Modulation of the Blood-Brain Barrier by Sigma-1R Activation.

Sigma non-opioid intracellular receptor 1 (Sigma-1R) is an intracellular chaperone protein residing on the endoplasmic reticulum at the mitochondrial-associated membrane (MAM) region. Sigma-1R is abundant in the brain and is involved in several physiological processes as well as in various disease states. The role of Sigma-1R at the blood-brain barrier (BBB) is incompletely characterized. In this study, the effect of Sigma-1R activation was investigated in vitro on rat brain microvascular endothelial cells (RBMVEC), an important component of the blood-brain barrier (BBB), and in vivo on BBB permeability in rats. The Sigma-1R agonist PRE-084 produced a dose-dependent increase in mitochondrial calcium, and mitochondrial and cytosolic reactive oxygen species (ROS) in RBMVEC. PRE-084 decreased the electrical resistance of the RBMVEC monolayer, measured with the electric cell-substrate impedance sensing (ECIS) method, indicating barrier disruption. These effects were reduced by pretreatment with Sigma-1R antagonists, BD 1047 and NE 100. In vivo assessment of BBB permeability in rats indicates that PRE-084 produced a dose-dependent increase in brain extravasation of Evans Blue and sodium fluorescein brain; the effect was reduced by the Sigma-1R antagonists. Immunocytochemistry studies indicate that PRE-084 produced a disruption of tight and adherens junctions and actin cytoskeleton. The brain microcirculation was directly visualized in vivo in the prefrontal cortex of awake rats with a miniature integrated fluorescence microscope (aka, miniscope; Doric Lenses Inc.). Miniscope studies indicate that PRE-084 increased sodium fluorescein extravasation in vivo. Taken together, these results indicate that Sigma-1R activation promoted oxidative stress and increased BBB permeability.

Blood-Brain Barrier Disruption Mediated by FFA1 Receptor-Evidence Using Miniscope.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), obtained from diet and dietary supplements, have been tested in clinical trials for the prevention or treatment of several diseases. n-3 PUFAs exert their effects by activation of free fatty acid (FFA) receptors. FFA1 receptor, expressed in the pancreas and brain, is activated by medium- to long-chain fatty acids. Despite some beneficial effects on cognition, the effects of n-3 PUFAs on the blood-brain barrier (BBB) are not clearly understood. We examined the effects of FFA1 activation on BBB permeability in vitro, using rat brain microvascular endothelial cells (RBMVEC), and in vivo, by assessing Evans Blue extravasation and by performing live imaging of brain microcirculation in adult rats. AMG837, a synthetic FFA1 agonist, produced a dose-dependent decrease in RBMVEC monolayer resistance assessed with Electric Cell-Substrate Impedance Sensing (ECIS); the effect was attenuated by the FFA1 antagonist, GW1100. Immunofluorescence studies revealed that AMG837 produced a disruption in tight and adherens junction proteins. AMG837 increased Evans Blue content in the rat brain in a dose-dependent manner. Live imaging studies of rat brain microcirculation with miniaturized fluorescence microscopy (miniscope) showed that AMG837 increased extravasation of sodium fluorescein. Taken together, our results demonstrate that FFA1 receptor activation reduced RBMVEC barrier function and produced a transient increase in BBB permeability.

Intra-Tumoral Nerve-Tracing in a Novel Syngeneic Model of High-Grade Serous Ovarian Carcinoma.

