Mild carotid stenosis creates gradual, progressive, lifelong brain, and eye damage: An experimental laboratory rat model.

In humans, carotid stenosis of 70% and above might be the cause of clinical symptoms such as transient ischemic attack and stroke. No clinical or animal studies have evaluated mild carotid occlusion, and few examined unilateral occlusion. Here, Westar rats underwent bilateral or unilateral carotid occlusion of 28-45%. Long-term effects were evaluated 9-11 months later. We conducted cognitive evaluation using spatial learning in a water maze and exploration behavior in an open field. Morphology of the brain was examined by MRI using diffusion-tensor imaging (DTI) and immunohistochemistry staining of the brain and eyes. Cognitive deficit was found in spatial memory and exploration behavior in both occluded groups. Brain and eyes histology presented severe damage in the bilateral group, compared to the unilateral one. DTI revealed an increase in mean diffusivity (MD) in the ventral thalamus and a decrease in fractional anisotropy in optic nerve and optic tract in bilateral rats, while unilateral rats showed only an increase in MD in the ventral pons. In those areas, a significant change in astrocytes, microglia, and number of apoptotic cells were found. Bilateral occlusion produced severe damage to both retinas, while unilateral occlusion produced damage mainly in the occluded side. We found that mild carotid stenosis, even in a unilateral occlusion, creates behavioral abnormalities presented by brain and eye histopathology. The results support our hypothesis that gradual formation of mild carotid stenosis along the life course leads to progressive damage that may create different degenerative diseases at a later age.

Increased extracellular free-water in adult male rats following in utero exposure to maternal immune activation.

BACKGROUND: In previous work, we applied novel in vivo imaging methods to reveal that white matter pathology in patients with first-episode psychosis (FEP) is mainly characterized by excessive extracellular free-water, and to a lesser extent by cellular processes, such as demyelination. Here, we apply a back-translational approach to evaluate whether or not a rodent model of maternal immune activation (MIA) induces patterns of white matter pathology that we observed in patients with FEP. To this end, we examined free-water and tissue-specific white matter alterations in rats born to mothers exposed to the viral mimic polyriboinosinic-polyribocytidylic acid (Poly-I:C) in pregnancy, which is widely used to produce alterations relevant to schizophrenia and is characterized by a robust neuroinflammatory response. METHOD: Pregnant dams were injected on gestational day 15 with the viral mimic Poly-I:C (4 mg/kg) or saline. Diffusion-weighted magnetic resonance images were acquired from 17 male offspring (9 Poly-I:C and 8 saline) on postnatal day 90, after the emergence of brain structural and behavioral abnormalities. The free-water fraction (FW) and tissue-specific fractional anisotropy (FAT), as well as conventional fractional anisotropy (FA) were computed across voxels traversing a white matter skeleton. Voxel-wise and whole-brain averaged white matter were tested for significant microstructural alterations in immune-challenged, relative to saline-exposed offspring. RESULTS: Compared to saline-exposed offspring, those exposed to maternal Poly-I:C displayed increased extracellular FW averaged across voxels comprising a white matter skeleton (t(15) = 2.74; p = 0.01). Voxel-wise analysis ascribed these changes to white matter within the corpus callosum, external capsule and the striatum. In contrast, no significant between-group differences emerged for FAT or for conventional FA, measured across average and voxel-wise white matter. CONCLUSION: We identified excess FW across frontal white matter fibers of rats exposed to prenatal immune activation, analogous to our "bedside" observation in FEP patients. Findings from this initial experiment promote use of the MIA model to examine pathological pathways underlying FW alterations observed in patients with schizophrenia. Establishing these mechanisms has important implications for clinical studies, as free-water imaging reflects a feasible biomarker that has so far yielded consistent findings in the early stages of schizophrenia.

The autism/neuroprotection-linked ADNP/NAP regulate the excitatory glutamatergic synapse.

