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SfN 2013—Interneuron Dysfunction Poster Tour

See Allison Curley's snapshots from the conference.

January 28, 2014. Inhibitory interneurons, especially the 25 percent that contain the calcium-binding protein parvalbumin (PV), have emerged as key characters in the ever unfolding schizophrenia story, although their exact role in the illness—and the extent to which they are primary drivers of symptoms or more downstream consequences of other pathological changes—remains to be uncovered (see SRF related news report). A large number of posters at the Neuroscience 2013 meeting held in San Diego explored the role of GABA neuron dysfunction in the impaired oscillations and cognitive deficits of schizophrenia. What follows is a small sample of the work presented on this topic.

At the first poster session of the meeting on Saturday afternoon, November 9, Kristen Delevich of Cold Spring Harbor Laboratory, New York, presented her study of prefrontal cortex circuit functioning in a DISC1 heterozygous mouse, a model of the chromosomal translocation associated with mental illness in humans (see SRF related news report). Using optogenetics, Delevich observed a lower paired pulse ratio of currents evoked from PV—but not somatostatin (SST)-containing interneurons—suggestive of a reduction in the GABA release probability in PV interneurons. As a consequence of this reduced release probability, pyramidal cells in the prefrontal cortex received less feed-forward inhibition from the mediodorsal thalamus. This reduced inhibitory input upset the pyramidal cells' normal inhibitory-to-excitatory balance, providing a possible circuit basis for the impaired executive function that is found in both DISC1 mutant mice and schizophrenia. (For more DISC1 news from the conference, see SRF related conference report.)

Going after GAD67
Although the GABA release probability of SST cells was not affected in the DISC1 mutants, on Sunday afternoon Brad Rocco of the University of Pittsburgh, Pennsylvania, presented immunofluorescence data from schizophrenia postmortem tissue suggesting that these cells are far from healthy. SST degrades very quickly after death, so Rocco and colleagues used the process of elimination to find them. By examining axon terminals that contained the GABA-synthesizing enzyme GAD67, but lacked PV or the other GABA maker GAD65 (which their prior research has shown marks cannabinoid 1 basket cells but not SST terminals), the researchers enriched for SST terminals. Rocco observed a 70 percent reduction in the density of the terminals, as well as lower levels of the GAD67 protein present in them. Given the role of these cells in synaptic integration, the researchers suggest that a deficit in these cells in schizophrenia may lead to improper input/output of large groups of neurons.

On Monday afternoon, Sivan Subburaju, McLean Hospital, Belmont, Massachusetts, described an extension of earlier findings on a network of genes associated with GAD67 regulation that are differentially expressed in schizophrenia and bipolar disorder. Using the HiB5 hippocampal cell culture model, the researchers previously showed that the inhibition of two of these genes—epigenetic regulators HDAC1 and Daxx—produced elevated GAD67 mRNA (Subburaju and Benes, 2012). To examine whether inhibition of the two genes in particular can influence other genes involved in the regulation of GAD67, Subburaju and colleagues knocked them down using lentiviral vectors carrying small hairpin RNAi sequences. The knockdown HDAC1 increased HDAC2 and RunX2 and decreased GluR6, while silencing of Daxx increased RunX2 and decreased Pax5, GluR6, and GluR7 expression. The authors concluded that genes in the GAD67 regulatory network have complex interactions with each other that may influence GABA neuron function.

On Tuesday afternoon, Lichao Chen, Harvard University, Boston, Massachusetts, described the use of RNA interference (RNAi) and the Cre-lox recombination system to examine whether the widely reported reduction in GAD67 mRNA is a cause of schizophrenia symptoms or a compensatory response to other cortical abnormalities. In adult mice, knockdown of GAD67 expression specifically in PV neurons in the prefrontal cortex (PFC) produced a significant reduction in the number of PV neurons that had detectable levels of GAD67, but did not affect the power or frequency of kainate-induced γ oscillations in vitro. In contrast, knockdown in adolescent mice showed a trend toward a reduction in γ band power, suggesting lower GAD67 may be a cause of the altered γ oscillations observed in schizophrenia, but a reduction in GAD67 prior to adulthood is needed to produce the effect.

The path to impaired cognition
On Saturday afternoon, Kathleen Cho of the University of California, San Francisco, used a mouse model to link the deficits in PV cell signaling to the cognitive difficulties of schizophrenia through impaired γ oscillations. She reported that mice heterozygous for both Dlx5 and Dlx6 (two transcription factors that are crucial to PV neuron development) exhibit behavioral and EEG abnormalities that are consistent with schizophrenia: a reduction in γ band power during a social exploration task that depends on the PFC, as well as an increase in baseline γ power. Dlx5/6 heterozygous mice also show impairments on some aspects of a cognitive flexibility task, and these deficits are accompanied by disturbed oscillatory activity.