Dense tumor innervation is associated with enhanced cancer progression and poor prognosis. We observed innervation in breast, prostate, pancreatic, lung, liver, ovarian, and colon cancers. Defining innervation in high-grade serous ovarian carcinoma (HGSOC) was a focus since sensory innervation was observed whereas the normal tissue contains predominantly sympathetic input. The origin, specific nerve type, and the mechanisms promoting innervation and driving nerve-cancer cell communications in ovarian cancer remain largely unknown. The technique of neuro-tracing enhances the study of tumor innervation by offering a means for identification and mapping of nerve sources that may directly and indirectly affect the tumor microenvironment. Here, we establish a murine model of HGSOC and utilize image-guided microinjections of retrograde neuro-tracer to label tumor-infiltrating peripheral neurons, mapping their source and circuitry. We show that regional sensory neurons innervate HGSOC tumors. Interestingly, the axons within the tumor trace back to local dorsal root ganglia as well as jugular-nodose ganglia. Further manipulations of these tumor projecting neurons may define the neuronal contributions in tumor growth, invasion, metastasis, and responses to therapeutics.

Choline-Sigma-1R as an Additional Mechanism for Potentiation of Orexin by Cocaine.

Orexin A, an endogenous peptide involved in several functions including reward, acts via activation of orexin receptors OX1 and OX2, Gq-coupled GPCRs. We examined the effect of a selective OX1 agonist, OXA (17-33) on cytosolic calcium concentration, [Ca2+]i, in neurons of nucleus accumbens, an important area in the reward circuit. OXA (17-33) increased [Ca2+]i in a dose-dependent manner; the effect was prevented by SB-334867, a selective OX1 receptors antagonist. In Ca2+-free saline, the OXA (17-33)-induced increase in [Ca2+]i was not affected by pretreatment with bafilomycin A1, an endo-lysosomal calcium disrupter, but was blocked by 2-APB and xestospongin C, antagonists of inositol-1,4,5-trisphosphate (IP3) receptors. Pretreatment with VU0155056, PLD inhibitor, or BD-1047 and NE-100, Sigma-1R antagonists, reduced the [Ca2+]i response elicited by OXA (17-33). Cocaine potentiated the increase in [Ca2+]i by OXA (17-33); the potentiation was abolished by Sigma-1R antagonists. Our results support an additional signaling mechanism for orexin A-OX1 via choline-Sigma-1R and a critical role for Sigma-1R in the cocaine-orexin A interaction in nucleus accumbens neurons.

Assessment of Blood-Brain Barrier Permeability Using Miniaturized Fluorescence Microscopy in Freely Moving Rats.

We report here the method of visualization of brain microcirculation and assessment of blood-brain barrier (BBB) permeability changes using the miniature integrated fluorescence microscope (i.e., miniscope) technology in awake, freely moving rats. The imaging cannula is implanted in the brain area of interest of anesthetized adult rats. After recovery and habituation, sodium fluorescein, a low-molecular-weight tracer, is injected i.v. Fluorescence intensity in the vicinity of microvessels, as an indicator of BBB permeability, is then recorded in vivo via the miniscope for extended periods of time. The method can be used to assess the changes in BBB permeability produced by pharmacologic agents; in this case, the drug of interest is administered after sodium fluorescein. An increase in the sodium fluorescein extravasation in brain microcirculation demonstrates an increase in BBB permeability. The method described here allows a high-resolution visualization of real-time changes in BBB permeability in awake, freely moving rats.

Sex differences in the effect of the FKBP5 inhibitor SAFit2 on anxiety and stress-induced reinstatement following cocaine self-administration.

Cocaine use and withdrawal prompt stress system responses. Stress and the negative affective state produced by cocaine withdrawal are major triggers for relapse. FKBP5 is a co-chaperone of the glucocorticoid receptor and regulates HPA axis negative feedback. The role of FKBP5 in cocaine-related behaviors has not been studied. The FKBP5 inhibitor SAFit2 was used to examine the role of FKBP5 in anxiety-like behavior during early cocaine withdrawal and in stress-induced reinstatement following cocaine self-administration in male and female rats. Withdrawal from cocaine self-administration resulted in heightened anxiety-like behavior in female rats, which was significantly attenuated by SAFit2 administration. SAFit2 pretreatment prior to stress-induced reinstatement to cocaine seeking significantly reduced active lever presses of males. In female rats, SAFit2 administration prevented stress-induced reinstatement for rats in metestrus or diestrus, but not proestrus or estrus phases at the time of reinstatement. These data suggest an important role for FKBP5 in stress-related behaviors following cocaine self-administration, particularly in females.

