Biochemical adaptations in the mesolimbic dopamine system in response to heroin self-administration

Previous studies have shown that chronic, forced exposure to opiates produces specific biochemical adaptations in the ventral tegmental area (VTA) and nucleus accumbens (NAc). The functional consequences of these adaptations have been hypothesized to contribute to certain motivational aspects of drug addiction. In this study, the possibility that similar adaptations could occur in response to intermittent heroin self-administration was tested by comparing homogenates of VTA and NAc from rats self-administering heroin, rats receiving yoked injections of heroin, and rats receiving yoked injections of saline (controls). Tyrosine hydroxylase (TH) immunoreactivity was increased (31–38%) in the VTA and decreased (11%) in the NAc of heroin-exposed rats relative to controls. Heroin exposure also increased cAMP-dependent protein kinase (PKA) activity in both particulate (19–27%) and soluble (17–20%) fractions of the NAc, and decreased (16–17%) the level of G_iα, immunoreactivity in this brain region. In contrast, no significant biochemical changes were found in the substantia nigra or caudate-puta-men, indicating a selective effect on the mesolimbic dopamine system. Overall, adapta-tions in the VTA and NAc of heroin-exposed rats were similar to, but generally smaller in magnitude than, adaptations produced by chronic morphine administration. However, in contrast to morphine-treated animals, heroin-exposed animals failed to display overt signs of opiate physical dependence, suggesting that adaptations in motivational systems may occur more readily than adaptations in brain regions associated with physical dependence. © 1995 Wiley-Liss, Inc.     

Differential behavioral responses to cocaine are associated with dynamics of mesolimbic dopamine proteins in Lewis and Fischer 344 rats

Differential behavioral and biochemical responses to drugs of abuse may reflect genetic makeup as suggested by studies of inbred Lewis (LEW) and Fischer 344 (F344) rats. We investigated locomotor activity, stereotypy signs, and levels of specific proteins in the nucleus accumbens (NAc) and ventral tegmental area (VTA) in these strains at baseline and following chronic administration of cocaine (30 mg/kg/day for 14 days). Using Western blot analysis, we replicated our previous findings of baseline strain differences and found lower levels of DeltaFosB immunoreactivity in NAc of F344 vs. LEW rats. F344 rats showed greater baseline locomotor activity, sniffing, and grooming compared to LEW rats. Chronic cocaine increased DeltaFosB levels in NAc in both strains, whereas adaptations in other proteins were induced in F344 rats only. These included reduced levels of tyrosine hydroxylase (TH) in NAc and increased TH and glial fibrillary acidic protein (GFAP) immunoreactivity in VTA. Chronic cocaine led to greater increases in overall stereotypy in F344 vs. LEW rats and decreased exploratory behaviors in LEW rats. Opposing effects by strain were seen in locomotor activity. Whereas F344 rats showed higher initial activity levels that decreased with cocaine exposure (tolerance), LEW rats showed increased activity over days (sensitization) with no strain differences seen at 14 days. Further, conditioned locomotor activation to vehicle injections was greater in F344 vs. LEW rats. These results suggest that behavioral responsiveness to chronic cocaine exposure may reflect dynamics of mesolimbic dopamine protein levels and demonstrate the role of genetic background in responsiveness to cocaine.     

Differential regulation of ionotropic glutamate receptor subunits following cocaine self-administration

Previous examination of binge cocaine self-administration and 2 week withdrawal from cocaine self-administration on ionotropic glutamate receptor subunit (iGluRs) protein levels revealed significant alterations in iGluR protein levels that differed between the mesocorticolimbic and nigrostriatal pathways. The present study was undertaken to extend the examination of cocaine-induced alterations in iGluR protein expression by assessing the effects of acute withdrawal (15-16 h) from limited access cocaine self-administration (8 h/day, 15 days). Western blotting was used to compare levels of iGluR protein expression (NR1-3B, GluR1-7, KA2) in the mesolimbic (ventral tegmental area, VTA; nucleus accumbens, NAc; and prefrontal cortex, PFC) and nigrostriatal pathways (substantia nigra, SN and dorsal caudate-putamen, CPu). Within the mesolimbic pathway, reductions were observed in NR1 and GluR5 immunoreactivity in the VTA although no significant alterations were observed in any iGluR subunits in the NAc. In the PFC, NR1 was significantly upregulated while GluR2/3, GluR4, GluR5, GluR6/7, and KA2 were decreased. Within the nigrostriatal pathway, NR1, NR2A, NR2B, GluR1, GluR6/7 and KA2 were increased in the dorsal CPu, whereas no significant changes were observed in the SN. The results demonstrate region- and pathway-specific alterations in iGluR subunit expression following limited cocaine self-administration and suggest the importance for the activation of pathways that are substrates of the reinforcing and motoric effects of cocaine.     

