improvement in increased frontal γ band activity induced during the task, although not meeting the criterion for statistical significance. POP requires subjects either to “go with the flow” (indicated by a green light) and respond in the same direction as an arrow, or when cued by a red light to “go upstream” and point in the opposite direction, a test previously used in the Cho et al. 2006 PNAS paper and found to be accompanied by increased induced γ band oscillations.

γ band activity has justifiably attracted considerable attention, since there is mounting evidence of its relevance to human cognition as well as to basic neuroscience studies of neuronal assembly communication. Its important basis in the GABA cortical neuronal interaction with pyramidal cells makes it especially fascinating in schizophrenia. However, an important caution light was recently flashed by Yuval-Greenberg et al. in an article in Neuron (2008) in which they presented strong evidence that apparent increases or decreases in the “induced γ band oscillations” (those not temporally linked to a response or stimulus) could be the result of the eye muscle activation associated with small saccadic eye movements, “a saccadic spike potential” that could be confused with γ band oscillations. The Yuval-Greenberg article appeared too late for the authors to discuss in the present paper, but its implications for future work using induced γ are important. For studies of induced γ, we all will have to begin using eye movement measures sensitive to mini-saccades. Those of us who measure γ phase-locked to measureable events, such as sensory stimuli or responses, appear to be off the hook since we condition on known events, unlike conditions where induced γ is measured.

References:

Cho RY, Konecky RO, Carter CS. Impairments in frontal cortical gamma synchrony and cognitive control in schizophrenia. Proc Natl Acad Sci U S A. 2006 Dec 26;103(52):19878-83. Abstract

Yuval-Greenberg S, Tomer O, Keren AS, Nelken I, Deouell LY. Transient induced gamma-band response in EEG as a manifestation of miniature saccades. Neuron. 2008 May 8;58(3):429-41. Abstract

View all comments by Robert McCarley

Related News: Special K: Primate-specific Potassium Channel Variant Implicated in Schizophrenia

Comment by:  Paul Shepard
Submitted 18 May 2009
Posted 19 May 2009
  I recommend the Primary Papers

The manuscript by Huffaker et al. extends the growing number of cardiac potassium channels that have found their way into the brain and onto the list of putative therapeutic targets for the treatment of neurological and psychiatric disease. In an extensive series of experiments, these investigators demonstrate an association between single nucleotide polymorphisms in a gene encoding an inwardly rectifying potassium channel (KCNH2), the expression of a previously unknown isoform (KCNH2-3.1), and schizophrenia. Named for the dance exhibited by ether-intoxicated fruit fly mutants in which the gene family was first identified, ether-a-go-go related gene or ERG K+ channels contribute to the repolarization of cardiac action potentials and the propensity of antipsychotic drugs to prolong the QT interval, a direct result of their ability to attenuate this current in the heart. The unique gating properties of ERG K+ channels (for review, see Shepard et al., 2007) give rise to a strong resurgent current that can profoundly alter both intermediate and slow components of neuronal signaling. Thus, ERG currents have been shown to alter spike timing (e.g., latency to first spike in a stimulus-evoked train, spike frequency adaptation) in cerebellar Purkinje (Sacco et al., 2003), medial vestibular nucleus (Pessia et al., 2008), and cultured cortical neurons, while in dopamine cells, they appear to underlie a slow afterhyperpolarization envisioned to contribute to the termination of plateau oscillations and the obligatory pause in firing after a burst of spikes (Canavier et al., 2007; Nedergaard, 2004).

Identification of a primate-specific KCNH2-3.1 isoform in hippocampus and cortex whose expression in brain alters the function of the channel begs a number of questions that will undoubtedly be the focus of subsequent research. Foremost among these is whether the therapeutic effects of antipsychotic drugs derive in some measure from their ability to block ERG channels containing the KCNH2-3.1 protein. Although the truncated KCNH2-3.1 isoform is unique to primates, phenotypic changes associated with expression of the protein result from loss of the PAS domain, a region of the protein responsible for the resurgent nature of the outward current. In addition to increasing the rate of ERG channel deactivation, expression of the truncated isoform may reduce the number of functional channels brought to the surface as suggested by the reported reduction in ERG current density in rat cortical neurons transfected with human KCNH2-3.1. The functional consequences associated with the loss of the PAS domain in individual cells can be characterized using dynamic clamp—a technique in which a computer simulation is used to introduce an artificial membrane conductance into individual neurons. However, the effects of the mutation on channel trafficking and assessment of the myriad of conductance states likely to result from heterologous expression with other ERG channel subunits will require a transgenic model, which if history serves, the Weinberger group has already begun constructing.

