Assessment of the inbred C57BL/6 and outbred CD1 mouse strains using a progressive ratio schedule during development.

Inbred strains have a genetic similarity of at least 98.6% compared to their outbred counterparts. Several studies have shown that inbred C57BL/6 mice and outbred ICR (CD1) mice differ in locomotion, cognitive flexibility, and aggression. However, their performance in operant paradigms is not well understood. A progressive ratio (PR) schedule of reinforcement is a method of quantitative estimation of the incentive state of an animal for a reward by increasing response requirements for reinforcer delivery, which is relevant to assess the breakpoint (amount of response effort an animal is willing to invest for a single unit of reward). This study tested male and female C57BL/6 and CD1 mice with an open field to analyze locomotion. Then, we used conditioning chambers with a PR3 schedule for ten consecutive days (P30-P40). PR performance was measured with the breakpoint, and the mathematical principles of reinforcement (MPR) were used to estimate motivation, impulsivity, and motor skills to manipulate the operandum. We found that CD1 mice showed higher locomotor activity than C57BL/6 independently of sex. CD1 mice had a higher breakpoint. However, male CD1 mice gradually increased breakpoint until the last session. In the MPR model, CD1 mice showed decreased fixed paused parameter (impulsivity) than C57BL/6, independent of sex. Our data suggest that the higher breakpoint in CD1 strain may partially be related to impulsivity. Therefore, the MPR model can help identify factors that affect performances, such as motivation, impulsivity, and motor skills during a PR in adolescent CD1 and C57BL/6 mice. These findings are essential to characterize the differences in the behavioral performance between C57BL/6 and CD1 strains and their potential as animal models.

Raman spectroscopy to assess the differentiation of bone marrow mesenchymal stem cells into a glial phenotype.

Background: Mesenchymal stem cells (MSCs) are multipotent precursor cells with the ability to self-renew and differentiate into multiple cell linage, including the Schwann-like fate that promotes regeneration after lesion. Raman spectroscopy provides a precise characterization of the osteogenic, adipogenic, hepatogenic and myogenic differentiation of MSCs. However, the differentiation of bone marrow mesenchymal stem cells (BMSCs) towards a glial phenotype (Schwann-like cells) has not been characterized before using Raman spectroscopy. Method: We evaluated three conditions: 1) cell culture from rat bone marrow undifferentiated (uBMSCs), and two conditions of differentiation; 2) cells exposed to olfactory ensheathing cells-conditioned medium (dBMSCs) and 3) cells obtained from olfactory bulb (OECs). uBMSCs phenotyping was confirmed by morphology, immunocytochemistry and flow cytometry using antibodies of cell surface: CD90 and CD73. Glial phenotype of dBMSCs and OECs were verified by morphology and immunocytochemistry using markers of Schwann-like cells and OECs such as GFAP, p75 NTR and O4. Then, the Principal Component Analysis (PCA) of Raman spectroscopy was performed to discriminate components from the high wavenumber region between undifferentiated and glial-differentiated cells. Raman bands at the fingerprint region also were used to analyze the differentiation between conditions. Results: Differences between Raman spectra from uBMSC and glial phenotype groups were noted at multiple Raman shift values. A significant decrease in the concentration of all major cellular components, including nucleic acids, proteins, and lipids were found in the glial phenotype groups. PCA analysis confirmed that the highest spectral variations between groups came from the high wavenumber region observed in undifferentiated cells and contributed with the discrimination between glial phenotype groups. Conclusion: These findings support the use of Raman spectroscopy for the characterization of uBMSCs and its differentiation in the glial phenotype.

Normal pressure hydrocephalus decreases the proliferation of oligodendrocyte progenitor cells and the expression of CNPase and MOG proteins in the corpus callosum before behavioral deficits occur.