Neurofilament proteins and the mesolimbic dopamine system: common regulation by chronic morphine and chronic cocaine in the rat ventral tegmental area.

The ventral tegmental area (VTA) and its dopaminergic projections appear to mediate some of the rewarding properties of opiates, cocaine, and other drugs of abuse. In a previous study, we demonstrated that chronic morphine and cocaine exert common actions on tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, in this dopaminergic brain reward region (Beitner-Johnson and Nestler, 1991). In the present study, we investigated the effects of chronic morphine and cocaine on other phosphoproteins in the VTA by back phosphorylation and two-dimensional electrophoretic analysis. It was found that a number of phosphoproteins, in addition to tyrosine hydroxylase, were regulated similarly by the two drug treatments in this brain region. Several of these morphine- and cocaine-regulated phosphoproteins were identified as neurofilament (NF) proteins. Chronic, but not acute, administration of either morphine or cocaine was found to decrease levels of the three NF proteins, NF-200 (NF-H), NF-160 (NF-M), and NF-68 (NF-L), by between 15% and 50% in the VTA by back phosphorylation, immunolabeling, and Coomassie blue staining. Such regulation of NF proteins was selective, in that no detectable changes were observed in the levels of eight other major cytoskeletal or cytoskeletal-associated proteins analyzed. Furthermore, NF levels were not altered by chronic treatment with either imipramine or haloperidol, two psychotropic drugs without reinforcing properties, or by chronic stress. Morphine and cocaine regulation of NFs showed regional specificity, as NF levels were not altered in the substantia nigra, or other parts of the brain or spinal cord, by these drug treatments. NFs are thought to function as determinants of neuronal morphology and to be associated with axonal transport. Thus, decreased NF levels in the VTA in response to chronic morphine and chronic cocaine could lead to drug-induced alterations in the structural and functional properties of this brain region, which may represent, in turn, part of a common biochemical basis of morphine and cocaine addiction and craving.     

Dopaminergic brain reward regions of Lewis and Fischer rats display different levels of tyrosine hydroxylase and other morphine- and cocaine-regulated phosphoproteins

We studied cyclic AMP-dependent protein phosphorylation in the mesolimbic and nigrostriatal dopamine systems of two genetically inbred rat strains, Lewis (LEW) and Fischer (F344) rats. These strains represent genetically divergent populations of rats that have been used to study possible genetic factors involved in a variety of biological processes. We found striking differences in levels of tyrosine hydroxylase, and several other phosphoproteins, in the mesolimbic, but not the nigrostriatal, dopamine system between the two rat strains. Interestingly, in Sprague-Dawley rats, these same phosphoproteins are altered by chronic morphine and chronic cocaine specifically in the mesolimbic dopamine system, generally thought to be a brain reward pathway that mediates some of the reinforcing actions of many drugs of abuse. As LEW and F344 rats have been reported to show different levels of preference for several types of drugs of abuse, the results are consistent with the possibility that these phosphoproteins may mediate aspects of drug reinforcement and contribute to individual differences in vulnerability to drug addiction.     

