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Deficits in arousal and stress responsiveness are a feature of numerous psychiatric disorders including depression and anxiety. Arousal is supported by norepinephrine (NE) released from specialized brainstem nuclei, including the locus coeruleus (LC) neurons into cortical and limbic areas. During development, the NE system matures in concert with increased exploration of the animal’s environment. While several psychiatric medications target the NE system, the possibility that its modulation during discreet developmental periods can have long-lasting consequences has not yet been explored. We used a chemogenetic strategy in mice to reversibly inhibit NE signaling during brief developmental periods and then evaluated any long-lasting impact of our intervention on adult NE circuit function and on emotional behavior. We also tested whether developmental exposure to the α2 receptor agonist guanfacine, which is commonly used in the pediatric population and is not contraindicated during pregnancy and nursing, recapitulates the effect seen with the chemogenetic strategy. Our results reveal that postnatal days 10–21 constitute a sensitive period during which alterations in NE signaling lead to changes in baseline anxiety, increased anhedonia, and passive coping behaviors in adulthood. Disruption of NE signaling during this sensitive period also caused altered LC autoreceptor function, along with circuit specific changes in LC-NE target regions at baseline, and in response to stress. Our findings indicate an early critical role for NE in sculpting brain circuits that support adult emotional function. Interfering with this role by guanfacine and similar clinically used drugs can have lasting implications for mental health.

Methamphetamine (METH) is a potent psychostimulant that exerts many of its physiological and psychomotor effects by increasing extracellular dopamine (DA) concentrations in limbic brain regions. While several studies have focused on how potent, neurotoxic doses of METH augment or attenuate DA transmission, the acute effects of lower and behaviorally-activating doses of METH on modulating DA regulation (release and clearance) through DA D2 autoreceptors and transporters remains to be elucidated. In this study, we investigated how systemic administration of escalating, sub-neurotoxic doses of METH (0.5 – 5 mg/kg, IP) alter extracellular DA regulation in the nucleus accumbens (NAc), in both anesthetized and awake-behaving rats through the use of in vivo fast-scan cyclic voltammetry. Pharmacological, electrochemical, and behavioral evidence show that lower doses (≤ 2.0 mg/kg, IP) of METH enhance extracellular phasic DA concentrations and locomotion as well as stereotypies. In contrast, higher doses (≥ 5.0 mg/kg) further increase both phasic and baseline DA concentrations and stereotypies but decrease horizontal locomotion. Importantly, our results suggest that acute METH-induced enhancement of extracellular DA concentrations dose-dependently activate D2 autoreceptors. Therefore, these different METH dose-dependent effects on mesolimbic DA transmission may distinctly impact METH induced behavioral changes. This study provides valuable insights regarding how low METH doses alter DA transmission and paves the way for future clinical studies on the reinforcing effects of METH.

Akathisia is an urgent need to move that is associated with treatment with dopamine receptor blocking agents (DRBAs) and with restless legs syndrome (RLS). The pathogenetic mechanism of akathisia has not been resolved. This article proposes that it involves an increased presynaptic dopaminergic transmission in the ventral striatum and concomitant strong activation of postsynaptic dopamine D1 receptors, which form complexes (heteromers) with dopamine D3 and adenosine A1 receptors. It also proposes that in DRBA-induced akathisia, increased dopamine release depends on inactivation of autoreceptors, whereas in RLS it depends on a brain iron deficiency-induced down-regulation of striatal presynaptic A1 receptors.

Rationale The prevalence of depression is ever-increasing throughout the population. However, available treatments are ineffective in around one-third of patients and there is a need for more effective and safer drugs. Objectives The antidepressant-like and procognitive effects of the “biased agonists” F15599 (also known as NLX-101) which preferentially targets postsynaptic 5-HT1A receptors and F13714, which targets 5-HT1A autoreceptors, were investigated in mice. Methods Antidepressant-like properties of the compounds and their effect on cognitive functions were assessed using the forced swim test (FST) and the novel object recognition (NOR), respectively. Next, we induced a depressive-like state by an unpredictable chronic mild stress (UCMS) procedure to test the compounds’ activity in the depression model, followed by measures of sucrose preference, FST, and locomotor activity. Levels of phosphorylated cyclic AMP response element-binding protein (p-CREB) and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) were also determined. Results F15599 reduced immobility time in the FST over a wider dose-range (2 to 16 mg/kg po) than F13714 (2 and 4 mg/kg po), suggesting accentuated antidepressant-like properties in mice. F15599 did not disrupt long-term memory consolidation in the NOR at any dose tested, while F13714 impaired memory formation, notably at higher doses (4–16 mg/kg). In UCMS mice, a single administration of F15599 and F13714 was sufficient to robustly normalize depressive-like behavior in the FST but did not rescue disrupted sucrose preference. Both F15599 and F13714 rescued cortical and hippocampal deficits in p-ERK1/2 levels of UCMS mice but did not influence the p-CREB levels. Conclusions Our studies showed that 5-HT1A receptor biased agonists such as F13714 and especially F15599, due to its less pronounced side effects, might have potential as fast-acting antidepressants.

