Methylazoxymethanol Research Articles

Prenatal MAM exposure raises kynurenic acid levels in the prefrontal cortex of adult rats.

Elevated brain levels of kynurenic acid (KYNA), a metabolite in the kynurenine pathway, are associated with cognitive dysfunctions, which are nowadays often considered as fundamental characteristics of several psychopathologies; however, the role of KYNA in mental illnesses, such as schizophrenia, is not fully elucidated. This study aimed to assess KYNA levels in the prefrontal cortex (PFC) of rats prenatally treated with methylazoxymethanol (MAM) acetate, i.e., a well-validated neurodevelopmental animal model of schizophrenia. The effects of an early pharmacological modulation of the endogenous cannabinoid system were also evaluated. Pregnant Sprague-Dawley rats were treated with MAM (22mg/kg, ip) or its vehicle at gestational day 17. Male offspring were treated with the cannabinoid CB1 receptor antagonist/inverse agonist AM251 (0.5mg/kg/day, ip) or with the typical antipsychotic haloperidol (0.6mg/kg/day, ip) from postnatal day (PND) 19 to PND39. The locomotor activity and cognitive performance were assessed in the novel object recognition test and the open field test in adulthood. KYNA levels in the PFC of prenatally MAM-treated rats were also assessed. A significant cognitive impairment was observed in prenatally MAM-treated rats (p < 0.01), which was associated with enhanced PFC KYNA levels (p < 0.05). The peripubertal AM251, but not haloperidol, treatment ameliorated the cognitive deficit (p < 0.05), by normalizing the PFC KYNA content in MAM rats. The present findings suggest that the cognitive deficit observed in MAM rats may be related to enhanced PFC KYNA levels which could be, in turn, mediated by the activation of cannabinoid CB1 receptor. These results further support the modulation of brain KYNA levels as a potential therapeutic strategy to ameliorate the cognitive dysfunctions in schizophrenia.

CNS autoimmune response in the MAM/pilocarpine rat model of epileptogenic cortical malformation

The development of seizures in epilepsy syndromes associated with malformations of cortical development (MCDs) has traditionally been attributed to intrinsic cortical alterations resulting from abnormal network excitability. However, recent analyses at single-cell resolution of human brain samples from MCD patients have indicated the possible involvement of adaptive immunity in the pathogenesis of these disorders. By exploiting the MethylAzoxyMethanol (MAM)/pilocarpine (MP) rat model of drug-resistant epilepsy associated with MCD, we show here that the occurrence of status epilepticus and subsequent spontaneous recurrent seizures in the malformed, but not in the normal brain, are associated with the outbreak of a destructive autoimmune response with encephalitis-like features, involving components of both cell-mediated and humoral immune responses. The MP brain is characterized by blood-brain barrier dysfunction, marked and persisting CD8+ T cell invasion of the brain parenchyma, meningeal B cell accumulation, and complement-dependent cytotoxicity mediated by antineuronal antibodies. Furthermore, the therapeutic treatment of MP rats with the immunomodulatory drug fingolimod promotes both antiepileptogenic and neuroprotective effects. Collectively, these data show that the MP rat could serve as a translational model of epileptogenic cortical malformations associated with a central nervous system autoimmune response. This work indicates that a preexisting brain maldevelopment predisposes to a secondary autoimmune response, which acts as a precipitating factor for epilepsy and suggests immune intervention as a therapeutic option to be further explored in epileptic syndromes associated with MCDs.

Unlocking the Therapeutic Potential of Exosomes Derived From Nasal Olfactory Mucosal Mesenchymal Stem Cells: Restoring Synaptic Plasticity, Neurogenesis, and Neuroinflammation in Schizophrenia.

