Sex differences in susceptibility to substance use disorder: Role for X chromosome inactivation and escape?

There is a sex-based disparity associated with substance use disorders (SUDs) as demonstrated by clinical and preclinical studies. Females are known to escalate from initial drug use to compulsive drug-taking behavior (telescoping) more rapidly, and experience greater negative withdrawal effects than males. Although these biological differences have largely been attributed to sex hormones, there is evidence for non-hormonal factors, such as the influence of the sex chromosome, which underlie sex disparities in addiction behavior. However, genetic and epigenetic mechanisms underlying sex chromosome influences on substance abuse behavior are not completely understood. In this review, we discuss the role that escape from X-chromosome inactivation (XCI) in females plays in sex-associated differences in addiction behavior. Females have two X chromosomes (XX), and during XCI, one X chromosome is randomly chosen to be transcriptionally silenced. However, some X-linked genes escape XCI and display biallelic gene expression. We generated a mouse model using an X-linked gene specific bicistronic dual reporter mouse as a tool to visualize allelic usage and measure XCI escape in a cell specific manner. Our results revealed a previously undiscovered X-linked gene XCI escaper (CXCR3), which is variable and cell type dependent. This illustrates the highly complex and context dependent nature of XCI escape which is largely understudied in the context of SUD. Novel approaches such as single cell RNA sequencing will provide a global molecular landscape and impact of XCI escape in addiction and facilitate our understanding of the contribution of XCI escape to sex disparities in SUD.

Porcine Epidemic Diarrhea Virus Deficient in RNA Cap Guanine-N-7 Methylation Is Attenuated and Induces Higher Type I and III Interferon Responses.

The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses.IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.

Influence of Serotonin Transporter SLC6A4 Genotype on the Effect of Psychosocial Stress on Cognitive Performance: An Exploratory Pilot Study.

BACKGROUND AND OBJECTIVE: Previous research has shown an effect of various psychosocial stressors on unconstrained cognitive flexibility, such as searching through a large set of potential solutions in the lexical-semantic network during verbal problem-solving. Functional magnetic resonance imaging has shown that the presence of the short (S) allele (lacking a 43-base pair repeat) of the promoter region of the gene (SLC6A4) encoding the serotonin transporter (5-HTT) protein is associated with a greater amygdalar response to emotional stimuli and a greater response to stressors. Therefore, we hypothesized that the presence of the S-allele is associated with greater stress-associated impairment in performance on an unconstrained cognitive flexibility task, anagrams. METHODS: In this exploratory pilot study, 28 healthy young adults were genotyped for long (L)-allele versus S-allele promoter region polymorphism of the 5-HTT gene, SLC6A4. Participants solved anagrams during the Trier Social Stress Test, which included public speaking and mental arithmetic stressors. We compared the participants' cognitive response to stress across genotypes. RESULTS: A Gene×Stress interaction effect was observed in this small sample. Comparisons revealed that participants with at least one S-allele performed worse during the Stress condition. CONCLUSIONS: Genetic susceptibility to stress conferred by SLC6A4 appeared to modulate unconstrained cognitive flexibility during psychosocial stress in this exploratory sample. If confirmed, this finding may have implications for conditions associated with increased stress response, including performance anxiety and cocaine withdrawal. Future work is needed both to confirm our findings with a larger sample and to explore the mechanisms of this proposed effect.

Paradoxical abatement of striatal dopaminergic transmission by cocaine and methylphenidate.

We combined in vitro amperometric, optical analysis of fluorescent false neurotransmitters and microdialysis techniques to unveil that cocaine and methylphenidate induced a marked depression of the synaptic release of dopamine (DA) in mouse striatum. In contrast to the classical dopamine transporter (DAT)-dependent enhancement of the dopaminergic signal observed at concentrations of cocaine lower than 3 µM, the inhibitory effect of cocaine was found at concentrations higher than 3 µM. The paradoxical inhibitory effect of cocaine and methylphenidate was associated with a decrease in synapsin phosphorylation. Interestingly, a cocaine-induced depression of DA release was only present in cocaine-insensitive animals (DAT-CI). Similar effects of cocaine were produced by methylphenidate in both wild-type and DAT-CI mice. On the other hand, nomifensine only enhanced the dopaminergic signal either in wild-type or in DAT-CI mice. Overall, these results indicate that cocaine and methylphenidate can increase or decrease DA neurotransmission by blocking reuptake and reducing the exocytotic release, respectively. The biphasic reshaping of DA neurotransmission could contribute to different behavioral effects of psychostimulants, including the calming ones, in attention deficit hyperactivity disorder.