Insulin in the ventral tegmental area reduces cocaine‐evoked dopamine in the nucleus accumbens in vivo

Mesolimbic dopamine circuits, implicated in incentive motivation, are sensitive to changes in metabolic state such as weight loss and diet‐induced obesity. These neurons are important targets for metabolic hormones such as leptin, glucagon‐like peptide‐1, ghrelin and insulin. Insulin receptors are located on dopamine neurons in the ventral tegmental area (VTA) and we have previously demonstrated that insulin induces long‐term depression of excitatory synapses onto VTA dopamine neurons. While insulin can decrease dopamine concentration in somatodendritic regions, it can increase dopamine in striatal slices. Whether insulin directly targets the VTA to alter dopamine release in projection areas, such as the nucleus accumbens (NAc), remains unknown. The main goal of the present experiments was to examine NAc dopamine concentration following VTA administration of insulin. Using in vivo FSCV to detect rapid fluctuations in dopamine concentration, we showed that intra‐VTA insulin via action at insulin receptors reduced pedunculopontine nucleus‐evoked dopamine release in the NAc. Furthermore, intra‐VTA insulin reduced cocaine‐potentiated NAc dopamine. Finally, intra‐VTA or intranasal insulin decreased locomotor responses to cocaine, an effect blocked by an intra‐VTA administered insulin receptor antagonist. Together, these data demonstrate that mesolimbic dopaminergic projections are important targets of the metabolic hormone, insulin.     

Lewis and Fischer rat strains display differences in biochemical, electrophysiological and behavioral parameters: studies in the nucleus accumbens and locus coeruleus of drug naive and morphine-treated animals

In previous studies, we demonstrated that tyrosine hydroxylase and neurofilament proteins are regulated by chronic morphine and chronic cocaine treatments in the ventral tegmental area in Sprague-Dawley rats and that the imbred Lewis and Fischer 344 rat strains, under drug-naive conditions, show different levels of these proteins specifically in this brain region. In the current study, we compared Lewis and Fischer rats with respect to levels of adenylate cyclase, cyclic AMP-dependent protein kinase and G-proteins in the nucleus accumbens (NAc) and locus coeruleus (LC), brain regions in Sprague-Dawley rats where these proteins are regulated by chronic exposure to morphine or to cocaine. We found that levels of adenylate cyclase and cyclic AMP-dependent protein kinase activity are higher in the NAc and LC of Lewis rats compared to Fischer rats, whereas levels of G_iα and G_β were lower. These strain differences were not seen in several other brain regions analyzed and no strain differences were detected in levels of other G-protein subunits. Lewis and Fischer rats also differed in the ability of chronic morphine to regulate adenylate cyclase and cyclic AMP-dependent protein kinase in the NAc and LC. In the NAc, chronic morphine increased levels of the two enzymes in the Fischer strain only, whereas in the LC chronic morphine increased levels of the enzymes in both strains, with more robust effects seen in the Lewis rat. To understand possible physiological consequences of these strain differences in the cyclic AMP pathway, we studied LC neuronal activity under basal and chronic morphine-treated conditions. LC neurons of Lewis rats showed higher spontaneous firing rates in brain slices in vitro than those of Fischer rats and also showed greater morphine-induced increases in responsiveness to bath-applied 8-bromo-cyclic AMP. These electrophysiological findings are generally consistent with the biochemical observations. Moreover, Lewis and Fischer rats displayed very different opiate withdrawal syndromes, with different types of behaviors elicited upon precipitation of opiate withdrawal with the opiate receptor antagonist, naltrexone. The possible relationship between these behavioral findings and the biochemical and electrophysiological data is discussed. These studies provide further support for the possibility that Lewis and Fischer rat strains provide a useful model system in which some of the genetic factors that contribute to drug-related behaviors can be investigated.     

Accelerated maternal responding following intra-VTA pertussis toxin treatment

Prior studies have supported a role for mesolimbic dopaminergic mechanisms in the regulation of maternal behavior. Accordingly, the ventral tegmental area (VTA) and its dopaminergic projections to the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) have been implicated in both the onset and maintenance of normal maternal behavior. To date, studies of direct manipulation of VTA neurochemistry at the onset of maternal behavior have been limited. The current study was undertaken to directly test the hypothesis that enhancement of dopaminergic transmission in the mesolimbic dopamine system can stimulate maternal activity using a pup-induced virgin model. Nulliparous female rats were stereotaxically infused with pertussis toxin (PTX 0, 0.1, or 0.3 μg/hemisphere) into the VTA to chronically stimulate the activity of dopaminergic projection neurons. After 3 days of recovery, maternal responding to donor pups was tested daily, and latency (in days) to full maternal behavior was recorded. Intra-VTA PTX treatment produced a robust dose-dependent decrease in maternal behavior latency, and a long-lasting increase in locomotor activity. These effects were associated with significantly decreased dopamine D1 receptor mRNA expression in the NAc. No effects of PTX treatment on mesolimbic dopamine utilization or mPFC receptor expression were observed. The findings indicate that chronic neural activation in the VTA accelerates the onset of maternal behavior in virgin female rats via modification of the NAc dopamine D1 receptor.     

