Judith M. Horowitz

Drugs of abuse and brain gene expression.

Addictive drugs like cocaine, ethanol, and morphine activate signal transduction pathways that regulate brain gene expression. Such regulation is modulated by the presence of certain transcription factor proteins present in a given neuron. This article summarizes the effects of several addictive drugs on transcriptional processes contributing to the development of a drug-dependent state. The characterization of drug-induced changes in gene expression shows promise for improving our understanding of drug-addiction phenomena and cellular modes of cocaine, ethanol, and morphine action.

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. Anat Rec, 2010.

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. Synapse, 2011.

Immunodetection of Parkin protein in vertebrate and invertebrate brains: a comparative study using specific antibodies.

Parkin is an intracellular protein that plays a significant role in the etiopathogenesis of autosomal recessive juvenile parkinsonism. Using immunoblot methods, we found Parkin isoforms varying from 54 to 58 kDa in rat, mouse, bird, frog and fruit-fly brains. Immunocytochemical studies carried out in rats, mice and birds demonstrated multiple cell types bearing the phenotype for Parkin throughout telencephalic, diencephalic, mesencephalic and metencephalic brain structures. While in some instances Parkin-containing neurons tended to be grouped into clusters, the majority of these labeled nerve cells were widely scattered throughout the neuraxis. The topographical distribution and organizational pattern of Parkin within major functional brain circuits was comparable in both rats and mice. However, the subcellular localization of Parkin was found to vary significantly as a function of antibody reactivity. A consistent cytoplasmic labeling for Parkin was observed in rodent tissue incubated with a polyclonal antibody raised against the human Parkin protein and having an identical amino-acid sequence with that of the rat. In contrast, rodent tissue alternately incubated with a polyclonal antibody raised against a different region of the same human Parkin protein but having 10 mismatched amino-acid sequence changes with those of the rat and mouse, resulted in nuclear labeling for Parkin in rat but not mouse neurons. This difference in epitope recognition, however, was reversed when mouse brain tissue was heated at 80 degrees C, apparently unmasking target epitopes against which the antisera were directed. Collectively, these results show a high degree of conservation in the cellular identity of Parkin in animals as different as drosophilids and mammals and points to the possibility that the biochemical specificities of Parkin, including analogous functional roles, may have been conserved during the course of evolution.

Identification and distribution of Parkin in rat brain

Mutations within the amino acid sequence of Parkin, the encoded protein of the parkin gene, appear to trigger the degeneration of dopaminergic neurons in the substantia nigra. Here, the presence and anatomical distribution of Parkin within the rat was examined. Immunoblot analysis of tissue homogenates showed two major bands at 50 and 44kDa. Within the brain, Parkin-containing neurons were identified in the basal ganglia, including the substantia nigra and caudate-putamen. Parkin was visualized in the raphe nucleus, which as in the substantia nigra, was closely localized to monoaminergic-encoding neurons. In addition, Parkin was detected in laminar structures such as the cortex and hippocampus; a substantial number of Parkin-immunoreactive neurons was seen in the cerebellum as well. Parkin therefore is widely distributed in brain pathways implicated in the pathology of Parkinsons disease.

Individual and combined effects of ethanol and cocaine on intracellular signals and gene expression

1. Ethanol and cocaine are drugs of abuse that can produce long-lived changes in behavior, including dependence. 2. A common set of neural pathways appears to mediate the addictive actions of ethanol and cocaine. 3. Many prominent aspects of drug dependence may be the result of alterations in intracellular signals as well as specific patterns of gene expression. 4. For instance, changes in G proteins and cAMP, phosphorylation of proteins and induction of c-fos and zif/268 in specific drug-sensitive brain regions may represent adaptive changes in response to a drug-dependent state. 5. The concurrent use of ethanol and cocaine is the most prevalent pattern of drug abuse in humans. However, the number of studies investigating the behavioral and molecular effects of this combination are few. 6. Emerging evidence indicates a possible antagonistic effect of ethanol and cocaine action on transcription factor function. In addition, cocaethylene (a psychoactive metabolite derived from combined ethanol and cocaine exposure) has significant effects on gene expression as well.