Cocaine increases quantal norepinephrine secretion through NET-dependent PKC activation in locus coeruleus neurons.

The norepinephrine neurons in locus coeruleus (LC-NE neurons) are essential for sleep arousal, pain sensation, and cocaine addiction. According to previous studies, cocaine increases NE overflow (the profile of extracellular NE level in response to stimulation) by blocking the NE reuptake. NE overflow is determined by NE release via exocytosis and reuptake through NE transporter (NET). However, whether cocaine directly affects vesicular NE release has not been directly tested. By recording quantal NE release from LC-NE neurons, we report that cocaine directly increases the frequency of quantal NE release through regulation of NET and downstream protein kinase C (PKC) signaling, and this facilitation of NE release modulates the activity of LC-NE neurons and cocaine-induced stimulant behavior. Thus, these findings expand the repertoire of mechanisms underlying the effects of cocaine on NE (pro-release and anti-reuptake), demonstrate NET as a release enhancer in LC-NE neurons, and provide potential sites for treatment of cocaine addiction.

Role of aberrant striatal dopamine D1 receptor/cAMP/protein kinase A/DARPP32 signaling in the paradoxical calming effect of amphetamine.

Attention deficit/hyperactivity disorder (ADHD) is characterized by inattention, impulsivity, and motor hyperactivity. Several lines of research support a crucial role for the dopamine transporter (DAT) gene in this psychiatric disease. Consistently, the most commonly prescribed medications in ADHD treatment are stimulant drugs, known to preferentially act on DAT. Recently, a knock-in mouse [DAT-cocaine insensitive (DAT-CI)] has been generated carrying a cocaine-insensitive DAT that is functional but with reduced dopamine uptake function. DAT-CI mutants display enhanced striatal extracellular dopamine levels and basal motor hyperactivity. Herein, we showed that DAT-CI animals present higher striatal dopamine turnover, altered basal phosphorylation state of dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP32) at Thr75 residue, but preserved D(2) receptor (D(2)R) function. However, although we demonstrated that striatal D(1) receptor (D(1)R) is physiologically responsive under basal conditions, its stimulus-induced activation strikingly resulted in paradoxical electrophysiological, behavioral, and biochemical responses. Indeed, in DAT-CI animals, (1) striatal LTP was completely disrupted, (2) R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) treatment induced paradoxical motor calming effects, and (3) SKF 81297 administration failed to increase cAMP/protein kinase A (PKA)/DARPP32 signaling. Such biochemical alteration selectively affected dopamine D(1)Rs since haloperidol, by blocking the tonic inhibition of D(2)R, unmasked a normal activation of striatal adenosine A(2A) receptor-mediated cAMP/PKA/DARPP32 cascade in mutants. Most importantly, our studies highlighted that amphetamine, nomifensine, and bupropion, through increased striatal dopaminergic transmission, are able to revert motor hyperactivity of DAT-CI animals. Overall, our results suggest that the paradoxical motor calming effect induced by these drugs in DAT-CI mutants depends on selective aberrant phasic activation of D(1)R/cAMP/PKA/DARPP32 signaling in response to increased striatal extracellular dopamine levels.

Loss of striatal cannabinoid CB1 receptor function in attention-deficit / hyperactivity disorder mice with point-mutation of the dopamine transporter.

Abnormal dopamine (DA) transmission in the striatum plays a pivotal role in attention-deficit/hyperactivity disorder (ADHD). As striatal DA signalling modulates the endocannabinoid system (ECS), the present study was aimed at investigating cannabinoid CB1 receptor (CB1R) function in a model of ADHD obtained by triple point-mutation in the dopamine transporter (DAT) gene in mice, making them insensitive to cocaine [DAT cocaine-insensitive (DAT-CI) mice]. DAT-CI mice had a marked hyperactive phenotype, and neurophysiological recordings revealed that the sensitivity of CB1Rs controlling GABA-mediated synaptic currents [CB1Rs((GABA)) ] in the striatum was completely lost. In contrast, CB1Rs modulating glutamate transmission [CB1Rs((Glu)) ], and GABA(B) receptors were not affected in this model of ADHD. In DAT-CI mice, the blockade of CB1R((GABA)) function was complete even after cocaine or environmental manipulations activating the endogenous DA-dependent reward system, which are known to sensitize these receptors in control animals. Conversely, the hedonic property of sucrose was intact in DAT-CI mice, indicating normal sweet perception in these animals. Our results point to CB1Rs as novel molecular players in ADHD, and suggest that therapeutic strategies aimed at interfering with the ECS might prove effective in this disorder.

