Schizophrenia Research Forum - A Catalyst for Creative Thinking

SfN 2007—Integrating Schizophrenia Hypotheses

4 February 2008. Dopaminergic, glutamatergic, GABAergic, cholinergic. Several hypotheses linking faulty neurotransmission to schizophrenia have emerged over the years. As is often the case with apparently conflicting theories, each may be correct in its own way. So how are they related? That question was addressed by a symposium at last November’s annual meeting of the Society for Neuroscience. As symposium chair John Lisman, Brandeis University, Waltham, Massachusetts, pointed out in his introduction, there is a need to come up with a model of neural networks that can explain not only how these various neurotransmitters tie in with the positive and negative symptoms of the disease, but how they relate to emerging schizophrenia risk genes.

Joseph Coyle, McLean Hospital, Belmont, Massachusetts, started the symposium by reviewing some of the links between glutamatergic hypofunction and novel risk genes for schizophrenia. The glutamatergic hypothesis of schizophrenia is well established (see SRF hypothesis) and stems from the observation that antagonists of the NMDA-type glutamate receptor, such as phencyclidine and ketamine (see SRF related news story), can induce psychosis, as well as some of the physiological signs found in schizophrenia patients, including eye tracking abnormalities (see Avila et al., 2002), frontal cortical hypofunction (see Hertzmann et al., 1990), elevated subcortical dopamine release (see Kegeles et al., 2000), impaired prepulse inhibition (see Braff et al., 2001), and mismatch negativity in auditory event-related potentials (ERPs; see Umbricht et al., 2002). But it is not clear how the glutamatergic hypothesis fits with patterns of inheritance. The explanation may lie not with the genes for NMDA receptors themselves, but in associated genes, suggested Coyle.

Coyle reviewed some of the evidence linking schizophrenia to genetic polymorphisms in genes that regulate NMDA co-agonists such as serine. Researchers reported in 2002 that genetic polymorphisms in two overlapping genes, G72 and G30, associated with schizophrenia (see Chumakov et al., 2002), and it turns out that G72 activates D-amino acid oxidase (DAO), which degrades serine. Thus, genetic links to schizophrenia may be within two degrees of separation from the NMDA receptor, noted Coyle. More recent studies support the link between G72/G30, DAO polymorphisms, and both schizophrenia and bipolar disorder (see Detera-Wadleigh and McMahon, 2006 and Corvin et al., 2007). Though polymorphisms in individual genes may not have large effects, a combination of genetic variations in multiple genes impacting NMDA function, including G72/G30, DAO, and serine racemase, which converts L-serine to the NMDA receptor relevant D-serine, could be significant, Coyle suggested.

Coyle has investigated the serine connection pharmacologically using D-cycloserine, an approved antibiotic which also happens to be a partial NMDA agonist. Though he cautioned that this drug also inhibits serine racemase and therefore might have a complex dose response curve, he has found that at intermediate doses the drug relieves some of the negative symptoms of schizophrenia (see Goff et al., 1995). Other researchers found that glycine, another NMDA co-agonist, also reduces negative symptoms of schizophrenia, Coyle said. His lab also found that there is enhanced activation of some regions of the brain when patients taking D-cycloserine perform a memory task (see Yurgelun-Todd et al., 2005). The data point to a role for NMDA co-agonists in disease pathology and potential treatments.

While several studies show NMDA co-agonists might improve schizophrenia symptoms, it is not clear which neurons in the brain mediate these effects. But a hint came from an earlier study from Robbie Greene’s lab, suggested Coyle. These researchers found that sensitivity to NMDA antagonists is different in pyramidal cells and GABAergic interneurons (see Grunze et al., 1996)—the latter are 10 times more sensitive to receptor antagonists, said Coyle. Coyle is now trying to model serine deficiency in mice to get a better handle on the neurons involved. In collaboration with colleagues at the Ottawa Health Research Institute, Canada, he has created a serine racemase knockout, which is predicted to limit the availability of D-serine. In a poster presented in San Diego, his colleague Chun Ma demonstrated that wild-type mice only get a mild boost in NMDA currents when perfused with serine, but serine racemase knockouts show a dramatic increase in current. In fact, in the knockout animal, long-term potentiation, a key facet of synaptic plasticity, cannot be elicited unless D-serine is applied. Behaviorally, these animals are less active than normal mice, though they seem to perform fairly well in a water maze test of learning and memory.

GABAergic dysfunction has also been linked to the pathology of schizophrenia and related diseases, including bipolar disorder. The GABAergic hypothesis dates back several decades, noted Francine Benes, also from McLean Hospital. In the 1970s it was reported that GABA and glutamate decarboxylase, which catalyzes a key step in GABA synthesis, are lower in brain tissue taken from schizophrenia patients. Those earlier studies were treated with some skepticism, however, because it was thought that the losses may be related to perimortem brain changes rather than the disease itself, Benes said. But further evidence, including decreased GABA uptake, loss of GABAergic interneurons, and compensatory increases in GABA receptors and GABA binding, helped to solidify the idea that GABAergic transmission is dysfunctional in schizophrenia. In addition, the GAD67 isoform of glutamate decarboxylase is reduced in the prefrontal cortex (see Guidotti et al., 2000), and both GAD67 and the smaller GAD65 isoform are reduced in the hippocampus of schizophrenia patients (see Heckers et al., 2002). Genetic polymorphisms in GAD genes have also been linked to schizophrenia (see SRF related news story).

