Characterization of Behavioral, Signaling and Cytokine Alterations in a Rat Neurodevelopmental Model for Schizophrenia, and Their Reversal by the 5-HT6 Receptor Antagonist SB-399885.

Post-weaning social isolation of rats produces neuroanatomical, neurochemical and behavioral alterations resembling some core features of schizophrenia. This study examined the ability of the 5-HT6 receptor antagonist SB-399885 to reverse isolation-induced cognitive deficits, then investigated alterations in hippocampal cell proliferation and hippocampal and frontal cortical expression of selected intracellular signaling molecules and cytokines. Male Lister hooded rats (weaned on post-natal days 21-24 and housed individually or in groups of 3-4) received six i.p. injections of vehicle (1% Tween 80, 1 mL/kg) or SB-399885 (5 or 10 mg/kg) over a 2-week period starting 40 days post-weaning, on the days that locomotor activity, novel object discrimination (NOD), pre-pulse inhibition of acoustic startle and acquisition, retention and extinction of a conditioned freezing response (CFR) were assessed. Tissue was collected 24 h after the final injection for immunohistochemistry, reverse-phase protein microarray and western blotting. Isolation rearing impaired NOD and cue-mediated CFR, decreased cell proliferation within the dentate gyrus, and elevated hippocampal TNFα levels and Cdc42 expression. SB-399885 reversed the NOD deficit and partially normalized CFR and cell proliferation. These effects were accompanied by altered expression of several members of the c-Jun N-terminal Kinase (JNK) and p38 MAPK signaling pathways (including TAK1, MKK4 and STAT3). Although JNK and p38 themselves were unaltered at this time point hippocampal TAK1 expression and phosphorylation correlated with visual recognition memory in the NOD task. Continued use of this neurodevelopmental model could further elucidate the neurobiology of schizophrenia and aid assessment of novel therapies for drug-resistant cognitive symptoms.

Behavioural and neurochemical comparison of chronic intermittent cathinone, mephedrone and MDMA administration to the rat.

The synthetic cathinone derivative, mephedrone, is a controlled substance across Europe. Its effects have been compared by users to 3,4-methylenedioxymethamphetamine (MDMA), but little data exist on its pharmacological properties. This study compared the behavioural and neurochemical effects of mephedrone with cathinone and MDMA in rats. Young-adult male Lister hooded rats received i.p. cathinone (1 or 4 mg/kg), mephedrone (1, 4 or 10mg/kg) or MDMA (10mg/kg) on two consecutive days weekly for 3 weeks or as a single acute injection (for neurochemical analysis). Locomotor activity (LMA), novel object discrimination (NOD), conditioned emotional response (CER) and prepulse inhibition of the acoustic startle response (PPI) were measured following intermittent drug administration. Dopamine, 5-hydroxytryptamine (5-HT) and their major metabolites were measured in striatum, frontal cortex and hippocampus by high performance liquid chromatography 7 days after intermittent dosing and 2h after acute injection. Cathinone (1, 4 mg/kg), mephedrone (10mg/kg) and MDMA (10mg/kg) induced hyperactivity following the first and sixth injections and sensitization to cathinone and mephedrone occurred with chronic dosing. All drugs impaired NOD and mephedrone (10mg/kg) reduced freezing in response to contextual re-exposure during the CER retention trial. Acute MDMA reduced hippocampal 5-HT and 5-HIAA but the only significant effect on dopamine, 5-HT and their metabolites following chronic dosing was altered hippocampal 3,4-dihydroxyphenylacetic acid (DOPAC), following mephedrone (4, 10mg/kg) and MDMA. At the doses examined, mephedrone, cathinone, and MDMA induced similar effects on behaviour and failed to induce neurotoxic damage when administered intermittently over 3 weeks.

Effects of 5-FU.

