Cocaine-Induced Expression of the Tetraspanin CD81 and Its Relation to Hypothalamic Function

doi:10.1006/mcne.2000.0942

Molecular and Cellular Neuroscience

Volume 17, Issue 2, February 2001, Pages 303-316

https://doi.org/10.1006/mcne.2000.0942 Get rights and content

Abstract

CD81, a tetraspanin transmembrane protein involved in cell adhesion, was found by differential display to be upregulated in the nucleus accumbens of rat brain following acute cocaine treatment (four injections of 30 mg/kg every 2 h followed by 24 h withdrawal). Cocaine-induced expression of CD81 in adult rat brain was confirmed by quantitative real-time RT-PCR. Its expression in neurons and its function in the brain are unknown. In situ hybridization shows a neuron-specific expression pattern in brain regions functionally related to the regulation of cardiovascular function and fluid homeostasis. CD81 displays codistribution to galanin and, to a lesser extent, to vasopressin. These findings add to data that suggest a connection between the brain reward pathway and the centers regulating endocrine and autonomic functions, in relation to neurochemical, behavioral, and somatic consequences of drug abuse.

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Cited by (47)

Chronic knockdown of the tetraspanin gene CD81 in the mouse nucleus accumbens modulates anxiety and ethanol-related behaviors
2022, Physiology and Behavior
Citation Excerpt :
In fact, we reported in our previous studies, using in situ hybridization and real-time PCR, that CD81 expression was upregulated in the Nacc rat brain following acute cocaine [56]. In addition, we showed that total ablation of CD81, in knockout mice, altered cocaine's sensitivity as measured using cocaine-induced place preference and cocaine-induced locomotor activity [18], suggesting that targeting CD81 signaling has potential therapeutic effects on psychostimulants-related disorders. Previously, using viral vectors, we found that accumbal CD81 overexpression increased cocaine-induced locomotor activity in the rat [7], whereas accumbal CD81 knockdown - using lentiviral vectors-expressing shRNAs - decreased the stimulating effects of cocaine [8].
CD81, a member of the tetraspanin family, plays important roles in many physiological processes, such as cell motility, attachment, and entry. Yet, CD81 functions in the brain remain unclear. In this study, we investigated the effects of CD81 knockdown, using lentiviral vectors (LV), on anxiety- and ethanol-related behaviors. For this purpose, mice were stereotaxically injected with CD81 shRNA-expressing LV into the nucleus accumbens (Nacc) and were assessed for anxiety-like behavior using the elevated plus maze (EPM) and open field (OF) tests. Alcohol's sedative effects were studied using loss-of-righting-reflex (LORR) and voluntary ethanol intake was assessed using a two-bottle choice (TBC) procedure. Results showed that mice depleted of CD81 exhibited an anxiolytic-like response in the EPM and OF tests with no effect on locomotor activity. In addition, genetic reduction of CD81 in the Nacc increased mice’ sensitivity to alcohol's sedative effects in the LORR test, although plasma alcohol concentrations were unaffected. Interestingly, CD81 loss-of-function-induced anxiolysis was accompanied by a significant decrease in ethanol, but not saccharin nor quinine, intake in the TBC procedure. Finally, and following CD81 mRNA quantification, Pearson's correlations showed a significant positive relationship between accumbal CD81 mRNA with anxiety and ethanol-related behaviors. Our data indicate that CD81 is implicated in the pathogenesis of anxiety and alcoholism. Indeed the targeted disruption of CD81, with the resultant decrease in CD81 mRNA in the Nacc, converted ethanol-“preferring” mice into ethanol “non-preferring” mice. Collectively, these findings demonstrate that future CD81-targeted pharmacotherapies may be beneficial for the treatment of anxiety and alcoholism.
CART in the brain of vertebrates: Circuits, functions and evolution
2014, Peptides
Cocaine- and amphetamine-regulated transcript peptide (CART) with its wide distribution in the brain of mammals has been the focus of considerable research in recent years. Last two decades have witnessed a steady rise in the information on the genes that encode this neuropeptide and regulation of its transcription and translation. CART is highly enriched in the hypothalamic nuclei and its relevance to energy homeostasis and neuroendocrine control has been understood in great details. However, the occurrence of this peptide in a range of diverse circuitries for sensory, motor, vegetative, limbic and higher cortical areas has been confounding. Evidence that CART peptide may have role in addiction, pain, reward, learning and memory, cognition, sleep, reproduction and development, modulation of behavior and regulation of autonomic nervous system are accumulating, but an integration has been missing. A steady stream of papers has been pointing at the therapeutic potentials of CART. The current review is an attempt at piecing together the fragments of available information, and seeks meaning out of the CART elements in their anatomical niche. We try to put together the CART containing neuronal circuitries that have been conclusively demonstrated as well as those which have been proposed, but need confirmation. With a view to finding out the evolutionary antecedents, we visit the CART systems in sub-mammalian vertebrates and seek the answer why the system is shaped the way it is. We enquire into the conservation of the CART system and appreciate its functional diversity across the phyla.
Chromatin immunoprecipitation assays revealed CREB and serine 133 phospho-CREB binding to the CART gene proximal promoter
2010, Brain Research