How do GABAergic deficits fit in with other theories of schizophrenia? For one thing, GABAergic dysfunction may be related to loss of NMDA receptor-positive GABAergic cells, which may be a consequence of increased glutamatergic innervation, suggested Benes, thus tying together the glutamatergic and GABAergic components of the disease. Important clues to the role of GABA might be come from studying exactly which GABAergic neurons are affected in schizophrenia.

Benes postulated that amygdalar-hippocampal circuits might play a role in regulating hippocampal GABAergic neurons. To test this, she has developed an in vivo rat model where amygdalar innervation to the hippocampus is disrupted by an intra-amygdalar infusion of picrotoxin, an antagonist for the GABAA receptor. The picrotoxin treatment causes a loss of various GABAergic cells in the CA2 and CA3 region of the hippocampus, including parvalbumin-, calretinin-, and calbindin-positive cells, suggesting innervation by amygdalar neurons is crucial for the well-being of hippocampal interneurons. In addition, inhibitory postsynaptic potentials in the CA3 region of the hippocampus, but not in the CA1, are disrupted by amygdalar picrotoxin treatment (see SRF related news story).

While this picrotoxin treatment may serve as a model for GABAergic dysfunction in schizophrenia, what might underlie amygdalar-hippocampal disruptions in the disease proper? To address this, Benes has used laser dissection microscopy followed by microarray analysis to identify expression changes in the CA2/3 sector in brain samples from schizophrenia patients. Last June she reported that GAD67 expression in the CA2 and CA3 layers of the hippocampus are dramatically lower in both bipolar and schizophrenia patients. She has also found altered expression, either up- or downregulation, of other genes, which can be linked to protein networks that may impinge on GAD expression. GRIK2 and GRIK3, coding for kainate receptor subunits, are upregulated in schizophrenia patients, for example, while genes that may affect GAD more indirectly, such as histone deacetylase 1 and DAXX, are also upregulated in the hippocampus (see SRF related news story). The findings suggest that activity-driven changes in afferent inputs to GABA cells may contribute to unique patterns of gene expression in GABA cells at different points along complex circuits, concluded Benes.

The involvement of the amygdalar-hippocampal circuitry may also help explain the timing of schizophrenia onset, which occurs predominantly during adolescence. Benes showed that amygdalar fibers infiltrate the rat cingulate cortex gradually after birth, reaching maximal innervation during adolescence. At around the same time, these fibers begin to form contacts with GABAergic soma and dendrites in the anterior cingulate cortex. Thus, the ingrowth of amygdalar fibers during adolescence may induce complex molecular changes in GABAergic cells receiving direct inputs from this region. Should this input go awry, perhaps together with altered dopaminergic input, the risk for schizophrenia could be elevated, she concluded.

The involvement of dopamine was addressed by Arvid Carlsson from the University of Gothenburg, Sweden. By one measure, the hypothesis of a dopamine dysfunction in schizophrenia may be considered the most valid—except for the recent clinical trial success of a metabotropic glutamate agonist (see SRF related news story), all antipsychotic drugs so far developed to treat the disease are dopaminergic antagonists. Though that might suggest that pharmacological exploitation of the dopaminergic system is nigh exhausted, Carlsson ventured that as far as dopamine receptor pharmacology is concerned “serious R & D has hardly started yet.” He suggested that the development of new high-throughput screening (HTS) techniques was at least partly to blame for the dearth of new drugs. HTS disregards entirely that the same D2 dopaminergic receptor in one cell may do something entirely different in another cell, he said. Among the shortcomings of HTS, he asserted that the most common version measures the competition of the test molecule with another D2 receptor antagonist (radioactively labeled), based on the assumption that it can predict competition with the physiological agonist dopamine. Also, sometimes the labeled ligand is a D2 receptor agonist other than dopamine, "a fairly hazardous procedure," according to Carlsson.

Almost without exception, Carlsson said, the existence of allosteric binding sites is disregarded. Thus, he concluded, HTS casts too wide a web, and the most interesting drug candidates may well be lost. To illustrate this he described drugs that bind to allosteric sites of the GABAA receptor, such as benzodiazepines, barbiturates, ethanol, and steroids. A similar situation may well exist in the case of dopamine receptors, he noted.

Carlsson reviewed some of the properties of a D2 receptor antagonist called OSU6162. This has low affinity for the D2 receptor, yet it can stabilize behavior by reducing locomotor activity in animals given amphetamines or exposed to a novel stimulating environment, while increasing locomotion in animals that have been habituated to the environment. OSU6162 has a biphasic response curve, enhancing the effect of dopamine at low doses but inhibiting it at higher doses. The drug seems to have complex actions on both orthosteric and allosteric sites. This all speaks to the value of looking at allosteric effects, suggested Carlsson.