References:

Canavier CC, Oprisan SA, Callaway JC, Ji H, Shepard PD. Computational model predicts a role for ERG current in repolarizing plateau potentials in dopamine neurons: implications for modulation of neuronal activity. J Neurophysiol . 2007 Nov 1 ; 98(5):3006-22. Abstract

Nedergaard S. A Ca2+-independent slow afterhyperpolarization in substantia nigra compacta neurons. Neuroscience . 2004 Jan 1 ; 125(4):841-52. Abstract

Pessia M, Servettini I, Panichi R, Guasti L, Grassi S, Arcangeli A, Wanke E, Pettorossi VE. ERG voltage-gated K+ channels regulate excitability and discharge dynamics of the medial vestibular nucleus neurones. J Physiol . 2008 Oct 15 ; 586(Pt 20):4877-90. Abstract

Sacco T, Bruno A, Wanke E, Tempia F. Functional roles of an ERG current isolated in cerebellar Purkinje neurons. J Neurophysiol . 2003 Sep 1 ; 90(3):1817-28. Abstract

Shepard PD, Canavier CC, Levitan ES. Ether-a-go-go-related gene potassium channels: what's all the buzz about? Schizophr Bull . 2007 Nov 1 ; 33(6):1263-9. Abstract

View all comments by Paul Shepard

Related News: Special K: Primate-specific Potassium Channel Variant Implicated in Schizophrenia

Comment by:  Szatmar Horvath
Submitted 11 May 2009
Posted 1 June 2009
  I recommend the Primary Papers

Related News: GABA Is Up in Prefrontal Cortex of Schizophrenia Subjects

Comment by:  Dost Ongur
Submitted 19 January 2012
Posted 19 January 2012

This news story by Allison Curley cogently and succinctly describes the current state of affairs in studies of parenchymal GABA levels in schizophrenia. Measuring GABA in vivo in the human brain has been challenging because this metabolite exists in relatively low concentration and its signal overlaps with that of other, more abundant metabolites. The literature has grown recently with the advent of higher-field MRI scanners and reliable MRS approaches for GABA measurement.

As outlined in the story, the several papers on parenchymal GABA levels in schizophrenia are about evenly split, with reductions and elevations both being reported. Although MRS is characterized by a relatively low signal-to-noise ratio and high variance in most datasets, all the recent studies used reliable MRS techniques such as MEGAPRESS.

In my opinion, the current state of the literature offers two insights:

1. If there was a significant and consistent abnormality in parenchymal GABA levels in schizophrenia, we would have found it and the studies would agree. Rather, it appears that there may be patient and treatment factors leading to differential GABA patterns. For example, to speculate: elevations in early illness may be replaced by reductions with chronic disease, or anticonvulsants may elevate GABA levels while antipsychotics reduce them. Larger datasets with more detailed phenotypic analyses may provide leads for developing a clearer picture. Alternatively, and less interestingly, there may be no or minor abnormalities which result in conflicting findings due to sampling error, technical differences, etc.

2. As a corollary to any of the possibilities above, it is clear that abnormal GABAergic neurotransmission is not necessarily associated with consistently reduced parenchymal GABA levels as measured by MRS. Postmortem and other lines of evidence are quite convincing of abnormalities in GABAergic interneurons in schizophrenia. However, the in-vivo MRS studies are much less consistent, suggesting a disconnect between the two lines of inquiry. Just to describe one possibility, it is possible that GABA is inappropriately stored in synaptic vesicles instead of being released into the synapse and subsequently metabolized, setting up elevated GABA levels but reduced GABAergic neurotransmission.

Although confusing at the moment, the optimistic view is that MRS studies of brain GABA levels in schizophrenia will ultimately offer a more sophisticated understanding of the relationship between metabolite levels measured using MRS and the brain functions we all care about.