Schizophrenia (SCZ) is a multifaceted mental disorder marked by a spectrum of symptoms, including hallucinations, delusions, cognitive deficits, and negative symptoms. Its etiology involves intricate interactions between genetic and environmental factors, posing significant challenges for effective treatment. We hypothesized that intranasal administration of exosomes derived from nasal olfactory mucosal mesenchymal stem cells (OM-MSCs-exos) could alleviate SCZ-like behaviors in a murine model induced by methylazoxymethanol (MAM). We conducted a comprehensive investigation to assess the impact of intranasally delivered OM-MSC-exos on SCZ-like behaviors in MAM-induced mice. This study encompassed behavioral assessments, neuroinflammatory markers, glial activation, synaptic protein expression, and neurogenesis within the hippocampus. Our findings demonstrated that intranasal administration of OM-MSC-exos effectively ameliorated SCZ-like behaviors, specifically addressing social withdrawal and sensory gating deficits in the MAM-induced murine model. Furthermore, OM-MSC-exos intervention yielded a reduction in neuroinflammatory markers and a suppression of microglial activation within the hippocampus. Simultaneously, we observed an upregulation of key synaptic protein expression, including PSD95 and TH, the rate-limiting enzyme for dopamine biosynthesis. Our study underscores the therapeutic potential of OM-MSC-exos in mitigating SCZ-like behavior. The OM-MSC-exos have the capacity to modulate glial cell activation, diminish neuroinflammation, and promote BDNF-associated synaptic plasticity and neurogenesis, thus ameliorating SCZ-like behaviors. In summary, intranasal administration of OM-MSC-exos offers a multifaceted approach to address SCZ mechanisms, promising innovative treatments for this intricate disorder.

Insulin-Like Growth Factor-1 Promotes Synaptogenesis Signaling, a Major Dysregulated Pathway in Malformation of Cortical Development, in a Rat Model.

Malformation of cortical development (MCD) is one of the main causes of intractable epilepsy in childhood. We explored a treatment based on molecular changes using an infant rat model of methylazoxymethanol (MAM)-induced MCD established by injecting MAM at gestational day 15. The offspring were sacrificed on postnatal day (P) 15 for proteomic analysis, which revealed significant downregulation in the synaptogenesis signaling pathway in the cortex of MCD rats. Recombinant human insulin-growth factor-1 (rhIGF-1) was injected from P12 to P14 twice daily and the effect of IGF1 on N-methyl-D-aspartate (NMDA)-induced spasms (15mg/kg of NMDA, i.p.) was tested; the onset of P15 single spasm was significantly delayed (p = 0.002) and the number of spasms decreased (p < 0.001) in rhIGF1-pretreated rats (n = 17) compared to those in VEH-treated rats (n = 18). Electroencephalographic monitoring during spasms showed significantly reduced spectral entropy and event-related spectral dynamics of fast oscillation in rhIGF-1 treated rats. Magnetic resonance spectroscopy of the retrosplenial cortex showed decreased glutathione (GSH) (p = 0.039) and significant developmental changes in GSH, phosphocreatine (PCr), and total creatine (tCr) (p = 0.023, 0.042, 0.015, respectively) after rhIGF1 pretreatment. rhIGF1 pretreatment significantly upregulated expression of cortical synaptic proteins such as PSD95, AMPAR1, AMPAR4, NMDAR1, and NMDAR2A (p < 0.05). Thus, early rhIGF-1 treatment could promote synaptic protein expression, which was significantly downregulated by prenatal MAM exposure, and effectively suppress NMDA-induced spasms. Early IGF1 treatment should be further investigated as a therapeutic strategy in infants with MCD-related epilepsy.

Methylation pattern and mRNA expression of synapse-relevant genes in the MAM model of schizophrenia in the time-course of adolescence.

Schizophrenia is highly heritable and aggregating in families, but genetics alone does not exclusively explain the pathogenesis. Many risk factors, including childhood trauma, viral infections, migration, and the use of cannabis, are associated with schizophrenia. Adolescence seems to be the critical period where symptoms of the disease manifest. This work focuses on studying an epigenetic regulatory mechanism (the role of DNA methylation) and its interaction with mRNA expression during development, with a particular emphasis on adolescence. The presumptions regarding the role of aberrant neurodevelopment in schizophrenia were tested in the Methyl-Azoxy-Methanol (MAM) animal model. MAM treatment induces neurodevelopmental disruptions and behavioral deficits in off-springs of the treated animals reminiscent of those observed in schizophrenia and is thus considered a promising model for studying this pathology. On a gestational day-17, adult pregnant rats were treated with the antimitotic agent MAM. Experimental animals were divided into groups and subgroups according to substance treatment (MAM and vehicle agent [Sham]) and age of analysis (pre-adolescent and post-adolescent). Methylation and mRNA expression analysis of four candidate genes, which are often implicated in schizophrenia, with special emphasis on the Dopamine hypothesis i.e., Dopamine receptor D2 (Drd2), and the "co-factors" Disrupted in schizophrenia 1 (DISC1), Synaptophysin (Syp), and Dystrobrevin-binding protein 1 (Dtnbp1), was performed in the Gyrus cingulum (CING) and prefrontal cortex (PFC). Data were analyzed to observe the effect of substance treatment between groups and the impact of adolescence within-group. We found reduced pre-adolescent expression levels of Drd2 in both brain areas under the application of MAM. The "co-factor genes" did not show high deviations in mRNA expression levels but high alterations of methylation rates under the application of MAM (up to ~20%), which diminished in the further time course, reaching a comparable level like in Sham control animals after adolescence. The pre-adolescent reduction in DRD2 expression might be interpreted as downregulation of the receptor due to hyperdopaminergic signaling from the ventral tegmental area (VTA), eventually even to both investigated brain regions. The notable alterations of methylation rates in the three analyzed co-factor genes might be interpreted as attempt to compensate for the altered dopaminergic neurotransmission.

