A behavioral and molecular analysis of ketamine in zebrafish.

Ketamine exerts powerful anesthetic, psychotic, and antidepressant effects in both healthy volunteers and clinically depressed patients. Although ketamine targets particular glutamate receptors, there is a dearth of evidence for additional, alternative molecular substrates for the behavioral actions of this N-methyl-D-aspartate (NMDA) receptor antagonist drug. Here, we provide behavioral and molecular evidence for the actions of ketamine using a new vertebrate model for psychiatric disorders: the zebrafish. Subanesthetic doses of ketamine produced a variety of abnormal behaviors in zebrafish that were qualitatively analogous to those previously measured in humans and rodents treated with drugs that produce transient psychosis. In addition, we revealed that the transcription factor Phox2b is a molecular substrate for the actions of ketamine, particularly during periods of hypoxic stress. Finally, we also show that SIRT1, a histone deacetylase widely recognized for its link to cell survival is also affected by hypoxia crises. These results establish a relevant assay system in which the effects of psychotomimetic drugs can rapidly be assessed, and provide a plausible and novel neuronal mechanism through which ketamine affects critical sensory circuits that monitor breathing behavior.

Distribution analysis of deacetylase SIRT1 in rodent and human nervous systems.

Sirtuins function with other biogenic molecules to promote adaptation to caloric restriction in a broad spectrum of eukaryotic species. Sirtuin pathways also converge in the mammalian brain where they appear to protect neurons from nutrient stress. However, few anatomical studies on sirtuins (e.g., SIRT1) are available, particularly those detailing the spatial distribution and subcellular localization pattern of SIRT1 in the brain parenchyma. Here, we report the characterization of a panel of SIRT1-specific antibodies within rodent (i.e., rat and mouse) and human central nervous systems. Immunocytochemical and Western blot analyses indicate that the subcellular localization of SIRT1 is predominantly nuclear throughout the rodent brain and spinal cord. A similar subcellular distribution pattern of SIRT1 was detected in human central nervous system material. SIRT1 is ubiquitously present in areas of the brain especially susceptible to age-related neurodegenerative states (e.g., the prefrontal cortex, hippocampus and basal ganglia). Further, we show no apparent species-specific differences in the subcellular localization pattern of rodent versus human SIRT1. Finally, we identify the chemical phenotype of SIRT1-containing neurons in a number of brain sites that are strongly compromised by aging. These data provide additional and important anatomical findings for the role of SIRT1 in the mammalian brain and suggest that SIRT1 pathways are broadly distributed in neurons most susceptible to senescence injury. Activating endogenous sirtuin pathways may, therefore, offer a therapeutic approach to delay and/or treat human age-related diseases.

The Vps33a gene regulates behavior and cerebellar Purkinje cell number.

A mutation in the Vps33a gene causes Hermansky-Pudlak Syndrome (HPS)-like-symptoms in the buff (bf) mouse mutant. The encoded product, Vps33a, is a member of the Sec1 and Class C multi-protein complex that regulates vesicle trafficking to specialized lysosome-related organelles. As Sec1 signaling pathways have been implicated in pre-synaptic function, we examined brain size, cerebellar cell number and the behavioral phenotype of bf mutants. Standardized behavioral tests (SHIRPA protocols) demonstrated significant motor deficits (e.g., grip strength, righting reflex and touch escape) in bf mutants, worsening with age. Histological examination of brain revealed significant Purkinje cell loss that was confirmed with staining for calbindin, a calcium binding protein enriched in Purkinje cells. This pathologic finding was progressive, as older bf mutants (13-14 months) showed a greater attrition of neurons, with their cerebella appearing to be particularly reduced (approximately 30%) in size relative to those of age-matched-control cohorts. These studies suggest that loss of Purkinje neurons is the most obvious neurological atrophy in the bf mutant, a structural change that generates motor coordination deficits and impaired postural phenotypes. It is conceivable therefore that death of cerebellar cells may also be a clinical feature of HPS patients, a pathological event which has not been reported in the literature. In general, the bf mutant may be a potentially new and useful model for understanding Purkinje cell development and function.

Magnetic resonance imaging and spectroscopy in a mouse model of schizophrenia.