Both over expression of cyclic AMP response element binding protein (CREB) in the nucleus accumbens (NAc), and intra-accumbal injection of cocaine- and amphetamine-regulated transcript (CART) peptides, have been shown to decrease cocaine reward. Also, over expression of CREB in the rat NAc increased CART mRNA and peptide levels, but it is not known if this was due to a direct action of P-CREB on the CART gene promoter. The goal of this study was to test if CREB and P-CREB bound directly to the CRE site in the CART promoter, using chromatin immunoprecipitation (ChIP) assays. ChIP assay with anti-CREB antibodies showed an enrichment of the CART promoter fragment containing the CRE region over IgG precipitated material, a non-specific control. Forskolin, which was known to increase CART mRNA levels in GH3 cells, was utilized to show that the drug increased levels of P-CREB protein and P-CREB binding to the CART promoter CRE-containing region. A region of the c-Fos promoter containing a CRE cis-regulatory element was previously shown to bind P-CREB, and it was used here as a positive control. These data suggest that the effects of CREB over expression on blunting cocaine reward could be, at least in part, attributed to the increased expression of the CART gene by direct interaction of P-CREB with the CART promoter CRE site, rather than by some indirect action.
Measuring levels of proteins by various technologies: Can we learn more by measuring turnover?
2010, Biochemical Pharmacology
In routine experiments, scientists measure the levels of various substances such as proteins after various treatments. Detection of a change in levels suggests an impact of treatment on that particular protein. However, we sometimes forget the importance of turnover in this process. Proteins have half-lives that may change in response to treatments (which is in fact why levels may change), and an examination of half-lives may yield better clues as to how treatment affects the protein. After an exploration of the quantitative aspects of protein turnover, several interesting conclusions may be drawn. (1) Even though levels of some proteins may NOT change after treatments, their half-lives and turnovers do change, and these may be more sensitive indicators of the impact of treatments on the proteins of interest. (2) Treatments can affect protein levels because they alter either the synthesis or degradation of the protein or both. But, the rate of change of the levels depends on the half-life of the protein. If the experimenter waits only a fraction of a half-life of the protein after treatment, no significant change in level may be found since it can take up to 5 half-lives for the protein level to adjust to about 97% of its new level after treatment. (3) Half-lives of the same protein can vary in different species and experimental conditions may have to be altered if using different species. These factors suggest that a consideration of protein turnover and half-lives will be useful for future studies of this type.
Regulation of cocaine- and amphetamine-regulated transcript mRNA expression by calcium-mediated signaling in GH3 cells
2009, Neuroscience
Cocaine- and amphetamine-regulated-transcript (CART) peptides are associated with multiple physiological processes, including, feeding, body weight, and the response to drugs of abuse. CART mRNA and peptide levels and the expression of the CART gene appears to be under the control of a number of extra- and intra-cellular factors including the transcription factor, cAMP response element binding protein (CREB). Similar to the effects of CART, Ca²⁺ signaling leads to the phosphorylation of CREB and has been associated with both feeding and the actions of psychostimulants; therefore, we hypothesized that Ca²⁺ may play a role in CART gene regulation. We used real-time PCR (rtPCR) and GH3 cells to examine the effect of ionomycin, which increases intracellular Ca²⁺, on CART mRNA levels. Ionomycin increased CART mRNA in a dose- and time-dependent manner. The effect of ionomycin appeared transient as CART mRNA had returned to control levels 3 h following treatment. Calmidazolium and KN93, inhibitors of calmodulin and Ca²⁺-modulated protein (CaM) kinases respectively, attenuated the effect of ionomycin (10 μM) on CART mRNA levels suggesting a calmodulin-dependent mechanism. Western immunoblotting indicated that ionomycin increased phosphorylated cAMP response element binding protein (pCREB) levels and electrophoretic mobility shift assay/supershift assay using antibodies against pCREB demonstrated increased levels of a CART oligo/pCREB protein complex. Finally, we showed that injection of ionomycin into the rat nucleus accumbens increases CART mRNA levels. To our knowledge, this is the first study providing evidence that the CART gene is, in part, regulated by Ca²⁺/CaM/CREB-dependent cell signaling.
Injection of CART (cocaine- and amphetamine-regulated transcript) peptide into the nucleus accumbens reduces cocaine self-administration in rats
2008, Behavioural Brain Research
Cocaine- and amphetamine-regulated transcript (CART) peptides appear to modulate various effects of psychostimulant drugs. Injections of CART peptide into the nucleus accumbens (NAcc) inhibit locomotion produced by systemic injections of the psychostimulants cocaine and amphetamine. Intra-NAcc injections of CART peptide also inhibit locomotion produced by microinfusions of dopamine into the NAcc, suggesting that the effects of CART peptides may be due to an interaction with the dopaminergic system in the NAcc. We sought to determine if this inhibitory effect of CART peptide generalizes to other measures of dopaminergic function such as reward/reinforcement by testing the effect of bilateral intra-NAcc CART infusions (0, 0.25, 1.0 and 2.5 μg per side) on cocaine and food self-administration. One group of rats self-administered cocaine (0.75 mg/kg per 140 μl IV infusion) on a progressive ratio schedule. A separate group received 45 mg food pellets on the same progressive ratio schedule. Bilateral intra-NAcc injections of CART peptide dose-dependently decreased the number of cocaine infusions, the breakpoint of cocaine self-administration, and the total number of bar presses on the cocaine-associated lever. There were no effects of CART injections on the breakpoint for food reward. Thus, we conclude that injections of CART into the NAcc appear to functionally antagonize a major site of action for cocaine self-administration in rats.