Could such dopaminergic modulators be of pharmacological value? Carlsson and colleagues have tested OSU6162 and ACR 16, a similar dopaminergic stabilizer, in schizophrenia patients and found that they reduce both positive and negative symptoms of the disease (in contrast, currently used antipsychotics only ameliorate the positive symptoms). These drugs have also shown promise in Phase 1 and 2 trials for Huntington’s disease, a broad spectrum neurodegenerative disorder with a variety of neurologic and psychiatric symptoms, including psychosis.

Lisman tied the glutamatergic, GABAergic, and dopaminergic components of schizophrenia together in his "halftime" talk. He suggested that the big picture is dominated by two loop circuits. The first loop is between the glutamatergic pyramidal cells of the hippocampus and the dopaminergic neurons they activate in the ventral tegmental area (VTA). Those dopaminergic neurons, in turn, feed back to the pyramidal cells. The second loop is between the pyramidal cells and the GABAergic inhibitory neurons in the hippocampus.

The idea that there is an interaction between the hippocampus, a major site for learning and memory in the brain, and the dopaminergic system came about by the discovery that novelty, such as simply opening a cage door, causes dopamine cells in the VTA to fire. It is not unexpected that the hippocampus would compute novelty, said Lisman, but the interesting thing is the relationship with the VTA. Dopamine release by the VTA has a strong effect in the transition between early and late long-term potentiation (LTP), he explained. Knocking out the D1 dopamine receptor, for example, blocks LTP in the CA1 region of the hippocampus. Lisman said he believes this is the circuit that is affected in schizophrenia, and he outlined growing evidence to support this contention (Lisman and Grace, 2005). The hippocampus becomes chronically activated in the disease, as evident by imaging analysis showing increased cerebral blood volume in the CA1 region of the hippocampus in schizophrenia patients (see Schobel et al., in press) Chemical stimulation of the ventral hippocampus is sufficient to activate dopaminergic neurons in the VTA (see Legault et al., 2000). And in an animal model of the disease, prenatal methylazoxymethanol acetate (MAM) administration, which leads to significantly greater number of spontaneously firing ventral tegmental dopaminergic neurons, disruption of hippocampal activity by tetrodotoxin reverses the elevated dopaminergic activity (see SRF related news story). Taking all the evidence together, the importance of the hippocampus-VTA loop is clear, Lisman said.

The second loop connects the glutamatergic and GABAergic components. What is the relationship between these two sets of neurons? asked Lisman. One key factor could be GAD67. Benes showed that GAD67 is reduced in schizophrenia, and it is well known that NMDA-type glutamate receptor activation stimulates interneurons and that NMDA antagonists lead to a reduction in GAD67. Why is that? Lisman asked. He ventured that there may be some homeostatic control over GAD67 synthesis. For example, he pointed out that Margarita Behrens’s group showed that NMDA antagonist-induced loss of GAD67 can be blocked if calcium levels are elevated (see Kinney et al., 2006). This may be in keeping with the role of fast spiking interneurons, which is to gather information and then process that information to stabilize firing. Calcium entry via NMDA activation may be a key sensor in this process, suggested Lisman. Blocking the NMDA receptor may lead the interneuron to “conclude” that firing is low and that inhibition needs to be relieved. Stopping synthesis of GABA by reducing GAD would be one way to re-balance the system.

One additional level of control over the pyramidal-interneuron loop may be via cholinergic innervation. Lorna Role, State University of New York, Stony Brook, reviewed some of the evidence linking nicotinic acetylcholine receptors with neuregulin, which has turned out to be one of the most studied genes in genetic association studies of schizophrenia. Role and colleagues discovered type 3 neuregulin when searching for regulators of nicotinic receptors. Type 3 neuregulin is essential for viability, since homozygous knockout mice die at birth. Type 3 neuregulin is found in the ventral hippocampus and also in the ventral subiculum, said Role. It is actively transported down axons and seems to colocalize with α7 nicotinic receptors. So what is it doing?

Clues come from heterozygous type 3 neuregulin knockouts. Though these animals do survive past birth, they have altered corticolimbic circuitry and altered behavior, including deficits in short-term memory and in gating sensory stimuli. “The phenotype is reminiscent of some schizophrenia endophenotypes,” said Role. The heterozygous mice perform poorly in a maze test of memory and in tests of prepulse inhibition. The latter is almost fully corrected by administration of nicotine (cigarette smoking is also very prevalent among schizophrenia patients).

What is the underlying cause of these behavioral deficits? Role showed that the animals have fewer parvalbumin-positive interneurons in the mouse equivalent of the human prefrontal cortex. In addition, pyramidal neurons do not seem quite normal. For example, the spines along the axons in the subiculum do not reach the same density as in wild-type mice, particularly in the middle of the axons, where spine levels are almost half wild-type levels. There are also functional consequences. The power of γ band oscillations is weaker than normal and gating-related LTP is lost. Also, unlike wild-type mice where nicotine can elicit sustained changes in the firing pattern in the ventral hippocampus and striatum, those changes are not sustained in type 3 neuregulin heterozygotes.