View all comments by Dost Ongur

Related News: GABA Is Up in Prefrontal Cortex of Schizophrenia Subjects

Comment by:  Jong H. YoonRichard J. Maddock
Submitted 8 February 2012
Posted 8 February 2012

The study by Kegeles et al. has added unique and important findings to the small but rapidly growing literature assessing in-vivo GABA levels in schizophrenia using MRS. In the context of these studies, the Kegeles publication also raises several challenging questions regarding the potential relevance and reliability of in-vivo GABA studies. Here, we would like to comment on two of these questions. The first pertains to the lack of convergence with the consistent postmortem studies. The second is the apparent lack of consistency across the recent in-vivo GABA studies in schizophrenia.

A starting point in the discussion of the first issue is to recognize the differences in what we are measuring with in-vivo spectroscopy as opposed to the postmortem studies. The latter have consistently demonstrated decreased mRNA levels for GAD67, one of the major synthetic enzymes for GABA, in a subset of GABAergic interneurons in the neocortex of schizophrenia. Based on this postmortem work and the important role GAD67 plays in determining whole cell content of GABA (Asada et al., 1997), many, including Kegeles and coauthors, had predicted MRS measurements of GABA would be decreased in schizophrenia. Spectroscopy measures bulk GABA, the largest fraction of which is cytoplasmic and not vesicular. The cytoplasmic fraction of GABA is synthesized by GAD67 (abnormal in postmortem studies of schizophrenia), while the vesicular fraction is synthesized in part by GAD65 (not apparently abnormal in schizophrenia) (Waagepetersen et al., 2007). While vesicular GABA is the source of GABA for synaptic neurotransmission, cytoplasmic GABA may play a role in both tonic and phasic inhibition mediated by extrasynaptic GABAergic signaling (Wu et al., 2007). One of the major limitations of MRS measurements of GABA, therefore, is that we currently do not really understand to what extent this bulk measurement relates to neural signaling. However, there are a growing number of studies (Edden et al., 2009; Sumner et al., 2010), including one by our group (Yoon et al., 2010), that suggest that bulk GABA measurement is a functionally meaningful measure. These studies have shown high correlations between MRS estimates of GABA and performance on tasks presumably dependent on the magnitude of GABA-mediated inhibition. In addition, animal studies have suggested that the concentrations of vesicular and non-vesicular pools of GABA appear to be in equilibrium (Waagepetersen et al., 1999), implying that bulk GABA levels reflect, to some degree, the vesicular fraction. Nonetheless, as others have pointed out, the diverse components of the GABA MRS measurements leave open a number of potential explanations as to why bulk GABA levels may not be decreased in schizophrenia in the setting of decreased GAD67 mRNA levels.

The second set of questions concerns the apparent lack of consistency among the recent set of in-vivo GABA studies. The potential reasons for this are many and diverse, and include clinical and neuroimaging-related factors that may have varied across the spectroscopy studies, including differences in illness severity, length of illness, brain regions assessed, and methods for GABA quantification. The Kegeles paper has identified medication status as an important clinical variable for which future studies should attempt to account. In-vivo GABA spectroscopy using MEGA PRESS is a relatively new method, particularly as applied to between-group studies. Consequently, there may be a number of neuroimaging-related variables that are important sources of noise or diminished signal, leading to false-negative findings of group differences, or bias, leading to false-positive findings of group differences. An example of the former relates to the phased array head coils frequently used in GABA studies. With these receive-only coils, signal strength decreases linearly as a function of the distance between the coil element that detects the spectroscopy signal and the brain region being sampled. Thus, the signal from brain regions farther away from these elements, for example, deep midline and subcortical regions, will be much lower than regions that are adjacent to these elements, for example, the occipital pole. Consequently, our ability to detect true differences between groups in these low-signal regions will be constrained. Another important variable may be in-scanner head movement. From our own work, we are coming to believe that in-scanner head movement may produce significant over- or underestimation of true GABA concentration, depending on the type of movement. The effect of head movement may be particularly important in between-group studies in which one group may exhibit a significantly different amount of movement compared to the other group. Even a few patients with excessive movement during a prolonged MRS acquisition could generate outlying and erroneous GABA values and lead to false-positive group differences.

In summary, we are in the very early stages of MRS studies of GABA in schizophrenia. There are many unanswered questions regarding the meaning of this signal and how it relates to GABA physiology, function, and their impairment in schizophrenia. The answers to these questions will require intense efforts relying on animal and human models to unravel the complex relationships between bulk GABA measurements and GABA signaling. As a methodology, much more work needs to be done to validly and reliably translate this method to clinical studies. In the immediate future, it will be critical to identify the important sources of noise and bias, and to develop methods controlling for these variables in clinical studies so that the true nature of GABA levels in schizophrenia may be established.