Activating properties of cocaine and cocaethylene in a behavioral preparation of Drosophila melanogaster

The use of Drosophila as a model to study the behavioral consequences of stimulant drugs was analyzed in an active preparation of decapitated Drosophila. Application of cocaine and cocaethylene to discrete nerve cord cells regulating motor programs of behavior produced striking patterns of behavioral activity in a concentration‐related manner. In general, intense circling behavior and significant wing buzzing activity were distinguishable behavioral markers in flies treated with mM concentrations of cocaine or cocaethylene. The significant changes in motor behavior induced by stimulant drugs in decapitated flies were not reproduced by the application of apomorphine, a direct dopamine (DA) agonist, or octopamine, a naturally occurring transmitter in arthropods. Because both cocaine and cocaethylene interfere with DA reuptake in mammals, we characterized the role of DA receptors mediating increased stereotypy and motor behavior in flies. Coadministration of SCH‐23390, a specific D1 receptor antagonist, significantly attenuated the behavior‐activating properties of cocaine and cocaethylene in this active experimental preparation. Therefore, the receptor protein mediating the behavioral responses to stimulant drugs in Drosophila is pharmacologically similar to the mammalian D1 subtype. In rats, cocaine‐ and cocaethylene‐induced behavioral activity is complex, with increasing evidence that the D1 receptor interacts significantly with N‐methyl‐D‐aspartate (NMDA) receptor pathways to produce an altered behavioral phenotype. To further characterize additional receptor subtypes targeted by the actions of cocaine and cocaethylene, we pretreated flies with MK‐801 and dextromethorphan. Both of these drugs are potent, selective noncompetitive NMDA receptor antagonists. Interestingly, MK‐801 and dextromethorphan profoundly reduced the behavior‐activating properties of cocaine and cocaethylene in Drosophila. Therefore, as in rats, the NMDA (and D1) receptor pathways in this arthropod represent obligatory targets for the behavioral effects of stimulant drugs. Synapse 29:148–161,1998.

Differential Behavioral Responses to Cocaethylene of Long-Evans and Sprague-Dawley Rats: Role of Serotonin

Cocaethylene is a neuroactive metabolite derived from the concurrent consumption of cocaine and ethanol. The effects of cocaethylene on locomotor activity, stereotypy, and rearing in Long‐Evans and Sprague‐Dawley rats were compared. A single cocaine injection (molar equivalent of 60 μmol/kg cocaethylene, intraperitoneal) elicited a robust series of motor output behaviors, including locomotion, stereotypy, and rearing over a 30‐minute testing period in Long‐Evans rats. In contrast, cocaethylene administration, under comparable testing conditions, produced no significant changes in locomotor and investigatory behaviors. Because cocaethylene has relatively little impact on serotonin (5‐HT) reuptake as opposed to reuptake of dopamine, we pretreated Long‐Evans rats with fluoxetine (10 mg/kg; IP), a selective 5‐HT reuptake inhibitor. Fluoxetine profoundly augmented cocaethylene‐stimulated behaviors in this rat phenotype. To examine whether other rat strains exhibit a similar response to cocaethylene, Sprague‐Dawley rats were injected (IP) with cocaethylene and their behavior patterns monitored over a 30‐minute testing period. Cocaethylene produced marked locomotor and exploratory behaviors in this strain, suggesting therefore that Long‐Evans and Sprague‐Dawley rats differ in their response to cocaethylene. To relate these behavioral differences to possible structural differences in the neuronal density of dopaminergic or serotonergic neurons, Long‐Evans and Sprague‐Dawley brains were evaluated for tyrosine hydroxylase and 5‐HT immunocytochemistry. No gross morphological differences in neuronal architecture or density were found in the ventral tegmental area or dorsal raphe nucleus of the two rat phenotypes. These results indicate that two commonly used rat strains show a differential response to cocaethylene and the neurochemical basis for this behavioral difference may be related to synaptic 5‐HT bioavailability. Synapse 26:11–21, 1997.

Silent information regulator 1 mediates hippocampal plasticity through presenilin1

Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase directly implicated in protecting a wide range of organisms against internal and external metabolic insults. However, the identification of SIRT1-specific DNA targets that confer such protection have remained elusive. Using human cells, we show that SIRT1 binds to, and transcriptionally regulates, a gene locus encoding presenilin1 (PSEN1), a protein intrinsically involved in the function of the γ-secretase protein complex. We also demonstrate that rats fed with resveratrol exhibit a significant increase in sirt1 and psen1 expression. Finally, dietary consumption of resveratrol also leads to an enhanced proliferative state of neuronal stem cells in the rat hippocampus. Our findings reveal a strong link between resveratrol-dependent SIRT1 signaling and hippocampal plasticity in the mammalian brain.

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

Apoptosis has been implicated in the pathophysiology of Parkinsons 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.