What are the molecular correlates of these functional changes? The bottom line is that neuregulin is important for targeting of α7-nicotinic receptors to enhance glutamate release in corticolimbic circuits, said Role. Decreased expression of neuregulin and/or α7 nicotinic receptors limits nicotine effects to transient facilitation, she said. In wild-type circuits nicotine effectively lowers the threshold for activity-dependent LTP, whereas in neuregulin heterozygotes LTP cannot be elicited even with tetanic stimulation.

Robert Freedman, from the University of Colorado, Denver, also addressed the involvement of nicotinic receptors in the pathology of schizophrenia. Freedman’s lab found that α-Bungarotoxin, a nicotinic receptor antagonist, affects sensory gating in animals, resulting in behaviors that resemble some endophenotypes found in schizophrenia patients. For example, patients respond almost as strongly to a second auditory stimulus as to one that briefly precedes it, while normal volunteers react much more weakly to the second stimulus. Inhibiting the nicotinic receptor in animals elevates the second sound-induced event-related potential (ERP) to rival the first, much like in patients.

Because the α7-nicotinic receptor gene (CHRNA7) has been cloned, Freedman and colleagues focused on that gene to tease out its relevance to sensory gating. They found that GABA and α-Bungarotoxin colocalize, showing that the nicotinic receptor is present in inhibitory neurons, and they reported that a loss of the receptor in CHRNA7 heterozygous null mice leads to an increase in the ratio of the second to first ERPs. But how do these findings relate to patients?

To test this, Freedman and colleagues have looked at genetic variation in the CHRNA7 gene. He reported that in a large family with two offspring that have schizophrenia, a common allele in the promoter of the gene is always associated with normal sensory gating, whereas a variant allele is associated with abnormal gating, even in individuals who did not have schizophrenia (see also Martin et al., 2007). In humans, there are also decreases in α-Bungarotoxin binding in postmortem brain tissue taken from schizophrenia patients, said Freedman, supporting the idea that nicotinic receptor levels may be linked to pathology.

These correlations between nicotinic receptor deficiency and schizophrenia-related phenotypes and behaviors may explain the prevalence of smoking among patients (by some estimates 90 percent of schizophrenia patients are smokers). But smoking is obviously not the best method of treatment, said Freedman. He has been involved in developing better nicotinic agonists. One of them, DMXB-A (3-[(2,4-dimethoxy)benzylidene]anabaseine), has been tried in a proof-of-concept trial, and Freedman reported that the drug improved scores in several measures including the RBANS (Repeatable Battery for the Assessment of Neuropsychological Status) and measures of auditory sensory gating. Patients also seemed to show improvement in scores of negative symptoms (SANS, Scale for Assessment of Negative Symptoms) and reported feeling better, said Freedman. In summary, there are a number of ways to get inhibitory neurons to function better, and α7-nicotinic receptor agonists may be one of them, he said.

Last but by no means least, Claudia Racca from Newcastle University, England, tied together the GABAergic and glutamatergic theories of schizophrenia. In this disorder there is both disruption of cortical γ oscillations (see Spencer et al., 2004) and reduction of parvalbumin-positive GABAergic interneurons, and the two are related because the γ oscillations depend on these fast-spiking interneurons. Racca is interested in modeling these losses in animals. She reported on two distinct models. One is an in vitro model of acute psychosis induced by ketamine, an NMDA receptor antagonist, while the other is a genetic/developmental animal model based on knocking out the LPA1 receptor gene. LPA1 is a receptor for lysophosphatidic acid, and though its biological role is not entirely clear, loss of the receptor leads to developmental problems that precipitate some phenotypes that are relevant to schizophrenia, including altered sensorimotor gating.

Racca’s findings have been covered in previous SRF news (see SRF related news story). Briefly, she showed that there is a drastic reduction in the power of γ oscillations in layer II of the entorhinal cortex in LPA1 knockout animals. In contrast, γ power in layers V-VI is unaltered. This reduction of γ power correlated with decrease in amplitude and frequency of GABA inhibitory postsynaptic potentials in stellate cells and their disinhibition, leading to increased spiking. This is accompanied by a specific loss of parvalbumin immunoreactivity in layer II of the entorhinal cortex. The reduction in γ power in layer II and the disinhibition of stellate cells are mimicked in the ketamine model. Other studies showed that chronic ketamine treatment also reduced parvalbumin immunoreactivity in neocortex (see Kinney et al., 2006 and SRF related news story). What seems to connect both these models is that GABAergic parvalbumin-positive interneurons in layer II of entorhinal cortex have a large NMDA receptor drive, noted Racca. The findings thus link glutamatergic systems and GABAergic systems and suggest that glutamatergic hypofunction may precipitate a disruption of γ oscillations, which is a proposed endophenotype of schizophrenia.

Lisman concluded the symposium by recounting the importance of the interaction between pyramidal cells and parvalbumin-positive interneurons. NMDAR hypofunction in the parvalbumin interneurons leads to disinhibition and reduced γ oscillations, perhaps contributing to the negative and cognitive symptoms of schizophrenia. The disinhibition in the hippocampus leads to hyperactivation of the hippocampal-VTA loop, leading to increased dopamine release and the positive symptoms of the disease. The beauty of having specific network models is that you can go at it with experimental tests, he suggested. In that vein, knocking out NMDA receptors on parvalbumin interneurons might be an excellent model for schizophrenia, Lisman suggested.—Tom Fagan.

