Getting Specific: Conditional Knockouts Address Glutamate Hypothesis
17 November 2009. Removing a subset of NMDA receptors during development in mice can disrupt neural circuits and behavior in ways that mirror some symptoms of schizophrenia, according to a study published online November 15 in Nature Neuroscience. Led by Kazu Nakazawa, the team of researchers based at the NIMH selectively eliminated the NMDA type of glutamate receptor from interneurons in the cortex and hippocampus of mice early in development and in adulthood.
When they induced NR1 loss early in development, a host of brain and behavior anomalies reminiscent of schizophrenia developed in late adolescence for the mice. These anomalies did not emerge, however, when NMDA receptor loss occurred in adulthood. This difference bolsters the idea that early problems in brain development may remain latent until later in life.
The approach tests some specifics of the glutamate hypothesis proposed for schizophrenia, which originally stemmed from the observation that drugs that block NMDA receptors throughout the brain can induce schizophrenia-like features in animals and humans (see SRF related hypothesis and SRF hypothesis). This led to the notion that underactive NMDA receptors (or "NMDA hypofunction") could be a primary problem in schizophrenia, and could then drive imbalances in other neurotransmitter systems throughout the brain (see SRF related news story). For example, underactive NMDA receptors lead to GABAergic malfunction in interneurons in mice, as shown by the loss of parvalbumin, a calcium binding protein, and GAD67, an enzyme that helps synthesize GABA (Behrens et al., 2007). Intriguingly, these two proteins are also reduced in some interneurons in postmortem brains of people who died with schizophrenia (Hashimoto et al., 2003).
Let’s make it conditional
When exploring the glutamate hypothesis, manipulations of NMDA function have been relatively coarse, affecting all NMDA receptors throughout the brain. Nakazawa and colleagues wanted to get more specific by testing the effects of eliminating NMDA receptors in a targeted cell type, in selected brain regions, and at precise times of development. They did this by engineering "conditional knockout" mice that did not have the essential NR1 subunit of the NMDA receptor in about half the interneurons located in the cortex and hippocampus.
First author Juan Belforte and colleagues verified that NR1 loss was restricted to interneurons. Under the microscope, brain tissue from mutant mice had the expected number of interneurons, but about half of these lacked NR1 mRNA. In controls, pretty much all interneurons—as inferred by staining for GAD67—were positive for NR1 mRNA. Yet, NR1 was unperturbed in other cell types, as judged by the normal number of GAD67-negative cells that also contained NR1 mRNA. Interneurons that lacked NR1 mRNA were found in medial prefrontal cortex, somatosensory cortex, hippocampus, but not striatum or basolateral amygdala. As expected, the loss of NR1 mRNA scrapped the NMDA receptor: when the team recorded from these interneurons in brain slices, they found a distinct lack of NMDA currents.
Those cells that lost NR1 early in development went on to mimic in late adolescence the reduced GAD67 and parvalbumin levels also found in schizophrenia. This suggests that these interneurons, while functional, are not up to snuff. Indeed, evidence for this came from in vivo recordings of pyramidal cells, which are inhibited by signals from interneurons: they fired at higher rates than usual, with less synchrony. These data suggest that the interneurons had trouble keeping a lid on pyramidal cell activity, and this kind of cortical "disinhibition" has been previously proposed as a downstream effect of NMDA hypofunction (Lisman et al., 2008).
In many ways these mice seemed normal behaviorally, but when stressed, they struggled. After a week or more of "social isolation stress" in which the mice were housed by themselves, they developed a number of unusual behaviors. They scored higher on measures of anxiety, for example, avoiding the center of an open-field environment more than controls did. In the social domain, they were not much good at building nests and they had social memory deficits, sniffing a mouse they had already interacted with as though for the first time. Short-term memory was impaired, with the mice failing to adapt an alternating pattern of entries between the two arms of a Y-shaped maze. And, as in some patients with schizophrenia, prepulse inhibition was substantially decreased.
The mice that lost the NR1 subunit as adults were a different story, however. When examined five weeks after losing NR1 (to match the delay between NR1 loss and testing in the previous group of mice), these mice showed no differences when compared to controls: their interneurons had the expected levels of GAD67 and parvalbumin, their excitatory cortical neurons fired normally, and they sailed through their battery of behavioral tests.
This indicates that NMDA receptors play a key role early in postnatal development for these interneurons, beyond merely providing a conduit for excitatory signals. The authors suggest that NMDA receptors influence interneuron maturation; without them, the interneurons and the neural circuits they participate in are disturbed, which may ultimately increase susceptibility to psychiatric illness. While this study in mice doesn't clarify whether schizophrenia is at its core a glutamate disorder, it does draw specific connections between NMDA hypofunction, neural circuits, and behavior that may inform further research on the glutamate hypothesis of schizophrenia.—Michele Solis.