Normal pressure hydrocephalus (NPH) compromises the morphology of the corpus callosum (CC). This study aims to determine whether 60- or 120-day NPH disrupts the cytoarchitecture and functioning of white matter (WM) and oligodendrocyte precursor cells (OPCs) and establish whether these changes are reversible after hydrocephalus treatment. NPH was induced in CD1 adult mice by inserting an obstructive lamina in the atrium of the aqueduct of Sylvius. Five groups were assembled: sham-operated controls (60 and 120 days), NPH groups (60 and 120 days), and the hydrocephalus-treated group (obstruction removal after 60-d hydrocephalus). We analyzed the cellular integrity of the CC by immunohistochemistry, TUNEL analysis, Western blot assays, and transmission electron microscopy (TEM). We found a reduction in the width of the CC at 60 and 120 days of NPH. TEM analysis demonstrated myelin abnormalities, degenerative changes in the WM, and an increase in the number of hyperdense (dark) axons that were associated with significant astrogliosis, and microglial reactivity. Hydrocephalus also caused a decrease in the expression of myelin-related proteins (MOG and CNPase) and reduced proliferation and population of OPCs, resulting in fewer mature oligodendrocytes. Hydrocephalus resolution only recovers the OPC proliferation and MOG protein density, but the rest of the WM abnormalities persisted. Interestingly, all these cellular and molecular anomalies occur in the absence of behavioral changes. The results suggest that NPH severely disrupts the myelin integrity and affects the OPC turnover in the CC. Remarkably, most of these deleterious events persist after hydrocephalus treatment, which suggests that a late treatment conveys irreversible changes in the WM of CC.

Phenytoin promotes the proliferation of oligodendrocytes and enhances the expression of myelin basic protein in the corpus callosum of mice demyelinated by cuprizone.

Oligodendrocyte loss and myelin sheet destruction are crucial characteristics of demyelinating diseases. Phenytoin promotes the proliferation of endogenous neural precursor cells in the ventricular-subventricular zone in the postnatal brain that help restore the oligodendroglial population. This study aimed to evaluate whether phenytoin promotes myelin recovery of the corpus callosum of demyelinated adult mice. CD1 male mice were exposed to a demyelinating agent (0.2% cuprizone) for 8 weeks. We assembled two groups: the phenytoin-treated group and the control-vehicle group. The treated group received oral phenytoin (10 mg/kg) for 4 weeks. We quantified the number of Olig2 + and NG2 + oligodendrocyte precursor cells (OPCs), Rip + oligodendrocytes, the expression level of myelin basic protein (MBP), and the muscle strength and motor coordination. The oligodendroglial lineage (Olig2 + cells, NG2 + cells, and RIP + cells) significantly increases by the phenytoin administration when compared to the control-vehicle group. The phenytoin-treated group also showed an increased expression of MBP in the corpus callosum and better functional scores in the horizontal bar test. These findings suggest that phenytoin stimulates the proliferation of OPCs, re-establishes the oligodendroglial population, promotes myelin recovery in the corpus callosum, and improves motor coordination and muscle strength.

Chronic exposure to cyclohexane induces stereotypic circling, hyperlocomotion, and anxiety-like behavior associated with atypical c-Fos expression in motor- and anxiety-related brain regions.

Recreational abuse of solvents continues, despite cyclohexane (CHX) is used as a safe replacement in gasoline or adhesive formulations. Increasing evidence indicates that CHX inhalation affects brain functioning; however, scanty information is available about its effects on behavior and brain activity upon drug removal. In this study, we used CD1 adult mice to mimic an intoxication period of recreational drugs for 30 days. During the CHX exposure (~30,000 ppm), we analyzed exploratory and biphasic behaviors, stereotypic circling, and locomotion. After CHX removal (24 h or a month later), we assessed anxiety-like behaviors and quantified c-Fos cells in motor- and anxiety-related brain regions. Our findings indicate that the repeated inhalation of CHX produced steady hyperactivity and reduced ataxia, sedation, and seizures as the exposure to CHX progressed. Also, CHX decreased grooming and rearing behaviors. In the first week of CHX inhalation, a stereotypic circling behavior emerged, and locomotion increased gradually. One month after CHX withdrawal, mice showed low activity in the center zone of the open field and more buried marbles. Twenty-four hours after CHX removal, c-Fos expression was low in the dorsal striatum, ventral striatum, motor cortex, dorsomedial prefrontal cortex, basolateral amygdala, lateral hypothalamus, and ventral hippocampus. One month later, c-Fos expression remained low in the ventral striatum and lateral hypothalamus but increased in the dorsomedial prefrontal cortex and primary motor cortex. This study provides a comprehensive behavioral characterization and novel histological evidence of the CHX effects on the brain when is administered in a recreational-like mode.