The dorsal striatum (DS) is implicated in behavioral and neural processes including action control and reinforcement. Alcohol alters these processes in rodents, and it is believed that the development of alcohol use disorder involves changes in DS dopamine signaling. In nonhuman primates, the DS can be divided into caudate and putamen subregions. As part of a collaborative effort examining the effects of long-term alcohol self-administration in rhesus macaques, we examined DS dopamine signaling using fast-scan cyclic voltammetry. We found that chronic alcohol self-administration resulted in several dopamine system adaptations. Most notably, dopamine release was altered in a sex- and region-dependent manner. Following long-term alcohol consumption, male macaques, regardless of abstinence status, had reduced dopamine release in putamen, while only male macaques in abstinence had reduced dopamine release in caudate. In contrast, female macaques had enhanced dopamine release in the caudate, but not putamen. Dopamine uptake was also enhanced in females, but not males (regardless of abstinence state). We also found that dopamine D2/3 autoreceptor function was reduced in male, but not female, alcohol drinkers relative to control groups. Finally, we found that blockade of nicotinic acetylcholine receptors inhibited evoked dopamine release in nonhuman primates. Altogether, our findings demonstrate that long-term alcohol consumption can sex-dependently alter dopamine release, as well as its feedback control mechanisms in both DS subregions.

Synaptic dopamine (DA) concentrations are largely determined by the activities of presynaptic D2 and D3 autoreceptors (D2R and D3R) and DA transporter (DAT). Furthermore, the activity of DAT is regulated by phosphorylation events and protein interactions that affect its surface expression. Because DA autoreceptors and DAT coordinately maintain synaptic DA homeostasis, we hypothesized that D3R might crosstalk with DAT to fine-tune synaptic DA concentrations. To test this hypothesis, we established [3H]DA uptake and DAT surface expression assays in hD3/rDAT-double-transfected HEK-293 cells or limbic forebrain synaptosomal preparations. Ropinirole, a preferential D3R agonist, reduced [3H]DA uptake in HEK-hD3/rDAT cells in a dose-dependent manner, an effect which could be blocked by the D2R/D3R antagonist, raclopride. Furthermore, ropinirole also reduced DAT surface expression in limbic forebrain synaptosomes, and this effect could be blocked by raclopride or the internalization inhibitor, concanavalin A. To identify potential mediators of this apparent D3R-DAT crosstalk, DAT-associated proteins were co-immunoprecipitated from limbic forebrain synaptosomes after D3R activation and identified by MALDI-TOF. From this analysis, the Hsc70 chaperone was identified as a DAT-associated protein. Interestingly, ropinirole induced the association of Hsc70/Hsp70 with DAT, and the Hsc70/Hsp70 inhibitor, apoptozole, prevented the ropinirole-induced reduction of DAT surface expression. Together, these results suggest that D3R negatively regulates DAT activity by promoting the association of DAT and Hsc70/Hsp70.

Background Serotonin (5-HT) 1B/1D receptor (5-HT1B/1DR) agonists undergo an abstinence-induced switch in their effects on cocaine-related behaviors, which may involve changes in modulation of dopamine (DA) neurons in the ventral tegmental area (VTA). However, it is unclear how 5-HT1B/1DRs affect VTA DA neuronal function and whether modulation of these neurons mediates the abstinence-induced switch after chronic cocaine exposure. Methods We examined the ability of 5-HT1B/1DRs to modulate D2 autoreceptors (D2ARs) and synaptic transmission in the VTA by slice recording and single unit recording in vivo in naive mice and in mice with chronic cocaine treatment. Results We report a bidirectional modulation of VTA DA neuronal firing through the interaction of VTA 5-HT1B/1DRs and D2ARs. In both VTA slices and the VTA of anesthetized mice, the 5-HT1B/1DR agonist CP94253 decreased DA neuronal firing rate and evoked excitatory postsynaptic currents to DA neurons in slice. Paradoxically, CP94253 decreased quinpirole-induced inhibition of DA neurons by reducing D2AR-mediated G protein–gated inwardly rectifying potassium current. This manifested decreased GABAA (gamma-aminobutyric acid A) receptor–mediated evoked inhibitory postsynaptic currents in slice, resulting in disinhibition of DA neurons, in opposition to the 5-HT1B/1DR-induced inhibition. This dual effect was verified in chronic cocaine-treated and mild stress-treated, male mice on days 1 and 20 posttreatment. Conclusions This study revealed dual effects of CP94253 on VTA DA neurons that are dependent on D2AR sensitivity, with anti-inhibition under normal D2AR sensitivity and inhibition under low D2AR sensitivity. These dual effects may underlie the ability of CP94253 to both enhance and inhibit cocaine-induced behaviors.

Background Serotonin is a neurotransmitter that has been linked to a wide variety of behaviors including feeding and body-weight regulation, social hierarchies, aggression and suicidality, obsessive compulsive disorder, alcoholism, anxiety, and affective disorders. Full understanding involves genomics, neurochemistry, electrophysiology, and behavior. The scientific issues are daunting but important for human health because of the use of selective serotonin reuptake inhibitors and other pharmacological agents to treat disorders. This paper presents a new deterministic model of serotonin metabolism and a new systems population model that takes into account the large variation in enzyme and transporter expression levels, tryptophan input, and autoreceptor function. Results We discuss the steady state of the model and the steady state distribution of extracellular serotonin under different hypotheses on the autoreceptors and we show the effect of tryptophan input on the steady state and the effect of meals. We use the deterministic model to interpret experimental data on the responses in the hippocampus of male and female mice, and to illustrate the short-time dynamics of the autoreceptors. We show there are likely two reuptake mechanisms for serotonin and that the autoreceptors have long-lasting influence and compare our results to measurements of serotonin dynamics in the substantia nigra pars reticulata. We also show how histamine affects serotonin dynamics. We examine experimental data that show very variable response curves in populations of mice and ask how much variation in parameters in the model is necessary to produce the observed variation in the data. Finally, we show how the systems population model can potentially be used to investigate specific biological and clinical questions. Conclusions We have shown that our new models can be used to investigate the effects of tryptophan input and meals and the behavior of experimental response curves in different brain nuclei. The systems population model incorporates individual variation and can be used to investigate clinical questions and the variation in drug efficacy. The codes for both the deterministic model and the systems population model are available from the authors and can be used by other researchers to investigate the serotonergic system.