Belforte J, Zsiros V, Sklar E, Jiang Z, Yu G, Li Y, Quinlan E, Nakazawa K. Postnatal NMDA receptor ablation in corticolimbic interneurons confers schizophrenia-like phenotypes. Nature Neuroscience 2009. Abstract
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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.
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
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Related News: Probing the Role of NMDA Receptor NR2B in Development and SchizophreniaComment by: Stefan Kolata
, Kazu Nakazawa
Submitted 21 February 2012
Posted 21 February 2012
The original NMDA receptor (NMDAR) hypofunction theory of schizophrenia was predicated on the discovery that, in adulthood, NMDAR antagonists mimicked disease symptomatology and exacerbated symptoms in schizophrenic patients (Javitt and Zukin, 1991). Recent advances have since shown that, in addition to this effect in adulthood, there may be a postnatal developmental sensitive period necessary for NMDAR hypofunction to later manifest as schizophrenia phenotypes. For instance, in mice, schizophrenia-like phenotypes were observed when NR1 (GluN1) was ablated selectively in corticolimbic interneurons after postnatal day 7, but not when the knockout occurred after adolescence (Belforte et al., 2010). Similarly, transient antagonism of NMDA during development later resulted in schizophrenia-like phenotypes in adult rats (Stefani and Moghaddam, 2005; Baier et al., 2009). In the present work by Wang et al. (2011), using an elegant molecular genetic technique, Benjamin Hall and his colleagues were able to show that it is perhaps the NR2B (GluN2B) subunit during this developmental period that is most critical for the later development of the symptomatology. NR2B is highly expressed during this postnatal sensitive period, and is only later replaced by NR2A (GluN2A) in most NMDA receptors. The present paper showed that an early replacement of NR2B with NR2A recapitulated some of the NMDA hypomorph phenotypes. While these results are very intriguing and dovetail nicely with the emerging thinking about the neurodevelopmental role of NMDARs, the possible involvement of NR2A itself in schizophrenia should not be lost. Impairment of NR2A results in several schizophrenia-like phenotypes, including a reduction in parvalbumin immunoreactivity, impaired fast-spiking interneuron maturation, altered dopamine metabolism, and a hyperlocomotion response in the open field that is rescued by antipsychotic treatment (Zhang and Sun, 2011; Miyamoto et al., 2001). Further studies of synapses, neurons, and neuronal networks regulated by NR2A and NR2B may lead to a better understanding of the mechanisms underlying the NMDAR hypofunction theory of schizophrenia.
Baier PC, Blume A, Koch J, Marx A, Fritzer G, Aldenhoff JB, Schiffelholz T. Early postnatal depletion of NMDA receptor development affects behaviour and NMDA receptor expression until later adulthood in rats--a possible model for schizophrenia. Behav Brain Res. 2009 Dec 14;205(1):96-101. 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. 2010 Jan;13(1):76-83. Abstract
Javitt DC, Zukin SR. Recent advances in the phencyclidine model of schizophrenia. Am J Psychiatry. 1991 Oct;148(10):1301-8. Abstract
Miyamoto Y, Yamada K, Noda Y, Mori H, Mishina M, Nabeshima T. Hyperfunction of dopaminergic and serotonergic neuronal systems in mice lacking the NMDA receptor epsilon1 subunit. J Neurosci. 2001 Jan 15;21(2):750-7. Abstract
Stefani MR, Moghaddam B. Transient N-methyl-D-aspartate receptor blockade in early development causes lasting cognitive deficits relevant to schizophrenia. Biol Psychiatry. 2005 Feb 15;57(4):433-6. Abstract
Zhang Z, Sun QQ. Development of NMDA NR2 subunits and their roles in critical period maturation of neocortical GABAergic interneurons. Dev Neurobiol. 2011 Mar;71(3):221-45. Abstract
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Related News: New Compounds May Help Probe the Glutamate Hypothesis of Schizophrenia
Comment by: John Krystal, SRF Advisor
Submitted 23 February 2016
Posted 23 February 2016
Positive allosteric modulators of NR2A-containing NMDA receptors (NMDARs) represents an important new pharmacologic advance for probing the role of NMDARs in cognition and behavior and the pathophysiology and treatment of psychiatric disorders. There is a long-standing interest in finding ways to enhance NMDAR function using allosteric modulators of NMDARs that might avoid the potential of direct agonists to cause neurotoxicity. There are a number of psychiatric disorders and addictions where deficits in NMDAR function are implicated in disturbances in network function or plasticity that compromise recovery. This has been a particular focus in the area of schizophrenia research, where compromised glutamate synaptic signaling has been mimicked by the administration of NMDA glutamate receptor antagonists. Since at least the late 1980s, investigators have been interested in the possibility that drugs that enhance NMDAR function might have value in the treatment of symptoms and functional impairment associated with schizophrenia.