Ficus dubia latex extract prevent DMH-induced rat early colorectal carcinogenesis through the regulation of xenobiotic metabolism, inflammation, cell proliferation and apoptosis

Ficus dubia latex is recognized as a remedy in Asian traditional medicine with various therapeutic effects. The present study aimed to determine the preventive action of Ficus dubia latex extract (FDLE) on 1,2-dimethylhydrazine (DMH)-induced rat colorectal carcinogenesis and its mechanisms. The experiment included an initiation model in which rats were orally administered with FDLE daily for 1 week before DMH injection until the end of the experiment, while only after DMH injection until the end in the post-initiation model. The results firstly indicated that FDLE treatment could reduce the level of methylazoxymethanol (MAM) in rat colonic lumen by inhibition of the activities of both phase I xenobiotic metabolizing enzymes in the liver and β-glucuronidase in the colon, leading to reduced DNA methylation in colonic mucosal cells, related to the number of ACF in the initiation stage. Besides, FDLE modulated the inflammation which could suppress the growth and induce apoptosis of aberrant colonic mucosal cells, leading to retardation of ACF multiplicity. Therefore, FDLE showed the ability to suppress the DMH-induced rat ACF formation and inflammation promoted growth of ACF. In conclusion, FDLE had the potential to prevent carcinogens-induced rat colorectal carcinogenesis in the initiation stage.

LY395756 promotes NR2B expression via activation of AKT/CREB signaling in the juvenile methylazoxymethanol mice model of schizophrenia.

IntroductionSynaptic N‐methyl‐d‐aspartate receptor subtype 2B(NR2B) is significantly reduced in prefrontal cortex (PFC) in the neurodevelopmental methylazoxymethanol (MAM) model of schizophrenia (SCZ). Recent research has shown that LY395756 can effectively restore NR2B levels and improve cognitive performance in juvenile MAM mice model. However, the underlying mechanisms of these beneficial effects remain unclear.Materials and methodsJuvenile MAM mice model of SCZ is used in our study. Synaptic membrane protein levels were examined by western blotting under different treatment conditions. Interaction of cAMP‐response element binding protein (CREB) and the promoter of NR2B was detected by the chromatin immunoprecipitation (ChIP) assay. Further examination of signaling pathway that mediates NR2B expression was also investigated by western blotting.ResultsIn the PFC of the juvenile MAM mice schizophrenia model, CREB was found to directly bind with the promoter of NR2B. LY395756 activated the phosphorylation of AKT. Phosphorylated AKT subsequently induced the phosphorylation of CREB, and the activated CREB promoted the expression of NR2B. Subsequent experiments showed that the dephosphorylation of CREB induced by protein phosphatase 1 (PP1) can inhibit NR2B levels. Taken together, these findings support that the AKT/CREB signaling pathway is essential for the promoting effect of LY395756 on synaptic NR2B in PFC in juvenile MAM mice SCZ model.ConclusionsOur investigation has identified a novel mechanism by which LY395756 increases NR2B expression in juvenile MAM mice SCZ model. The AKT/CREB signaling pathway warrants further research as a potential direction for clinical treatment of SCZ.

Genotoxic Damage During Brain Development Presages Prototypical Neurodegenerative Disease.