Fluorescence-based evaluation of shRNA efficacy.

RNA interference is a cellular mechanism regulating levels of mRNAs. It has been widely exploited to knock down specific protein targets. The selected interfering RNA sequence greatly influences its ability to knock down the target. Here we present a method for constructing multiple testing plasmids which express small hairpin RNAs (shRNA) targeting different regions of an mRNA. A simple fluorescence test in cultured cells allows convenient evaluation of mRNA knockdown by many different shRNAs on 96-well plates. We show that software predicted shRNAs have varying efficacies and only 2 of the 7 tested shRNAs significantly knocked down their targets.

Dopamine transporter inhibition is necessary for cocaine-induced increases in dendritic spine density in the nucleus accumbens.

Repeated exposure to cocaine produces changes in the nervous system that facilitate drug-seeking behaviors. These drug-seeking behaviors have been studied with animal models, such as cocaine-induced locomotor sensitization. Cocaine is hypothesized to induce locomotor sensitization by neural changes, including an increase in the density of spines on the dendrites of neurons in the nucleus accumbens (NAC). However, how cocaine increases dendritic spine density in the NAC has been difficult to discern because cocaine inhibits the function of multiple targets, including the transporters for dopamine, serotonin, and norepinephrine. Previously, our lab created a tool that is useful for determining how inhibiting the dopamine transporter (DAT) contributes to the effects of cocaine by generating mice that express a cocaine-insensitive DAT (DAT-CI mice). In this study, we used DAT-CI mice to determine the contribution of DAT inhibition in cocaine-induced increases in dendritic spine density in the NAC. We repeatedly injected DAT-CI mice with either cocaine or saline, and measured both dendritic spine density in the NAC and locomotor activity. Unlike wild-type mice, DAT-CI mice did not show an increase in dendritic spine density in the NAC or in locomotor activity in response to repeated injections of cocaine. These data show that cocaine-induced increases in dendritic spine density in the NAC require DAT inhibition. Thus, DAT-inhibition may play a role in mediating the long-lasting neural changes associated with drug addiction.

Lack of cocaine self-administration in mice expressing a cocaine-insensitive dopamine transporter.

Cocaine addiction is a worldwide public health problem for which there are no established treatments. The dopamine transporter (DAT) is suspected as the primary target mediating cocaine's abuse-related effects based on numerous pharmacological studies. However, in a previous study, DAT knockout mice were reported to self-administer cocaine, generating much debate regarding the importance of the DAT in cocaine's abuse-related effects. Here, we show that mice expressing a "knockin" of a cocaine-insensitive but functional DAT did not self-administer cocaine intravenously despite normal food-maintained responding and normal intravenous self-administration of amphetamine and a direct dopamine agonist. Our results have three implications. First, they imply a crucial role for high-affinity DAT binding of cocaine in mediating its reinforcing effects, reconciling mouse genetic engineering approaches with data from classic pharmacological studies. Second, they demonstrate the usefulness of knockin strategies that modify specific amino acid sequences within a protein. Third, they show that it is possible to alter the DAT protein sequence in such a way as to selectively target its interaction with cocaine, while sparing other behaviors dependent on DAT function. Thus, molecular engineering technology could advance the development of highly specialized compounds such as a dopamine-sparing "cocaine antagonist."

Functional mutations in mouse norepinephrine transporter reduce sensitivity to cocaine inhibition.

The transporters of dopamine, norepinephrine and serotonin are molecular targets of cocaine, amphetamine, and therapeutic antidepressants. The residues involved in binding these drugs are unknown. We have performed several rounds of random and site-directed mutagenesis in the mouse norepinephrine transporter and screened for mutants with altered sensitivity to cocaine inhibition of substrate uptake. We have identified a triple mutation that retains close to wild-type transport function but displays a 37-fold decrease in cocaine sensitivity and 24-fold decrease in desipramine sensitivity. In contrast, the mutant's sensitivities to amphetamine, methamphetamine, and methylphenidate are only slightly changed. Our data reveal critical residues contributing to the potent uptake inhibitions by these important drugs. Furthermore, this drug-resistant triple mutant can be used to generate a unique knock-in mouse line to study the role of norepinephrine transporter in the addictive effects of cocaine and the therapeutic effects of desipramine.