Activity-dependent neuroprotective protein (ADNP), essential for brain formation, was discovered as a leading de novo mutated gene causing the autism-like ADNP syndrome. This syndrome is phenotypically characterized by global developmental delays, intellectual disabilities, speech impediments, and motor dysfunctions. The Adnp haploinsufficient mouse mimics the human ADNP syndrome in terms of synapse density and gene expression patterns, as well as in developmental, motor, and cognitive abilities. Peripheral ADNP was also discovered as a biomarker for Alzheimer's disease and schizophrenia, with nasal administration of the ADNP snippet peptide NAP (enhancing endogenous ADNP activity) leading to partial cognitive and functional protection at the cellular, animal and clinical settings. Here, a novel formulation for effective delivery of NAP is provided with superior brain penetration capabilities. Also provided are methods for treating pertinent clinical implications such as autism, cognitive impairments, olfactory deficits, and muscle strength using the formulation in the Adnp haploinsufficient mouse. Results showed a dramatically specific increase in brain/body bioavailability with the new formulation, without breaching the blood brain barrier. Additional findings included improvements using daily intranasal treatments with NAP, at the behavioral and brain structural levels, diffusion tensor imaging (DTI), translatable to clinical practice. Significant effects on hippocampal and cerebral cortical expression of the presynaptic Slc17a7 gene encoding vesicular excitatory glutamate transporter 1 (VGLUT1) were observed at the RNA and immunohistochemical levels, explaining the DTI results. These findings tie for the first time a reduction in presynaptic glutamatergic synapses with the autism/Alzheimer's/schizophrenia-linked ADNP deficiency coupled with amelioration by NAP (CP201).

Immune activation in lactating dams alters sucklings' brain cytokines and produces non-overlapping behavioral deficits in adult female and male offspring: A novel neurodevelopmental model of sex-specific psychopathology.

Early immune activation (IA) in rodents, prenatal through the mother or early postnatal directly to the neonate, is widely used to produce behavioral endophenotypes relevant to schizophrenia and depression. Given that maternal immune response plays a crucial role in the deleterious effects of prenatal IA, and lactation is a critical vehicle of immunological support to the neonate, we predicted that immune activation of the lactating dam will produce long-term abnormalities in the sucklings. Nursing dams were injected on postnatal day 4 with the viral mimic poly-I:C (4mg/kg) or saline. Cytokine assessment was performed in dams' plasma and milk 2h, and in the sucklings' hippocampus, 6h and 24h following poly-I:C injection. Male and female sucklings were assessed in adulthood for: a) performance on behavioral tasks measuring constructs considered relevant to schizophrenia (selective attention and executive control) and depression (despair and anhedonia); b) response to relevant pharmacological treatments; c) brain structural changes. Maternal poly-I:C injection caused cytokine alterations in the dams' plasma and milk, as well as in the sucklings' hippocampus. Lactational poly-I:C exposure led to sex-dimorphic (non-overlapping) behavioral abnormalities in the adult offspring, with male but not female offspring exhibiting attentional and executive function abnormalities (manifested in persistent latent inhibition and slow reversal) and hypodopaminergia, and female but not male offspring exhibiting despair and anhedonia (manifested in increased immobility in the forced swim test and reduced saccharine preference) and hyperdopaminergia, mimicking the known sex-bias in schizophrenia and depression. The behavioral double-dissociation predicted distinct pharmacological profiles, recapitulating the pharmacology of negative/cognitive symptoms and depression. In-vivo imaging revealed hippocampal and striatal volume reductions in both sexes, as found in both disorders. This is the first evidence for the emergence of long-term behavioral and brain abnormalities after lactational exposure to an inflammatory agent, supporting a causal link between early immune activation and disrupted neuropsychodevelopment. That such exposure produces schizophrenia- or depression-like phenotype depending on sex, resonates with notions that risk factors are transdiagnostic, and that sex is a susceptibility factor for neurodevelopmental psychopathologies.

Maternal immune activation produces neonatal excitability defects in offspring hippocampal neurons from pregnant rats treated with poly I:C.