Western Pacific Amyotrophic Lateral Sclerosis and Parkinsonism-Dementia Complex (ALS/PDC) is a disappearing prototypical neurodegenerative disorder (tau-dominated polyproteinopathy) linked with prior exposure to phytogenotoxins in cycad seed used for medicine and/or food. The principal cycad genotoxin, methylazoxymethanol (MAM), forms reactive carbon-centered ions that alkylate nucleic acids in fetal rodent brain and, depending on the timing of systemic administration, induces persistent developmental abnormalities of the cortex, hippocampus, cerebellum, and retina. Whereas administration of MAM prenatally or postnatally can produce animal models of epilepsy, schizophrenia or ataxia, administration to adult animals produces little effect on brain structure or function. The neurotoxic effects of MAM administered to rats during cortical brain development (specifically, gestation day 17) are used to model the histological, neurophysiological and behavioral deficits of human schizophrenia, a condition that may precede or follow clinical onset of motor neuron disease in subjects with sporadic ALS and ALS/PDC. While studies of migrants to and from communities impacted by ALS/PDC indicate the degenerative brain disorder may be acquired in juvenile and adult life, a proportion of indigenous cases shows neurodevelopmental aberrations in the cerebellum and retina consistent with MAM exposure in utero. MAM induces specific patterns of DNA damage and repair that associate with increased tau expression in primary rat neuronal cultures and with brain transcriptional changes that parallel those associated with human ALS and Alzheimer’s disease. We examine MAM in relation to neurodevelopment, epigenetic modification, DNA damage/replicative stress, genomic instability, somatic mutation, cell-cycle reentry and cellular senescence. Since the majority of neurodegenerative disease lacks a solely inherited genetic basis, research is needed to explore the hypothesis that early-life exposure to genotoxic agents may trigger or promote molecular events that culminate in neurodegeneration.

Nicotine Administration Normalizes Behavioral and Neurophysiological Perturbations in the MAM Rodent Model of Schizophrenia.

BackgroundThe present study utilized the methylazoxymethanol (MAM) neurodevelopmental rodent model of schizophrenia (SCZ) to evaluate the hypothesis that individuals with SCZ smoke in an attempt to “self-medicate” their symptoms through nicotine (NIC) intake.MethodsTo explore this question, we examined the effects of acute and chronic administration of NIC in 2 established behavioral tests known to be disrupted in the MAM model: prepulse inhibition of startle and novel object recognition. Additionally, we assessed the effects of acute and chronic NIC on 2 indices of the pathophysiology of SCZ modeled by MAM, elevated dopamine neuron population activity in the ventral tegmental area and neuronal activity in the ventral hippocampus, using in vivo electrophysiological recordings.ResultsOur findings demonstrated that both acute and chronic administration of NIC significantly improved deficits in prepulse inhibition of startle and novel object recognition among MAM rats and normalized elevated ventral tegmental area and ventral hippocampal neuronal activity in these animals.ConclusionTogether, these findings of NIC-induced improvement of deficits lend support for a “self-medication” hypothesis behind increased cigarette smoking in SCZ and illustrate the potential utility of nicotinic modulation in future pharmacotherapies for certain SCZ symptoms.

Inhibition of BET Proteins during Adolescence Affects Prefrontal Cortical Development: Relevance to Schizophrenia.

Background: The present study investigated the role of proteins from the bromodomain and extra-terminal (BET) family in schizophrenia-like abnormalities in a neurodevelopmental model of schizophrenia induced by prenatal methylazoxymethanol (MAM) administration (MAM-E17). Methods: An inhibitor of BET proteins, JQ1, was administered during adolescence on postnatal days (P) 23–P29, and behavioural responses (sensorimotor gating, recognition memory) and prefrontal cortical (mPFC) function (long-term potentiation (LTP), molecular and proteomic analyses) studies were performed in adult males and females. Results: Deficits in sensorimotor gating and recognition memory were observed only in MAM-treated males. However, adolescent JQ1 treatment affected animals of both sexes in the control but not MAM-treated groups and reduced behavioural responses in both sexes. An electrophysiological study showed LTP impairments only in male MAM-treated animals, and JQ1 did not affect LTP in the mPFC. In contrast, MAM did not affect activity-dependent gene expression, but JQ1 altered gene expression in both sexes. A proteomic study revealed alterations in MAM-treated groups mainly in males, while JQ1 affected both sexes. Conclusions: MAM-induced schizophrenia-like abnormalities were observed only in males, while adolescent JQ1 treatment affected memory recognition and altered the molecular and proteomic landscape in the mPFC of both sexes. Thus, transient adolescent inhibition of the BET family might prompt permanent alterations in the mPFC.

