Matthew D. Puhl

Multiple risk pathways for schizophrenia converge in serine racemase knockout mice, a mouse model of NMDA receptor hypofunction

Significance We sought to determine whether the diverse hippocampal neuropathology observed in schizophrenia could be recapitulated in an animal model of NMDA receptor (NMDAR) hypofunction. Serine racemase-deficient (SR−/−) mice, which lack one of the NMDAR coagonists d-serine, display impaired hippocampal plasticity, as well as the morphological, neurochemical, and cognitive abnormalities consistent with what is observed in schizophrenia. Importantly, treatment in adulthood with d-serine reversed the electrophysiological, neurochemical, and cognitive deficits. These results demonstrate that NMDAR hypofunction can reproduce the hippocampal deficits associated with schizophrenia and point to potential interventions for the currently untreatable negative and cognitive symptoms of this disorder. Schizophrenia is characterized by reduced hippocampal volume, decreased dendritic spine density, altered neuroplasticity signaling pathways, and cognitive deficits associated with impaired hippocampal function. We sought to determine whether this diverse pathology could be linked to NMDA receptor (NMDAR) hypofunction, and thus used the serine racemase-null mutant mouse (SR−/−), which has less than 10% of normal brain d-serine, an NMDAR coagonist. We found that d-serine was necessary for the maintenance of long-term potentiation in the adult hippocampal dentate gyrus and for full NMDAR activity on granule cells. SR−/− mice had reduced dendritic spines and hippocampal volume. These morphological changes were paralleled by diminished BDNF/Akt/mammalian target of rapamycin (mTOR) signaling and impaired performance on a trace-conditioning memory task. Chronic d-serine treatment normalized the electrophysiological, neurochemical, and cognitive deficits in SR−/− mice. These results demonstrate that NMDAR hypofunction can reproduce the numerous hippocampal deficits associated with schizophrenia, which can be reversed by chronic peripheral d-serine treatment.

Viral over-expression of D1 dopamine receptors in the prefrontal cortex increase high-risk behaviors in adults: Comparison with adolescents

RationaleAdolescents are often described as “lacking brakes” resulting in an increase in several behaviors associated with risk for addiction. Prefrontal cortex dopamine and cortico-limbic interaction play an important role in addiction, and we have previously shown that the dopamine D1 receptor is elevated on prelimbic prefrontal output neurons in adolescent rats. We hypothesized that a constellation of risk-related behaviors is mediated by prefrontal output neuron expression of D1.ObjectivesWe aimed to determine the role of the dopamine D1 receptor in behavioral and neural correlates of risk for addiction that are often observed in adolescents. Therefore, high-risk behaviors as well as subcortical D2 receptor expression were investigated in adult animals with experimentally elevated D1 on prefrontal glutamatergic neurons.MethodsA lentiviral vector that selectively expressed the D1 receptor within glutamate neurons was injected in the prelimbic prefrontal cortex of adult male rats. Place conditioning to cocaine, alcohol, and nicotine, as well as delay discounting, novelty preferences, anxiety, cocaine self-administration, and sucrose preferences were assessed.ResultsVirally mediated D1 over-expression in adults leads to stronger drug-cue associations and greater consumption of sweet solutions, elevates bias towards immediate satisfaction rather than delaying gratification, decreases anxiety, and causes rats to work harder for and take more cocaine. Furthermore, elevated cortical D1 reduces D2 receptors in the accumbens (a putative risk marker).ConclusionsTogether, these data suggest a common mechanism for increased motivational drive to seek and consume substances with hedonic value, consistent with adolescent addictive processes.