Alcohol is the third most important risk factor for disease in Russia and throughout the world. WHO data indicate that alcohol abuse kills 2.5 million people a year around the world, which is the population of a large city; of these, 600–700,000 occur in Russia. Despite the development of science and medicine, the mechanisms underlying the development of alcohol dependence remain unclear. An understanding of these mechanisms will help create novel approaches to the treatment of alcohol dependence. It is now known that the mesolimbic dopamine system plays a key role in forming addictive disorders, particularly alcohol dependence. Fast scanning cyclic voltammetry (FSCV) is a widely used method for studying dopamine. An advantage of this method is its high time and spatial resolution, making FSCV a powerful method for detecting changes in monoamine neurotransmitter concentrations in vivo. Our studies using FSCV in freely moving animals demonstrated that electrical stimulation of the ventral tegmental area (VTA) leads to detection of dopamine release in the nucleus accumbens (NAcc). We showed that acute ethanol injections (0.5 g/kg) lead to significant decreases in dopamine release in the NAcc induced by electrical stimulation of the VTA in freely moving rats. Use of the selective dopamine D2 autoreceptor antagonist raclopride significantly increased the measured signal, indicating that the incoming signal measured in the NAcc is dopaminergic rather than noradrenergic.

The effect of benzodiazepine anxiolytic diazepam in Danio rerio was investigated. Methods. A stress test on novelty situation was used: a fish was placed first in a beaker with a dissolved pharmacological substance (or water) and then into a novel tank for 6 min, where the trajectory, the path length, the number of movements to the upper part of the novel tank, the number and time of the pattern of freezing of the experiment were measured. Results. In response to the novelty of tank, the fish was shown to react by submerging to the bottom, increasing the freezing, and reducing the number of movements to the upper half of the novel tank. After diazepam exposure (administration), the fish was not only in the lower, but also in the upper part of the novel tank. A pharmacological analysis of diazepam effect in Danio rerio showed that in a certain dose range of 110 mg/l anxiolytic reduces (in comparison with the control group of fish) the number and time of freezing, increases the number of movements in the upper half of the tank and the swimming time in upper part of the tank. Diazepam causes a disinhibition of motor activity at doses of 1 and 5 mg/l, which may be explained by the effect of small doses of tranquilizers on presynaptic GABA-A autoreceptors. Diazepam 20 mg/l has a depriving effect. Conclusion. Diazepam acts in a higher dose range (110 mg/l) than phenazepam (0.11 mg/l) in Danio rerio. At the same time, diazepam is characterized by a domed dose-dependent effect, in contrast to the action of phenazepam. The prospect of using Danio rerio as an animal model in behavioral pharmacology is not inferior to studies in rodents.

We previously reported that the serotonergic system is important for the antidepressant-like effects of ketamine, a non-competitive N-methyl-D-aspartate receptor antagonist, which produces rapid and long-lasting antidepressant effects in patients with major depressive disorder (MDD). In particular, selective stimulation of the 5-HT1A receptor in the medial prefrontal cortex (mPFC), as opposed to the somatic 5-HT1A autoreceptor, has been shown to play a critical role in the antidepressant-like actions of ketamine. However, the detailed mechanisms underlying mPFC 5-HT1A receptor-mediated antidepressant-like effects are not fully understood. Here we examined the involvement of the glutamate AMPA receptor and brain-derived neurotrophic factor (BDNF) in the antidepressant-like effects of 5-HT1A receptor activation in the mPFC. The results show that intra-mPFC infusion of the 5-HT1A receptor agonist 8-OH-DPAT induces rapid and long-lasting antidepressant-like effects in the forced swim, novelty-suppressed feeding, female urine sniffing, and chronic unpredictable stress tests. In addition, the results demonstrate that the antidepressant-like effects of intra-mPFC infusion of 8-OH-DPAT are blocked by co-infusion of an AMPA receptor antagonist or an anti-BDNF neutralizing antibody. In addition, mPFC infusion of 8-OH-DPAT increased the phosphorylation of signaling proteins downstream of BDNF, including mTOR, ERK, 4EBP1, and p70S6K. Finally, selective stimulation of the 5-HT1A receptor increased levels of synaptic proteins and synaptic function in the mPFC. Collectively, these results indicate that selective stimulation of 5-HT1A receptor in the mPFC exerts rapid and sustained antidepressant-like effects via activation of AMPA receptor/BDNF/mTOR signaling in mice, which subsequently increase synaptic function in the mPFC, and provide evidence for the 5-HT1A receptor as a target for the treatment of MDD.