Metabolic brain abnormalities, as demonstrated by (1)H-magnetic resonance spectroscopy, are common occurrences in adult schizophrenia. As mice share important biochemical and genomic similarities with humans, we tested whether brain metabolic abnormalities also occur in a transgenic mouse model of schizophrenia. In vivo(1)H-magnetic resonance spectroscopy at 4.7T of the chakragati mouse brain revealed abnormalities in relative levels of choline and N-acetylaspartate compounds. These results are consistent with a prior proposal that deficits in metabolite ratios may be common features of psychotic disorders. Thus, chakragati mice recapitulate certain aspects of the human disease phenotype and further support the utility of this animal model for understanding causal factors underlying uniquely human brain diseases.

Blood content modulates the induction of heat shock proteins in the neurovascular network.

Heat shock proteins are ubiquitous members of a family of molecular chaperones that protect various cell populations from injury. Up-regulation of heat shock proteins, particularly the 70 kDa species, bind selectively to denatured or partially damaged polypeptides that would otherwise perturb cell function and initiate cell death programs. In this regard, induction of heat shock proteins provides protection from cerebral ischemia in animal models of stroke. Endothelial cells, in particular, are intimately involved in the above protective event as these cells mount a stress response with induction of the 70 kDa heat shock protein. However, the coupling of heat shock proteins and the neurovascular response are not yet known. Here we show that blood content is an important factor in this stress response as rats devoid of blood content do not display a heat shock response in the microvasculature of the hippocampal formation. This lack of stress response, however, is reversed when rats are reperfused with exogenous rat or human blood content. We propose a new ischemic-sensing role for blood that serves to integrate information about protein-damaging conditions and heat shock protein levels in the neurovascular network. Further characterization of this sensing role could represent an attractive new approach to treatment of global ischemia and other microvascular pathologies.

Expression profile of Bag 1 in the postmortem brain.

Bag 1 is a protein intimately involved in signaling pathways that regulate cell survival. Here we examined the expression profile of Bag 1 in the brain to consider issues associated with the sampling of anti-apoptotic proteins in a rat model of the human postmortem process. Following a 4h postmortem interval, we analyzed the hippocampus of rats maintained at 24 or 4 degrees C storage temperatures using immunocytochemical and Western blotting techniques. Remarkably, postmortem tissue (up to 4h) showed a significant and prominent up-regulation of Bag 1 in CA1 and CA3 subfields of the hippocampal formation. Over-expression of Bag 1, however, could only be traced down to a storage temperature of 24 degrees C. These data suggest that storage temperatures, but not postmortem intervals, significantly affect the expression profile and cellular stability of Bag 1 proteins.

Distribution of constitutively expressed MEF-2A in adult rat and human nervous systems.

Myocyte enhancer factor 2A (MEF-2A) is a calcium-regulated transcription factor that promotes cell survival during nervous system development. To define and further characterize the distribution pattern of MEF-2A in the adult mammalian brain, we used a specific polyclonal antiserum against human MEF-2A to identify nuclear-localized MEF-2A protein in hippocampal and frontal cortical regions. Western blot and immunocytochemical analyses showed that MEF-2A was expressed not only in laminar structures but also in blood vessels of rat and human brains. MEF-2A was colocalized with doublecortin (DCX), a microtubule-associated protein expressed by migrating neuroblasts, in CA1 and CA2 boundaries of the hippocampus. MEF-2A was expressed heterogeneously in additional structures of the rat brain, including the striatum, thalamus, and cerebellum. Furthermore, we found a strong nuclear and diffuse MEF-2A labeling pattern in spinal cord cells of rat and human material. Finally, the neurovasculature of adult rats and humans not only showed a strong expression of MEF-2A but also labeled positive for hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels. This study further characterizes the distribution pattern of MEF-2A in the mammalian nervous system, demonstrates that MEF-2A colocalizes with DCX in selected neurons, and finds MEF-2A and HCN1 proteins in the neurovasculature network.

Preliminary evidence for reduced social interactions in Chakragati mutants modeling certain symptoms of schizophrenia.

Rodent models of schizophrenia provide powerful experimental tools for elucidating certain manifestations of the brain disease. The chakragati (ckr) mouse mutant, for instance, reproduces aberrant neuroanatomical and behavioral phenotypes observed in the corresponding human condition. To further investigate the utility of this mouse in the context of social behavior, we compared spontaneous behavioral activity and social interactions recorded during the subjective night among wild-type, heterozygous, and homozygous ckr mice. We found that both heterozygous and homozygous ckr animals failed to show appropriate norms of social behavior, including proximity, approach, huddling, and anogenital investigation in response to novel conspecifics. We further found that the anatomical distribution, topography, and connectivity of the neuropeptides oxytocin and vasopressin in the anterior hypothalamus did not differ among wild-type, heterozygous, or homozygous ckr animals. These latter findings suggest that although oxytocin and vasopressin influence social behavior, connectivity of such cells may not be phenotypically relevant for the observed social deficits seen in heterozygous and homozygous ckr mice. Collectively, ckr mice and their heterozygote kin are valuable experimental tools for pre-clinical studies involving disruptions of social behavior (e.g., social withdrawal).