Maternal immune activation (MIA) resulting from prenatal exposure to infectious pathogens or inflammatory stimuli is increasingly recognized to play an important etiological role in neuropsychiatric disorders with neurodevelopmental features. MIA in pregnant rodents induced by injection of the synthetic double-stranded RNA, Poly I:C, a mimic of viral infection, leads to a wide spectrum of behavioral abnormalities as well as structural and functional defects in the brain. Previous MIA studies using poly I:C prenatal treatment suggested that neurophysiological alterations occur in the hippocampus. However, these investigations used only juvenile or adult animals. We postulated that MIA-induced alterations could occur earlier at neonatal/early postnatal stages. Here we examined the neurophysiological properties of cultured pyramidal-like hippocampal neurons prepared from neonatal (P0-P2) offspring of pregnant rats injected with poly I:C. Offspring neurons from poly I:C-treated mothers exhibited significantly lower intrinsic excitability and stronger spike frequency adaptation, compared to saline. A similar lower intrinsic excitability was observed in CA1 pyramidal neurons from hippocampal slices of two weeks-old poly I:C offspring. Cultured hippocampal neurons also displayed lower frequency of spontaneous firing, higher charge transfer of IPSCs and larger amplitude of miniature IPSCs. Thus, maternal immune activation leads to strikingly early neurophysiological abnormalities in hippocampal neurons.

Maturation- and sex-sensitive depression of hippocampal excitatory transmission in a rat schizophrenia model.

Schizophrenia is associated with behavioral and brain structural abnormalities, of which the hippocampus appears to be one of the most consistent region affected. Previous studies performed on the poly I:C model of schizophrenia suggest that alterations in hippocampal synaptic transmission and plasticity take place in the offspring. However, these investigations yielded conflicting results and the neurophysiological alterations responsible for these deficits are still unclear. Here we performed for the first time a longitudinal study examining the impact of prenatal poly I:C treatment and of gender on hippocampal excitatory neurotransmission. In addition, we examined the potential preventive/curative effects of risperidone (RIS) treatment during the peri-adolescence period. Excitatory synaptic transmission was determined by stimulating Schaffer collaterals and monitoring fiber volley amplitude and slope of field-EPSP (fEPSP) in CA1 pyramidal neurons in male and female offspring hippocampal slices from postnatal days (PNDs) 18-20, 34, 70 and 90. Depression of hippocampal excitatory transmission appeared at juvenile age in male offspring of the poly I:C group, while it expressed with a delay in female, manifesting at adulthood. In addition, a reduced hippocampal size was found in both adult male and female offspring of poly I:C treated dams. Treatment with RIS at the peri-adolescence period fully restored in males but partly repaired in females these deficiencies. A maturation- and sex-dependent decrease in hippocampal excitatory transmission occurs in the offspring of poly I:C treated pregnant mothers. Pharmacological intervention with RIS during peri-adolescence can cure in a gender-sensitive fashion early occurring hippocampal synaptic deficits.

Maternal immune activation induces changes in myelin and metabolic proteins, some of which can be prevented with risperidone in adolescence.

BACKGROUND: Maternal infection is a risk factor for schizophrenia but the molecular and cellular mechanisms are not fully known. Myelin abnormalities are amongst the most robust neuropathological changes observed in schizophrenia, and preliminary evidence suggests that prenatal inflammation may play a role. METHODS: Label-free liquid chromatography-mass spectrometry was performed on the prefrontal cortex (PFC) of adult rat offspring born to dams that were exposed on gestational day 15 to the viral mimic polyinosinic:polycytidylic acid [poly(I:C), 4 mg/kg] or saline and treated with the atypical antipsychotic drug risperidone (0.045 mg/kg) or saline in adolescence. Western blotting was employed to validate protein changes. RESULTS: Over 1,000 proteins were quantified in the PFC with pathway analyses implicating changes in core metabolic pathways, following prenatal poly(I:C) exposure. Some of these protein changes were absent in the PFC of poly(I:C)-treated offspring that subsequently received risperidone treatment in adolescence. Particularly interesting reductions in the expression of the myelin-related proteins myelin basic protein isoform 3 (MBP1) and rhombex 29 were observed, which were reversed by risperidone treatment. Validation by Western blotting confirmed changes in MBP1 and mitogen-activated kinase 1 (MAPK1). Western blotting was extended to assess the MAPK signalling proteins due to their roles in inflammation, namely phosphorylated MAPK1 and phosphorylated MAPK-activated protein kinase 2. Both were upregulated by poly(I:C) treatment and reversed by risperidone treatment. CONCLUSIONS: Overall, our data suggest that maternal inflammation may contribute to an increased risk for schizophrenia through mechanisms involving metabolic function and myelin formation and that risperidone in adolescence may prevent or reverse such changes.