Muscarinic receptor blockade in the ventral tegmental area attenuates cocaine enhancement of laterodorsal tegmentum stimulation‐evoked accumbens dopamine efflux in the mouse

The reinforcing properties of cocaine have been related to increased extracellular concentrations of dopamine in the nucleus accumbens (NAc). M5 muscarinic acetylcholine receptors (mAChRs) on dopamine cells in the ventral tegmental area (VTA) facilitate mesoaccumbens dopamine transmission and are critically involved in mediating natural and drug reinforcement. We investigated the effects of pharmacological blockade of mAChRs in the VTA on cocaine's ability to enhance electrically evoked NAc dopamine efflux. Using fixed potential amperometry together with carbon fiber recording microelectrodes positioned in the NAc core, we quantified dopamine oxidation currents (dopamine efflux) evoked by brief stimulation (15 monophasic pulses at 50 Hz every 30 s) of the laterodorsal tegmentum (LDT) in urethane (1.5 g/kg, i.p.) anesthetized mice. Compared to predrug baseline responses, cocaine (5 or 10 mg/kg, i.p.) dose‐dependently enhanced LDT stimulation‐evoked NAc dopamine efflux, whereas the nonsubtype selective mAChR antagonist scopolamine (10 μg/0.5 μl) microinfused into the VTA diminished LDT‐evoked NAc dopamine efflux. Preinfusion of scopolamine into the VTA diminished the facilitatory actions of cocaine on LDT stimulation‐evoked NAc dopamine efflux, and when infused at the peak effect of cocaine attenuated LDT‐evoked dopamine efflux to below predrug baseline levels. These findings suggest that LDT cholinergic inputs to dopamine neurons in the VTA, via activation of mAChRs (probably of the M5 subtype), are involved in modulating the facilitatory effects of cocaine on NAc dopamine neurotransmission. They also suggest that the development of antagonists aimed at selectively disrupting M5 receptor function may be valuable in reducing abuse liability of psychostimulants. Synapse 64:216–223, 2010. © 2009 Wiley‐Liss, Inc.     

Fischer and Lewis rat strains differ in basal levels of neurofilament proteins and their regulation by chronic morphine in the mesolimbic dopamine system.

We studied levels of neurofilament (NF) proteins in the ventral tegmental area (VTA), and other regions of the central nervous system, of two genetically inbred rat strains, Lewis (LEW) and Fischer (F344) rats. These strains represent genetically divergent populations of rats that have been used to study possible genetic factors involved in a variety of biological processes, including drug addiction: compared to F344 rats, LEW rats show a much higher preference for several classes of drugs of abuse. We found 30-50% lower levels of three NF proteins, NF-200 (NF-H), NF-160 (NF-M), and NF-68 (NF-L), in the VTA of LEW compared to F344 rats by use of immunolabeling and Coomassie blue staining. These strain differences were highly specific to this brain region, with no differences observed elsewhere in brain or spinal cord. Interestingly, chronic treatment of F344 rats with morphine decreased levels of these three NF proteins in the VTA, as found previously in outbred Sprague-Dawley rats (Beitner-Johnson, D., Guitart, X., and Nestler, E.J.:J. Neurosci., 12:2165-2176, 1992), whereas morphine had no effect on NF levels in the VTA of LEW rats. A similar strain difference was observed in chronic morphine regulation of tyrosine hydroxylase, with morphine increasing enzyme immunoreactivity in the VTA of F344 rats (as has been observed previously in Sprague-Dawley rats [Beitner-Johnson, D., and Nestler, E.J.:J. Neurochem., 57:344-347, 1991]), but not in LEW rats. In view of the observations that LEW and F344 rats show different levels of preference for several types of drugs of abuse, and of the evidence supporting a central role of the mesolimbic dopamine system in drug reward mechanisms, the results of the current study suggest the possibility that levels of NFs and tyrosine hydroxylase may mediate some aspects of drug reinforcement and contribute to individual genetic differences in vulnerability to drug addiction.     