5-fluorouracil (5-FU) is a chemotherapeutical agent used to treat cancers including breast and colorectal. Working as an antimetabolite to prevent cell proliferation, it primarily inhibits the enzyme thymidylate synthase blocking the thymidine formation required for DNA synthesis. Although having a relatively short half-life (< 30 mins) it readily enters the brain by passive diffusion. Clinically, it is used both as a single agent or in combination with other chemotherapies and has been associated with the long-term side effects of cognitive impairment, known as "chemo brain" or "chemo fog" These accounts have come primarily from patients undergoing treatment for breast cancer who report symptoms including confusion and memory impairment, which can last for months to years. Psychometric studies of patients have suffered from confounding variables, which has led to the use of rodent models to assess the cognitive effects of this drug. Researchers have used behavioral and physiological tests including the Morris water maze, novel object location/recognition tests, shock motivated T-maze, sensory gating and conditioning, to investigate the effect of this drug on cognition. The variety of cognitive tests and the difference in dosing and administration of 5-FU has led to varied results, possibly due to the different brain regions associated with each test and the subtlety of the drug's effect, but overall these studies indicates that 5-FU has a negative effect on memory, executive function and sensory gating. 5-FU has also been demonstrated to have biochemical and structural changes on specific regions of the brain. Evidence shows it can induce apoptosis and depress cell proliferation in the neurogenic regions of the adult brain including the sub granular zone (SGZ) within the hippocampus and in oligodendrocyte precursor populations within white matter tracts. Furthermore, investigations indicate levels ofdoublecortin, a marker for newly formed neurons and brain derived neurotrophic factor, a cell survival modulator, are also reduced by 5-FU in the SGZ. Thus, 5-FU appears to have a lasting negative impact on cognition and to affect cellular and biochemical markers in various brain regions. Further work is needed to understand the exact mechanisms involved and to devise strategies for the prevention or recovery from these symptoms.

Fluoxetine improves the memory deficits caused by the chemotherapy agent 5-fluorouracil.

Cancer patients who have been treated with systemic adjuvant chemotherapy have described experiencing deteriorations in cognition. A widely used chemotherapeutic agent, 5-fluorouracil (5-FU), readily crosses the blood-brain barrier and so could have a direct effect on brain function. In particular this anti mitotic drug could reduce cell proliferation in the neurogenic regions of the adult brain. In contrast reports indicate that hippocampal dependent neurogenesis and cognition are enhanced by the SSRI antidepressant Fluoxetine. In this investigation the behavioural effects of chronic (two week) treatment with 5-FU and (three weeks) with Fluoxetine either separately or in combination with 5-FU were tested on adult Lister hooded rats. Behavioural effects were tested using a context dependent conditioned emotional response test (CER) which showed that animals treated with 5-FU had a significant reduction in freezing time compared to controls. A separate group of animals was tested using a hippocampal dependent spatial working memory test, the object location recognition test (OLR). Animals treated only with 5-FU showed significant deficits in their ability to carry out the OLR task but co administration of Fluoxetine improved their performance. 5-FU chemotherapy caused a significant reduction in the number of proliferating cells in the sub granular zone of the dentate gyrus compared to controls. This reduction was eliminated when Fluoxetine was co administered with 5-FU. Fluoxetine on its own had no effect on proliferating cell number or behaviour. These findings suggest that 5-FU can negatively affect both cell proliferation and hippocampal dependent working memory and that these deficits can be reversed by the simultaneous administration of the antidepressant Fluoxetine.

5-Fluorouracil chemotherapy affects spatial working memory and newborn neurons in the adult rat hippocampus.

Chemotherapy-associated memory deficits in adults are prevalent with systemic treatment utilizing 5-fluorouracil (5-Fu). 5-Fu disrupts cell proliferation and readily crosses the blood-brain barrier. Proliferating cells within the adult dentate gyrus of the hippocampus give rise to new neurons involved in memory and learning and require neurotrophic factors such as brain-derived neurotrophic factor (BDNF) to nurture this process of adult neurogenesis. Some of these proliferating cells are anatomically and functionally supported by vascular endothelial cells. We propose that systemically administered 5-Fu chemotherapy will cause deficits in hippocampal memory that are associated with altered BDNF levels and proliferating cells (particularly vascular-associated cells) in the dentate gyrus. This was tested by determining the effect of 5-Fu on spatial working memory as modelled by the object location recognition test. Numbers of vascular-associated (VA) and non-vascular-associated (NVA) proliferating cells in the dentate gyrus were measured using double-labelling immunohistochemistry with markers of proliferation (Ki67) and endothelial cells (RECA-1). 5-Fu-induced changes in hippocampal BDNF and doublecortin (DCX) protein levels were quantified using Western immunoblotting. 5-Fu chemotherapy caused a marginal disruption in spatial working memory and did not alter the total proliferating cell counts or the percentage of VA and NVA proliferating cells in the dentate gyrus. In contrast, 5-Fu significantly reduced BDNF and DCX levels in the hippocampus, indicating alterations in neurotrophin levels and neurogenesis. These findings highlight the usefulness of animal models of 'chemobrain' for understanding the mechanisms that underlie chemotherapy-associated declines in cognitive performance and memory.

