Cocaine increases stimulation-evoked serotonin efflux in the nucleus accumbens

Cocaine is one of the most common illicit drugs globally, but the role of serotonin in its mechanism of action is insufficiently characterised. Consequently, we investigated the acute effects of the psychomotor stimulant cocaine on electrical stimulation-evoked serotonin (phasic) release in the nucleus accumbens core (NAcc) of urethane-anesthetized (1.5 g/kg i.p.) male Sprague-Dawley rats using N-shaped fast-scan cyclic voltammetry (N-FSCV). A single carbon fiber microelectrode was first implanted in the NAcc. Stimulation was applied to the medial forebrain bundle using 60 Hz, 2 ms, 0.2 mA, 2 s biphasic pulses before and after cocaine (2 mg/kg i.v.) was administered. Stimulation-evoked serotonin release significantly increased 5 minutes after cocaine injection compared to baseline (153±21 nM vs 257±12 nM; p = 0.0042; n = 5) but was unaffected by saline injection (1 ml/kg i.v.; n = 5). N-FSCV's selective measurement of serotonin release in vivo was confirmed pharmacologically via administration of the selective serotonin reuptake inhibitor escitalopram (10 mg/kg i.p.) which effectively increased the signal in a separate group of rats (n = 5). Selectivity to serotonin was further confirmed in vitro in which dopamine was minimally detected by N-FSCV with a serotonin to dopamine response ratio of 1:0.04 (200 nM of serotonin:1 mM dopamine ratio; p = 0.0048; n = 5 electrodes). This study demonstrates a noteworthy influence of cocaine on serotonin dynamics, and confirms that N-FSCV can effectively and selectively measure phasic serotonin release in the NAcc.

Global Cerebral Ischemia in Male Long Evans Rats Impairs Dopaminergic/ΔFosB Signalling in the Mesocorticolimbic Pathway Without Altering Delay Discounting Rates

Global cerebral ischemia (GCI) in rats has been shown to promote exploration of anxiogenic zones of the Elevated-Plus Maze (EPM) and Open Field Test (OFT). This study investigated changes in impulsive choice and/or defensive responses as possible contributors of heightened anxiogenic exploration observed after ischemia. Impulsivity was assessed using delay discounting (DD) paradigms, while the Predator Odour Test (PO) served to assess changes in defensive responses towards a naturally aversive stimulus. Male Long Evans rats underwent 9 days of autoshaping training and 24 days of DD training prior to GCI or sham surgery (n = 9/group). Post-surgery, rats completed the OFT, EPM, and PO, followed by 6 days of DD sessions. Blood droplets served to evaluate corticosterone secretion associated with PO exposure. With impulsivity being regulated through mesocorticolimbic monoaminergic pathways, we also characterised post-ischemic changes in the expression of dopamine D2 receptors (DRD2), dopamine transporters (DAT), and 1FosB in the basolateral amygdala (BLA), nucleus accumbens core (NAcC) and shell (NAcS), and ventromedial prefrontal cortex (vmPFC) using immunohistofluorescence. Our findings revealed no impact of GCI on delay discounting rates, while PO approach behaviours were minimally affected. Nonetheless, GCI significantly reduced DRD2 and ΔFosB-ir in the NAcS and NAcC, respectively, while DAT-ir was diminished in both NAc subregions. Collectively, our findings refine the understanding of cognitive-behavioural and biochemical responses following stroke or cardiac arrest. They support significant alterations to the dopaminergic mesocorticolimbic pathway after ischemia, which are not associated with altered impulsive choice in a DD task but may influence locomotor exploration of the OFT and EPM.