Direct evidence of bradycardic effect of omega-3 fatty acids acting on nucleus ambiguus.

Docosahexaenoic acid (DHA) an omega-3 polyunsaturated fatty acid, is an agonist of FFA1 receptor. DHA administration reduces the heart rate via unclear mechanisms. We examined the effect of DHA on neurons of nucleus ambiguus that provide the parasympathetic control of heart rate. DHA produced a dose-dependent increase in cytosolic Ca2+ concentration in cardiac-projecting nucleus ambiguus neurons; the effect was prevented by GW1100, a FFA1 receptor antagonist. DHA depolarized cultured nucleus ambiguus neurons via FFA1 activation. Bilateral microinjection of DHA into nucleus ambiguus produced bradycardia in conscious rats. Our results indicate that DHA decreases heart rate by activation of FFA1 receptor on cardiac-projecting nucleus ambiguus neurons.

Glycogen synthase kinase-3 signaling in cellular and behavioral responses to psychostimulant drugs.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase implicated in numerous physiological processes and cellular functions through its ability to regulate the function of many proteins, including transcription factors and structural proteins. GSK-3ß has been demonstrated to function as a regulator of multiple behavioral processes induced by drugs of abuse, particularly psychostimulant drugs. In this review, we provide an overview of the regulation of GSK-3ß activity produced by psychostimulants, and the role of GSK-3ß signaling in psychostimulant-induced behaviors including drug reward, associative learning and memory which play a role in the maintenance of drug-seeking. Evidence supports the conclusion that GSK-3ß is an important component of the actions of psychostimulant drugs and that GSK-3ß is a valid target for developing novel therapeutics. Additional studies are required to examine the role of GSK-3ß in distinct cell types within the mesolimbic and memory circuits to further elucidate the mechanisms related to the acquisition, consolidation, and recall of drug-related memories, and potentially countering neuroadaptations that reinforce drug-seeking behaviors that maintain drug dependence.

Glycogen Synthase Kinase 3ß in the Ventral Hippocampus is Important for Cocaine Reward and Object Location Memory.

The ventral hippocampus is a component of the neural circuitry involved with context-associated memory for reward and generation of appropriate behavioral responses to context. Glycogen synthase kinase 3 beta (GSK3ß) has been linked to the maintenance of synaptic plasticity, contextual memory retrieval, and is involved in the reconsolidation of cocaine-associated contextual memory. In this study, the effects of targeted downregulation of GSK3ß in the ventral hippocampus were examined on a series of behavioral tests for assessing drug reward-context association and non-reward related memory. The Cre/loxP site-specific recombination system was used to knockdown GSK3ß through bilateral stereotaxic delivery of an adeno-associated virus expressing Cre-recombinase (AAV-Cre) into the ventral hippocampus of adult mice homozygous for a floxed GSK3ß allele. GSK3ß floxed mice injected with AAV-Cre had a loss of 56-75% of GSK3ß in the ventral hippocampus and displayed diminished development of cocaine conditioned place preference, but not morphine place preference as compared with wild-type mice injected with AAV-Cre or GSK3ß floxed mice injected with a control virus, AAV-GFP. Impaired object location memory was observed in mice with GSK3ß downregulation in the ventral hippocampus, but novel object recognition remained intact. These results indicate that GSK3ß signaling in the ventral hippocampus is differentially involved in the formation of place-drug reward association dependent upon drug class. Additionally, ventral hippocampal GSK3ß signaling is important in detection of discrete spatial cues, but not recognition memory for objects.

Acute cocaine administration alters permeability of blood-brain barrier in freely-moving rats- Evidence using miniaturized fluorescence microscopy.