In Vivo Magnetic Resonance Studies Reveal Neuroanatomical and Neurochemical Abnormalities in the Serine Racemase Knockout Mouse Model of Schizophrenia

BACKGROUND Decreased availability of the N-methyl-D-aspartate receptor (NMDAR) co-agonist D-serine is thought to promote NMDAR hypofunction and contribute to the pathophysiology of schizophrenia, including neuroanatomical abnormalities, such as cortical atrophy and ventricular enlargement, and neurochemical abnormalities, such as aberrant glutamate and γ-aminobutyric acid (GABA) signaling. It is thought that these abnormalities directly relate to the negative symptoms and cognitive impairments that are hallmarks of the disorder. Because of the genetic complexity of schizophrenia, animal models of the disorder are extremely valuable for the study of genetically predisposing factors. Our laboratory developed a transgenic mouse model lacking serine racemase (SR), the synthetic enzyme of d-serine, polymorphisms of which are associated with schizophrenia. Null mutants (SR-/-) exhibit NMDAR hypofunction and cognitive impairments. We used 9.4 T magnetic resonance imaging (MRI) and proton spectroscopy (MRS) to compare in vivo brain structure and neurochemistry in wildtype (WT) and SR-/- mice. METHODS Mice were anesthetized with isoflurane for MRI and MRS scans. RESULTS Compared to WT controls, SR-/- mice exhibited 23% larger ventricular volumes (p<0.05). Additionally, in a medial frontal cortex voxel (15 μl), SR-/- mice exhibited significantly higher glutamate/water (12%, t=1.83, p<0.05) and GABA/water (72%, t=4.10, p<0.001) ratios. CONCLUSIONS Collectively, these data demonstrate in vivo neuroanatomical and neurochemical abnormalities in the SR-/- mouse comparable to those previously reported in humans with schizophrenia.

Global Biochemical Profiling Identifies β-Hydroxypyruvate as a Potential Mediator of Type 2 Diabetes in Mice and Humans

Glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 are incretins secreted by respective K and L enteroendocrine cells after eating and amplify glucose-stimulated insulin secretion (GSIS). This amplification has been termed the “incretin response.” To determine the role(s) of K cells for the incretin response and type 2 diabetes mellitus (T2DM), diphtheria toxin–expressing (DT) mice that specifically lack GIP-producing cells were backcrossed five to eight times onto the diabetogenic NONcNZO10/Ltj background. As in humans with T2DM, DT mice lacked an incretin response, although GLP-1 release was maintained. With high-fat (HF) feeding, DT mice remained lean but developed T2DM, whereas wild-type mice developed obesity but not diabetes. Metabolomics identified biochemicals reflecting impaired glucose handling, insulin resistance, and diabetes complications in prediabetic DT/HF mice. β-Hydroxypyruvate and benzoate levels were increased and decreased, respectively, suggesting β-hydroxypyruvate production from d-serine. In vitro, β-hydroxypyruvate altered excitatory properties of myenteric neurons and reduced islet insulin content but not GSIS. β-Hydroxypyruvate–to–d-serine ratios were lower in humans with impaired glucose tolerance compared with normal glucose tolerance and T2DM. Earlier human studies unmasked a neural relay that amplifies GIP-mediated insulin secretion in a pattern reciprocal to β-hydroxypyruvate–to–d-serine ratios in all groups. Thus, K cells may maintain long-term function of neurons and β-cells by regulating β-hydroxypyruvate levels.

Availability of N-Methyl-d-Aspartate Receptor Coagonists Affects Cocaine-Induced Conditioned Place Preference and Locomotor Sensitization: Implications for Comorbid Schizophrenia and Substance Abuse