The α2a-adrenergic receptor (α2a-AR) agonist guanfacine has been investigated as a potential treatment for substance use disorders. While decreasing stress-induced reinstatement of cocaine seeking in animal models and stress-induced craving in human studies, guanfacine has not been reported to decrease relapse rates. Although guanfacine engages α2a-AR autoreceptors, it also activates excitatory Gi-coupled heteroreceptors in the bed nucleus of the stria terminalis (BNST), a key brain region in driving stress-induced relapse. Thus, BNST α2a-AR heteroreceptor signaling might decrease the beneficial efficacy of guanfacine. We aimed to determine the role of α2a-AR heteroreceptors and BNST Gi-GPCR signaling in stress-induced reinstatement of cocaine conditioned place preference (CPP) and the effects of low dose guanfacine on BNST activity and stress-induced reinstatement. We used a genetic deletion strategy and the cocaine CPP procedure to first define the contributions of α2a-AR heteroreceptors to stress-induced reinstatement. Next, we mimicked BNST Gi-coupled α2a-AR heteroreceptor signaling using a Gi-coupled designer receptor exclusively activated by designer drug (Gi-DREADD) approach. Finally, we evaluated the effects of low-dose guanfacine on BNST cFOS immunoreactivity and stress-induced reinstatement. We show that α2a-AR heteroreceptor deletion disrupts stress-induced reinstatement and that BNST Gi-DREADD activation is sufficient to induce reinstatement. Importantly, we found that low-dose guanfacine does not increase BNST activity, but prevents stress-induced reinstatement. Our findings demonstrate a role for α2a-AR heteroreceptors and BNST Gi-GPCR signaling in stress-induced reinstatement of cocaine CPP and provide insight into the impact of dose on the efficacy of guanfacine as a treatment for stress-induced relapse of cocaine use.

We reported recently that activators of AMP-activated protein kinase (AMPK) slow the rundown of current evoked by the D2 autoreceptor agonist quinpirole in rat substantia nigra compacta (SNC) dopamine neurons. The present study examined the effect of AMPK on current generated by dopamine, which unlike quinpirole, is a substrate for the dopamine transporter (DAT). Using whole-cell patch-clamp, we constructed current–voltage (I–V) plots while superfusing brain slices with dopamine (100 μM) for 25 min. Two minutes after starting superfusion, dopamine evoked a peak current with an average slope conductance of 0.97 nS and an estimated reversal potential (Erev) of −113 mV, which is near that expected for K+. But after 10 min of superfusion, dopamine-evoked currents had shifted to more depolarized values with a slope conductance of 0.64 nS and an Erev of −83 mV. This inward shift in current was completely blocked by the DAT inhibitor GBR12935. However, an AMPK blocking agent (dorsomorphin) permitted the emergence of inward current despite the continued presence of the DAT inhibitor. When D2 autoreceptors were blocked by sulpiride, I–V plots showed that dopamine evoked an inward current with an estimated slope conductance of 0.45 nS with an Erev of −57 mV. Moreover, this inward current was completely blocked by the trace amine-associated receptor 1 (TAAR1) antagonist EPPTB. These results suggest that dopamine activates a TAAR1-dependent non-selective cation current that is regulated by AMPK.

Polyphenol ellagic acid (EA) possesses antioxidant, anti-inflammatory, anti-carcinogenic, anti-diabetic and cardio protection activities, making it an interesting multi-targeting profile. EA also controls the central nervous system (CNS), since it was proven to reduce the immobility time of mice in both the forced swimming and the tail-suspension tests, with an efficiency comparable to that of classic antidepressants. Interestingly, the anti-depressant-like effect was almost nulled by the concomitant administration of selective antagonists of the noradrenergic receptors, suggesting the involvement of these cellular targets in the central effects elicited by EA and its derivatives. By in silico and in vitro studies, we discuss how EA engages with human α2A-ARs and α2C-AR catalytic pockets, comparing EA behaviour with that of known agonists and antagonists. Structurally, the hydrophobic residues surrounding the α2A-AR pocket confer specificity on the intermolecular interactions and hence lead to favourable binding of EA in the α2A-AR, with respect to α2C-AR. Moreover, EA seems to better accommodate within α2A-ARs into the TM5 area, close to S200 and S204, which play a crucial role for activation of aminergic GPCRs such as the α2-AR, highlighting its promising role as a partial agonist. Consistently, EA mimics clonidine in inhibiting noradrenaline exocytosis from hippocampal nerve endings in a yohimbine-sensitive fashion that confirms the engagement of naïve α2-ARs in the EA-mediated effect.

SUMMARY Neuromodulation mediated by synaptically released endogenous transmitters acting in G-protein-coupled receptors (GPCRs) is slow primarily because of multistep downstream signaling. What is less well understood is the spatial and temporal kinetics of transmitter and receptor interaction. The present work uses the combination of the dopamine sensor, dLight, to detect the spatial release and diffusion of dopamine and a caged form of a D2-dopamine receptor antagonist, CyHQ-sulpiride, to rapidly block the D2 autoreceptors. Photoactivation of the CyHQ-sulpiride blocks receptors in milliseconds such that the time course of dopamine/receptor interaction is mapped onto the downstream signaling. The results show that highly localized release, but not dopamine diffusion, defines the time course of the functional interaction between dopamine and D2 autoreceptors, which determines downstream inhibition., Graphical Abstract, In brief Somatodendritic release of dopamine via D2 autoreceptors activates an inhibitory postsynaptic current. The synaptic current is dependent on a transiently high concentration of dopamine. Condon et al. use a combination of optical and electrical approaches to show the spatial and temporal substrates of dendrodendritic transmission in the substantia nigra.