References:

Asada H, Kawamura Y, Maruyama K, Kume H, Ding RG, Kanbara N, et al (1997): Cleft Palate and Decreased Brain Gamma-aminobutyric Acid in Mice Lacking the 67-kDa Isoform of Glutamic Acid Decarboxylase. Proc Natl Acad Sci U S A 94:6496-6499. Abstract

Edden RAE, Muthukumaraswamy SD, Freeman TCA, Singh KD. (2009) Orientation Discrimination Performance Is Predicted by GABA Concentration and Gamma Oscillation Frequency in Human Primary Visual Cortex. Journal of Neuroscience 29(50):15721-15726. Abstract

Sumner P, Edden RAE, Bompas A, Evans JC, Singh KD (2010) More GABA, Less Distraction: a Neurochemical Predictor of Motor Decision Speed. Nature Neuroscience 13:825-827. Abstract

Waagepetersen HS, Sonnewald U, Larsson OM, Schousboe A. (1999) Synthesis of Vesicular GABA From Glutamine Involves TCA Cycle Metabolism in Neocortical Neurons. Journal of Neuroscience Research 57:342-349. Abstract

Waagepetersen HS, Sonnewald U, Schousboe A (2007) Glutamine, Glutamate, and GABA: Metabolic Aspects. In: Lajtha A, Oja S, Schousboe A, Saransaari P (eds) Handbook of Neurochemistry and Molecular Neurobiology: Amino Acids and Peptides in the Nervous System. Springer, New York, pp 1-21.

Wu Y, Wang W, Diez-Sampedro A, Richerson GB (2007) Nonvesicular Inhibitory Neurotransmission Via Reversal of the GABA Transporter GAT-1. Neuron 56:851-865. Abstract

Yoon JH, Maddock RJ, Rokem AS, Silver MA, Minzenberg MJ, Ragland JD, Carter CS. (2010) Gamma-aminobutyric Acid Concentration is Reduced in Visual Cortex in Schizophrenia and Correlates with Orientation-Specific Surround Suppression. Journal of Neuroscience 10;30(10):3777-81. Abstract

View all comments by Jong H. Yoon
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Related News: GABA Is Up in Prefrontal Cortex of Schizophrenia Subjects

Comment by:  Robert McCarleyMargaret NiznikiewiczMartina M. VoglmaierKevin Spencer (Disclosure), Nick BoloAlexander P. LinYouji HiranoElisabetta del ReIsrael MolinaVicky LiaoSai Merugumala
Submitted 13 February 2012
Posted 14 February 2012
  I recommend the Primary Papers

The important and elegantly controlled work by Kegeles et al., and the informed comments of Ongur, Yoshimura, and Yoon and Maddock, on GABA in schizophrenia raise a series of potentially key factors about the sources of variability of MRS findings in this disorder (medication, stage of illness, and region of interest [ROI]). They also point out the need for association of MRS GABA findings with physiologic measures such as γ oscillations (40 Hz), a functional measure particularly relevant because of the involvement of GABA interneurons interacting with pyramidal neurons in generating this oscillation.

We would like to call the reader's attention to a potentially informative schizophrenia spectrum disorder, schizotypal personality disorder (SPD), that may help shed light on and respond to these issues. As has been documented by Kendler (Kendler et al., 1993; Fanous et al., 2007), SPD shares a genetic relationship with schizophrenia. Although sharing the symptoms of schizophrenia, SPD individuals have an attenuated version of the symptoms and are not psychotic. One thus can recruit SPD individuals who are living in the community, have never been neuroleptic medicated, who have no current medication, and who do not show the profound lifestyle disturbance of individuals with schizophrenia.

We have begun MRS evaluations on SPD subjects with these characteristics, choosing ROI in the superior temporal gyrus (STG) because of the strong evidence of the association of this region with the auditory steady-state (ASSR) γ oscillation response, as well as structural MRI evidence for left STG reduced gray matter volume. Our still quite preliminary data showed, compared with matched healthy controls, a mean reduction in GABA levels and an increase in glutamate. Although the levels were not yet statistically significantly different in our preliminary data, what was notable, and statistically significant, was the very high correlation of the left STG glutamate and GABA levels with the levels of the ASSR γ oscillation, measured as the strength of the phase locking factor (PLF) over left-sided electrodes. As predicted, GABA levels were positively correlated with the PLF, while glutamate levels were inversely (negatively) correlated with the PLF. Obviously, more data are needed, but these initial findings suggest the promise of using SPD subjects with both MRS and γ oscillation measurements in the STG.