The first area of focus was amino acids that enhanced NMDAR function by stimulating the glycine/D-serine co-agonist site of the NMDAR. The initial approach was to orally administer naturally occurring glycine or D-amino acids, D-serine, or D-alanine. Studies with these agents produced small and somewhat inconsistent benefits across studies, raising concerns that this approach could not be successful. However, these amino acids were not really developed as drugs. The optimal dose level and pattern of administration was never established with these agents. From this perspective, any positive data provided tantalizing support for the hypothesis even though there were ongoing efforts to find alternatives with superior pharmacologic properties to enhance NMDAR function.
The first of these alternative agents was D-cycloserine, a partial agonist of the glycine site with a complex pharmacology. D-cycloserine has 30 to 50 percent of the activity of glycine at NR2A- and NR2B-containing NMDARs, meaning that in many behavioral assays, high-dose D-cycloserine produces effects (weak sedation, weak euphoria, memory impairment, etc.) that resemble low doses of NMDAR antagonists. Importantly, at high doses, D-cycloserine seemed to worsen symptoms in some patients diagnosed with schizophrenia. However, D-cycloserine is a nearly full or even super (greater activity than glycine) agonist at NR2C- and NR2D-containing NMDARs, and it is able to stimulate NR3-containing NMDARs that are not directly stimulated by glutamate. It is likely that by stimulating NR2C-, NR2D-, or NR3-containing receptors, beneficial NMDAR agonist-like effects attributed to D-cycloserine emerge. But the complex pharmacology of D-cycloserine limits its therapeutic potential.
The next class of drugs developed was glycine transporter-1 antagonists (GlyT1 inhibitors). These drugs are based on the premise that GlyT1 is a high-activity transporter that controls synaptic glycine levels below the saturation level, allowing receptors to be stimulated by exogenous glycine or GlyT1 inhibition. Some early agents had limited pharmacologic development but showed promising clinical results. Later agents received systematic study, and Roche initially developed exciting initial data, but positive results could not be replicated. Many questions remain, including the following:
1. Is glycine the right amino acid to target relative to D-serine? D-serine appears to have greater synaptic function relative to glycine, which may be more important for extrasynaptic signaling.
2. Is tonic inhibition of GlyT1 detrimental due to its potential to stimulate downregulation of NMDAR function? Instead, should these agents be administered intermittently to enhance network function and plasticity? (
3. Do GlyT1 inhibitors have an inverted U dose response to avoid stimulating NMDAR downregulation and to optimally modulate network functions?
There are a number of reasons that NR2A-containing NMDARs are an interesting target. NR2A-containing NMDARs are predominately synaptic receptors, while NR2B-containing NMDARs are widely distributed in synaptic and extrasynaptic spaces. When "overstimulated," extrasynaptic NR2B-containing NMDARs suppress BDNF levels and cause the pruning of dendritic spines and even dendrites themselves. They also have the potential to produce even more severe forms of toxicity. NR2A-containing NMDARs also emerge early in life and gradually displace NR2B-containing receptors during childhood and adolescence in many circuit elements. Since the risk for many psychiatric disorders also emerges during this period, the NR2A subunit is an intriguing target. In addition, while overstimulation of NR2B-containing receptors has negative effects on synaptic connectivity, NR2A-containing NMDARs are implicated in some neurotrophic processes. Further, there is interest in drugs that facilitate NR2A-mediated signaling (positive allosteric modulation) rather than stimulating NMDARs in order to avoid neurotoxicity that might be associated with directly stimulating these receptors. In this way, one might think of NR2A-positive allosteric modulators (PAMs) as analogous to the way that benzodiazepines enhance the activation of GABAA receptors.
The exciting paper by Hackos et al. in Neuron represents an important advance in the effort to develop NR2A PAMs. The researchers present an elegant story about the identification and validation of these drugs. Schizophrenia is an obvious disorder where these medications might play a role. But there are many other conditions where modulating NMDAR function via NR2A could be predicted to produce benefits. I think that this paper will stimulate interest and discussion in the field.
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