Neurotransmitter release is a highly controlled process by which synapses can critically regulate information transfer within neural circuits. While presynaptic receptors - typically activated by neurotransmitters and modulated by neuromodulators - provide a powerful way of fine-tuning synaptic function, their contribution to activity-dependent changes in transmitter release remains poorly understood. Here, we report that presynaptic NMDA receptors (preNMDARs) at mossy fiber boutons in the rodent hippocampus can be activated by physiologically relevant patterns of activity and selectively enhance short-term synaptic plasticity at mossy fiber inputs onto CA3 pyramidal cells and mossy cells, but not onto inhibitory interneurons. Moreover, preNMDARs facilitate brain-derived neurotrophic factor release and contribute to presynaptic calcium rise. Taken together, our results indicate that by increasing presynaptic calcium, preNMDARs fine-tune mossy fiber neurotransmission and can control information transfer during dentate granule cell burst activity that normally occur in vivo.

We previously reported that the inactivation (cKO) or the stabilization (cST) of β-catenin in cells expressing the astrocyte-specific glutamate aspartate transporter (GLAST) is associated with the vulnerability or resilience to exhibit anxious/depressive-like behaviors, respectively, and to changes in hippocampal proliferation. Here, we used these cKO and cST β-catenin mice to study the serotonergic system functionality associated with their behavioral/molecular phenotype. The activity of 5-HT receptors was assessed by (+)-8-OH-DPAT-induced hypothermia and [S]GTPγS binding autoradiography. The animals’ response to acute stress and the levels of extracellular serotonin (5-HT) in the medial prefrontal cortex (mPFC) were also assessed. cKO mice presented higher 5-HT autoreceptor functionality, lower 5-HT heteroreceptor functionality, and a decrease in extracellular 5-HT levels in the mPFC. These neurochemical changes were accompanied with a blunted physiological response to stress-induced hyperthermia. In contrast, cST mice showed a reduced 5-HT autoreceptor functionality and higher extracellular 5-HT levels in the mPFC after fluoxetine administration. Moreover, cST mice subjected to chronic corticosterone administration did not show a blunted response to fluoxetine. Our findings suggest the existence of a link between β-catenin levels and 5-HT receptor functionality, which may be relevant to understand the neurobiological bases underlying the vulnerability or resilience to stress-related disorders., This research was supported by the Spanish Ministry of Economy and Competitiveness (SAF2011-25020 and SAF2015-67457-R MINECO/FEDER) and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM CB/07/09/0029). E F-Z and L G-A were supported by a predoctoral fellowship from the University of Cantabria (Spain) and a JAE-Intro (CSIC) fellowship, respectively.

The risky decision-making task (RDT) measures risk-taking in a rat model by assessing preference between a small, safe reward and a large reward with increasing risk of punishment (mild foot shock). It is well-established that dopaminergic drugs modulate risk-taking; however, little is known about how differences in baseline phasic dopamine signaling drive individual differences in risk preference. Here, we usedin vivofixed potential amperometry in male Long-Evans rats to test if phasic nucleus accumbens shell (NACs) dopamine dynamics are associated with risk-taking. We observed a positive correlation between medial forebrain bundle-evoked dopamine release in the NACs and risky decision-making, suggesting that risk-taking is associated with elevated dopamine sensitivity. Moreover, “risk-taking” subjects were found to demonstrate greater phasic dopamine release than “risk-averse” subjects. Risky decision-making also predicted enhanced sensitivity to nomifensine, a dopamine reuptake inhibitor, quantified as elevated latency for dopamine to clear from the synapse. Importantly, this hyperdopaminergic phenotype was selective for risky decision-making, as delay discounting performance was not predictive of phasic dopamine release or dopamine supply. These data identify phasic NACs dopamine release as a possible therapeutic target for alleviating the excessive risk-taking observed across multiple forms of psychopathology.Significance StatementExcessive risky decision-making is a hallmark of addiction, promoting ongoing drug seeking despite the risk of social, financial, and physical consequences. However, punishment-driven risk-taking is understudied in preclinical models. Here, we examined the relationship between individual differences in risk-taking and dopamine release properties in the rat nucleus accumbens shell, a brain region associated with motivation and decision-making. We observed that high risk taking predicted elevated phasic dopamine release and sensitivity to the dopamine transporter blocker nomifensine. This hypersensitive dopamine system was not observed in rats with high impulsive choice, another behavior associated with substance use disorder. This provides critical information about neurobiological factors underlying a form of decision-making that promotes vulnerability to substance abuse.