Purinergic modulation of neuronal activity in the mesolimbic dopaminergic system in vivo

ATP and its metabolite adenosine activate membrane receptors, termed P2 and P1, respectively. In the present study, the modulation of the mesolimbic neuronal circuit by ATPergic and adenosinergic mechanisms was investigated by microdialysis in the nucleus accumbens (NAc) and by telemetrically recorded EEG from both the NAc and the ventral tegmental area (VTA) of freely moving rats. The basal extracellular dopamine concentration was enhanced after accumbal perfusion with the ATP analog 2-methylthio ATP (2-MeSATP; 100 microM); by contrast, adenosine (100 microM) caused a reduction of extracellular dopamine. When given alone, the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 20 microM) decreased the concentration of dopamine, whereas the P1 receptor antagonist 8-(p-sulfophenyl)theophylline (8-SPT; 100 microM) increased it. In the same animals, P2 receptor stimulation by 2-MeSATP caused neuronal activation, indicated by an elevation of the absolute power in the EEG of the NAc mainly by enhancement of the relative power in the alpha band (8-13 Hz) of the EEG spectrum. By contrast, adenosine led to a depression of the absolute power in the VTA accompanied by an elevation of the delta-band power (0.4-6 Hz) in the NAc corresponding to a slowing of neuronal activity. When given alone, PPADS reduced the absolute EEG power in the NAc accompanied by a decrease in the high-frequency power, but had no effects on the VTA. 8-SPT on its own enhanced the total power in both the NAc and the VTA, reflected by an enhancement in the slow and the high-frequency bands. Whereas the 8-SPT-evoked changes of EEG pattern as well as of dopamine concentration in the NAc were abolished by the co-application of PPADS, the 8-SPT-induced EEG changes in the VTA persisted under these conditions. In conclusion, the accumbal neuronal output, reflected by both dopamine release and neuronal electrical activity, is modulated in a functionally antagonistic manner by P2 and P1 receptor stimulation. It is suggested that an inhibitory GABAergic feedback projection to the VTA is stimulated by adenosine, either directly or indirectly via glutamate release.     

Chronic nicotine and smoke treatment modulate dopaminergic activities in ventral tegmental area and nucleus accumbens and the gamma-aminobutyric acid type B receptor expression of the rat prefrontal cortex

Dopaminergic afferents from the mesencephalic areas, such as ventral tegmental area (VTA), synapse with the gamma-aminobutyric acid (GABA)-ergic interneurons in the prefrontal cortex (PFC). Pharmacological and electrophysiological data show that the reinforcement, the dependence-producing properties, as well as the psychopharmacologic effects of nicotine depend to a great extent on activation of nicotinic receptors within the mesolimbocortical dopaminergic projection. To explore further the relationship between the mesencephalic dopaminergic neurons and PFC GABAergic neurons, we investigated the effects of nicotine and passive exposure to cigarette smoke on the regulation of tyrosine hydroxylase (TH) in VTA and substantia nigra (SNC) and dopamine (DA) D1 receptor levels in nucleus accumbens (NAc) and caudate-putamen (CPu). Also, the simultaneous changes in GABAB receptors mRNAs in the PFC were studied. The results showed that chronic nicotine and smoking treatment differentially changed the levels of TH protein in VTA and SNC and DA D1 receptor levels in Nac and CPu. GABAB1 and GABAB2 receptor mRNA levels also showed different change patterns. Ten and thirty minutes of smoke exposure increased GABAB1 receptor mRNA to a greater extent than that of GABAB2, whereas GABAB2 was greatly enhanced after 1 hr of smoke exposure. The TH levels in VTA were closely related to DA D1 receptor levels in NAc and with GABAB receptor mRNA changes in PFC. These results suggest that the mesolimbic pathway and GABAB receptor mRNA in PFC are modulated by nicotine and cigarette smoke, implying an important role in nicotine's psychopharmacological effects.     