Treatment with olanzapine increases cell proliferation in the subventricular zone and prefrontal cortex.

The present study examines the effect of chronic treatment with two atypical neuroleptics, commonly used to treat schizophrenia. Adult rats were given either risperidone or olanzapine in their drinking water for 21 days. Memory was assessed on the first and last day of treatment using an object discrimination test, and the rate of cell proliferation in the subventricular zone (SVZ), dentate gyrus (DG) and prefrontal cortex (PFC) was quantified by immuno staining for Ki-67. The results show that both risperidone and olanzapine significantly improved performance in object discrimination after 21 days, and additionally, olanzapine significantly increased cell proliferation in the SVZ and PFC but not the DG.

Gene transfer into intact fetal skeletal muscle grown in vitro.

The development of an organ culture system for growing prenatal intercostal muscle in vitro and its use to study gene function is described. Fetal skeletal muscle is relatively inaccessible during the key stages of its development, and this method enables DNA transfections and other manipulations to be carried out. The system allows cell proliferation and differentiation to continue and also maintains the morphology and fiber types of developing muscle. Gene transfer into cultured embryonic intercostal muscle was achieved by square-pulse electroporation of intact pieces of tissue. Expression of a marker gene (GFP) was found within 5 h and maintained for 2 days in muscle fibers and cells. The technique should enable the function of genes implicated in muscle development and disease to be studied at stages when access is difficult and in a controlled environment.

Novel strategy to study gene expression and function in developing cerebellar granule cells.

The advent of techniques for global analyses of cell biology, such as genomics and proteomics, opens the way to rapid progress in understanding the molecular control of developing tissues. However, such studies in the CNS are hindered by the complexity of this tissue. In particular, few approaches allow cells to be isolated that are enriched for specific stages of their maturation. We describe a new strategy to study gene expression and function in cerebellar granule cells. In these experiments, we have used square pulse electroporation to introduce fluorescent dye or DNA constructs into immature granule cell precursors in situ. This method only labels granule cell precursors in the superficial part of the external granule layer. Combining this labelling with fluorescent activated cell sorting (FACS) allows the transfected cells to be isolated at any time during their subsequent development, thus providing a means of analysing granule cells as they undergo maturation. This transfection method can be used to study events in the normal maturation of granule cells or the effects of introduced transgenes. Such studies can be carried out on cells purified from primary cultures or cells in situ using cerebellar slice cultures. Our strategy provides a new route to detailed analysis of the role of genes in controlling many aspects of granule cell biology. These approaches will allow recent global analyses to be more readily applied to subpopulations of cells in complex tissues.

Molecular and cellular mechanisms involved in the generation of fiber diversity during myogenesis.

Skeletal muscles have a characteristic proportion and distribution of fiber types, a pattern which is set up early in development. It is becoming clear that different mechanisms produce this pattern during early and late stages of myogenesis. In addition, there are significant differences between the formation of muscles in head and those found in rest of the body. Early fiber type differentiation is dependent upon an interplay between patterning systems which include the Wnt and Hox gene families and different myoblast populations. During later stages, innervation, hormones, and functional demand increasingly act to determine fiber type, but individual muscles still retain an intrinsic commitment to form particular fiber types. Head muscle is the only muscle not derived from the somites and follows a different development pathway which leads to the formation of particular fiber types not found elsewhere. This review discusses the formation of fiber types in both head and other muscles using results from both chick and mammalian systems.