Ventricular size mapping in a transgenic model of schizophrenia.

Genetically engineered mice have been generated to model a variety of neurological disorders. The chakragati (ckr) mouse is beginning to provide valuable insights into the structural brain changes underlying certain manifestations of schizophrenia. For instance, these mice show enlargement of the lateral ventricles, an abnormality frequently reported as a structural aberration in the schizophrenic brain. As neither the anatomical pattern nor the timing of this ventricular enlargement is known, we used magnetic resonance imaging (MRI) techniques to non-invasively visualize the development of the ventricular system in 5-, 10- and 30-day-old ckr pups. High-resolution MR images obtained from these mutants showed a progressive enlargement of the lateral ventricles, starting at day 5 of postnatal life. These emerging deficits were associated with abnormalities in mid-saggital corpus callosum area and thickness, particularly in 30-day-old adolescent animals. At this time of development, aberrant behaviors that mimic certain symptoms of schizophrenia also appeared in ckr mice suggesting that structural changes in ventricular size predates the onset of psychotic-like behaviors. These results are viewed as further indication that pre- and peri-natal disturbances of the ventricular system and adjacent neural regions may be important pathogenic factors in schizophrenia. Application of MRI to the ckr mouse is relatively new but has great potential for clarifying the relationship between brain structure changes and genetically induced vulnerabilities to psychoses.

Weight loss dynamics during combined fluoxetine and olanzapine treatment.

BACKGROUND: Fluoxetine and olanzapine combination therapy is rapidly becoming an effective strategy for managing symptoms of treatment-resistant depression. Determining drug-drug interactions, drug metabolism and pharmacokinetics is of particular interest for revealing potential liabilities associated with drug augmentation in special patient populations. In the current studies, we chronically administered fluoxetine and olanzapine in non-stressed rats to extend our previous findings regarding body weight dynamics. RESULTS: Chronic fluoxetine (10 mg/kg) and olanzapine (5 mg/kg and 0.5 mg/kg) treatment decreased weight gain irrespective of olanzapine dosing. At the 10 mg/kg and 5 mg/kg dose, respectively, fluoxetine and olanzapine also significantly reduced food and water consumption. This pharmacodynamic event-related effect, however, was not observed at the 10 mg/kg and 0.5 mg/kg dosing paradigm suggesting differences in tolerability rates as a function of olanzapine dose. The decrease in weight gain was not associated with apparent changes in glucose metabolism as vehicle- and drug-treated rats showed undistinguishable serum glucose levels. The combination of fluoxetine and olanzapine in rats yielded drug plasma concentrations that fell within an expected therapeutic range for these drugs in psychiatric patients. CONCLUSIONS: These data suggest that fluoxetine and olanzapine treatment decreases weight gain in rats; a pharmacodynamic event-related effect that differs considerably from what is observed in the clinical condition. The possibility of mismatched models regarding body weight changes during drug augmentation therapy should be seriously considered.

A neurobehavioral screening of the ckr mouse mutant: implications for an animal model of schizophrenia.

A model of schizophrenia, the chakragati (ckr) mouse was serendipitously created as a result of a transgenic insertional mutation. The apparent loss-of-function of an endogenous gene produced mice that, when homozygous, displayed an abnormal circling behavior phenotype. To determine whether this phenotype could be corrected by atypical antipsychotics, we compared the effects of clozapine and olanzapine on rotational turns and hyperactivity. Both of these drugs successfully ameliorated circling behavior and hyperactivity in homozygous mice. The increased motor activity of these mutant mice was both qualitatively and quantitatively similar to that observed in wild-type animals treated with dizocilpine, an N-methyl-D-aspartate receptor antagonist that produces behaviors resembling positive symptoms of schizophrenia. Mice either homozygous or heterozygous for the mutation also displayed enlargement of the lateral ventricles, which was accompanied only in the homozygous genotype by a loss of individual myelinated axons in the striatum and agenesis of the corpus callosum. These structural brain deficits were selective in that the nigro-striatal dopamine system was normal in these homozygous mice. In addition, two types of interneurons in the neostriatum, namely those producing acetylcholine or nitric-oxide synthase were also devoid of significant structural abnormalities. These results indicate that the ckr mouse mutant could be used as a possible animal model to study the pathophysiology of schizophrenia and suggest possible strategies for treating the behavioral aspects of this brain disease.