The serotonergic 5-HT1A receptor is known to be involved in both impulsivity and anxiety-related behavior. Although anxiety and impulsivity are different constructs, it has been shown that anxiogenesis can result in impulsiveness. It is therefore important to determine if the 5-HT1A receptor is involved in the commission of impulsive actions independent of its effects on anxiety. The 5-HT1A agonist 8-OH-DPAT (0.0125–0.1 mg/kg subcutaneous) increased impulsive action at low doses, but decreased it at higher doses, on the novel paced variable consecutive number with discriminative stimulus task (VCN). Neither the 5-HT1A antagonist WAY 100,635 (0.2–1.2 mg/kg subcutaneous), nor the noradrenergic antagonist and pharmacological stressor yohimbine (1–2 mg/kg intraperitoneal) altered measures of impulsivity. Stress induced by yohimbine was sufficient to produce anxiety-like behavior in the elevated zero maze, confirming that the VCN task is a selective assay of impulsive action that is not affected by anxiety. We hypothesize that the biphasic effect of 8-OH-DPAT is due to actions on presynaptic raphe 5-HT1A autoreceptors, and also postsynaptic 5-HT1A receptors. These results suggest that this receptor mediates impulsive action and that this is not secondary to its role in anxiety.

Regulation of neurotransmitter release in the central nervous system is complex. Here, we investigated regulatory mechanisms for acetylcholine (ACh) release from cholinergic neurons by performing superfusion experiments with rat striatal segments after labelling the cellular ACh pool with [3 H]choline. Electrical stimulation-evoked pronounced [3 H]ACh release from cholinergic neurons. The estimated quantity of [3 H]ACh release per pulse of electrical stimulation was reduced by an increase in stimulus frequency, showing an inverse correlation between release probability of ACh and neuronal excitation. ACh release was also negatively regulated by pre-synaptic muscarinic ACh receptors (mAChRs). The autoinhibition induced by released ACh was predominantly suppressed by the M2 -selective antagonist AF-DX 116, partially inhibited by M3 -selective darifenacin, and minimally by M4 -selective PD 102807. Other subtype-selective antagonists had no effect at subtype-selective concentrations. ACh esterase (AChE) inhibitors (diisopropylfluorophosphate, donepezil and galantamine) at concentrations that mostly inhibit esterase activity reduced [3 H]ACh release, and the reduction was abolished by treatment with atropine. This implies that pre-synaptic autoreceptors are activated more after blockade of ACh hydrolysis, leading to autoinhibition of ACh release and consequent reduction in synaptic ACh concentrations. [3 H]efflux was also enhanced by ACh uptake inhibitors (100 μM hemicholinium-3 and physostigmine), regardless of ACh hydrolysis. This study shows that synaptic ACh concentrations in striatal cholinergic neurons are regulated in a complex manner by many factors such as release probability, pre-synaptic M2 /M3 /M4 mAChRs, AChE and post-synaptic ACh uptake, and provides important information about cholinergic neurotransmission for future exploration of therapeutic strategies for Alzheimer's and other central nervous system diseases. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/openscience-badges/.

Melanin-concentrating hormone (MCH) is a neuropeptide present in neurons located in the hypothalamus that densely innervate serotonergic cells in the dorsal raphe nucleus (DRN). MCH administration into the DRN induces a depressive-like effect through a serotonergic mechanism. To further understand the interaction between MCH and serotonin, we used primary cultured serotonergic neurons to evaluate the effect of MCH on serotonergic release and metabolism by HPLC-ED measurement of serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels. We confirmed the presence of serotonergic neurons in the E14 rat rhombencephalon by immunohistochemistry and showed for the first time evidence of MCHergic fibers reaching the area. Cultures obtained from rhombencephalic tissue presented 2.2 ± 0.7% of serotonergic and 48.9 ± 5.4% of GABAergic neurons. Despite the low concentration of serotonergic neurons, we were able to measure basal cellular and extracellular levels of 5-HT and 5-HIAA without the addition of any serotonergic-enhancer drug. As expected, 5-HT release was calcium-dependent and induced by depolarization. 5-HT extracellular levels were significantly increased by incubation with serotonin reuptake inhibitors (citalopram and nortriptyline) and a monoamine-oxidase inhibitor (clorgyline), and were not significantly modified by a 5-HT1A autoreceptor agonist (8-OHDPAT). Even though serotonergic cells responded as expected to these pharmacological treatments, MCH did not induce significant modifications of 5-HT and 5-HIAA extracellular levels in the cultures. Despite this unexpected result, we consider that assessment of 5-HT and 5-HIAA levels in primary serotonergic cultures may be an adequate approach to study the effect of other drugs and modulators on serotonin release, uptake and turnover.

Many conventional antidepressants can quickly raise the levels of extracellular serotonin, yet their positive effects on mood ensues only weeks later. This delay in efficacy is a clinical problem that has proven difficult to overcome. Early investigation noted that the initial increases in extracellular serotonin engaged strong feedback inhibition of serotonin neurons via 5-HT(1A) autoreceptors, resulting in a profound reduction in their firing rate. Over the course of chronic treatment, however, firing rate returned to normal and the inhibition via 5-HT(1A) receptor agonists was attenuated. The coincident timeline of these phenomena led to the influential hypothesis that the relationship was causal and that gradual loss of feedback inhibition mediated by 5-HT(1A) receptors was critical to the delayed therapeutic onset. Simple and appealing, the desensitization hypothesis has taken strong hold, yet much of the supporting evidence is circumstantial and there are several observations that would refute a causal relationship. In particular, even though 5-HT(1A) receptors may desensitize, there is evidence that feedback inhibition mediated by remaining receptors persists. That is, baseline serotonin firing rate returns to normal not because of 5-HT(1A) desensitization but rather despite ongoing feedback inhibition. Thus, while 5-HT(1A) receptors remain important for emotional behavior, it may be other slow-adaptive changes triggered by antidepressants that allow for therapeutic effects, such as those involving glutamatergic synaptic plasticity.