BACKGROUND: Cocaine has a variety of negative effects on the central nervous system, including reports of decreased barrier function of brain microvascular endothelial cells. However, few studies have directly shown the effects of cocaine on blood-brain barrier (BBB) function in vivo. The miniature integrated fluorescence microscope (i.e., miniscope) technology was used to visualize cocaine-induced changes in BBB permeability in awake, freely-moving rats. METHODS: The miniscope was implanted in the prefrontal cortex of adult male rats. After recovery and acclimation, rats received an injection of cocaine (5-20 mg/kg ip) 15 minutes following iv infusion of sodium fluorescein, a low molecular weight tracer. Fluorescence intensity was recordedin vivo via the miniscope for 30 minutes or 24 hours post cocaine administration and served as an indicator of BBB permeability. RESULTS: Results demonstrate that cocaine increased the sodium fluorescein extravasation in brain microcirculation in a dose-dependent manner 30 minutes, but not 24 hours after administration. CONCLUSION: We report for the first time using direct visualization of brain microcirculation with the miniscope technology in awake, freely-moving rats, that acute cocaine administration produced a transient increase in the BBB permeability.

Differential Roles of Accumbal GSK3ß in Cocaine versus Morphine-Induced Place Preference, U50,488H-Induced Place Aversion, and Object Memory.

Previous research has demonstrated that activity of glycogen synthase kinase-3 (GSK3) is necessary for the rewarding effects of cocaine. In the present study, a conditional GSK3ß gene knockdown model was used to determine if GSK3ß activity specifically in the nucleus accumbens is important for cocaine conditioned reward. The roles of accumbal GSK3ß in morphine conditioned reward, trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide methanesulfonate salt (U50,488H)-induced conditioned place aversion, and cognitive function were also studied. Adult male and female GSK3ß-floxed or wild-type mice were injected with adeno-associated virus/Cre into the nucleus accumbens to reduce expression of GSK3ß and underwent behavioral testing 4 weeks later. The development of cocaine-induced conditioned place preference was significantly attenuated in mice with reduced levels of GSK3ß in the nucleus accumbens, whereas the development of morphine-induced place preference remained intact. Conditional knockdown of GSK3ß in the accumbens prevented the development of conditioned aversion produced by U50,488H, a κ-opioid receptor agonist. Cognitive memory tests revealed deficits in object location memory, but not novel object recognition in mice with accumbal GSK3ß knockdown. These data demonstrate that GSK3ß in the nucleus accumbens is required for cocaine conditioned place preference and U50,488H conditioned place aversion, as well as spatial memory in object location task, indicating differential roles of GSK3ß in the psychostimulant and opiate reward process, as well as in memory for spatial locations and object identity. SIGNIFICANCE STATEMENT: Knockdown of GSK3ß in the nucleus accumbens attenuated the development of cocaine-induced place preference, as well as conditioned place aversion to U50,488H, a κ-opioid receptor agonist. In contrast, the development of morphine place preference was not altered by GSK3ß knockdown. GSK3ß knockdown in nucleus accumbens impaired performance in the object location task, but not the novel object recognition task. These results elucidate different physiological roles of accumbal GSKß in conditioned reward, aversion, and memory.

Activation of GSK3ß induced by recall of cocaine reward memories is dependent on GluN2A/B NMDA receptor signaling.