Effect of repeated MDMA exposure on rat brain and behaviour

<p>Rationale. ±3,4-Methylenedioxymethamphetamine (MDMA; ‘ecstasy’) is a popular recreational drug of abuse. Like other drugs of abuse, a proportion of users develop symptoms that are characteristic of a Substance Use Disorder (SUD). The behavioural and neurobiological consequences of repeated misuse of MDMA are not well understood, however. Objectives. The purpose of the present thesis was to investigate behaviourally relevant neuroadaptations that develop with repeated MDMA exposure in laboratory rats. Methods. First, the effect of chronic, long-access (6 hour) self-administration of MDMA on the accumulation of the transcription factor, ΔFosB, in the nucleus accumbens (core, shell), dorsal striatum (dorsomedial, dorsolateral, ventromedial, ventrolateral), prefrontal cortex (anterior cingulate, prelimbic, infralimbic, orbitofrontal), amygdala (central, basolateral), ventral tegmental area (anterior, posterior), and raphe (dorsal, median) was measured using immunohistochemistry. Second, the behavioural relevance of these findings was determined by examining the effect of bi-lateral intra-striatal (nucleus accumbens, dorsomedial striatum, dorsolateral striatum) microinjections of MDMA (200 μg/1 μL/side) on the expression of behavioural sensitisation following two days of withdrawal from a regimen of repeated, systemic MDMA exposure (10 mg/kg/day, i.p., for 5 days). Third, a procedure was developed to examine neurochemical correlates of sensitised MDMA-produced behaviour (0, 5, 10 mg/kg, i.p.) following the same regimen of repeated MDMA exposure. Samples were collected from the medial striatum using in vivo microdialysis and the extracellular concentrations of serotonin, dopamine, MDMA, and their metabolites were quantified using liquid chromatography coupled with quadrupole time-of-flight (Q-TOF) mass spectrometry. Lastly, a unique untargeted metabolomics procedure was developed to further analyse these microdialysis samples and to identify novel or unexpected metabolites that were relevant to the sensitised behavioural response produced by MDMA. Results. MDMA self-administration produced region-dependant increases in ΔFosB. Significant increases in ΔFosB were observed in the nucleus accumbens core, the medial areas of the dorsal striatum, as well as all areas of the prefrontal cortex and amygdala. Small, but significant increases were also observed in the dorsal raphe. Increases were observed in the nucleus accumbens shell and the posterior tail of the ventral tegmental area, but these increases were not significant following statistical correction for multiple comparisons. Acute exposure to MDMA increased locomotor activity only when the drug was infused into the nucleus accumbens. Following repeated systemic exposure, behavioural sensitisation was expressed when MDMA was infused into both the nucleus accumbens or the dorsomedial striatum, but not the dorsolateral striatum. Analysis of microdialysates from the medial striatum indicated that behavioural sensitisation was accompanied by small increases in baseline levels of extracellular serotonin and decreased MDMA-produced increases in serotonin, but these changes were not statistically significant. Behavioural sensitisation was also accompanied by increased extracellular concentrations of dopamine at baseline and following acute MDMA exposure, but these data were not statistically analysed due to small sample sizes. MDMA-produced extracellular concentrations of MDMA did not change with repeated exposure. Untargeted metabolomics revealed potential changes in MDMA and dopamine metabolism that might be relevant to the sensitised behavioural response. Conclusions. The findings of the current research suggest that repeated MDMA exposure results in many of the same neuroadaptations that result from repeated exposure to other drugs of abuse. These included increased ΔFosB expression in many brain regions that are relevant to addiction, such as the nucleus accumbens, dorsal striatum, and prefrontal cortex. Dopaminergic mechanisms also appeared to be influenced and were associated with sensitised MDMA-produced behaviour. Surprisingly, serotonergic mechanisms were not significantly impacted by repeated MDMA exposure under the current conditions. Some of the procedures developed in this thesis are unique and may be of value for future research investigating the neurochemical underpinnings of addictive behaviour or other disease states.</p>