Infusion of brain‐derived neurotrophic factor into the ventral tegmental area switches the substrates mediating ethanol motivation

Recent work has shown that infusion of brain‐derived neurotrophic factor (BDNF) into the ventral tegmental area (VTA) promotes a switch in the mechanisms mediating morphine motivation, from a dopamine‐independent to a dopamine‐dependent pathway. Here we showed that a single infusion of intra‐VTA BDNF also promoted a switch in the mechanisms mediating ethanol motivation, from a dopamine‐dependent to a dopamine‐independent pathway (exactly opposite to that seen with morphine). We suggest that intra‐VTA BDNF, via its actions on TrkB receptors, precipitates a switch similar to that which occurs naturally when mice transit from a drug‐naive, non‐deprived state to a drug‐deprived state. The opposite switching of the mechanisms underlying morphine and ethanol motivation by BDNF in previously non‐deprived animals is consistent with their proposed actions on VTA GABA_A receptors.     

Oxytocin receptors are expressed on dopamine and glutamate neurons in the mouse ventral tegmental area that project to nucleus accumbens and other mesolimbic targets

The mesolimbic dopamine (DA) circuitry determines which behaviors are positively reinforcing and therefore should be encoded in the memory to become a part of the behavioral repertoire. Natural reinforcers, like food and sex, activate this pathway, thereby increasing the likelihood of further consummatory, social, and sexual behaviors. Oxytocin (OT) has been implicated in mediating natural reward and OT‐synthesizing neurons project to the ventral tegmental area (VTA) and nucleus accumbens (NAc); however, direct neuroanatomical evidence of OT regulation of DA neurons within the VTA is sparse. To phenotype OT‐receptor (OTR) expressing neurons originating within the VTA, we delivered Cre‐inducible adeno‐associated virus that drives the expression of fluorescent marker into the VTA of male mice that had Cre‐recombinase driven by OTR gene expression. OTR‐expressing VTA neurons project to NAc, prefrontal cortex, the extended amygdala, and other forebrain regions but less than 10% of these OTR‐expressing neurons were identified as DA neurons (defined by tyrosine hydroxylase colocalization). Instead, almost 50% of OTR‐expressing cells in the VTA were glutamate (GLU) neurons, as indicated by expression of mRNA for the vesicular GLU transporter (vGluT). About one‐third of OTR‐expressing VTA neurons did not colocalize with either DA or GLU phenotypic markers. Thus, OTR expression by VTA neurons implicates that OT regulation of reward circuitry is more complex than a direct action on DA neurotransmission. J. Comp. Neurol. 525:1094–1108, 2017. © 2016 Wiley Periodicals, Inc.     

Laterodorsal tegmental stimulation elicits dopamine efflux in the rat nucleus accumbens by activation of acetylcholine and glutamate receptors in the ventral tegmental area

Cholinergic and glutamatergic neurons in the laterodorsal tegmentum (LDT) and neighbouring mesopontine nuclei are thought to influence mesolimbic dopaminergic neuronal activity involved in goal-directed behaviours. We measured the changes in dopamine oxidation current (corresponding with dopamine efflux) in the nucleus accumbens (NAc) in response to electrical stimulation of the LDT using in vivo chronoamperometry in urethane-anaesthetized rats. LDT stimulation (35 Hz pulse trains for 60 s, 1 s intertrain interval) evoked a three-component change in dopamine efflux in the NAc: (i) an initial stimulation time-locked increase in the dopamine signal above baseline, followed by (ii) an immediate decrease below baseline, and thereafter by (iii) a prolonged increase in the dopamine signal above baseline. Intra-VTA infusion of the nicotinic receptor antagonist mecamylamine (5 microg/0.5 microL) or the ionotropic glutamate receptor antagonist kynurenate (10 microg/microL) attenuated the first LDT-elicited component. The second suppressive component was abolished by intra-LDT infusions of either the nonselective or the M2-selective muscarinic receptor antagonists scopolamine (100 microg/microL) and methoctramine (50 microg/microL), respectively. In contrast, intra-VTA infusions of scopolamine (200 microg/microL) resulted in a selective attenuation of the third facilitatory component, whereas both second and third components were abolished by systemic injections of scopolamine (5 mg/kg). These results suggest that the initial increase, subsequent decrease, and final prolonged increase in extracellular dopamine levels in the NAc are selectively mediated by LDT-elicited activation of (i) nicotinic and glutamatergic receptors in the VTA, (ii) muscarinic M2 autoreceptors on LDT cell bodies, and (iii) muscarinic receptors in the VTA, respectively.     