Characterization of a mouse model of chronic hydrocephalus induced by partial occlusion of the aqueduct of Sylvius in the adult brain.

BACKGROUND: Hydrocephalus is a neurologic disturbance produced by the abnormal production, circulation, and absorption of cerebrospinal fluid (CSF). Late-onset idiopathic aqueductal stenosis induces normal pressure hydrocephalus (NPH) in adults. To date, no animal model replicating chronic NPH is available to study the pathophysiological changes observed in these subjects. NEW METHOD: We performed and characterized a model that induces chronic hydrocephalus in the adult mouse brain by producing a pre-aqueductal semiobstruction using an acetate lamina inserted into the atrium of the aqueduct of Sylvius. After surgical procedure, we analyzed the hydrocephalus development on days 60 and 120 and sham-operated animals were used as controls. We included an additional group of hydrocephalus resolution in which we removed the obstruction and analyzed the morphological changes in the brain. RESULTS: The hydrocephalus was fully established on day 60 after the obstruction and remained stable for 120 days. In all animals, the intracranial pressure remained ~4.08 mmHg and we did not find statistically significant differences between the hydrocephalus groups and controls. We did not find motor impairments and anxiety-like behaviors among groups and the analysis of microglia and astrogliosis revealed mild glial reactivity. COMPARISON WITH EXISTING METHODS: This model generates a long-term ventricular enlargement with normal intracranial pressure and moderate glial reactivity. Importantly, this model allows the reversibility of ventricular enlargement after the removal of the obstructive film from the brain. CONCLUSIONS: This mouse model may be useful to study the long-term cerebral alterations that occur during NPH or after its surgical resolution.

Cyclohexane Inhalation Produces Long-Lasting Alterations in the Hippocampal Integrity and Reward-Seeking Behavior in the Adult Mouse.

Cyclohexane (CHX) is an organic solvent commonly used as a drug-of-abuse. This drug increases the oxidative stress and glial reactivity in the hippocampus, which suggests that this brain region is vulnerable to CHX effects. This study aimed to establish the behavioral changes and the pathological alterations that occur in the Cornu Ammonis 3 (CA3) and Dentate Gyrus (DG) after a long-lasting exposure to CHX. We exposed CD1 mice to a recreational-like dose of CHX (~ 30,000 ppm) for 30 days and explored its consequences in motor skills, reward-seeking behavior, and the CA3 and DG hippocampal subfields. Twenty-four hours after the last administration of CHX, we found a significant decrease in the number of c-Fos+ cells in the hippocampal CA3 and DG regions. This event coincided with an increased in NMDAR1 expression and apoptotic cells in the CA3 region. At day 13th without CHX, we found a persistent reduction in the number of c-Fos+ and TUNEL+ cells in DG. At both time points, the CHX-exposed mice showed a strong overexpression of neuropeptide Y (NPY) in the CA3 stratum lucidum and the hippocampal hilus. In parallel, we used an operant-based task to assess motor performance and operant conditioning learning. The behavioral analysis indicated that CHX did not modify the acquisition of operant conditioning tasks, but affected some motor skills and increased the reward-seeking behavior. Altogether, this evidence reveals that CHX exposure provokes long-lasting changes in the hippocampal subfields, induces motor impairments and increases the motivation-guided behavior. These findings can help understand the deleterious effect of CHX into the adult hippocampus and unveil its potential to trigger addiction-like behaviors.