Comments on Related News


Related News: Gamma Band Plays a Sour Note in Entorhinal Cortex of Schizophrenia Models

Comment by:  Bita Moghaddam, SRF Advisor
Submitted 3 April 2006
Posted 3 April 2006

Cortical dysfunction in schizophrenia has been attributed to both inhibitory GABA and excitatory glutamate neurotransmission. Abnormalities in cortical GABA neurons have been observed primarily in the subset of GABA interneurons that contain the calcium-binding protein parvalbumin (PV). The glutamatergic dysfunction is suspected primarily because reducing glutamate neurotransmission at the NMDA receptors produces behavioral deficits that resemble symptoms of schizophrenia. These two mechanisms have been generally treated as separate conjectures when conceptualizing theories of schizophrenia. The paper by Cunningham et al. demonstrates that, in fact, disruptions in PV positive cortical GABA neurons and blockade of NMDA receptors produce similar disruptions to the function of cortical networks.

The authors used lysophosphatidic acid 1 receptor (LPA-1)-deficient mice which, they argue, are a relevant model of schizophrenia because these animals display sensorimotor gating deficits, a critical feature of schizophrenia. They demonstrate that, similar to schizophrenia, the number of PV positive GABA neurons is significantly reduced in LPA-1-deficient mice. Furthermore, the γ frequency network oscillation disruptions they observe in these animals are similar to those seen in wild-type mice treated with the NMDA antagonist ketamine. (γ oscillations have been associated with sensory processing and deficits in γ rhythm generation have been reported in patients with schizophrenia during performance of sensory processing tasks.) The disruptive effect of ketamine on γ oscillations was mediated by a decrease in the output of fast-spiking GABA interneurons causing a disinhibition (i.e., increased firing) of glutamate neurons. These findings are significant because they suggest that cortical NMDA hypofunction may cause the reported GABA interneuron deficits in schizophrenia.

View all comments by Bita Moghaddam

Related News: Gamma Band Plays a Sour Note in Entorhinal Cortex of Schizophrenia Models

Comment by:  Patricio O'Donnell, SRF Advisor
Submitted 7 April 2006
Posted 7 April 2006

Animal models of schizophrenia and other psychiatric disorders are receiving increasing interest, as they provide useful tools to test possible pathophysiological scenarios. Some models have been tested with a wide array of approaches and many others continue to develop. If one focuses on possible cortical alterations, a critical issue emerging from many different lines of research using several different models is the apparent contradiction between the hypo-NMDA concept and the data suggesting a loss of cortical interneurons. Is there a hypo- or a hyperactive cortex?

This conundrum has been present since earlier days in the postmortem and clinical research literature, but with the advent of more refined animal models, it may be time to provide a possible way in which these discrepant sets of data can be reconciled. Whether this was the authors’ intention or not, the article by Cunningham and colleagues is an excellent step in that direction. This study used mice deficient in lysophosphatidic acid 1 receptor, a manipulation that reduced the GABA and parvalbumin-containing interneuron population by about 40 percent and disrupted γ (rapid) oscillations in the entorhinal cortex. A key element in this study was the finding that a similar alteration in rapid cortical oscillations was observed with the noncompeting NMDA antagonist ketamine. There is a large body of evidence indicating that interneurons (in particular, the fast-spiking type that include parvalbumin-positive neurons) are critical for synchronization of fast cortical oscillatory activity. As fast oscillations can be envisioned as phenomena with deep impact on cognitive functions, these findings may have bearing on possible pathophysiological scenarios underlying cognitive deficits in schizophrenia. This article does provide a strong indication that antagonism of NMDA receptors may selectively target cortical interneurons. This is in agreement with the work of Bita Moghaddam, who has shown that noncompeting NMDA antagonists can indeed increase pyramidal cell firing and glutamate levels in the prefrontal cortex. Thus, it is conceivable that psychotomimetic agents such as PCP or ketamine exert their cognitive effects by impairing interneuronal activity, hampering the fine-tuning of pyramidal cell firing that is expressed as fast cortical oscillations.

View all comments by Patricio O'Donnell

Related News: Genetics, Expression Profiling Support GABA Deficits in Schizophrenia

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 Mirnics

Related News: Genetics, Expression Profiling Support GABA Deficits in Schizophrenia

Comment 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

Related News: Studies Explore Glutamate Receptors as Target for Schizophrenia Monotherapy

Comment by:  Dan Javitt, SRF Advisor
Submitted 3 September 2007
Posted 3 September 2007

A toast to success, or new wine in an old skin?
Patil et al. present a landmark study. It is the kind of study that represents the best of how science should work. It pulls together the numerous strands of schizophrenia research from the last 50 years, from the development of PCP psychosis as a model for schizophrenia in the late 1950s, through the links to glutamate, the discovery of metabotropic receptors, and the seminal discovery in 1998 by Moghaddam and Adams that metabotropic glutamate 2/3 receptor (mGluR2/3) agonists reverse the neurochemical and behavioral effects of PCP in rodents (Moghaddam and Adams, 1998. The story would not be possible without the elegant medicinal chemistry of Eli Lilly, which provided the compounds needed to test the theories; the research support of NIMH and NIDA, who have been consistent supporters of the “PCP theory”; or the hard work of academic investigators, who provided the theories and the platforms for testing. The study is large and the effects robust. Assuming they replicate (and there is no reason to suspect that they will not), this compound, and others like it, will represent the first rationally developed drugs for schizophrenia. Patients will benefit, drug companies will benefit, and academic investigators and NIH can feel that they have played their role in new treatment development.