A single high dose of cocaine induces behavioural sensitization and modifies mRNA encoding GluR1 and GAP-43 in rats

Neuroadaptive changes underlying repeated exposure to cocaine-induced behavioural sensitization have been related to modification in the pattern of synaptic connectivity and excitatory transmission. Remarkably, even a single exposure to abused drugs is sufficient to elicit lasting behavioural sensitization. The present study investigated whether in Sprague-Dawley rats a single, behavioural sensitizing dose of cocaine is sufficient to induce changes in the mRNA levels of growth-associated protein 43 (GAP-43), an important protein in mediating experience-dependent plasticity and synaptic reorganization, and of glutamate receptor 1 (GluR1), a subunit of AMPA glutamate receptors, a protein that is up-regulated with repeated cocaine. Single exposure to 20, but not 10 mg/kg cocaine induced locomotor sensitization to a second injection of 10 mg/kg cocaine, observed at 24 h, 48 h and 7 days. Single dose of 20 but not 10 mg/kg cocaine 48 h before scheduled death significantly enhanced GluR1 and GAP-43 mRNA expression in the nucleus accumbens (NAc), both shell and core subregions, and ventral tegmental area (VTA). No changes were found in the levels of mRNA for GluR1 and GAP-43 in the frontal cortex, caudate putamen, dentate gyrus of hippocampus and basolateral nucleus of the amygdala after the single dose of 20 mg/kg cocaine. These results further strengthen the involvement of NAc and VTA in the behavioural sensitization and suggest a role of GAP-43 in the synaptic reorganization associated to drug abuse.     

Diurnal rhythms in neural activation in the mesolimbic reward system: critical role of the medial prefrontal cortex

Previous evidence suggests a circadian modulation of drug‐seeking behavior and responsiveness to drugs of abuse. To identify potential mechanisms for rhythmicity in reward, a marker of neural activation (cFos) was examined across the day in the mesolimbic reward system. Rats were perfused at six times during the day [zeitgeber times (ZTs): 2, 6, 10, 14, 18, and 22], and brains were analysed for cFos and tyrosine hydroxylase (TH)‐immunoreactive (IR) cells. Rhythmic expression of cFos was observed in the nucleus accumbens (NAc) core and shell, in the medial prefrontal cortex (mPFC), and in TH‐IR and non‐TH‐IR cells in the ventral tegmental area (VTA), with peak expression during the late night and nadirs during the late day. No significant rhythmicity was observed in the basolateral amgydala or the dentate gyrus. As the mPFC provides excitatory input to both the NAc and VTA, this region was hypothesised to be a key mediator of rhythmic neural activation in the mesolimbic system. Hence, the effects of excitotoxic mPFC lesions on diurnal rhythms in cFos immunoreactivity at previously observed peak (ZT18) and nadir (ZT10) times were examined in the NAc and VTA. mPFC lesions encompassing the prelimbic and infralimbic subregions attenuated peak cFos immunoreactivity in the NAc, eliminating the diurnal rhythm, but had no effect on VTA rhythms. These results suggest that rhythmic neural activation in the mesolimbic system may contribute to diurnal rhythms in reward‐related behaviors, and indicate that the mPFC plays a critical role in mediating rhythmic neural activation in the NAc.     

Effects of repeated cocaine exposure and withdrawal on voluntary ethanol drinking,and the expression of glial glutamate transporters in mesocorticolimbic system of P rats