Glycogen synthase kinase-3ß (GSK3ß) is a critical regulator of the balance between long-term depression and long-term potentiation which is essential for learning and memory. Our previous study demonstrated that GSK3ß activity is highly induced during cocaine memory reactivation, and that reconsolidation of cocaine reward memory is attenuated by inhibition of GSK3ß. NMDA receptors and protein phosphatase 1 (PP1) are activators of GSK3ß. Thus, this study investigated the roles of NMDA receptor subtypes and PP1in the reconsolidation of cocaine contextual reward memory. Cocaine contextual memories were established and evaluated using cocaine conditioned place preference methods. The regulation of GSK3ß activity in specific brain areas was assessed by measuring its phosphorylation state using immunoblot assays. Mice underwent cocaine place conditioning for 8 days and were tested for place preference on day 9. Twenty-four hours later, mice were briefly confined to the compartment previous paired with cocaine to reactivate cocaine-associated memories. Administration of the GluN2A- and GluN2B-NMDA receptor antagonists, NVP-AAM077 and ifenprodil, respectively, immediately following recall abrogated an established cocaine place preference, while preventing the activation of GSK3ß in the amygdala, nucleus accumbens, and hippocampus during cocaine memory reactivation. PP1 inhibition with okadaic acid also blocked the activation of GSK3ß and attenuated a previously established cocaine place preference. These findings suggest that the dephosphorylation of GSK3ß that occurred upon activation of cocaine-associated reward memories may be initiated by the activation of PP1 during the induction of NMDA receptor-dependent reconsolidation of cocaine mnemonic traces. Moreover, the importance of NMDA receptors and PP1 in reconsolidation of cocaine memory makes them potential therapeutic targets in treatment of cocaine use disorder and prevention of relapse.

Choline Is an Intracellular Messenger Linking Extracellular Stimuli to IP3-Evoked Ca2+ Signals through Sigma-1 Receptors.

Sigma-1 receptors (Sig-1Rs) are integral ER membrane proteins. They bind diverse ligands, including psychoactive drugs, and regulate many signaling proteins, including the inositol 1,4,5-trisphosphate receptors (IP3Rs) that release Ca2+ from the ER. The endogenous ligands of Sig-1Rs are unknown. Phospholipase D (PLD) cleaves phosphatidylcholine to choline and phosphatidic acid (PA), with PA assumed to mediate all downstream signaling. We show that choline is also an intracellular messenger. Choline binds to Sig-1Rs, it mimics other Sig-1R agonists by potentiating Ca2+ signals evoked by IP3Rs, and it is deactivated by metabolism. Receptors, by stimulating PLC and PLD, deliver two signals to IP3Rs: IP3 activates IP3Rs, and choline potentiates their activity through Sig-1Rs. Choline is also produced at synapses by degradation of acetylcholine. Choline uptake by transporters activates Sig-1Rs and potentiates Ca2+ signals. We conclude that choline is an endogenous agonist of Sig-1Rs linking extracellular stimuli, and perhaps synaptic activity, to Ca2+ signals.

Modulation of cardiac vagal tone by bradykinin acting on nucleus ambiguus.

Bradykinin (BK), a component of the kallikrein-kininogen-kinin system exerts multiple effects via B1 and B2 receptor activation. In the cardiovascular system, bradykinin has cardioprotective and vasodilator properties. We investigated the effect of BK on cardiac-projecting neurons of nucleus ambiguus, a key site for the parasympathetic cardiac regulation. BK produced a dose-dependent increase in cytosolic Ca2+ concentration. Pretreatment with HOE140, a B2 receptor antagonist, but not with R715, a B1 receptor antagonist, abolished the response to BK. A selective B2 receptor agonist, but not a B1 receptor agonist, elicited an increase in cytosolic Ca2+ similarly to BK. Inhibition of N-type voltage-gated Ca2+ channels with ω-conotoxin GVIA had no effect on the Ca2+ signal produced by BK, while pretreatment with ω-conotoxin MVIIC, a blocker of P/Q-type of Ca2+ channels, significantly diminished the effect of BK. Pretreatment with xestospongin C and 2-aminoethoxydiphenyl borate, antagonists of inositol 1,4,5-trisphosphate receptors, abolished the response to BK. Inhibition of ryanodine receptors reduced the BK-induced Ca2+ increase, while disruption of lysosomal Ca2+ stores with bafilomycin A1 did not affect the response. BK produced a dose-dependent depolarization of nucleus ambiguus neurons, which was prevented by the B2 receptor antagonist. In vivo studies indicate that microinjection of BK into nucleus ambiguus elicited bradycardia in conscious rats via B2 receptors. In summary, in cardiac vagal neurons of nucleus ambiguus, BK activates B2 receptors promoting Ca2+ influx and Ca2+ release from endoplasmic reticulum, and membrane depolarization; these effects are translated in vivo by bradycardia.