Tracing the development of psychosis and its prevention: what can be learned from animal models.

Schizophrenia (SCZ) is a neurodevelopmental disorder manifested symptomatically after puberty whose pharmacotherapy remains unsatisfactory. In recent years, longitudinal structural neuroimaging studies have revealed that neuroanatomical aberrations occur in this disorder and in fact precede symptom onset, raising the exciting possibility that SCZ can be prevented. There is some evidence that treatment with atypical antipsychotic drugs (APDs) prior to the development of the full clinical phenotype reduces the risk of transition to psychosis, but results remain controversial. It remains unknown whether progressive structural brain aberrations can be halted. Given the diagnostic, ethical, clinical and methodological problems of pharmacological and imaging studies in patients, getting such information remains a major challenge. Animal neurodevelopmental models of SCZ are invaluable for investigating such questions because they capture the notion that the effects of early brain damage are progressive. In recent years, data derived from such models have converged on key neuropathological and behavioral deficits documented in SCZ attesting to their strong validity, and making them ideal tools for evaluating progression of pathology following in-utero insults as well as its prevention. We review here our recent studies that use longitudinal in vivo structural imaging to achieve this aim in the prenatal immune stimulation model that is based on the association of prenatal infection and increased risk for SCZ. Pregnant rats were injected on gestational day 15 with the viral mimic polyriboinosinic-polyribocytidylic acid (poly I:C) or saline. Male and female offspring were imaged and tested behaviorally on postnatal days (PNDs) 35, 46, 56, 70 and 90. In other experiments, offspring of poly I:C- and saline-treated dams received the atypical antipsychotic drugs (APDs) clozapine or risperidone in two developmental windows: PND 34-47 and PND 48-61, and underwent behavioral testing and imaging at adulthood. Prenatal poly I:C-induced interference with fetal brain development led to aberrant postnatal brain development as manifested in structural abnormalities in the hippocampus, the striatum, the prefrontal cortex and lateral ventricles (LV), as seen in SCZ. The specific trajectories were region-, age- and sex-specific, with females having delayed onset of pathology compared to males. Brain pathology was accompanied by development of behavioral abnormalities phenotypic of SCZ, attentional deficit and hypersensitivity to amphetamine, with same sex difference. Hippocampal volume loss and LV volume expansion as well as behavioral abnormalities were prevented in the offspring of poly I:C mothers who received clozapine or risperidone during the asymptomatic period of adolescence (PND 34-47). Administration at a later window, PNDs 48-61, exerted sex-, region- and drug- specific effects. Our data show that prenatal insult leads to progressive postnatal brain pathology, which gradually gives rise to "symptoms"; that treatment with atypical APDs can prevent both brain and behavioral pathology; and that the earlier the intervention, the more pathological outcomes can be prevented.

Effects of risperidone treatment in adolescence on hippocampal neurogenesis, parvalbumin expression, and vascularization following prenatal immune activation in rats.