The effects of methylphenidate on knockin mice with a methylphenidate-resistant dopamine transporter.

Methylphenidate (Ritalin) is one of the most commonly abused prescription drugs. It is a psychostimulant that inhibits the dopamine and norepinephrine transporters with high affinity. In mice, methylphenidate stimulates locomotor activity, is self-administered, and produces conditioned place preference, typical properties of an addictive drug. We have generated a knockin mouse line bearing a mutant dopamine transporter that is approximately 80-fold less sensitive to cocaine inhibition than wild type. It is interesting to note that this mutant is also almost 50-fold less sensitive to methylphenidate inhibition, suggesting similarities in the binding site for cocaine and methylphenidate. Because methylphenidate is not effective at inhibiting the mutant dopamine transporter, we hypothesized that it would not stimulate locomotor activity or produce reward in the knockin mice. In these knockin mice, doses up to 40 mg/kg methylphenidate either inhibit or fail to stimulate locomotor activity and do not produce conditioned place preference. Doses up to 40 mg/kg methylphenidate also fail to produce stereotypy in the knockin mice. Nisoxetine and desipramine, selective norepinephrine transporter inhibitors, also reduce locomotor activity in wild-type and knockin mice. These results indicate that enhanced dopaminergic neurotransmission is required for methylphenidate's stimulating and rewarding effects. In addition, we observed that drugs enhancing noradrenergic neurotransmission inhibit locomotor activity in mice, which is consistent with the notion that methylphenidate's ability to inhibit the norepinephrine transporter may contribute to its efficacy in treating attention deficit hyperactivity disorder.

Dopamine transporter inhibition is required for cocaine-induced stereotypy.

The primary mechanism by which cocaine induces stereotypy has been difficult to discern because cocaine has three high-affinity targets: the reuptake transporters for dopamine (DAT), norepinephrine, and serotonin. To dissect out the role of DAT in cocaine effects, we generated a knock-in mouse line with a cocaine-insensitive DAT (DAT-CI mice). DAT-CI mice provide a powerful tool that can directly test whether DAT inhibition is important for cocaine-induced stereotypy. We found that acute cocaine failed to produce stereotypy in DAT-CI mice. In fact, 40 mg/kg cocaine suppressed stereotypy in DAT-CI mice but produced profound stereotypy in wild-type mice. These findings suggest that DAT inhibition is necessary for cocaine-induced stereotypy. Furthermore, mechanisms independent of DAT inhibition appear to inhibit stereotypy.

Cocaine reward and locomotion stimulation in mice with reduced dopamine transporter expression.

BACKGROUND: The dopamine transporter (DAT) plays a critical role in regulating dopamine neurotransmission. Variations in DAT or changes in basal dopaminergic tone have been shown to alter behavior and drug responses. DAT is one of the three known high affinity targets for cocaine, a powerful psychostimulant that produces reward and stimulates locomotor activity in humans and animals. We have shown that cocaine no longer produces reward in knock-in mice with a cocaine insensitive mutant DAT (DAT-CI), suggesting that cocaine inhibition of DAT is critical for its rewarding effect. However, in DAT-CI mice, the mutant DAT has significantly reduced uptake activity resulting in elevated basal dopaminergic tone, which might cause adaptive changes that alter responses to cocaine. Therefore, the objective of this study is to determine how elevated dopaminergic tone affects how mice respond to cocaine. RESULTS: We examined the cocaine induced behavior of DAT knockdown mice that have DAT expression reduced by 90% when compared to the wild type mice. Despite a dramatic reduction of DAT expression and marked elevation in basal dopamine tone, cocaine produced reward, as measured by conditioned place preference, and stimulated locomotor activity in these mice. CONCLUSION: A reduction in DAT expression and elevation of dopaminergic tone do not lead to adaptive changes that abolish the rewarding and stimulating effects of cocaine. Therefore, the lack of reward to cocaine observed in DAT-CI mice is unlikely to have resulted from the reduced DAT activity but instead is likely due to the inability of cocaine to block the mutated DAT and increase extracellular dopamine. This study supports the conclusion that the blockade of DAT is required for cocaine reward and locomotor stimulation.

Molecular cloning and functional characterization of the dopamine transporter from Eloria noyesi, a caterpillar pest of cocaine-rich coca plants.