Nevertheless, it is always the prerogative of the academic investigator to ask for more. In this case, we do not yet know if this will be a revolution in the treatment of schizophrenia, or merely a platform shift. What is striking about the study, aside from the effectiveness of LY2140023, is the extremely close parallel in both cross-sectional and temporal pattern of response between it and olanzapine. Both drugs change positive and negative symptoms in roughly equal proportions, despite their different pharmacological targets. Both drugs show approximately equal slopes over a 4-week period. There is no intrinsic reason why symptoms should require 4 or more weeks to resolve, or why negative and positive symptoms should change in roughly the same proportion with two medications from two such different categories, except that evidently they do.

There are many things about mGluR2/3 agonists that we do not yet know. The medication used here was administered at a single, fixed dose. It is possible that a higher dose might have been better, and that optimal results have not yet been achieved. The medications were used in parallel. It is possible that combined medication might be more effective than treatment with either class alone. The study was stopped at 4 weeks, with the trend lines still going down. It is possible that longer treatment duration in future studies might lead to even more marked improvement and that the LY and olanzapine lines might separate. No cognitive data are reported. It is possible that marked cognitive improvement will be observed with these compounds when cognition is finally tested, in which case a breakthrough in pharmacotherapy will clearly have been achieved.

If one were to look at the glass as half empty, then the question is why the metabotropic agonist did not beat olanzapine, and why the profiles of response were so similar. If these compounds work, as suggested in the article by modulating mesolimbic dopamine, then it is possible that metabotropic agonists will share the same therapeutic limitations as current antipsychotics—good drugs certainly and without the metabolic side effects of olanzapine, but not “cures.” The recent study with the glycine transport inhibitor sarcosine by Lane and colleagues showed roughly similar overall change in PANSS total (-17.1 pts) to that reported in this study, but larger change in negative symptoms (-5.5 pts), and less change in positive symptoms (-2.3 pts) in a similar type of patient population. Onset of effect in the sarcosine study also appeared somewhat faster. The sarcosine study was smaller (n = 20) and did not include a true placebo group. As with the Lilly study, it was only 4 weeks in duration, and did not include cognitive measures. It also included only two, possibly non-optimized doses. As medications become increasingly available to test a variety of mechanisms, side-by-side comparisons will become increasingly important.

There are also causes for concern and effects to be watched. For example, a side effect signal was observed for affect lability in the LY group, at about the same prevalence rate as weight increase in the olanzapine group. What this means for the mechanism and how this will effect treatment remains to be determined. Since these medications are agonists, there is concern that metabotropic receptors may downregulate over time. Thus, whether treatment effects increase, decrease, or remain constant over the course of long-term treatment will need to be determined. Nevertheless, 50 years since the near-contemporaneous discovery of both PCP and chlorpromazine, it appears that glutamatergic drugs for schizophrenia may finally be on the horizon.

References:

Moghaddam B, Adams BW. Reversal of phencyclidine effects by a group II metabotropic glutamate receptor agonist in rats. Science. 1998 Aug 28;281(5381):1349-52. Abstract

View all comments by Dan Javitt

Related News: Studies Explore Glutamate Receptors as Target for Schizophrenia Monotherapy

Comment by:  Gulraj Grewal
Submitted 4 September 2007
Posted 4 September 2007
  I recommend the Primary Papers

Related News: Dopamine Problems? Blame the Hippocampus

Comment by:  Anissa Abi-Dargham, SRF Advisor
Submitted 29 November 2007
Posted 29 November 2007

What struck me most about the paper of Lodge and Grace is the overall consistency of the body of work between the preclinical and clinical observations, even down to the effect size for the dopaminergic alteration. Dopamine release in schizophrenia is at least double that in controls; whether measured after amphetamine (on average 17 percent displacement of the benzamide radiotracer versus 7 percent in controls) (Laruelle et al., 1999) or at baseline (19 percent D2 occupancy by dopamine in patients versus 9 percent in controls) (Abi-Dargham et al., 2000), the increase in dopamine activity in VTA of the MAM rats reported here is also a doubling of what is measured in saline-treated rats.

This work presents an important contribution to the field because it clarifies the role of the hippocampus in one of the cardinal features of the disorder as modeled in MAM rats. The fact that MAM treatment is one of the most valid animal models of schizophrenia—it replicates many of the disturbances, neurochemical, cellular, dendritic, morphometric, and behavioral, observed in schizophrenia—makes the finding very compelling.