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¹: To whom correspondence should be addressed at the Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland. Fax: ++41-26-300'9735. E-mail: [email protected].

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Regular ArticleCocaine-Induced Expression of the Tetraspanin CD81 and Its Relation to Hypothalamic Function

Abstract

J. Chem. Neuroanat.

Psychoneuroendocrinology

Brain Res. Bull.

Neurosci. Lett.

Trends Neurosci.

Cell. Immunol.

Peptides

Physiol. Behav.

Brain Res.

Biol. Psychiatry

Association of four antigens of the tetraspans family (CD37, CD53, TAPA-1, and R2/C33) with MHC class II glycoproteins

Immunogenetics

Engaging CD19 or target of an antiproliferative antibody 1 on human B lymphocytes induces binding of B cells to the interfollicular stroma of human tonsils via integrin alpha 4/beta 1 and fibronectin

J. Exp. Med.

Characterization of integrin–tetraspanin adhesion complexes: Role of tetraspanins in integrin signaling

J. Cell Biol.

A role for CD81 in early T cell development

Science

A simplified in situ hybridisation protocol using non-radioactively labeled probes to detect abundant and rare mRNAs on tissue sections

Biochemica

Modification of primer design facilitates the use of differential display

Biotechniques

Cocaine- and amphetamine-regulated transcript (CART) peptide immunoreactivity in myenteric plexus neurons of the rat ileum and co-localization with choline acetyltransferase

Synapse

PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine

J. Neurosci.

Mesolimbic gender differences in peptide CART mRNA expression: Effects of cocaine

NeuroReport

Cocaine addiction: Psychology and neurophysiology

Science

Astrocyte growth, reactivity, and the target of the antiproliferative antibody, TAPA

J. Neurosci.

The Human Brain and Spinal Cord: Functional Neuroanatomy and Dissection Guide

Regular Article
Cocaine-Induced Expression of the Tetraspanin CD81 and Its Relation to Hypothalamic Function