Effect of repeated MDMA exposure on rat brain and behaviour

<p>Rationale. ±3,4-Methylenedioxymethamphetamine (MDMA; ‘ecstasy’) is a popular recreational drug of abuse. Like other drugs of abuse, a proportion of users develop symptoms that are characteristic of a Substance Use Disorder (SUD). The behavioural and neurobiological consequences of repeated misuse of MDMA are not well understood, however. Objectives. The purpose of the present thesis was to investigate behaviourally relevant neuroadaptations that develop with repeated MDMA exposure in laboratory rats. Methods. First, the effect of chronic, long-access (6 hour) self-administration of MDMA on the accumulation of the transcription factor, ΔFosB, in the nucleus accumbens (core, shell), dorsal striatum (dorsomedial, dorsolateral, ventromedial, ventrolateral), prefrontal cortex (anterior cingulate, prelimbic, infralimbic, orbitofrontal), amygdala (central, basolateral), ventral tegmental area (anterior, posterior), and raphe (dorsal, median) was measured using immunohistochemistry. Second, the behavioural relevance of these findings was determined by examining the effect of bi-lateral intra-striatal (nucleus accumbens, dorsomedial striatum, dorsolateral striatum) microinjections of MDMA (200 μg/1 μL/side) on the expression of behavioural sensitisation following two days of withdrawal from a regimen of repeated, systemic MDMA exposure (10 mg/kg/day, i.p., for 5 days). Third, a procedure was developed to examine neurochemical correlates of sensitised MDMA-produced behaviour (0, 5, 10 mg/kg, i.p.) following the same regimen of repeated MDMA exposure. Samples were collected from the medial striatum using in vivo microdialysis and the extracellular concentrations of serotonin, dopamine, MDMA, and their metabolites were quantified using liquid chromatography coupled with quadrupole time-of-flight (Q-TOF) mass spectrometry. Lastly, a unique untargeted metabolomics procedure was developed to further analyse these microdialysis samples and to identify novel or unexpected metabolites that were relevant to the sensitised behavioural response produced by MDMA. Results. MDMA self-administration produced region-dependant increases in ΔFosB. Significant increases in ΔFosB were observed in the nucleus accumbens core, the medial areas of the dorsal striatum, as well as all areas of the prefrontal cortex and amygdala. Small, but significant increases were also observed in the dorsal raphe. Increases were observed in the nucleus accumbens shell and the posterior tail of the ventral tegmental area, but these increases were not significant following statistical correction for multiple comparisons. Acute exposure to MDMA increased locomotor activity only when the drug was infused into the nucleus accumbens. Following repeated systemic exposure, behavioural sensitisation was expressed when MDMA was infused into both the nucleus accumbens or the dorsomedial striatum, but not the dorsolateral striatum. Analysis of microdialysates from the medial striatum indicated that behavioural sensitisation was accompanied by small increases in baseline levels of extracellular serotonin and decreased MDMA-produced increases in serotonin, but these changes were not statistically significant. Behavioural sensitisation was also accompanied by increased extracellular concentrations of dopamine at baseline and following acute MDMA exposure, but these data were not statistically analysed due to small sample sizes. MDMA-produced extracellular concentrations of MDMA did not change with repeated exposure. Untargeted metabolomics revealed potential changes in MDMA and dopamine metabolism that might be relevant to the sensitised behavioural response. Conclusions. The findings of the current research suggest that repeated MDMA exposure results in many of the same neuroadaptations that result from repeated exposure to other drugs of abuse. These included increased ΔFosB expression in many brain regions that are relevant to addiction, such as the nucleus accumbens, dorsal striatum, and prefrontal cortex. Dopaminergic mechanisms also appeared to be influenced and were associated with sensitised MDMA-produced behaviour. Surprisingly, serotonergic mechanisms were not significantly impacted by repeated MDMA exposure under the current conditions. Some of the procedures developed in this thesis are unique and may be of value for future research investigating the neurochemical underpinnings of addictive behaviour or other disease states.</p>

Dopamine release in nucleus accumbens is under tonic inhibition by adenosine A1 receptors regulated by astrocytic ENT1 and dysregulated by ethanol