Schizophrenia is associated with high prevalence of substance abuse. Recent research suggests that dysregulation of N-methyl-d-aspartate receptor (NMDAR) function may play a role in the pathophysiology of both schizophrenia and drug addiction, and thus, may account for this high comorbidity. Our laboratory has developed two transgenic mouse lines that exhibit contrasting NMDAR activity based on the availability of the glycine modulatory site (GMS) agonists d-serine and glycine. Glycine transporter 1 knockdowns (GlyT1+/−) exhibit NMDAR hyperfunction, whereas serine racemase knockouts (SR−/−) exhibit NMDAR hypofunction. We characterized the behavior of these lines in a cocaine-induced (20 mg/kg) conditioned place preference (CPP) and locomotor sensitization paradigm. Compared with wild-type mice, GlyT1+/− mice displayed hastened extinction of CPP and robust cocaine-induced reinstatement. SR−/− mice appeared to immediately “forget” the learned preference, because they did not exhibit cocaine-induced reinstatement and also displayed attenuated locomotor sensitization. Treatment of GlyT1+/− mice with gavestinel (10 mg/kg on day 1; 5 mg/kg on days 2–17), a GMS antagonist, attenuated cocaine-induced CPP and caused them to immediately “forget” the learned preference. Treatment of SR−/− mice with d-serine (300 mg/kg on day 1; 150 mg/kg on days 2–17) to normalize brain levels caused them to avoid the cocaine-paired side of the chamber during extinction. These results highlight NMDAR dysfunction as a possible neural mechanism underlying comorbid schizophrenia and substance abuse. Also, these findings suggest drugs that directly or indirectly activate the NMDAR GMS could be an effective treatment of cocaine abuse.

D-serine deficiency attenuates the behavioral and cellular effects induced by the hallucinogenic 5-HT2A receptor agonist DOI

Both the serotonin and glutamate systems have been implicated in the pathophysiology of schizophrenia, as well as in the mechanism of action of antipsychotic drugs. Psychedelic drugs act through the serotonin 2A receptor (5-HT2AR), and elicit a head-twitch response (HTR) in mice, which directly correlates to 5-HT2AR activation and is absent in 5-HT2AR knockout mice. The precise mechanism of this response remains unclear, but both an intrinsic cortico-cortical pathway and a thalamo-cortical pathway involving glutamate release have been proposed. Here, we used a genetic model of NMDAR hypofunction, the serine racemase knockout (SRKO) mouse, to explore the role of glutamatergic transmission in regulating 5-HT2AR-mediated cellular and behavioral responses. SRKO mice treated with the 5-HT2AR agonist (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) showed a clearly diminished HTR and lower induction of c-fos mRNA. These altered functional responses in SRKO mice were not associated with changes in cortical or hippocampal 5-HT levels or in 5-HT2AR and metabotropic glutamate-2 receptor (mGluR2) mRNA and protein expression. Together, these findings suggest that D-serine-dependent NMDAR activity is involved in mediating the cellular and behavioral effects of 5-HT2AR activation.

Endogenous co-agonists of the NMDA receptor modulate contextual fear in trace conditioning

We have used mutant mice to probe the roles of the endogenous co-agonists of the NMDA receptor (NMDAR), D-serine and glycine, in fear learning and memory. Serine racemase knockout (SR-/-) mice have less than 15% of wild type forebrain levels of D-serine, whereas glycine transporter 1 heterozygous knockout (GlyT1+/-) mice have elevated synaptic glycine. While cued fear was normal in both delay and trace conditioned mice of both mutant genotypes, contextual fear was affected in trace conditioned subjects: SR-/- mice showed decreased contextual freezing, whereas GlyT1+/- mice showed elevated contextual freezing. These results indicate that endogenous co-agonists of the NMDAR modulate the conditioning of contextual fear responses, particularly in trace conditioning. They further suggest that endogenous glycine can compensate for the D-serine deficiency in cued and contextual fear following delay conditioning.

3.2 PARVALBUMIN INTERNEURON IMPAIRMENT INDUCED BY OXIDATIVE STRESS AS A COMMON PATHOLOGICAL MECHANISM IN ANIMAL MODELS OF SCHIZOPHRENIA