Two Sunday afternoon posters from Tracie Paine’s group at Oberlin College, Ohio, also used a rodent model to probe the role of GABA transmission on specific aspects of cognitive function in schizophrenia. Work presented by Avery O’Hara found that blockade of GABAA receptors, but not GABA synthesis, impaired decision making on a gambling task in rats. Paine presented a second poster suggesting that blocking GABA synthesis also had no effect on attention as measured by the five-choice serial reaction time task. Together, these data suggest that the widely reported deficit in GAD67 in schizophrenia does not contribute to the decision making and attention deficits found in schizophrenia.

In the same session, Rachel So of the University of Connecticut in Storrs presented her work examining neural synchrony patterns and levels of GABA and glutamate/glutamine during a working memory task in schizophrenia. The researchers found that the strength of the synchronization between the dorsolateral PFC and the occipital lobe increased with task difficulty in the schizophrenia subjects but not controls, and that the synchronization was correlated with GABA (but not glutamate/glutamine) levels in the left dorsolateral prefrontal cortex (DLPFC). So and colleagues suggest that the altered neural synchrony patterns in schizophrenia could represent a compensatory strategy to preserve working memory performance.

The excitatory side
On Sunday afternoon, a poster by Tsung-Ung Woo of Harvard Medical School in Boston, Massachusetts, described the mRNA and microRNA expression profiling of neuronal subtypes in schizophrenia. Pyramidal neurons in the PFC of cases exhibited differentially expressed mRNAs in the transforming growth factor β and bone morphogenetic protein signaling pathways, as well as those involved in the cytoskeleton, extracellular matrix, apoptosis, and oxidative stress. PV neurons from the PFC in schizophrenia displayed differentially expressed genes in pathways such as Wnt, Notch, and prostaglandin E2 pathways, along with transcripts that control the cell cycle and apoptosis. Woo and colleagues also observed a predominant downregulation of microRNAs in the illness.

In addition to receiving inputs from other GABA neurons, PV cells also receive synaptic contacts from excitatory pyramidal cells. One hypothesis is that glutamate receptor hypofunction may be the cause of PV dysfunction in schizophrenia (Gonzalez-Burgos and Lewis, 2012), and several Neuroscience 2013 posters modeled this proposed reduction of excitatory inputs onto PV neurons. On Sunday afternoon, Martha Hvoslef-Eide, Cambridge University, U.K., presented a cognitive characterization of mice with a conditional knockout of NR1 glutamate receptors on nearly half of cortical interneurons early in development (see SRF related news report). She reported that NR1 knockouts were significantly impaired on the continuous performance task, a test of attention on which schizophrenia subjects perform poorly, suggesting that conditional NR1 knockouts are a valuable model for studying cognitive dysfunction in schizophrenia.

Recent in vitro data suggest that synaptic events onto PV neurons may not have a strong NMDA component during adulthood. However, the presence of significant tonic NMDA current in PV neurons suggests a role for extrasynaptic NMDA receptors. On the Tuesday morning session, Eastman Lewis presented his work testing this prediction, finding that functional NMDA receptors are present in adult rat PFC. Consistent with the presence of extrasynaptic NMDA receptors, inhibition of glutamate reuptake enhanced the NMDA currents in PV neurons. The results suggest that modulation of extrasynaptic NMDA currents could be used to restore the balance of cortical excitation and inhibition in schizophrenia.

A trio of posters on Tuesday afternoon from the laboratory of Terry Sejnowski at the Salk Institute in La Jolla, California, examined the effects of postnatal ablation of mGluR5 glutamate receptors in PV neurons. The group has previously shown that PV-specific mGluR5 knockout mice exhibit lower levels of GAD67 and fewer inhibitory synapses onto pyramidal cells than controls. Aaron Kappe examined the neural circuit abnormalities by recording epidural auditory event-related potentials. He reported an increased amplitude of P20 and P80 components in both sexes and a decrease in the amplitude of the N40 component in females. Stephanie Barnes examined the behavioral effects of mGluR5 ablation, finding deficits in recognition (but not spatial) memory and social function. The mice also showed a reduced sensitivity to PCP-induced behavioral abnormalities, hinting at NMDA receptor hypofunction. António Pinto-Duarte found that although the mice seemed to develop normally and lacked any obvious gross abnormalities, PV-specific mGluR5 knockouts showed a reduction in the number of PV synaptic contacts in multiple brain areas such as the hippocampus. Basal synaptic transmission was also impaired, suggesting a deficit in the maturation of PV cells. Together, these results suggest that abnormal mGluR5 modulation of PV neurons may play a role in schizophrenia, and provide evidence of their utility as a therapeutic target in the illness (see SRF related conference report).—Allison A. Curley.

Comments on Related News


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

Related News: SfN 2013—Different Roads to Dopamine Dysfunction in Schizophrenia

Comment by:  Melkaye Melka
Submitted 5 December 2013
Posted 9 December 2013

Atypical antipsychotics have been used to treat psychiatric disorders such as schizophrenia. However, the mechanisms of action of antipsychotics remain poorly understood. On the other hand, dopamine neurons form the focus of attention in the etiology and pathophysiology of schizophrenia. As noted by Michele Solis’ snapshot from the conference, the work of Grace and colleagues showed that prenatal injections of methylazoxymethanol acetate (MAM), a DNA-methylating agent, lead to hyperactive dopamine signaling (Moore et al., 2006). Focusing on the mechanisms of action, previous studies have suggested that antipsychotic drugs may cause promoter methylation of genes involved in psychosis (Dong et al., 2009). DNA methylation changes have also been associated with major psychosis (Mill et al., 2008).