Alterations of Growth Factors in Autism and Attention-Deficit/Hyperactivity Disorder.

Growth factors (GFs) are cytokines that regulate the neural development. Recent evidence indicates that alterations in the expression level of GFs during embryogenesis are linked to the pathophysiology and clinical manifestations of attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorders (ASD). In this concise review, we summarize the current evidence that supports the role of brain-derived neurotrophic factor, insulin-like growth factor 2, hepatocyte growth factor (HGF), glial-derived neurotrophic factor, nerve growth factor, neurotrophins 3 and 4, and epidermal growth factor in the pathogenesis of ADHD and ASD. We also highlight the potential use of these GFs as clinical markers for diagnosis and prognosis of these neurodevelopmental disorders.

Growth factors as clinical biomarkers of prognosis and diagnosis in psychiatric disorders.

The psychiatric disorders are one of the most disabling illnesses in the world and represent a major problem for public health. These disorders are characterized by neuroanatomical or biochemical changes and it has been suggested that such changes may be due to inadequate neurodevelopment. Diverse alterations in the gene expression and/or serum level of specific growth factors have been implicated in the etiology, symptoms and progression of some psychiatric disorders. Herein, we summarize the latest information regarding the role of brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), fibroblast growth factor (FGF), Insulin-like growth factor (IGF-1), neuroregulin-1 (NGR-1), erythropoietin (EPO), vascular growth factor (VEGF), transforming growth factor beta (TGF-ß), nerve growth factor (NGF) and others cytokines in the pathogenesis of schizophrenia, depression, bipolar and anxiety disorders. Focusing on the role of these growth factors and their relationship with the main impairments (cognitive, emotional and social) of these pathologies. Some of these signaling molecules may be suitable biological markers for diagnosis and prognosis in cognitive, mood and social disabilities across different mental disorders.

Phenytoin enhances the phosphorylation of epidermal growth factor receptor and fibroblast growth factor receptor in the subventricular zone and promotes the proliferation of neural precursor cells and oligodendrocyte differentiation.

Phenytoin is a widely used antiepileptic drug that induces cell proliferation in several tissues, such as heart, bone, skin, oral mucosa and neural precursors. Some of these effects are mediated via fibroblast growth factor receptor (FGFR) and epidermal growth factor receptor (EGFR). These receptors are strongly expressed in the adult ventricular-subventricular zone (V-SVZ), the main neurogenic niche in the adult brain. The aim of this study was to determine the cell lineage and cell fate of V-SVZ neural progenitors expanded by phenytoin, as well as the effects of this drug on EGFR/FGFR phosphorylation. Male BALB/C mice received 10 mg/kg phenytoin by oral cannula for 30 days. We analysed the proliferation of V-SVZ neural progenitors by immunohistochemistry and western blot. Our findings indicate that phenytoin enhanced twofold the phosphorylation of EGFR and FGFR in the V-SVZ, increased the number of bromodeoxyuridine (BrdU)+/Sox2+ and BrdU+/doublecortin+ cells in the V-SVZ, and expanded the population of Olig2-expressing cells around the lateral ventricles. After phenytoin removal, a large number of BrdU+/Receptor interacting protein (RIP)+ cells were observed in the olfactory bulb. In conclusion, phenytoin enhanced the phosphorylation of FGFR and EGFR, and promoted the expression of neural precursor markers in the V-SVZ. In parallel, the number of oligodendrocytes increased significantly after phenytoin removal.

Cyclohexane produces behavioral deficits associated with astrogliosis and microglial reactivity in the adult hippocampus mouse brain.