Glutamatergic neurotransmission within the brain's reward circuits plays a major role in the reinforcing properties of both ethanol and cocaine. Glutamate homeostasis is regulated by several glutamate transporters, including glutamate transporter type 1 (GLT-1), cystine/glutamate transporter (xCT), and glutamate aspartate transporter (GLAST). Cocaine exposure has been shown to induce a dysregulation in glutamate homeostasis and a decrease in the expression of GLT-1 and xCT in the nucleus accumbens (NAc). In this study, alcohol preferring (P) rats were exposed to free-choice of ethanol (15% and 30%) and/or water for five weeks. On Week 6, rats were administered (i.p.) cocaine (10 and 20 mg/kg) or saline for 12 consecutive days. This study tested two groups of rats: the first group was euthanized after seven days of repeated cocaine i.p. injection, and the second group was deprived from cocaine for five days and euthanized at Day 5 after cocaine withdrawal. Only repeated cocaine (20 mg/kg, i.p.) exposure decreased ethanol intake from Day 3 through Day 8. Co-exposure of cocaine and ethanol decreased the relative mRNA expression and the expression of GLT-1 in the NAc but not in the medial prefrontal cortex (mPFC). Importantly, co-exposure of cocaine and ethanol decreased relative expression of xCT in the NAc but not in the mPFC. Our findings demonstrated that chronic cocaine exposure affects ethanol intake; and ethanol and cocaine co-abuse alters the expression of glial glutamate transporters.     

Effect of cocaine self-administration on striatal dopamine D1 receptors in rhesus monkeys

An array of evidence indicates that long-term exposure to cocaine alters several components of the brain dopamine system. Because the release of dopamine in the nucleus accumbens (NAc) has been implicated in mediating the reinforcing effects of cocaine, changes in dopamine function can have profound effects on drug-seeking and drug-taking behavior. The present study examined the effects of the chronic self-administration of cocaine on the D₁ family of dopamine receptors in the rhesus monkey. The brains of three rhesus monkeys that had intravenously self-administered an average of 1.35 mg/kg cocaine per day for 18–22 months were compared to the brains of three cocaine-naive controls. The in vitro quantitative autoradiographic technique was used to quantify binding densities of the D₁ ligand [³H]SCH-23390 on cryostat-cut sections of fresh frozen tissue. In animals that self-administered cocaine, the density of D₁ binding was significantly lower in the regions of the striatum at the level where the nucleus accumbens is most fully developed. The shell of the NAc showed the largest difference with significantly lower D₁ binding also detected in adjacent regions of the caudate nucleus and the putamen. No differences were found in the rostral pole of the NAc or the dorsal striatum at that level. These findings suggest that chronic self-administration of cocaine can modulate the density of dopamine D₁ receptors in specific portions of the primate striatum. Such changes might underlie some of the behavioral consequences, like drug dependence and craving, of long-term cocaine use. Synapse 28:1–9, 1998. © 1998 Wiley-Liss, Inc.     

Region-specific tolerance to cocaine-regulated cAMP-dependent protein phosphorylation following chronic self-administration

Chronic cocaine self-administration can produce either tolerance or sensitization to certain cocaine-regulated behaviours, but whether differential alterations develop in the biochemical response to cocaine is less clear. We measured cocaine-induced phosphorylation of multiple cAMP-dependent and -independent protein substrates in mesolimbic dopamine terminal regions following chronic self-administration. Changes in self-administering rats were compared to changes produced by passive yoked injection to identify reinforcement-related regulation, whereas acute and chronic yoked groups were compared to identify the development tolerance or sensitization in the biochemical response to cocaine. Microwave-fixed brain tissue was collected immediately following 4 h of intravenous cocaine administration, and subjected to Western blot analysis of phosphorylated and total protein substrates. Chronic cocaine produced region- and substrate-specific tolerance to cAMP-dependent protein phosphorylation, including GluR1(S845) phosphorylation in striatal and amygdala subregions and NR1(S897) phosphorylation in the CA1 subregion of the hippocampus. Tolerance also developed to cAMP-independent GluR1(S831) phosphorylation in the prefrontal cortex. In contrast, sensitization to presynaptic regulation of synapsin(S9) phosphorylation developed in the hippocampal CA3 subregion while cAMP-dependent tyrosine hydroxylase(S40) phosphorylation decreased in striatal dopamine terminals. Cocaine-induced ERK and CREB(S133) phosphorylation were dissociated in many brain regions and failed to develop either tolerance or sensitization with chronic administration. Positive reinforcement-related correlations between cocaine intake and protein phosphorylation were found only in self-administering animals, while negative dose-related correlations were found primarily with yoked administration. These regional- and substrate-specific adaptations in cocaine-induced protein phosphorylation are discussed in view of their potential impact on the development of cocaine addiction.