Interacting effects of the MAM model of schizophrenia and antipsychotic treatment: Untargeted proteomics approach in adipose tissue

Schizophrenia is a severe neuropsychiatric disease associated with substantially higher mortality. Reduced life expectancy in schizophrenia relates to an increased prevalence of metabolic disturbance, and antipsychotic medication is a major contributor. Molecular mechanisms underlying adverse metabolic effects of antipsychotics are not fully understood; however, adipose tissue homeostasis deregulation appears to be a critical factor. We employed mass spectrometry-based untargeted proteomics to assess the effect of chronic olanzapine, risperidone, and haloperidol treatment in visceral adipose tissue of prenatally methylazoxymethanol (MAM) acetate exposed rats, a well-validated neurodevelopmental animal model of schizophrenia. Bioinformatics analysis of differentially expressed proteins was performed to highlight the pathways affected by MAM and the antipsychotics treatment. MAM model was associated with the deregulation of the TOR (target of rapamycin) signalling pathway. Notably, alterations in protein expression triggered by antipsychotics were observed only in schizophrenia-like MAM animals where we revealed hundreds of affected proteins according to our two-fold threshold, but not in control animals. Treatments with all antipsychotics in MAM rats resulted in the downregulation of mRNA processing and splicing, while drug-specific effects included among others upregulation of insulin resistance (olanzapine), upregulation of fatty acid metabolism (risperidone), and upregulation of nucleic acid metabolism (haloperidol). Our data indicate that deregulation of several energetic and metabolic pathways in adipose tissue is associated with APs administration and is prominent in MAM schizophrenia-like model but not in control animals.

P.548 The effects of clozapine on cognitive functioning in schizophrenia

Evidence indicates that the disruption of epigenetic processes might play an important role in the development of schizophrenia symptoms. The present study investigated the role of histone acetylation in the development of sensorimotor gating deficits in a neurodevelopmental model of schizophrenia based on prenatal administration of methylazoxymethanol (MAM) at embryonic day 17.Valproic acid (VPA), an inhibitor of class I histone deacetylases, was administered (250 mg/kg, twice a day for 7 consecutive days) in early adolescence (23rd–29th day) or early adulthood (63rd–69th day) to rats. The effect of VPA treatment on the sensorimotor gating deficits induced by prenatal MAM administration was analyzed in adult rats at postnatal day 70 (P70). In addition, the effects of VPA administration (at the same doses) on MAM-induced changes in the levels of histone H3 acetylation at lysine 9 (H3K9ac) and histone deacetylase 2 (HDAC2) in the medial prefrontal cortex (mPFC) were determined at P70 using Western blot.VPA administration in either adolescence or early adulthood prevented the sensorimotor gating deficits induced by MAM. However, VPA administration in early adolescence or early adulthood did not alter H3K9ac levels induced by MAM. In contrast, VPA administration in either adolescence or adulthood prevented the increase in HDAC2 level evoked by MAM.Prenatal MAM administration impaired histone acetylation in the mPFC, which might be involved in the development of some of the neurobehavioral deficits (i.e., sensorimotor gating deficits) associated with schizophrenia. Blockade of HDAC2 might prevent the disruption of sensorimotor gating in adulthood.

Metabolic profile of methylazoxymethanol model of schizophrenia in rats and effects of three antipsychotics in long-acting formulation