HIV Tat excites D1 receptor-like expressing neurons from rat nucleus accumbens.

BACKGROUND: HIV-1 infection and drug abuse are frequently co-morbid and their association greatly increases the severity of HIV-1-induced neuropathology. While nucleus accumbens (NAcc) function is severely perturbed by drugs of abuse, little is known about how HIV-1 infection affects NAcc. METHODS: We used calcium and voltage imaging to investigate the effect of HIV-1 trans-activator of transcription (Tat) on rat NAcc. Based on previous neuronal studies, we hypothesized that Tat modulates intracellular Ca2+ homeostasis of NAcc neurons. RESULTS: We provide evidence that Tat triggers a Ca2+ signaling cascade in NAcc medium spiny neurons (MSN) expressing D1-like dopamine receptors leading to neuronal depolarization. Firstly, Tat induced inositol 1,4,5-trisphsophate (IP3) receptor-mediated Ca2+ release from endoplasmic reticulum, followed by Ca2+ and Na+ influx via transient receptor potential canonical channels. The influx of cations depolarizes the membrane promoting additional Ca2+ entry through voltage-gated P/Q-type Ca2+ channels and opening of tetrodotoxin-sensitive Na+ channels. By activating this mechanism, Tat elicits a feed-forward depolarization increasing the excitability of D1-phosphatidylinositol-linked NAcc MSN. We previously found that cocaine targets NAcc neurons directly (independent of the inhibition of dopamine transporter) only when IP3-generating mechanisms are concomitantly initiated. When tested here, cocaine produced a dose-dependent potentiation of the effect of Tat on cytosolic Ca2+. CONCLUSION: We describe for the first time a HIV-1 Tat-triggered Ca2+ signaling in MSN of NAcc involving TRPC and depolarization and a potentiation of the effect of Tat by cocaine, which may be relevant for the reward axis in cocaine-abusing HIV-1-positive patients.

Mechanisms of activation of nucleus accumbens neurons by cocaine via sigma-1 receptor-inositol 1,4,5-trisphosphate-transient receptor potential canonical channel pathways.

Cocaine promotes addictive behavior primarily by blocking the dopamine transporter, thus increasing dopamine transmission in the nucleus accumbens (nAcc); however, additional mechanisms are continually emerging. Sigma-1 receptors (σ1Rs) are known targets for cocaine, yet the mechanisms underlying σ1R-mediated effects of cocaine are incompletely understood. The present study examined direct effects of cocaine on dissociated nAcc neurons expressing phosphatidylinositol-linked D1 receptors. Endoplasmic reticulum-located σ1Rs and inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) were targeted using intracellular microinjection. IP3 microinjection robustly elevated intracellular Ca(2+) concentration, [Ca(2+)]i. While cocaine alone was devoid of an effect, the IP3-induced response was σ1R-dependently enhanced by cocaine co-injection. Likewise, cocaine augmented the [Ca(2+)]i increase elicited by extracellularly applying an IP3-generating molecule (ATP), via σ1Rs. The cocaine-induced enhancement of the IP3/ATP-mediated Ca(2+) elevation occurred at pharmacologically relevant concentrations and was mediated by transient receptor potential canonical channels (TRPC). IP3 microinjection elicited a slight, transient depolarization, further converted to a greatly enhanced, prolonged response, by cocaine co-injection. The cocaine-triggered augmentation was σ1R-dependent, TRPC-mediated and contingent on [Ca(2+)]i elevation. ATP-induced depolarization was similarly enhanced by cocaine. Thus, we identify a novel mechanism by which cocaine promotes activation of D1-expressing nAcc neurons: enhancement of IP3R-mediated responses via σ1R activation at the endoplasmic reticulum, resulting in augmented Ca(2+) release and amplified depolarization due to subsequent stimulation of TRPC. In vivo, intra-accumbal blockade of σ1R or TRPC significantly diminished cocaine-induced hyperlocomotion and locomotor sensitization, endorsing a physio-pathological significance of the pathway identified in vitro.