Cyclohexane is a volatile substance that has been utilized as a safe substitute of several organic solvents in diverse industrial processes, such as adhesives, paints, paint thinners, fingernail polish, lacquers, and rubber industry. A number of these commercial products are ordinarily used as inhaled drugs. However, it is not well known whether cyclohexane has noxious effects in the central nervous system. The aim of this study was to analyze the effects of cyclohexane inhalation on motor behavior, spatial memory, and reactive gliosis in the hippocampus of adult mice. We used a model that mimics recreational drug use in male Balb/C mice (P60), divided into two groups: controls and the cyclohexane group (exposed to 9,000 ppm of cyclohexane for 30 days). Both groups were then evaluated with a functional observational battery (FOB) and the Morris water maze (MWM). Furthermore, the relative expression of AP endonuclease 1 (APE1), and the number of astrocytes (GFAP+ cells) and microglia (Iba1+ cells) were quantified in the hippocampal CA1 and CA3 areas. Our findings indicated that cyclohexane produced severe functional deficits during a recreational exposure as assessed by the FOB. The MWM did not show statistically significant changes in the acquisition and retention of spatial memory. Remarkably, a significant increase in the number of astrocytes and microglia cells, as well as in the cytoplasmic processes of these cells were observed in the hippocampal CA1 and CA3 areas of cyclohexane-exposed mice. This cellular response was associated with an increase in the expression of APE1 in the same brain regions. In summary, cyclohexane exposure produces functional deficits that are associated with an important increase in the APE1 expression as well as the number of astrocytes and microglia cells and their cytoplasmic complexity in the CA1 and CA3 regions of the adult hippocampus.

Cyclohexane, a Potential Drug of Abuse with Pernicious Effects on the Brain.

Cyclohexane is a volatile solvent used as a harmless substitute for dangerous organic solvents in several products, such as paint thinners, gasoline and adhesives. Many of these products are used as drugs of abuse and can severely damage neural tissue and impair neurological functions. However, there is very little information on the effects of cyclohexane on the brain. In humans, cyclohexane produces headaches, sleepiness, dizziness, limb weakness, motor changes, and verbal memory impairment. Recent studies in mice have demonstrated behavioral alterations, reactive gliosis, microglial reactivity, and oxidative stress in the brains of cyclohexane-exposed animals. This indicates that cyclohexane may represent a potential problem for public health. Therefore, studies are needed to clarify the neurobiological effects of this volatile compound, including the cellular and molecular mechanisms of neurotoxicity, and to minimize the human health risk posed by the intentional or accidental inhalation of this potential drug of abuse.

Long-term hydrocephalus alters the cytoarchitecture of the adult subventricular zone.

Hydrocephalus can develop secondarily to a disturbance in production, flow and/or absorption of cerebrospinal fluid. Experimental models of hydrocephalus, especially subacute and chronic hydrocephalus, are few and limited, and the effects of hydrocephalus on the subventricular zone are unclear. The aim of this study was to analyze the effects of long-term obstructive hydrocephalus on the subventricular zone, which is the neurogenic niche lining the lateral ventricles. We developed a new method to induce hydrocephalus by obstructing the aqueduct of Sylvius in the mouse brain, thus simulating aqueductal stenosis in humans. In 120-day-old rodents (n=18 per group), the degree of ventricular dilatation and cellular composition of the subventricular zone were studied by immunofluorescence and transmission electron microscopy. In adult patients (age>18years), the sizes of the subventricular zone, corpus callosum, and internal capsule were analyzed by magnetic resonance images obtained from patients with and without aqueductal stenosis (n=25 per group). Mice with 60-day hydrocephalus had a reduced number of Ki67+ and doublecortin+cells on immunofluorescence, as well as decreased number of neural progenitors and neuroblasts in the subventricular zone on electron microscopy analysis as compared to non-hydrocephalic mice. Remarkably, a number of extracellular matrix structures (fractones) contacting the ventricular lumen and blood vessels were also observed around the subventricular zone in mice with hydrocephalus. In humans, the widths of the subventricular zone, corpus callosum, and internal capsule in patients with aqueductal stenosis were significantly smaller than age and gender-matched patients without aqueductal stenosis. In summary, supratentorial hydrocephalus reduces the proliferation rate of neural progenitors and modifies the cytoarchitecture and extracellular matrix compounds of the subventricular zone. In humans, this similar process reduces the subventricular niche as well as the width of corpus callosum and internal capsule.