Mortality in psychiatric patients with severe mental illnesses reaches a 2–3 times higher mortality rate compared to the general population, primarily due to somatic comorbidities. A high prevalence of cardiovascular morbidity can be attributed to the adverse metabolic effects of atypical antipsychotics (atypical APs), but also to metabolic dysregulation present in drug-naïve patients. The metabolic aspects of neurodevelopmental schizophrenia-like models are understudied. This study evaluated the metabolic phenotype of a methylazoxymethanol (MAM) schizophrenia-like model together with the metabolic effects of three APs [olanzapine (OLA), risperidone (RIS) and haloperidol (HAL)] administered via long-acting formulations for 8 weeks in female rats. Body weight, feed efficiency, serum lipid profile, gastrointestinal and adipose tissue-derived hormones (leptin, ghrelin, glucagon and glucagon-like peptide 1) were determined. The lipid profile was assessed in APs-naïve MAM and control cohorts of both sexes.Body weight was not altered by the MAM model, though cumulative food intake and feed efficiency was lowered in the MAM compared to CTR animals. The effect of the APs was also present; body weight gain was increased by OLA and RIS, while OLA induced lower weight gain in the MAM rats. Further, the MAM model showed lower abdominal adiposity, while OLA increased it. Serum lipid profile revealed MAM model-induced alterations in both sexes; total, HDL and LDL cholesterol levels were increased. The MAM model did not exert significant alterations in hormonal parameters except for elevation in leptin level. The results support intrinsic metabolic dysregulation in the MAM model in both sexes, but the MAM model did not manifest higher sensitivity to metabolic effects induced by antipsychotic treatment.

Delayed Functional Networks Development and Altered Fast Oscillation Dynamics in a Rat Model of Cortical Malformation.

Malformations of cortical development (MCD) is associated with a wide range of developmental delay and drug resistant epilepsy in children. By using resting-state functional magnetic resonance imaging (RS-fMRI) and event-related spectral perturbation (ERSP) of cortical electroencephalography (EEG) data, we tried to investigate the neural changes of spatiotemporal functional connectivity (FC) and fast oscillation (FO) dynamics in a rat model of methylazoxymethanol (MAM)-induced MCD. A total of 28 infant rats with prenatal exposure to MAM and those of age matched 28 controls with prenatal saline exposure were used. RS-fMRI were acquired at postnatal day 15 (P15) and 29 (P29), and correlation coefficient analysis of eleven region of interests (ROI) was done to find the differences of functional networks between four groups. Two hour-cortical EEGs were also recorded at P15 and P29 and the ERSP of gamma (30–80 Hz) and ripples (80–200 Hz) were analyzed. The rats with MCD showed significantly delayed development of superior colliculus-brainstem network compared to control rats at P15. In contrast to marked maturation of default mode network (DMN) in controls from P15 to P29, there was no clear development in MCD rats. The MCD rats showed significantly higher cortical gamma and ripples-ERSP at P15 and lower cortical ripples-ERSP at P29 than those of control rats. This study demonstrated delayed development of FC and altered cortical FO dynamics in rats with malformed brain. The results should be further investigated in terms of the epileptogenesis and cognitive dysfunction in patients with MCD.

In vivo MRI Successfully Reveals the Malformation of Cortical Development in Infant Rats.

Objective: Malformations of cortical development (MCDs) are major causes of intractable epilepsies. To characterize the early neuroimaging findings of MCDs, we tried to identify the MRI features consistent with pathological findings in an infant rat MCD model, prenatally exposed to methylazoxymethanol (MAM), by using newly developed MRI techniques.Methods: At gestational day 15, two doses of MAM (15 mg/kg intraperitoneally) or normal saline were injected into pregnant rats. The offspring underwent in vivo MRI, including glutamate chemical exchange saturation transfer (GluCEST), 1H-MR spectroscopy, and diffusion tensor imaging, at postnatal day (P) 15 using a 7T small-animal imaging system. Another set of prenatally MAM-exposed rats were sacrificed for histological staining.Results: At P15, the retrosplenial cortex (RSC) of rats with MCDs showed decreased neuronal nuclei, parvalbumin, and reelin expressions. Moreover, dendritic arborization of pyramidal cells in the RSC significantly decreased in infant rats with MCDs. In vivo MRI showed significantly decreased GluCEST (%) in the RSC of rats with MCDs (p = 0.000) and a significant correlation between GluCEST (%) and RSC thickness (r = 0.685, p = 0.003). The rats with MCDs showed reduced glutamate (p = 0.002), N-acetylaspartate (p = 0.002), and macromolecule and lipid levels (p = 0.027) and significantly reduced fractional anisotropy values in the RSC.Conclusion: In vivo MRI revealed reduced neuronal population and dendritic arborization in the RSC of infant rats with MCDs during the early postnatal period. These pathological changes of the cortex could serve as clinical imaging biomarkers of MCDs in infants.