References:

Preliminary results to be presented at the 3rd Biennial Schizophrenia International Research Society Conference 14-18 April 2012, Florence, Italy, as a poster and an oral presentation, and at the 20th Annual Meeting of the International Society of Magnetic Resonance in Medicine 5-11 May 2012, Melbourne, Australia.

Kendler KS, McGuire M, Gruenberg AM, O'Hare A, Spellman M, Walsh D. (1993). The Roscommon Family Study. III. Schizophrenia-related personality disorders in relatives. Arch Gen Psychiatry, 50(10):781-788. Abstract

Fanous AH, Neale MC, Gardner CO, Webb BT, Straub RE, O'Neill FA, Walsh D, Riley BP, Kendler KS. Significant correlation in linkage signals from genome-wide scans of schizophrenia and schizotypy. Mol Psychiatry. 2007 Oct;12(10):958-65. Abstract

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Related News: GABA Is Up in Prefrontal Cortex of Schizophrenia Subjects

Comment by:  Lawrence KegelesDikoma C. Shungu
Submitted 4 April 2012
Posted 5 April 2012

The news story by Allison Curley on our recent paper gives a concise and insightful overview of in-vivo studies of GABA levels in schizophrenia. As the story notes, for those keeping score, studies measuring GABA in schizophrenia are evenly split in that two showed increases, two found decreases, and one reported no change. A major theme running through the thoughtful commentaries by Ongur, Yoshimura, Yoon and Maddock, and McCarley and colleagues is how to understand the variability across studies.

Some regularities can already be found in these and similar studies of the glutamate system. If we confine the scorekeeping to GABA in the prefrontal cortex (PFC), the studies are more uniform: two showed increases (Ongur et al., 2010; Kegeles et al., 2012) and two showed no change (Goto et al., 2009; Tayoshi et al., 2010). If we further limit attention to unmedicated patients, but broaden the review to include the glutamatergic system as well as GABA in the PFC, the studies all agree: glutamine, glutamate-glutamine (Glx), or GABA is increased in the medial PFC (Bartha et al., 1997; Théberge et al., 2002; Théberge et al., 2007; Kegeles et al., 2012), but unchanged in the dorsolateral PFC (Stanley et al., 1996; Ohrmann et al., 2007; Kegeles et al., 2012).

It is encouraging to find patterns where we can, but so far these are limited. We still need (and have begun) to investigate other important brain regions, and it is essential to understand the effects of antipsychotic medication. The commentary by Yoshimura describes the subjects studied by Goto et al. (2009) as medicated and unmedicated, and we wonder if a comparison between those subsamples, as we did in our study, might be informative.

Besides brain region and medication status, the commentaries suggest other patient, treatment, or technical measurement factors contributing to the variability. These include chronicity or duration of illness, medications other than antipsychotics such as benzodiazepines and anticonvulsants, and specifics of MRS methodology. We share these views and encourage any efforts to find systematic impacts of these variables.

Yoon and Maddock raise technical cautions: use of phased-array head coils can limit signal detection in deeper brain regions, and movement artifacts might introduce spurious group differences. As their commentary notes, regions adjacent to the coil elements, such as the occipital lobe (or the dorsolateral PFC) will yield greater signal than deeper structures. In our study, it was the surface region, the dorsolateral PFC, where no group difference was detected, and the slightly deeper medial PFC that showed differences, suggesting adequate sensitivity in the deeper region. Acquisition parameters can be used to offset the coil depth effect. In our study, we enhanced the medial PFC signal by doubling the volume, tending to offset the greater distance from the coil array. Head movement might raise special concerns as a source of artifact in a technique such as MEGA PRESS that relies on subtraction of sequentially acquired spectra, and Yoon and Maddock raise the possibility of resulting over- or underestimation of GABA concentration. Evidence that this may not have occurred in our study is the agreement of our Glx data with prior studies in both medial (Bartha et al., 1997; Théberge et al., 2002; Théberge et al., 2007) and dorsolateral PFC (Stanley et al., 1996; Ohrmann et al., 2007) that did not use MEGA PRESS. Our Glx and GABA measurements that did use MEGA PRESS were correlated and were both elevated in medial PFC, so the agreement with prior methodologies seems to lessen the likelihood of artifacts specific to subtraction methodology. Also, the deeper region (medial PFC) would be expected to undergo less movement than the surface region, yet showed the elevations. Additional evidence that movement artifact may not be a confounder in MEGA PRESS measurements is a recent study by Hasler et al., (2007) in major depression, where a very different pattern of abnormalities was seen in medial PFC (decreased Glx and unchanged GABA). It seems unlikely that patients with depression and schizophrenia would exhibit movement patterns systematically different from controls, yet so different from each other as to have generally opposite impacts on the outcome measures. However, these are all indirect considerations. Systematic characterization of movement effects in MEGA PRESS and other acquisition sequences could add important specific data on potential artifacts, and these issues deserve further study.

