Pharmacological Rescue of Hippocampal Fear Learning Deficits in Fragile X Syndrome.

Fragile X Syndrome (FXS) is the leading cause of autism spectrum disorder and intellectual disability and results from loss of Fragile X mental retardation protein (FMRP). In neurons, FMRP controls the translation of synaptic plasticity proteins that are implicated in learning and memory. FMRP also regulates development- and experience-dependent actin cytoskeleton remodeling within dendritic spines through the small Rho GTPase Rac1. Modulation of Rac1 activity is critical during synaptic plasticity as well as learning and memory. We have previously shown that FXS mouse models exhibit learning and memory deficits as well as hyperactive Rac1 in the hippocampus. To determine whether pharmacological inhibition of Rac1 in FXS improves cognitive impairment, FXS mice were treated with the specific Rac1 inhibitor NSC23766, followed by fear conditioning. Whereas non-cognitive functions were unaffected, hippocampus-related memory improved in FXS mice treated with the Rac1 inhibitor. Furthermore, long-term potentiation in hippocampal slices from FXS mice was increased after incubation with the Rac1 inhibitor. Together, these observations indicate that modulation of Rac1 may provide a novel therapeutic target in the treatment of cognitive impairment in FXS.

Increased Training Intensity Induces Proper Membrane Localization of Actin Remodeling Proteins in the Hippocampus Preventing Cognitive Deficits: Implications for Fragile X Syndrome.

Behavioral intervention therapy has proven beneficial in the treatment of autism and intellectual disabilities (ID), raising the possibility of certain changes in molecular mechanisms activated by these interventions that may promote learning. Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by autistic features and intellectual disability and can serve as a model to examine mechanisms that promote learning. FXS results from mutations in the fragile X mental retardation 1 gene (Fmr1) that prevents expression of the Fmr1 protein (FMRP), a messenger RNA (mRNA) translation regulator at synapses. Among many other functions, FMRP organizes a complex with the actin cytoskeleton-regulating small Rho GTPase Rac1. As in humans, Fmr1 KO mice lacking FMRP display autistic-like behaviors and deformities of actin-rich synaptic structures in addition to impaired hippocampal learning and synaptic plasticity. These features have been previously linked to proper function of actin remodeling proteins that includes Rac1. An important step in Rac1 activation and function is its translocation to the membrane, where it can influence synaptic actin cytoskeleton remodeling during hippocampus-dependent learning. Herein, we report that Fmr1 KO mouse hippocampus exhibits increased levels of membrane-bound Rac1, which may prevent proper learning-induced synaptic changes. We also determine that increasing training intensity during fear conditioning (FC) training restores contextual memory in Fmr1 KO mice and reduces membrane-bound Rac1 in Fmr1 KO hippocampus. Increased training intensity also results in normalized long-term potentiation in hippocampal slices taken from Fmr1 KO mice. These results point to interventional treatments providing new therapeutic options for FXS-related cognitive dysfunction.

Moderate treadmill exercise prevents oxidative stress-induced anxiety-like behavior in rats.

Recent work has suggested correlation of oxidative stress with anxiety-like behavior. There also is evidence for anxiolytic effects of physical exercise. However, a direct role of oxidative stress in anxiety is not clear and a protective role of physical exercise in oxidative stress-mediated anxiety has never been addressed. In this study, we have utilized rats to test direct involvement of oxidative stress with anxiety-like behavior and have identified oxidative stress mechanisms likely involved in anxiolytic effects of physical exercise. Intraperitoneal injections at non-toxic dose of l-buthionine-(S,R)-sulfoximine (BSO), an agent that increases oxidative stress markers, increased anxiety-like behavior of rats compared to vehicle-treated control rats. Prior 2 weeks treatment with the antioxidant, tempol attenuated BSO-induced anxiety-like behavior of rats suggesting a role of oxidative stress in this phenomenon. Moreover, moderate treadmill exercise prevented BSO-induced anxiety-like behavior of rats and also prevented BSO-mediated increase in oxidative stress markers in serum, urine and brain tissue homogenates from hippocampus, amygdala and locus coeruleus. Thus increasing oxidative stress increases anxiety-like behavior of rats. Moreover, antioxidant or treadmill exercise training both reduce oxidative stress in the rat brain regions implicated in anxiety response and prevent anxiety-like behavior of rats.