Maternal infection in pregnancy is an environmental risk factor for the development of schizophrenia and related disorders in the offspring, and this association is recapitulated in animal models using gestational infection or immune stimulation. We have recently shown that behavioral abnormalities and altered hippocampal morphology emerging in adult offspring of dams treated with the viral mimic polyriboinosinic-polyribocytidilic acid (poly I:C) are prevented by treatment with the atypical antipsychotic drug risperidone (RIS) in adolescence. Here we used a battery of cellular markers and Nissl stain to morphometrically analyze different hippocampal cell populations in the offspring of poly I:C and saline-treated mothers that received saline or RIS in adolescence, at different time points of postnatal development. We report that impaired neurogenesis, disturbed micro-vascularization and loss of parvalbumin-expressing hippocampal interneurons, are found in the offspring of poly I:C-treated dams. Most, but not all, of these neuropathological changes are not present in poly I:C offspring that had been treated with RIS. These effects may be part of the complex processes underlying the capacity of RIS treatment in adolescence to prevent structural and behavioral abnormalities deficits in the poly I:C offspring.

Abnormal trajectories of neurodevelopment and behavior following in utero insult in the rat.

BACKGROUND: Environmental or genetic disturbances of early brain development are suggested to underlie the pathophysiology of several adult-onset neuropsychiatric disorders. We traced the developmental trajectories of brain structural and behavioral abnormalities from adolescence to young adulthood in rats born to mothers exposed to the viral mimic polyriboinosinic-polyribocytidylic acid (poly-I:C) in pregnancy. METHODS: Pregnant rats were injected on gestational day 15 with poly-I:C (4 mg/kg) or saline. Volumes of lateral ventricles, hippocampus, striatum, and prefrontal cortex in male and female offspring were assessed longitudinally at postnatal days 35, 46, 56, 70, and 90 using in vivo magnetic resonance imaging. At parallel time windows, groups of offspring from the same litters underwent behavioral testing (latent inhibition and amphetamine-induced activity) and magnetic resonance imaging (cross-sectional assessment). RESULTS: The specific developmental trajectories of volumetric changes in both control and poly-I:C offspring were region-, age-, and sex-specific, but overall, poly-I:C offspring had smaller volumes of the hippocampus, striatum and prefrontal cortex, and larger ventricular volume. Structural pathology in different regions had different times of onset and was gradually accompanied by behavioral deficits, disrupted latent inhibition, and excessive amphetamine-induced activity. The onset of structural frontocortical and ventricular abnormalities and behavioral abnormalities was delayed in females. In both sexes, hippocampal and striatal volume reduction predated the appearance of behavioral abnormalities. CONCLUSIONS: Prenatal insult interferes with postnatal brain maturation, which in turn may result in behavioral abnormalities.

Dynamic changes in neurexins' alternative splicing: role of Rho-associated protein kinases and relevance to memory formation.

The three neurexins genes (NRXN1/2/3) encode polymorphic synaptic membrane proteins that are involved in cognitive functioning. Neurexins' selectivity of function is presumably conferred through differential use of 2 promoters and 5 alternative splicing sites (SS#1/2/3/4/5). In day-old rat brain neurons grown in culture, activation (depolarization) induces reversible, calcium dependent, repression of NRXN2α SS#3 insert. The effects of depolarization on NRXN1/2/3α splicing and biochemical pathways mediating them were further studied in these neurons. NRXN1/2/3α splicing in the course of memory formation in vivo was also explored, using fear conditioning paradigm in rats in which the animals were trained to associate an aversive stimulus (electrical shock) with a neutral context (a tone), resulting in the expression of fear responses to the neutral context.In the cultured neurons depolarization induced, beside NRXN2α SS#3, repression of SS#3 and SS#4 exons in NRXN3α but not NRXN1α. The repressions were mediated by the calcium/protein kinase C/Rho-associated protein kinase (ROCK) pathway. Fear conditioning induced significant and transient repressions of the NRXN1/2/3α SS#4 exons in the rat hippocampus. ROCK inhibition prior to training attenuated the behavioral fear response, the NRXN1/2/3α splicing repressions and subsequent recovery and the levels of excitatory (PSD95) and inhibitory (gephyrin) synaptic proteins in the hippocampus. No such effects were observed in the prefrontal cortex. Significant correlations existed between the fear response and hippocampal NRXN3α and NRXN2α SS#4 inserts as well as PSD95 protein levels. Hippocampal NRXN1α SS#4 insert and gephyrin levels did not correlate with the behavioral response but were negatively correlated with each other.These results show for the first time dynamic, experience related changes in NRXN1/2/3α alternative splicing in the rat brain and a role for ROCK in them. Specific neurexins' transcripts may be involved in synaptic remodeling occurring at an intermediate (hours) time scale in the course of memory formation.