Cocaine is produced by coca plants as a chemical defense to deter feeding by insects. It has been shown that cocaine sprayed on tomato leaves reduces insect feeding, causes abnormal behaviors at low doses and kills feeding insects at doses equivalent to that in coca leaves [Nathanson, J.A., Hunnicutt, E.J., Kantham, L., Scavone, C., 1993. Cocaine as a naturally occurring insecticide. Proc. Natl. Acad. Sci. U. S. A. 90, 9645-9648.]. Most insects avoid coca leaves except the larvae of Eloria noyesi, a caterpillar pest of coca plants, which feeds preferentially on coca leaves. In the current study, we cloned and characterized the dopamine transporters (DATs) from caterpillars of E. noyesi (enDAT) and the silkworm, Bombyx mori (B. mori, bmDAT). The two insect DATs shared 88% amino acid sequence homology and functional similarity. Although enDAT and bmDAT showed the highest affinity for dopamine among endogenous amines, they were more sensitive to mammalian NET-selective inhibitors than to mammalian DAT-selective inhibitors. Despite a high cocaine content in the food source for E. noyesi, cocaine sensitivity of enDAT was similar to that of bmDAT, suggesting that mechanisms other than DAT insensitivity to cocaine, such as cocaine sequestration, might be responsible for cocaine resistance in this species. Given the significant differences in pharmacological profile from mammalian DATs, invertebrate DATs provide excellent tools for identifying regions and residues in the transporters that contribute to high-affinity binding of psychostimulants and antidepressants.

Comparison of the monoamine transporters from human and mouse in their sensitivities to psychostimulant drugs.

BACKGROUND: The plasma membrane neurotransmitter transporters terminate neurotransmissions by the reuptake of the released neurotransmitters. The transporters for the monoamines dopamine, norepinephrine, and serotonin (DAT, NET, and SERT) are targets for several popular psychostimulant drugs of abuse. The potencies of the psychostimulant on the monoamine transporters have been studied by several laboratories. However, there are significant discrepancies in the reported data with differences up to 60-fold. In addition, the drug potencies of the 3 monoamine transporters from mouse have not been compared in the same experiments or along side the human transporters. Further studies and systematic comparisons are needed. RESULTS: In this study, we compared the potencies of five psychostimulant drugs to inhibit human and mouse DAT, SERT and NET in the same cellular background. The KI values of cocaine to inhibit the 3 transporters are within a narrow range of 0.2 to 0.7 microM. In comparison, methylphenidate inhibited DAT and NET at around 0.1 microM, while it inhibited SERT at around 100 microM. The order of amphetamine potencies was NET (KI = 0.07-0.1 microM), DAT (KI approximately 0.6 microM), and SERT (KI between 20 to 40 microM). The results for methamphetamine were similar to those for amphetamine. In contrast, another amphetamine derivative, MDMA (3-4 methylenedioxymethamphetamine), exhibited higher potency at SERT than at DAT. The human and mouse transporters were similar in their sensitivities to each of the tested drugs (KI values are within 4-fold). CONCLUSION: The current and previous studies support the following conclusions: 1) cocaine blocks all 3 monoamine transporters at similar concentrations; 2) methylphenidate inhibits DAT and NET well but a 1000-fold higher concentration of the drug is required to inhibit SERT; 3) Amphetamine and methamphetamine are most potent at NET, while being 5- to 9-fold less potent at DAT, and 200- to 500-fold less potent at SERT; 4) MDMA has moderately higher apparent affinity for SERT and NET than for DAT. The relative potencies of a drug to inhibit DAT, NET and SERT suggest which neurotransmitter systems are disrupted the most by each of these stimulants and thus the likely primary mechanism of drug action.

Inactivation of the catalytic phosphatase domain of PTPRT/RPTPρ increases social interaction in mice.

Receptor protein tyrosine phosphatase rho (RPTPρ, gene symbol PTPRT) is a transmembrane protein expressed at high levels in the developing hippocampus, olfactory bulb, cortex, and cerebellum. It has an extracellular domain that interacts with other cell adhesion molecules, and it has two intracellular phosphatase domains, one of which is catalytically active. In a recent genome-wide association study, PTPRT was identified as a potential candidate gene for autism spectrum disorder (ASD) susceptibility. Mutation of a critical aspartate to alanine (D1046A) in the PTPRT catalytic domain inactivates phosphatase function but retains substrate binding. We have generated a knockin mouse line carrying the PTPRT D1046A mutation. The D1046A mutation in homozygous knockin mice did not significantly change locomotor activities or anxiety-related behaviors. In contrast, male homozygous mice had significantly higher social approach scores than wild-type animals. Our results suggest that PTPRT phosphatase function is important in modulating neural pathways involved in mouse social behaviors relevant to the symptoms in human ASD patients.