Another theme of the commentary is the apparent discrepancy between postmortem markers of GABA function and parenchymal GABA measured in vivo with MRS. There is a clear indication of diminished GABA function associated with fast-spiking, parvalbumin-positive GABA interneurons in the postmortem findings, yet we reported an elevation of parenchymal GABA concentration in vivo in the medial PFC. Ongur’s commentary raises the interesting possibility of abnormally increased storage in synaptic vesicles, while Yoon and Maddock cite evidence from animal studies of equilibrium between vesicular and non-vesicular GABA pools. Possibilities are a disruption of this normal equilibrium in schizophrenia and, alternatively, a compensatory increase in GABA signaling from the non-parvalbumin interneurons. These speculative possibilities raise the questions of detectable postmortem markers of abnormal vesicular function or heightened signaling by the non-fast-spiking interneurons.

Finally, the commentaries offered important observations on the functional role of total tissue GABA levels. Since neurotransmission is only one of several compartments contributing to parenchymal GABA, it is reasonable to wonder whether this MRS measurement has any detectable functional significance at all. Our study found no relation between elevated parenchymal GABA and working memory performance. We did find a relationship to positive symptoms that did not survive multiple comparisons correction, but suggests a focus for future testing. Yoon and Maddock cite several studies documenting functional importance of total GABA (Edden et al., 2009; Sumner et al., 2010; Yoon et al., 2010). McCarley and colleagues note in their commentary that relationships to physiological measures such as gamma oscillations suggest that bulk GABA is functionally meaningful (see also Muthukumaraswamy et al., 2009).

In the end, if we can develop a consistent picture of GABA abnormalities in schizophrenia, the primary motivation for all of these studies is to establish their functional relevance, and to raise the possibility of interventions designed to restore not only normal levels, but also, more importantly, normal function.

References:

Bartha R, Williamson PC, Drost DJ, Malla A, Carr TJ, Cortese L, Canaran G, Rylett RJ, Neufeld RWJ (1997) Measurement of glutamate and glutamine in the medial prefrontal cortex of never-treated schizophrenic patients and healthy controls by proton magnetic resonance spectroscopy. Arch Gen Psychiatry 54:959-65. Abstract

Edden RA, Muthukumaraswamy SD, Freeman TC, Singh KD (2009) Orientation discrimination performance is predicted by GABA concentration and gamma oscillation frequency in human primary visual cortex. J Neurosci 29:15721-6. Abstract

Goto N, Yoshimura R, Moriya J, Kakeda S, Ueda N, Ikenouchi-Sugita A, Umene-Nakano W, Hayashi K, Oonari N, Korogi Y, Nakamura J (2009) Reduction of brain gamma-aminobutyric acid (GABA) concentrations in early-stage schizophrenia patients: 3T Proton MRS study. Schizophr Res 112:192-3. Abstract

Hasler G, van der Veen JW, Tumonis T, Meyers N, Shen J, Drevets WC (2007) Reduced prefrontal glutamate/glutamine and gamma-aminobutyric acid levels in major depression determined using proton magnetic resonance spectroscopy. Arch Gen Psychiatry 64:193-200. Abstract

Muthukumaraswamy SD, Edden RA, Jones DK, Swettenham JB, Singh KD (2009) Resting GABA concentration predicts peak gamma frequency and fMRI amplitude in response to visual stimulation in humans. Proc Natl Acad Sci U S A 106:8356-61. Abstract

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