Activity-regulated gene expression in immature neurons in the dentate gyrus following re-exposure to a cocaine-paired environment.

Intense craving for drug and relapse are observed in addicts who are exposed to environmental stimuli associated with drug-taking behavior even after long periods of abstinence. The hippocampus is a brain region known to be involved in contextual processing, taking place predominantly in the septal hippocampus, and emotional processing, taking place predominantly in the temporal hippocampus. Conditioned place preference is an animal model of context-conditioned reward. The dentate gyrus is a hippocampal sub-region particularly important for the acquisition of cocaine-induced place preference and is a site of continuous neurogenesis, which has been implicated in the vulnerability to drug-taking behavior. Therefore, these experiments explored the role of newly generated neurons in drug reward-context association by examining the activation, as determined by expression of the immediate early gene cfos, of young and mature granule cells in the septal and temporal dentate gyrus of adult rats that were re-exposed to a drug-paired environment following the development of cocaine place preference. The overall level of cfos expression was increased in both the septal and temporal dentate gyrus of animals that developed place preference and were re-exposed to the drug paired environment compared with re-exposure to a neutral environment. Overall level of neurogenesis, as detected by the S-phase marker 5'-bromo-2'-deoxyuridine (BrdU) and the immature neuron marker doublecortin (DCX), was unaltered by cocaine conditioning. However, the number of activated new neurons (DCX + cfos) was greater in the temporal dentate gyrus of cocaine-conditioned rats re-exposed to the drug-paired environment as compared to those re-exposed to a neutral environment. Further understanding of the role of dentate gyrus neurogenesis on the conditioned effects of drugs of abuse may provide new insights into the role of this process in the expression of addictive behaviors.

Repeated cocaine enhances ventral hippocampal-stimulated dopamine efflux in the nucleus accumbens and alters ventral hippocampal NMDA receptor subunit expression.

Dopaminergic neurotransmission in the nucleus accumbens is important for various reward-related cognitive processes including reinforcement learning. Repeated cocaine enhances hippocampal synaptic plasticity, and phasic elevations of accumbal dopamine evoked by unconditioned stimuli are dependent on impulse flow from the ventral hippocampus. Therefore, sensitized hippocampal activity may be one mechanism by which drugs of abuse enhance limbic dopaminergic activity. In this study, in vivo microdialysis in freely moving adult male Sprague-Dawley rats was used to investigate the effect of repeated cocaine on ventral hippocampus-mediated dopaminergic transmission within the medial shell of the nucleus accumbens. Following seven daily injections of saline or cocaine (20 mg/kg, ip), unilateral infusion of N-methyl-d-aspartate (NMDA, 0.5 µg) into the ventral hippocampus transiently increased both motoric activity and ipsilateral dopamine efflux in the medial shell of the nucleus accumbens, and this effect was greater in rats that received repeated cocaine compared to controls that received repeated saline. In addition, repeated cocaine altered NMDA receptor subunit expression in the ventral hippocampus, reducing the NR2A : NR2B subunit ratio. Together, these results suggest that repeated exposure to cocaine produces maladaptive ventral hippocampal-nucleus accumbens communication, in part through changes in glutamate receptor composition. A behaviorally sensitizing regimen of cocaine (20 mg/kg, ip 7 days) also sensitized ventral hippocampus (hipp)-mediated dopaminergic transmission within the nucleus accumbens (Nac) to NMDA stimulation (bolts). This was associated with reduced ventral hippocampal NR2A:NR2B subunit ratio, suggesting that repeated exposure to cocaine produces changes in hippocampal NMDA receptor composition that lead to enhanced ventral hippocampus-nucleus accumbens communication.