Restoration of cocaine stimulation and reward by reintroducing wild type dopamine transporter in adult knock-in mice with a cocaine-insensitive dopamine transporter.

In previous studies, we generated knock-in mice with a cocaine-insensitive dopamine transporter (DAT-CI mice) and found cocaine does not stimulate locomotion or produce reward in these mice, indicating DAT inhibition is necessary for cocaine stimulation and reward. However, DAT uptake is reduced in DAT-CI mice and thus the lack of cocaine responses could be due to adaptive changes. To test this, we used adeno-associated virus (AAV) to reintroduce the cocaine-sensitive wild type DAT (AAV-DATwt) back into adult DAT-CI mice, which restores cocaine inhibition of DAT in affected brain regions but does not reverse the adaptive changes. In an earlier study we showed that AAV-DATwt injections in regions covering the lateral nucleus accumbens (NAc) and lateral caudate-putamen (CPu) restored cocaine stimulation but not cocaine reward. In the current study, we expanded the AAV-DATwt infected areas to cover the olfactory tubercle (Tu) and the ventral midbrain (vMB) containing the ventral tegmental area (VTA) and substantia nigra (SN) in addition to CPu and NAc with multiple injections. These mice displayed the restoration of both locomotor stimulation and cocaine reward. We further found that AAV-DATwt injection in the vMB alone was sufficient to restore both cocaine stimulation and reward in DAT-CI mice. AAV injected in the VTA and SN resulted in DATwt expression and distribution to the DA terminal regions. In summary, cocaine induced locomotion and reward can be restored in fully developed DAT-CI mice, and cocaine inhibition of DAT expressed in dopaminergic neurons originated from the ventral midbrain mediates cocaine reward and stimulation.

Cocaine inhibition of nicotinic acetylcholine receptors influences dopamine release.

Nicotinic acetylcholine receptors (nAChRs) potently regulate dopamine (DA) release in the striatum and alter cocaine's ability to reinforce behaviors. Since cocaine is a weak nAChR inhibitor, we hypothesized that cocaine may alter DA release by inhibiting the nAChRs in DA terminals in the striatum and thus contribute to cocaine's reinforcing properties primarily associated with the inhibition of DA transporters. We found that biologically relevant concentrations of cocaine can mildly inhibit nAChR-mediated currents in midbrain DA neurons and consequently alter DA release in the dorsal and ventral striatum. At very high concentrations, cocaine also inhibits voltage-gated Na channels in DA neurons. Furthermore, our results show that partial inhibition of nAChRs by cocaine reduces evoked DA release. This diminution of DA release via nAChR inhibition more strongly influences release evoked at low or tonic stimulation frequencies than at higher (phasic) stimulation frequencies, particularly in the dorsolateral striatum. This cocaine-induced shift favoring phasic DA release may contribute to the enhanced saliency and motivational value of cocaine-associated memories and behaviors.

Effect of serotonin on platelet function in cocaine exposed blood.

5-hydroxytryptamine (5-HT) reuptake inhibitors counteract the pro-thrombotic effect of elevated plasma 5-HT by down-regulating the 5-HT uptake rates of platelets. Cocaine also down-regulates the platelet 5-HT uptake rates but in contrast, the platelets of cocaine-injected mice show a much higher aggregation rate than the platelets of control mice. To examine the involvement of plasma 5-HT in cocaine-mediated platelet aggregation, we studied the function of platelets isolated from wild-type and transgenic, peripheral 5-HT knock-out (TPH1-KO) mice, and cocaine-insensitive dopamine transporter knock in (DAT-KI) mice. In cocaine-injected mice compared to the control mice, the plasma 5-HT level as well as the surface level of P-selectin was elevated; in vitro platelet aggregation in the presence of type I fibrillar collagen was enhanced. However, cocaine injection lowered the 5-HT uptake rates of platelets and increased the plasma 5-HT levels of the DAT-KI mice but did not change their platelets aggregation rates further which are already hyper-reactive. Furthermore, the in vitro studies supporting these in vivo findings suggest that cocaine mimics the effect of elevated plasma 5-HT level on platelets and in 5-HT receptor- and transporter-dependent pathways in a two-step process propagates platelet aggregation by an additive effect of 5-HT and nonserotonergic catecholamine.