Neurochemical, behavioral and architectural changes after chronic inactivation of NMDA receptors in mice.

Schizophrenia is a psychotic illness characterized by problems in perception, learning, and memory. Post-mortem clinical data revealed abnormalities in neuronal organization, reduced soma and dendritic tree size. In rodents, reduction of glutamatergic neurotransmission by NMDA receptor antagonists mimics symptoms of schizophrenia. However, the dosage, treatment and species used in previous studies have not been consistent, leading to a lack of correlation between the findings reported in low-dose, long-term treatment models and the results in acute or chronic high dose administration. Thus, the present study investigates whether long-term, low-dose blockade of NMDA receptors with MK-801 in the early postnatal period results in molecular, cellular, morphological and behavioral changes in the mouse, alterations that have been singly described by using different drugs and dosages in either mice or rats. We found that early postnatal administration of 0.1mg/kg MK-801 for 15 days altered protein translation, synapse formation, hippocampus-dependent learning and neuronal development, resembling findings reported in schizophrenia. These results suggest that there are strong parallels between this animal model and schizophrenia, which validates it as an animal model for this condition and lends further strength of the NMDA receptor hypofunction as a useful model for the study of psychosis.

Rapid isolation of synaptoneurosomes and postsynaptic densities from adult mouse hippocampus.

Previous postsynaptic density (PSD) isolation methodologies have utilized either whole brain or discrete brain regions of relatively large mammals such as dogs and rats. The present report details a simple and highly effective procedure for the rapid isolation of PSDs from small amounts of adult mouse hippocampus that has several advantages. First, by substituting synaptoneurosomes for synaptosomes as starting material, we have decreased the steps, time, and amount of tissue required to isolate PSDs. Second, by modifying critical steps in the synaptic isolation protocols we were able to isolate PSDs from less than 200 mg of mouse hippocampi in 3 h. Electron micrographs of isolated synaptoneurosomes showed presynaptic vesicles and densely stained membranes representing PSDs. Morphological examination of these PSDs by electron microscopy revealed a preparation that seems to be quite pure, with little or no membrane contamination. A comparison by Western blot analysis of synaptoneurosome and PSD fractions suggests that this technique yields a purified sample. Moreover, two different protocols using swing and fixed bucket rotors were used for this small-scale PSD isolation and both resulted in a very pure partition, supporting the idea that this procedure is reliable and consistent.

Comparative Detection of Fumonisin by HPLC, ELISA, and Immunocytochemical Localization in Fusarium Cultures.

Fumonisins are a group of mycotoxins that are elaborated by Fusarium moniliforme and Fusarium proliferatum and that have recently been associated with animal and human disease. In this study, the time course of fumonisin B1 (FB1) production in corn was monitored in five Fusarium cultures using high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and in situ localization by an enzyme-linked immunocytochemical technique (ELICT). Using HPLC on culture extracts prepared with 50% (vol/vol) acetonitrile in water, FB1 was detectable at 3 days with maximal FB1 (ranging from 230 to 3,000 ppm) occurring between 14 and 28 days. Although there was a positive correlation between FB1 detected by HPLC and ELISA, the latter consistently yielded higher results than HPLC. Maximal FB1 "equivalents" detected by ELISA ranged from 12,000 to 35,000 ppm. Following fixation of Fusarium from cultures, ELICT revealed the presence of large deposits indicative of fumonisin or fumonisin-like cross-reacting compounds in mycelia, microconidia, and microconidia. Prior to fixation, these compounds were extractable in 50% (vol/vol) acetonitrile in water. ELICT results qualitatively correlated with HPLC and ELISA over the time course of the cultures. Taken together, the results suggest that (a) ELISA or ELICT could be used for qualitative screening of FB1-producing cultures, and (b) in addition to FB1, the monoclonal antibody-based ELISA detected one or more compounds that structurally resemble FB1 and occur concurrently with FB1.