Regulation of hippocampal parkin protein by corticosteroids.

Parkin is a protein that when mutated leads to an inherited form of Parkinson's disease. Under normal conditions, this molecule has multiple functions in different cell types, including protein degradation and tumor suppression. To understand the relationship between parkin and circulating corticosteroid hormones, we studied the long-term depletion of corticosterone due to bilateral adrenalectomy in rats. We show that adrenalectomy deletes the expected expression of nuclear parkin in hippocampal neurons. Notably, the effect of adrenalectomy on parkin was prevented by corticosterone hormone replacement therapy. This finding suggests that adrenal hormones may be critical in sustaining parkin ubiquitinating activity in the rat hippocampus.

BAX protein-immunoreactivity in midbrain neurons of Parkinson's disease patients.

Apoptosis has been implicated in the pathophysiology of Parkinson's disease (PD). Components of signaling pathways that initiate cell death are highly concentrated in vulnerable substantia nigra (SN) neurons and may therefore contribute to the relentless demise of dopamine cells. Here, we report the distribution and organizational pattern of the pro-apoptotic protein BAX in the parkinsonian brain. Coronal sections (60 microm) of SN material from control and PD patients showed identical expression of BAX-immunoreactivity (IR) in all cases examined. Neurons positive for BAX-IR exhibited a discrete cytoplasmic and dendritic labeling that was conspicuously interspersed with previously unrecognized axonal spheroid-like inclusions. Direct comparisons revealed a difference in the aggregation of BAX-rich inclusions, with the parkinsonian brain containing more SN inclusions than control cases. BAX expression by midbrain neurons was confirmed by immunoblot analysis on SN extracts showing a specific band of approximately 21kDa, which is consistent with the known molecular weight of native BAX. These results suggest that apoptosis or programmed cell death may play an indirect role in idiopathic PD.

Characterization of fluoxetine plus olanzapine treatment in rats: a behavior, endocrine, and immediate-early gene expression analysis.

A large number of individuals afflicted with psychiatric disorders, particularly depression with psychotic features, do not respond to conventional drug therapy. An option for this phenomenon is to augment a standard selective serotonin (5-HT) reuptake inhibitor with an atypical antipsychotic agent. In this regard, fluoxetine and olanzapine have been used concomitantly for treatment-resistant depression and bipolar depression. Although highly efficacious in terms of producing superior improvement of symptoms across a variety of psychological measures, the motor patterns, endocrine profiles, and intracellular signaling pathways affected by drug augmentation have not been determined. Here we show that fluoxetine (10 mg/kg) plus olanzapine (5 mg/kg) given to rats for 7 consecutive days (i.e., subchronic treatment) alters motor activity and diminishes spontaneous behaviors as measured by spatial position and angular path analyses. In addition, the same drug combination pattern sensitizes peak adrenal corticosterone secretion without altering serum glucose levels. We also show that subchronic fluoxetine and olanzapine exposure suppresses the induction of two immediate-early gene transcription factors (e.g., pCREB and FOS) that are associated with long-lasting changes in synaptic efficacy and structural modifications in the prefrontal cortex, piriform cortex, and hippocampus. These results suggest that fluoxetine plus olanzapine can interact in a fashion not predicted by the currently accepted model of fluoxetine monotherapy and provide insight into the synergistic actions of drug augmentation in patients with treatment-resistant depression.

Rat strain differences to fluoxetine in striatal Fos-like proteins.

Humans afflicted with mood disorders respond differently to antidepressant drug therapy. Different responses to psychoactive drugs are also observed in rats, and specific strains exhibit substantial differences in gene expression following synaptic activity. We examined the effects of fluoxetine on the induction of Fos-like proteins in Long-Evans and Sprague-Dawley rat brains. Fluoxetine elicited a strong induction of Fos in the striatum of Long-Evans but not Sprague-Dawley rats following acute drug exposure. This effect was specific to fluoxetine as two highly selective serotonin reuptake inhibitors, HD-31 and HD-50, as well as treatment with fenfluramine failed to replicate the observed differences in Fos. These differences in Fos expression between rat strains may represent variability in post-receptor pathways that ultimately mediate the therapeutic actions of fluoxetine.