Abstract Background Parvalbumin inhibitory interneurons (PVIs) are crucial for maintaining proper excitatory/inhibitory balance and high-frequency neuronal synchronization. Their activity supports critical developmental trajectories, sensory and cognitive processing, and social behavior. Despite heterogeneity in the etiology across schizophrenia and autism spectrum disorder, PVI circuits are altered in these psychiatric disorders. Identifying mechanism(s) underlying PVI deficits is essential to establish treatments targeting in particular cognition. Based on our previous publications and new data, we propose oxidative stress as a common pathological mechanism leading to PVI impairment in schizophrenia and some forms of autism. Methods Using immunohistochemistry technique and confocal imaging analysis, we assessed the relationship between oxidative stress (as revealed by 8-oxo-DG immunolabeling) and PVI and their perineuronal net (PNN) in twelve established animal models relevant to autism (i.e., the fmr1 KO and CNV 15q13.3) and schizophrenia (CNV: 22q11, 15q13.3, 1q21, serine racemase (SR) KO, GRIN2A KO, Gclm KO) with or without additional insult (e.g., environmental: Gclm KO + GBR12909, GRIN2A KO + GBR12909, neonatal ventral hippocampal lesion (NVHL), methylazoxymethanol acetate developmental rodent model (MAM) and poly:IC). Results When PVI deficits in the anterior cingulate cortex were found in these animal models carrying genetic and/or environmental risks relevant to diverse etiological aspects of these disorders, oxidative stress was always present. Specifically, oxidative stress was negatively correlated with the integrity of PVIs and the extracellular perineuronal net enwrapping these interneurons. Oxidative stress may result from dysregulation of systems typically affected in schizophrenia, including glutamatergic, dopaminergic, immune, and antioxidant signaling. As convergent endpoint, redox dysregulation has successfully been targeted to protect PVIs with antioxidants/redox regulators across several animal models (e.g., Gclm KO, NVHL rats, GRIN2A KO and SR KO mice). D-serine, an allosteric modulator of brain NMDA receptor also protected PVIs and PNN against oxidative stress in SR KO mice. Discussion In view of the fact that the established pathophysiological processes dopamine excess, immune dysregulation and NMDA receptor hypofunction could all induce oxidative stress and are potentiated by additional oxidative insults, this mechanism could be central to damage of the highly metabolically active PVIs and the PNN surrounding them. Antioxidant systems are therefore potential therapeutic targets, assuming that redox regulators could be applied early, during environmental impacts, long before the clinical emergence of the disease.

N -Methyl-d -aspartate receptor co-agonist availability affects behavioral and neurochemical responses to cocaine: insights into comorbid schizophrenia and substance abuse: Role of NMDARs in SZ and cocaine addiction

Both schizophrenia (SZ) and substance abuse (SA) exhibit significant heritability. Moreover, N‐methyl‐d‐aspartate receptors (NMDARs) have been implicated in the pathophysiology of both SZ and SA. We hypothesize that the high prevalence of comorbid SA in SZ is due to dysfunction of NMDARs caused by shared risk genes. We used transgenic mice with a null mutation of the gene encoding serine racemase (SR), the enzyme that synthesizes the NMDAR co‐agonist d‐serine and an established risk gene for SZ, to recreate the pathology of SZ. We determined the effect of NMDAR hypofunction resulting from the absence of d‐serine on motivated behavior by using intracranial self‐stimulation and neurotransmitter release in the nucleus accumbens by using in vivo microdialysis. Compared with wild‐type mice, SR−/− mice exhibited similar baseline intracranial self‐stimulation thresholds but were less sensitive to the threshold‐lowering (rewarding) and the performance‐elevating (stimulant) effects of cocaine. While basal dopamine (DA) and glutamate release were elevated in the nucleus accumbens of SR−/− mice, cocaine‐induced increases in DA and glutamate release were blunted. γ‐Amino‐butyric acid efflux was unaffected in the SR−/− mice. Together, these findings suggest that the impaired NMDAR function and a consequent decrease in sensitivity to cocaine effects on behavior are mediated by blunted DA and glutamate responses normally triggered by the drug. Projected to humans, NMDAR hypofunction due to mutations in SR or other genes impacting glutamatergic function in SZ may render abused substances less potent and effective, thus requiring higher doses to achieve a hedonic response, resulting in elevated drug exposure and increased dependence/addiction.