The GSK3 signaling pathway is activated by cocaine and is critical for cocaine conditioned reward in mice.

The Akt - GSK3 signaling pathway has been recently implicated in psychostimulant-induced behavioral and cellular effects. Here, the ability of cocaine to regulate the activity of Akt and GSK3 was investigated by measuring the phosphorylation states of the two kinases. The anatomical specificity of the response was determined, as was the contributions of dopamine and NMDA receptors to the actions of cocaine. As GSK3 activity was found to be increased by cocaine, subsequent experiments investigated the importance of GSK3 activation in cocaine conditioned reward. Adult male CD-1 mice were injected with cocaine or saline, and levels of phosphorylated Akt and GSK3α/ß were measured 30 minutes later. Acute administration of cocaine significantly decreased the phosphorylation of Akt-Thr308 (pAkt-Thr308) and GSK3ß in the caudate putamen and nucleus accumbens core, without altering pAkt-Ser473 and pGSK3α. To investigate the role of dopamine and NMDA receptors in the regulation of Akt and GSK3 by cocaine, specific receptor antagonists were administered prior to cocaine. Blockade of dopamine D2 receptors with eticlopride prevented the reduction of pAkt-Thr308 produced by cocaine, whereas antagonists at dopamine D1, dopamine D2 or glutamatergic NMDA receptors each blocked cocaine-induced reductions in pGSK3ß. The potential importance of GSK3 activity in the rewarding actions of cocaine was determined using a cocaine conditioned place preference procedure. Administration of the selective GSK3 inhibitor, SB 216763, prior to cocaine conditioning sessions blocked the development of cocaine place preference. In contrast, SB 216763 did not alter the acquisition of a contextual fear conditioning response, demonstrating that SB 216763 did not globally inhibit contextual learning processes. The results of this study indicate that phosphorylation of GSK3ß is reduced, hence GSK3ß activity is increased following acute cocaine, an effect that is contingent upon both dopaminergic and glutamatergic receptors. Further, GSK3 activity is required for the development of cocaine conditioned reward.

Decreased prefrontal cortex dopamine activity following adolescent social defeat in male rats: role of dopamine D2 receptors.

RATIONALE: Adverse social experience in adolescence causes reduced medial prefrontal cortex (mPFC) dopamine (DA) and associated behavioral deficits in early adulthood. OBJECTIVE: This study aims to determine whether mPFC DA hypofunction following social stress is specific to adolescent experience and if this results from stress-induced DA D2 receptor activation. MATERIALS AND METHODS: Male rats exposed to repeated social defeat during adolescence or adulthood had mPFC DA activity sampled 17 days later. Separate experiments used freely moving microdialysis to measure mPFC DA release in response to adolescent defeat exposure. At P40, 49 and 56 mPFC DA turnover was assessed to identify when DA activity decreased in relation to the adolescent defeat experience. Finally, nondefeated adolescent rats received repeated intra-mPFC infusions of the D2 receptor agonist quinpirole, while another adolescent group received intra-mPFC infusions of the D2 antagonist amisulpride before defeat exposure. RESULTS: Long-term decreases or increases in mPFC DA turnover were observed following adolescent or adult defeat, respectively. Adolescent defeat exposure elicits sustained increases in mPFC DA release, and DA turnover remains elevated beyond the stress experience before declining to levels below normal at P56. Activation of mPFC D2 receptors in nondefeated adolescents decreases DA activity in a similar manner to that caused by adolescent defeat, while defeat-induced reductions in mPFC DA activity are prevented by D2 receptor blockade. CONCLUSIONS: Both the developing and mature PFC DA systems are vulnerable to social stress, but only adolescent defeat causes DA hypofunction. This appears to result in part from stress-induced activation of mPFC D2 autoreceptors.