Striatal adenosine A1 receptor (A1R) activation can inhibit dopamine release. A1Rs on other striatal neurons are activated by an adenosine tone that is limited by equilibrative nucleoside transporter 1 (ENT1) that is enriched on astrocytes and is ethanol-sensitive. We explored whether dopamine release in nucleus accumbens core is under tonic inhibition by A1Rs, and is regulated by astrocytic ENT1 and ethanol. In ex vivo striatal slices from male and female mice, A1R agonists inhibited dopamine release evoked electrically or optogenetically and detected using fast-scan cyclic voltammetry, most strongly for lower stimulation frequencies and pulse numbers, thereby enhancing the activity-dependent contrast of dopamine release. Conversely, A1R antagonists reduced activity-dependent contrast but enhanced evoked dopamine release levels, even for single optogenetic pulses indicating an underlying tonic inhibition. The ENT1 inhibitor NBTI reduced dopamine release and promoted A1R-mediated inhibition, and conversely, virally-mediated astrocytic overexpression of ENT1 enhanced dopamine release and relieved A1R-mediated inhibition. By imaging the genetically encoded fluorescent adenosine sensor GRAB-Ado, we identified a striatal extracellular adenosine tone that was elevated by the ENT1 inhibitor and sensitive to gliotoxin fluorocitrate. Finally, we identified that ethanol (50 mM) promoted A1R-mediated inhibition of dopamine release, through diminishing adenosine uptake via ENT1. Together, these data reveal that dopamine output dynamics are gated by a striatal adenosine tone, limiting amplitude but promoting contrast, regulated by ENT1, and promoted by ethanol. These data add to the diverse mechanisms through which ethanol modulates striatal dopamine, and to emerging datasets supporting astrocytic transporters as important regulators of striatal function.

Stress deceleration theory: chronic adolescent stress exposure results in decelerated neurobehavioral maturation

Normative development in adolescence indicates that the prefrontal cortex is still under development thereby unable to exert efficient top-down inhibitory control on subcortical regions such as the basolateral amygdala and the nucleus accumbens. This imbalance in the developmental trajectory between cortical and subcortical regions is implicated in expression of the prototypical impulsive, compulsive, reward seeking and risk-taking adolescent behavior. Here we demonstrate that a chronic mild unpredictable stress procedure during adolescence in male Wistar rats arrests the normal behavioral maturation such that they continue to express adolescent-like impulsive, hyperactive, and compulsive behaviors into late adulthood. This arrest in behavioral maturation is associated with the hypoexcitability of prelimbic cortex (PLC) pyramidal neurons and reduced PLC-mediated synaptic glutamatergic control of BLA and nucleus accumbens core (NAcC) neurons that lasts late into adulthood. At the same time stress exposure in adolescence results in the hyperexcitability of the BLA pyramidal neurons sending stronger glutamatergic projections to the NAcC. Chemogenetic reversal of the PLC hypoexcitability decreased compulsivity and improved the expression of goal-directed behavior in rats exposed to stress during adolescence, suggesting a causal role for PLC hypoexcitability in this stress-induced arrested behavioral development.

5-HT2C receptor perturbation has bidirectional influence over instrumental vigour and restraint

The serotonin (5-HT) system, particularly the 5-HT2C receptor, has consistently been implicated in behavioural control. However, while some studies have focused on the role 5-HT2C receptors play in regulating motivation to work for reward, others have highlighted its importance in response restraint. To date, it is unclear how 5-HT transmission at this receptor regulates the balance of response invigoration and restraint in anticipation of future reward. In addition, it remains to be established how 5-HT2C receptors gate the influence of internal versus cue-driven processes over reward-guided actions. To elucidate these issues, we investigated the effects of administering the 5-HT2C receptor antagonist SB242084, both systemically and directly into the nucleus accumbens core (NAcC), in rats performing a Go/No-Go task for small or large rewards. The results were compared to the administration of d-amphetamine into the NAcC, which has previously been shown to promote behavioural activation. Systemic perturbation of 5-HT2C receptors—but crucially not intra-NAcC infusions—consistently boosted rats’ performance and instrumental vigour on Go trials when they were required to act. Concomitantly, systemic administration also reduced their ability to withhold responding for rewards on No-Go trials, particularly late in the holding period. Notably, these effects were often apparent only when the reward on offer was small. By contrast, inducing a hyperdopaminergic state in the NAcC with d-amphetamine strongly impaired response restraint on No-Go trials both early and late in the holding period, as well as speeding action initiation. Together, these findings suggest that 5-HT2C receptor transmission, outside the NAcC, shapes the vigour of ongoing goal-directed action as well as the likelihood of responding as a function of expected reward.