In a recent in-vivo study, we have observed organ-specific (hippocampus, cerebellum, and liver) changes in DNA methylation following a therapeutic dose of a model antipsychotic drug (olanzapine) (Melka et al., 2013, in press). In particular, we noted that the dopamine signaling pathway was one of the most significant networks affected by olanzapine-induced DNA methylation changes. Specifically, the results showed that olanzapine significantly alters promoter DNA methylation of genes involved in dopamine synthesis, transport, receptors, and metabolism. These results support a dopamine hypothesis of psychosis and a role for epigenetic mechanisms in the development of psychosis, as well as its treatment with antipsychotic drugs. Given that some of the genes affected are tissue specific and affect a variety of networks, our results could also explain the delayed therapeutic response of antipsychotics as well as their patient-specific efficacy and side effects.

References:

Dong E, Grayson DR, Guidotti A, Costa E. Antipsychotic subtypes can be characterized by differences in their ability to modify GABAergic promoter methylation: Epigenomics 2009; 1: 201-1. Abstract

Melka MG, Castellani CA, Laufer BI, Rajakumar N, O’Reilly R and Singh SM. Olanzapine induced DNA methylation changes support the dopamine hypothesis of psychosis. J Mol Psychiatry; 2013; 1:19. (In press)

Mill J, Tang T, Kaminsky Z, Khare T, Yazdanpanah S, Bouchard L, Jia P, Assadzadeh A, Flanagan J, Schumacher A, Wang SC, Petronis A. Epigenomic profiling reveals DNA-methylation changes associated with major psychosis. Am J Hum Genet . 2008 Mar ; 82(3):696-711. Abstract

Moore H, Jentsch JD, Ghajarnia M, Geyer MA, Grace AA. A neurobehavioral systems analysis of adult rats exposed to methylazoxymethanol acetate on E17: implications for the neuropathology of schizophrenia. Biol Psychiatry . 2006 Aug 1 ; 60(3):253-64. Abstract

View all comments by Melkaye Melka

Related News: SfN 2013—New Tools for Rational Drug Design

Comment by:  Hugo Geerts
Submitted 29 January 2014
Posted 5 February 2014

Multi-target drug discovery has typically been neglected in the world of genetics and high-throughput screening because of the difficulty of rationally defining a pharmacological profile, but it has major advantages for treating complex disorders such as schizophrenia. It is no wonder that the currently approved antipsychotics do have a rich pharmacology and substantially improve the clinical phenotype. With so many different genotypes defining individual patients, focusing on only one target is likely to have small effects that might disappear in clinical trials with larger patient populations. Even over all indications (not only CNS), more than half of the first-in-class medicines approved in the last decade have been found by using phenotypic assays and have typically multi-target pharmacology (Swinney and Anthony, 2011).

The approach presented here suggests a rational way to identify 1) a set of targets and 2) chemical structures that might serve as hits for further medical chemistry development. It might therefore alleviate the concerns of many medical chemistry departments in pharmaceutical companies.

Changing the mindset from developing the next extremely specific and potent inhibitor to pursuing multi-target pharmacology is urgently needed to break the deadlock of unsuccessful new drug development in schizophrenia.

References:

Swinney DC, Anthony J. How were new medicines discovered? Nat Rev Drug Discov . 2011 Jul ; 10(7):507-19. Abstract

View all comments by Hugo Geerts

Related News: Research Roundup: PV Interneurons and Neural Circuit (Dys)Function

Comment by:  Takao Hensch
Submitted 15 February 2014
Posted 15 February 2014

Our Conte Center is focused on the transcriptome, connectome, and plasticity of PV cells as the neurodevelopmental basis for mental illness. Their maturational state dictates the degree of plasticity in developmental critical periods, and now we know, from Donato et al., in adult learning. Once plasticity is opened by PV cells' function, it closes when they mature ("high PV" state), including the tightening of the perineuronal nets (PNNs) around them.

In schizophrenia, PV cells may remain in the "low PV" weak PNN state for some time longer than normal, suggesting, interestingly, that developmental plasticity may be prolonged (i.e., neural circuits fail to stabilize when they normally should). PV cell maturation may potentially be controlled by Otx2 secreted from the choroid plexus, which would link enlarged ventricles to impaired PV cells in the brain in schizophrenia (see Spatazza et al., 2013).

References:

Spatazza J, Lee HH, Di Nardo AA, Tibaldi L, Joliot A, Hensch TK, Prochiantz A. Choroid-plexus-derived Otx2 homeoprotein constrains adult cortical plasticity. Cell Rep. 2013 Jun 27;3(6):1815-23. Abstract

View all comments by Takao Hensch