Risperidone administered during asymptomatic period of adolescence prevents the emergence of brain structural pathology and behavioral abnormalities in an animal model of schizophrenia.

Schizophrenia is a disorder of a neurodevelopmental origin manifested symptomatically after puberty. Structural neuroimaging studies show that neuroanatomical aberrations precede onset of symptoms, raising a question of whether schizophrenia can be prevented. Early treatment with atypical antipsychotics may reduce the risk of transition to psychosis, but it remains unknown whether neuroanatomical abnormalities can be prevented. We have recently shown, using in vivo structural magnetic resonance imaging, that treatment with the atypical antipsychotic clozapine during an asymptomatic period of adolescence prevents the emergence of schizophrenia-like brain structural abnormalities in adult rats exposed to prenatal immune challenge, in parallel to preventing behavioral abnormalities. Here we assessed the preventive efficacy of the atypical antipsychotic risperidone (RIS). Pregnant rats were injected on gestational day 15 with the viral mimic polyriboinosinic-polyribocytidylic acid (poly I:C) or saline. Their male offspring received daily RIS (0.045 or 1.2 mg/kg) or vehicle injection in peri-adolescence (postnatal days [PND] 34-47). Structural brain changes and behavior were assessed at adulthood (from PND 90). Adult offspring of poly I:C-treated dams exhibited hallmark structural abnormalities associated with schizophrenia, enlarged lateral ventricles and smaller hippocampus. Both of these abnormalities were absent in the offspring of poly I:C dams that received RIS at peri-adolescence. This was paralleled by prevention of schizophrenia-like behavioral abnormalities, attentional deficit, and hypersensitivity to amphetamine in these offspring. We conclude that pharmacological intervention during peri-adolescence can prevent the emergence of behavioral abnormalities and brain structural pathology resulting from in utero insult. Furthermore, highly selective 5HT(2A) receptor antagonists may be promising targets for psychosis prevention.

Clozapine administration in adolescence prevents postpubertal emergence of brain structural pathology in an animal model of schizophrenia.

BACKGROUND: Schizophrenia is a neuropsychiatric disorder of a neurodevelopmental origin manifested symptomatically after puberty. Structural neuroimaging studies show that neuroanatomical aberrations occur before onset of symptoms, raising a question of whether schizophrenia can be prevented. Treatment with atypical antipsychotic drugs before the development of the full clinical phenotype might reduce the risk of transition to psychosis, but it remains unknown whether neuroanatomical abnormalities can be prevented. We used a neurodevelopmental animal model of schizophrenia to assess the efficacy of the atypical antipsychotic clozapine to prevent neuroanatomical deterioration. METHODS: Pregnant rats received injection on gestational day 15 with the viral mimic polyriboinosinic-polyribocytidylic acid (PolyI:C) or saline. Structural brain changes in the male offspring were assessed at adolescence and adulthood (35 days and 120 days) with structural neuroimaging. In the second part, male offspring of PolyI:C- and saline-treated dams received daily clozapine (7.5 mg/kg) or saline injection in adolescence (days 34-47) and underwent behavioral testing and imaging at adulthood (from 90 days onward). RESULTS: In utero exposure to maternal infection led in the offspring to postpubertal emergence of hallmark structural abnormalities associated with schizophrenia, enlarged ventricles, and smaller hippocampus. These abnormalities were not observed in the offspring of mothers who received PolyI:C that were treated with clozapine in adolescence. This was paralleled by prevention of behavioral abnormalities phenotypic of schizophrenia, attentional deficit, and hypersensitivity to amphetamine. CONCLUSIONS: This is the first demonstration that pharmacological intervention during adolescence can prevent the emergence of brain structural changes resulting from in-utero insult.