Targeting PSD95-nNOS interaction by Tat-N-dimer peptide during status epilepticus is neuroprotective in MAM-pilocarpine rat model

Glutamate receptors play a crucial pathogenic role in brain damage induced by status epilepticus (SE). SE may initiate NMDAR-dependent excitotoxicity through the production of oxidative damage mediated by the activation of a ternary complex formed by the NMDA receptor, the post-synaptic density scaffolding protein 95 (PSD95) and the neuronal NO synthase (nNOS). The inhibition of the protein-protein-interaction (PPI) of the NMDAR-PSD95-nNOS complex is one of the most intriguing challenges recently developed to reduce neuronal death in both animal models and in patients with cerebral ischemia. We took advantage of this promising approach to verify whether early administration of a neuroprotective NMDAR-PSD95-nNOS PPI inhibitor preserves the brain from SE-induced damage in a model of acquired cortical dysplasia, the methylazoxymethanol (MAM)/pilocarpine rat.Pilocarpine-induced SE rapidly determined neurodegenerative changes mediated by a NMDAR-downstream neurotoxic pathway in MAM rats. We demonstrated that SE rapidly induces NMDAR activation, nNOS membrane translocation, PSD95-nNOS molecular interaction associated with neuronal and glial peroxynitrite accumulation in the neocortex of MAM-pilocarpine rats. These changes were paralleled by rapid c-fos overexpression and by progressive spectrin proteolysis, suggestive of calpain activity and irreversible cytoskeletal damage. Early administration of a cell-penetrating Tat-N-dimer peptide inhibitor of NMDAR-PSD95-nNOS PPI during SE significantly rescued the MAM-pilocarpine rats from SE-induced mortality, reduced the number of degenerating neurons, decreased neuronal c-fos activation, peroxynitrite formation and cytoskeletal degradation and prevented astrogliosis.Our findings suggest an overall neuroprotective effect of blocking PSD95-nNOS protein-protein-interaction against SE insult.

Prenatal treatment with methylazoxymethanol acetate as a neurodevelopmental disruption model of schizophrenia in mice

Methylazoxymethanol (MAM)-treated pregnant rat at gestation day (GD) 17 has been shown to be a valuable developmental animal model for schizophrenia. Yet, this model remains to be established in mice. In the present study, we examined behavioral, cytoarchitectural, and neurochemical changes in the offspring of MAM-treated mice and validated the model's face, construct and predictive validities. We found that in contrast to a single injection of MAM to dams at GD 15, 16 or 17, its daily administration from GD 15 to 17 led to deficits in prepulse inhibition (PPI) of startle in the post-pubertal offspring. In addition, we observed behavioral deficits in working memory and social interactions, as well as an increase in locomotor activity induced by the NMDA antagonist MK-801 in GD15-17 MAM offspring. These animals also showed a reduction in the volume of the prefrontal cortex (PFC) and hippocampus, neuroanatomical changes such as discontinuities and heterotopias in the hippocampus, and an increase of DA level and DOPAC/DA ratio in the medial PFC. Atypical antipsychotic drugs clozapine, risperidone, and aripiprazole, but not the typical drug haloperidol, reversed the deficit in PPI and social withdrawal in the offspring of MAM-treated dams. In contrast, MK-801-induced hyperactivity in MAM mice was reversed by both and typical or atypical antipsychotic drugs. Taken together, the treatment of pregnant mice with MAM during GD 15–17 offers a new approach to study neurobiological mechanisms involved in the pathogenesis of schizophrenia.

Medial septum activation produces opposite effects on dopamine neuron activity in the ventral tegmental area and substantia nigra in MAM vs. normal rats