The role of an abnormal hippocampal node in an important circuit central to the pathophysiology of schizophrenia has face validity: there are now many converging lines of evidence in patients with schizophrenia for alterations in hippocampal volume, cytoarchitecture, function, and neurochemical indices. What this paper presents that is unique is evidence, in a valid model of schizophrenia, for an etiological link between the faulty hippocampus and the faulty VTA. The next step will be to test an association between pathology of the hippocampus and that of the VTA and related striatal output in patients with schizophrenia. This is a study we currently are conducting, and is an example of translational research where a theory gets support and contributions by going back and forth between preclinical and clinical testing. If there is an association in the same patients between the hippocampal pathology and dopamine dysregulation, it will suggest that what is described for the MAM model here may be true for schizophrenia, too, i.e., that the pathology of the dopamine system is driven by a faulty hippocampal input.

References:

Laruelle M, Abi-Dargham A, Gil R, Kegeles L, Innis R. Increased dopamine transmission in schizophrenia: relationship to illness phases. Biol Psychiatry. 1999;46:56-72. Abstract

Abi-Dargham A, Rodenhiser J, Printz D, Zea-Ponce Y, Gil R, Kegeles LS, Weiss R, Cooper TB, Mann JJ, Van Heertum RL, Gorman JM, Laruelle M. Increased baseline occupancy of D2 receptors by dopamine in schizophrenia. Proc Natl Acad Sci U S A. 2000;97:8104-8109. Abstract

View all comments by Anissa Abi-Dargham

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

Comment by:  John Krystal, SRF Advisor
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

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

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

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

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

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

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
View all comments by James I. Koenig

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

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

Related News: Dopamine Problems? Blame the Hippocampus

Comment by:  Elizabeth Tunbridge
Submitted 20 December 2007
Posted 20 December 2007
  I recommend the Primary Papers

In their recent paper Lodge and Grace elegantly demonstrate that hyperactivity of the ventral hippocampus underlies the elevated number of spontaneously active ventral tegmental dopamine neurons, and the concomitant increase in amphetamine-induced locomotor activity, found in MAM-treated rats. Since neonatal MAM treatment recapitulates some of the neurochemical, anatomical, and behavioral abnormalities associated with schizophrenia, these findings raise the possibility that the abnormal subcortical dopamine function associated with this disorder might also result from hippocampal dysfunction.

These findings are consistent with a wealth of evidence suggesting that the hippocampus is a prominent site of dysfunction in the schizophrenic brain (reviewed in Harrison, 2004), and it will be exciting to see the results of the clinical studies described by Anissa Abi-Dargham above.

In the future, it will be important to try to integrate these findings with other models aiming to explain the subcortical dopaminergic hyperactivity seen in schizophrenia. One well-known hypothesis is that these abnormalities might result from hypofunction of the prefrontal cortex (PFC; Weinberger, 1987; Bertolino et al., 2000). Animal studies demonstrate that PFC activity impacts on striatal dopamine function (e.g., Shim et al., 1996) and vice versa (Kellendonk et al., 2006). Thus, it will be of interest to assess the relative contributions of hippocampal and prefrontal dysfunction to these subcortical abnormalities in schizophrenia. Such investigations will necessarily involve the use of both patient populations and appropriate animal model systems. A difficult question will be to establish whether any one of these three regions represents a site of a primary “lesion” in schizophrenia or, perhaps more likely, whether their dysfunction reflects abnormalities in the circuits that link them.

References:

Bertolino A, Breier A, Callicott JH, Adler C, Mattay VS, Shapiro M, Frank JA, Pickar D, Weinberger DR. The relationship between dorsolateral prefrontal neuronal N-acetylaspartate and evoked release of striatal dopamine in schizophrenia. Neuropsychopharmacology. 2000 Feb;22(2):125-32. Abstract

Harrison PJ. The hippocampus in schizophrenia: a review of the neuropathological evidence and its pathophysiological implications. Psychopharmacology (Berl). 2004 Jun;174(1):151-62. Epub 2004 Mar 6. Review. Abstract

Kellendonk C, Simpson EH, Polan HJ, Malleret G, Vronskaya S, Winiger V, Moore H, Kandel ER. Transient and selective overexpression of dopamine D2 receptors in the striatum causes persistent abnormalities in prefrontal cortex functioning. Neuron. 2006 Feb 16;49(4):603-15. Abstract

Shim SS, Bunney BS, Shi WX. Effects of lesions in the medial prefrontal cortex on the activity of midbrain dopamine neurons. Neuropsychopharmacology. 1996 Nov;15(5):437-41. Abstract

Weinberger DR. Implications of normal brain development for the pathogenesis of schizophrenia. Arch Gen Psychiatry. 1987 Jul;44(7):660-9. Abstract

View all comments by Elizabeth Tunbridge

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

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

Related News: Studies Explore Glutamate Receptors as Target for Schizophrenia Monotherapy

Comment by:  Shoreh Ershadi
Submitted 8 June 2008
Posted 9 June 2008
  I recommend the Primary Papers

Related News: Getting Specific: Conditional Knockouts Address Glutamate Hypothesis

Comment by:  Margarita Behrens
Submitted 17 November 2009
Posted 17 November 2009