The medial septum (MS) differentially impacts midbrain dopamine (DA) neuron activity via the ventral hippocampus, a region implicated in DA-related disorders. However, whether MS regulation of ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) is disrupted in a developmental disruption model of schizophrenia is unknown. Male Sprague-Dawley rats were exposed at gestational day 17 to methylazoxymethanol (MAM) or saline. As adults, NMDA (0.75 µg/0.2 µL) was infused into the MS, and either DA neuron activity in the VTA and SNc (7–9 anesthetized rats per group) or amphetamine-induced hyperlocomotion (AIH, 11-13 rats per group) was measured. MS activation produced a 58% increase in the number of spontaneously active DA neurons in VTA and a 37% decrease in SNc in saline rats. However, MS activation produced opposite effects on DA population activity in MAM rats, decreasing VTA DA activity by 51% and increasing SNc DA activity by 47%. MS activation also increased AIH by 113% in MAM rats, opposite of what is seen in intact rats. The effect in behavioral output may be due to disrupted GABAergic regulation of SNc as bicuculline infusion into vSub, which selectively prevented the MS activation-induced decrease in SNc DA activity in intact rats, prevented the increase in AIH and SNc DA activity in MAM rats. These findings demonstrate that the regulation of midbrain DA neurons by the MS is disrupted in this well-validated animal model, suggesting that it could be a potential locus for pharmacological intervention in disorders such as schizophrenia.

Abnormalities in behaviour, histology and prefrontal cortical gene expression profiles relevant to schizophrenia in embryonic day 17 MAM-Exposed C57BL/6 mice

Gestational and perinatal disruption of neural development increases the risk of developing schizophrenia (SCZ) later in life. Embryonic day 17 (E17) methylazoxymethanol (MAM) treatment leads to histological, physiological and behavioural abnormalities in post-puberty rats that model the neuropathological and cognitive deficits reported in SCZ patients. However, the validity of E17 MAM-exposed mice to model SCZ has not been explored. Here we treated E17 C57BL/6 mouse dams with various dosages of MAM. We found that this mouse strain was more vulnerable to MAM treatment than rats and there were gender differences in behavioural abnormalities, histological changes and prefrontal cortical gene expression profiles in MAM (7.5 mg/kg)-exposed mice. Both male and female MAM-exposed mice had deficits in prepulse inhibition. Female MAM-exposed mice exhibited mildly increased spontaneous locomotion activity and social recognition deficits, while male mice were normal. Consistently, only female MAM-exposed mice exhibited reduced brain weight, decreased size of prefrontal cortex (PFC) and enlarged lateral ventricles. Transcriptome analysis of the PFC revealed that there were more differentially expressed genes in female MAM-exposed mice than those in male mice. Moreover, expression of Pvalb, Arc and genes in their association networks were downregulated in the PFC of female MAM-exposed mice. These results indicate that E17 MAM-exposure in C57BL/6 mice leads to behavioural changes that model certain deficits reported in SCZ patients. MAM-exposed female mice may be used to study gene expression changes, inhibitory neural circuit dysfunction and glutamatergic synaptic plasticity deficits with a possible relation to those in the brains of SCZ patients.

Altered brain cannabinoid 1 receptor mRNA expression across postnatal development in the MAM model of schizophrenia

Altered cannabinoid 1 receptor (CB1R) expression has been reported in the brain of subjects with schizophrenia, a developmental mental illness that usually emerges in late adolescence/early adulthood. However, the developmental period at which changes in the CB1R expression appear in schizophrenia is unknown. To gain insight into this factor, we assessed the postnatal developmental trajectory of CB1R expression in the methylazoxymethanol (MAM) model of schizophrenia. Using in situ hybridization with film and grain analyses, CB1R messenger RNA (mRNA) levels were quantified in multiple brain regions, including the medial prefrontal cortex (mPFC), secondary motor cortex, dorsomedial and dorsolateral striatum, dorsal subregions and ventral subiculum of the hippocampus, of MAM-treated rats and normal controls at three developmental periods [juvenile - postnatal day (PD) 30; adolescence - PD45; and adulthood - PD85]. In all brain regions studied, CB1R mRNA levels were highest in juveniles and then decreased progressively toward adolescent and adult levels in control and MAM-treated rats. However, in MAM-treated rats, CB1R mRNA levels were lower in the mPFC at PD85 and higher in the dorsolateral striatum at PD45 and PD85 relative to controls. Cellular analyses confirmed the changes in CB1R mRNA expression in MAM-treated rats. These findings are in accordance with previous studies showing a decrease in the CB1R mRNA expression from juvenile period to adolescence to adulthood in cortical, striatal, and hippocampal regions. Additionally, similar to most of the schizophrenia-like signs observed in the MAM model, embryonic exposure to MAM leads to schizophrenia-related changes in CB1R mRNA expression that only emerge later in development.