Since the discovery that phencyclidine and its analog ketamine exert their pro-psychotic effects through antagonism of NMDA receptors (Javitt and Zukin, 1991), the mechanisms by which these drugs exert these effects have been the subject of intensive research. These studies led to the hypo-NMDA theory of schizophrenia by Olney and collaborators that proposed that “blockade of NMDA receptors triggers a complex network disturbance featuring inactivation of inhibitory neurons and consequent disinhibition of excitatory pathways…” (Olney et al., 1999). Based on the effects of prolonged exposure of primary cultured neurons to selective and non-selective NMDAR antagonists, it was proposed that NMDARs expressed by the subpopulation of parvalbumin-positive (PV) fast spiking interneurons were the target of the antagonists, and that these glutamate receptors played a fundamental role in the maintenance of the GABAergic phenotype of the interneurons (Kinney et al., 2006). Using the Cre-LoxP system to produce the selective ablation of NMDARs in mouse corticolimbic interneurons, Kazu Nakasawa and colleagues now elegantly support this hypothesis in the latest issue of Nature Neuroscience (Belforte et al., 2009). Furthermore, they demonstrate the neurodevelopmental origin of schizophrenia-like behaviors by showing that it is the dysfunction of NMDARs during the period of active maturation of PV-interneurons that increases the chance of behavioral disruptions in late adolescence/early adulthood. These results give strong support to the hypothesis that disruption of the normal maturation of PV-interneurons will produce permanent changes of the inhibitory circuitry in cortex, thus profoundly affecting cortical network function (Behrens and Sejnowski, 2009).

An interesting outcome of Belforte’s results is that, per se, the diminished activity of NMDARs in PV-interneurons does not lead to behavioral disruption, but when these animals undergo the stress of being reared in isolation they manifest the schizophrenia-like behavior. The effects of isolation rearing on PV-interneurons and behavior were recently related to the activation of the superoxide producing enzyme NADPH-oxidase (Nox2) in brain (Schiavone et al., 2009). Treatment of these animals with the Nox2 inhibitor apocynin prevented the loss of GABAergic phenotype of PV-interneurons as well as the behavioral derangements produced by the isolation rearing.

These results have bearing on the effects of NMDAR antagonist exposure, where it was shown that activation of this same enzyme (Nox2) is responsible for the effects of the antagonists on the GABAergic phenotype of PV-interneurons (Behrens et al., 2007; Behrens et al., 2008). Therefore, we can speculate that the pro-psychotic effects of NMDAR-antagonists occur by a double-hit mechanism: first, blocking NMDAR activity in PV-interneurons leads to the loss of their GABAergic phenotype; and, second, inducing the activation of the IL-6/Nox2 pathway further promotes this loss even in the absence of the antagonist. However, it is still not clear why diminished activity of NMDARs in PV-interneurons is only consequential during the period of active maturation of PV-interneuronal circuits, and renders the cortical circuitry vulnerable to the sustained activation of the IL-6/Nox2 pathway. One possible answer is that inactivation of NMDARs in PV-interneurons during early postnatal development disrupts the development of PV-interneuronal synaptic contacts. This could lead to cortical networks that have all neurons in place but with a subset dysfunctional. In turn, this faulty network may be more vulnerable to the effects of activation of the IL-6/Nox2 pathway, such that when this pathway is activated, i.e., by social isolation, it leads to aberrant oscillatory activity in brain and cognitive disruption as observed in schizophrenia.

References:

Javitt DC, Zukin SR. Recent advances in the phencyclidine model of schizophrenia. Am J Psychiatry. 1991 Oct 1;148(10):1301-8. Abstract

Olney JW, Newcomer JW, Farber NB. NMDA receptor hypofunction model of schizophrenia. J Psychiatr Res. 1999 Nov-Dec ;33(6):523-33. 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

Belforte JE, Zsiros V, Sklar ER, Jiang Z, Yu G, Li Y, Quinlan EM, Nakazawa K. Postnatal NMDA receptor ablation in corticolimbic interneurons confers schizophrenia-like phenotypes. Nat Neurosci. 2009 Nov 15. Abstract

Behrens MM, Sejnowski TJ. Does schizophrenia arise from oxidative dysregulation of parvalbumin-interneurons in the developing cortex? Neuropharmacology. 2009 Sep 1;57(3):193-200. Abstract

Schiavone S, Sorce S, Dubois-Dauphin M, Jaquet V, Colaianna M, Zotti M, Cuomo V, Trabace L, Krause KH. Involvement of NOX2 in the development of behavioral and pathologic alterations in isolated rats. Biol Psychiatry. 2009 Aug 15;66(4):384-92. Abstract

Behrens MM, Ali SS, Dao DN, Lucero J, Shekhtman G, Quick KL, Dugan LL. Ketamine-induced loss of phenotype of fast-spiking interneurons is mediated by NADPH-oxidase. Science. 2007 Dec 7;318(5856):1645-7. Abstract

Behrens MM, Ali SS, Dugan LL. Interleukin-6 mediates the increase in NADPH-oxidase in the ketamine model of schizophrenia. J Neurosci. 2008 Dec 17;28(51):13957-66. Abstract

View all comments by Margarita Behrens