Alcohol is the third leading risk factor for disease in Russia and around the world. According to WHO, as a result of alcohol abuse, 2.5 million people die every year in the world, which is comparable to the population of a large city. Despite the development of science and medicine, the mechanisms for the development of alcohol dependence are not completely clear. Understanding these mechanisms will help create new approaches to the treatment of alcohol dependence. Today it is well known that the mesolimbic system of dopamine plays a key role in the formation of addictive disorders, in particular alcohol dependence. Fast-scan cyclic voltammetry (FSCV) is a widespread electrochemical method for detection of neurotransmitters such as dopamine. The advantages of this technics are a high temporal and spatial resolution that makes FSCV a powerful technique for detecting changing chemical concentrations in vivo. Using FSCV on freely moving animals, we demonstrated that electrical stimulation of the ventral tegmental area (VTA) triggers detectable dopamine release in the nucleus accumbens (NAcc). We showed that acute injection of ethanol (0,5g/kg) on freely moving rats produces a significant decrease in dopamine efflux in NAcc induced by electrical stimulation. Selective D2 DA autoreceptor antagonist raclopride significantly increased measured signal that indicated that the neurotransmitter release measured in the NAcc is dopaminergic but not noradrenergic.

Neurotransmitter release is a highly controlled process by which synapses can critically regulate information transfer within neural circuits. While presynaptic receptors – typically activated by neurotransmitters and modulated by neuromodulators – provide a powerful way of fine-tuning synaptic function, their contribution to activity-dependent changes in transmitter release remains poorly understood. Here, we report that presynaptic NMDA receptors (preNMDARs) at mossy fiber boutons in the rodent hippocampus can be activated by physiologically relevant patterns of activity and selectively enhance short-term synaptic plasticity at mossy fiber inputs onto CA3 pyramidal cells and mossy cells, but not onto inhibitory interneurons. Moreover, preNMDARs facilitate brain-derived neurotrophic factor release and contribute to presynaptic calcium rise. Taken together, our results indicate that by increasing presynaptic calcium, preNMDARs fine-tune mossy fiber neurotransmission and can control information transfer during dentate granule cell burst activity that normally occur in vivo.

G-Protein coupled D2 receptors expressed on dopamine neurons, i.e. D2-autoreceptors (D2Rs), negatively regulate transmitter release and cell firing in axonal terminals and somatodendritic compartments. However, as the membrane distribution of D2Rs at somatodendritic signaling sites has not been fully characterized, functional mechanistic details remain uncertain. This study utilized a knockin mouse to examine surface D2Rs linked at the N-terminus to a superecliptic pHlourin green fluorescent protein epitope (SEP) in dopamine neurons of the substantia nigra. By incubating live slices with a highly specific anti-SEP antibody, the selective labeling of plasma membrane associated receptors was achieved. The SEP-D2Rs appeared as puncta like structures apposed to the surface of dendrites and soma of dopamine neurons. The percentage of D2R expression apposed to TH positive structures varied linearly with the density of TH fibers observed. TH-associated SEP-D2Rs displayed a cell surface density of 0.180 puncta per µm2, which corresponds to an average frequency of 1 punctum every 1.326 µm using nearest neighbor analysis. The distinct punctate appearance of the anti-SEP staining indicates there is a population of D2Rs organized in discrete clusters along the plasma membrane, an arrangement that can spatially localize signaling and influence GPCR efficacy. The results also indicate that D2Rs are spatially distributed on the plasma membrane at a frequency higher than previously reported.

TOR1A-associated dystonia, otherwise known as DYT1 dystonia, is an inherited dystonia caused by a three base-pair deletion in the TOR1A gene (TOR1AΔE). Although the mechanisms underlying the dystonic movements are largely unknown, abnormalities in striatal dopamine and acetylcholine neurotransmission are consistently implicated whereby dopamine release is reduced while cholinergic tone is increased. Because striatal cholinergic neurotransmission mediates dopamine release, it is not known if the dopamine release deficit is mediated indirectly by abnormal acetylcholine neurotransmission or if Tor1a(ΔE) acts directly within dopaminergic neurons to attenuate release. To dissect the microcircuit that governs the deficit in dopamine release, we conditionally expressed Tor1a(ΔE) in either dopamine neurons or cholinergic interneurons in mice and assessed striatal dopamine release using ex vivo fast scan cyclic voltammetry or dopamine efflux using in vivo microdialysis. Conditional expression of Tor1a(ΔE) in cholinergic neurons did not affect striatal dopamine release. In contrast, conditional expression of Tor1a(ΔE) in dopamine neurons reduced dopamine release to 50% of normal, which is comparable to the deficit in Tor1a(+/ΔE) knockin mice that express the mutation ubiquitously. Despite the deficit in dopamine release, we found that the Tor1a(ΔE) mutation does not cause obvious nerve terminal dysfunction as other presynaptic mechanisms, including electrical excitability, vesicle recycling/refilling, Ca(2+) signaling, D2 dopamine autoreceptor function and GABA(B) receptor function, are intact. Although the mechanistic link between Tor1a(ΔE) and dopamine release is unclear, these results clearly demonstrate that the defect in dopamine release is caused by the action of the Tor1a(ΔE) mutation within dopamine neurons.

The nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show by application of several single-molecule sensitive super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the dopamine transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and by DA D2-autoreceptor activation in manner dependent on Ca2+-influx via N-type voltage-gated Ca2+-channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2-autoreceptor, syntaxin-1 and clathrin nanodomains. By demonstrating that nanoscopic reorganizations with putative major impact on transmitter homeostasis can take place in dopaminergic varicosities, the data have important implications for understanding modulatory neurotransmitter physiology.

The dorsal region of the bed nucleus of the stria terminalis (dBNST) receives substantial dopaminergic input which overlaps with norepinephrine input implicated in stress responses. Using ex vivo fast scan cyclic voltammetry in male C57BL6 mouse brain slices, we demonstrate that electrically stimulated dBNST catecholamine signals are of substantially lower magnitude and have slower uptake rates compared with caudate signals. Dopamine terminal autoreceptor activation inhibited roughly half of the catecholamine transient, and noradrenergic autoreceptor activation produced an ∼30% inhibition. Dopamine transporter blockade with either cocaine or GBR12909 significantly augmented catecholamine signal duration. We optogenetically targeted dopamine terminals in the dBNST of transgenic (TH:Cre) mice of either sex and, using ex vivo whole-cell electrophysiology, we demonstrate that optically stimulated dopamine release induces slow outward membrane currents and an associated hyperpolarization response in a subset of dBNST neurons. These cellular responses had a similar temporal profile to dopamine release, were significantly reduced by the D2/D3 receptor antagonist raclopride, and were potentiated by cocaine. Using in vivo fiber photometry in male C57BL/6 mice during training sessions for cocaine conditioned place preference, we show that acute cocaine administration results in a significant inhibition of calcium transient activity in dBNST neurons compared with saline administration. These data provide evidence for a mechanism of dopamine-mediated cellular inhibition in the dBNST and demonstrate that cocaine augments this inhibition while also decreasing net activity in the dBNST in a drug reinforcement paradigm. SIGNIFICANCE STATEMENT The dorsal bed nucleus of the stria terminalis (dBNST) is a region highly implicated in mediating stress responses; however, the dBNST also receives dopaminergic inputs from classically defined drug reward pathways. Here we used various techniques to demonstrate that dopamine signaling within the dBNST region has inhibitory effects on population activity. We show that cocaine, an abused psychostimulant, augments both catecholamine release and dopamine-mediated cellular inhibition in this region. We also demonstrate that cocaine administration reduces population activity in the dBNST, in vivo. Together, these data support a mechanism of dopamine-mediated inhibition within the dBNST, providing a means by which drug-induced elevations in dopamine signaling may inhibit dBNST activity to promote drug reward.

Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor efficiency to controlling transmitter release in isolated nerve endings (e.g., synaptosomes) indirectly confirming their presence in these particles but also allowing to speculate on their subunit composition. Western blot analysis and confocal microscopy unveiled the presence of the GluA1, GluA2, GluA3, and GluA4 receptor subunits in cortical synaptosomes. Functional studies confirmed the presence of presynaptic release-regulating AMPA autoreceptors in these terminals, whose activation releases [3H]D-aspartate ([3H]D-Asp, here used as a marker of glutamate) in a NBQX-dependent manner. The AMPA autoreceptors traffic in a constitutive manner, since entrapping synaptosomes with the pep2-SVKI peptide (which interferes with the GluA2-GRIP1/PICK1 interaction) amplified the AMPA-evoked releasing activity, while the inactive pep2-SVKE peptide was devoid of activity. Incubation of synaptosomes with antibodies recognizing the NH2 terminus of the GluA2 and the GluA3 subunits increased, although to a different extent, the GluA2 and 3 densities in synaptosomal membranes, also amplifying the AMPA-evoked glutamate release in a NBQX-dependent fashion. We then analyzed the releasing activity of complement (1:300) from both treated and untreated synaptosomes and found that the complement-induced overflow occurred in a DL-t-BOA-sensitive, NBQX-insensitive fashion. We hypothesized that anti-GluA/GluA complexes in neuronal membranes could trigger the classic pathway of activation of the complement, modifying its releasing activity. Accordingly, the complement-evoked release of [3H]D-Asp from antiGluA2 and anti-GluA3 antibody treated synaptosomes was significantly increased when compared to untreated terminals and facilitation was prevented by omitting the C1q component of the immunocomplex. Antibodies recognizing the NH2 terminus of the GluA1 or the GluA4 subunits failed to affect both the AMPA and the complement-evoked tritium overflow. Our results suggest the presence of GluA2/GluA3-containing release-regulating AMPA autoreceptors in cortical synaptosomes. Incubation of synaptosomes with commercial anti-GluA2 or anti-GluA3 antibodies amplifies the AMPA-evoked exocytosis of glutamate through a complement-independent pathway, involving an excessive insertion of AMPA autoreceptors in plasma membranes but also affects the complement-dependent releasing activity, by promoting the classic pathway of activation of the immunocomplex. Both events could be relevant to the development of autoimmune diseases typified by an overproduction of anti-GluA subunits.

The addictive effects of cocaine are regulated by an autophagy protein and can be prevented by autophagy inhibitors., Drug abuse is a foremost public health problem. Cocaine is a widely abused drug worldwide that produces various reward-related behaviors. The mechanisms that underlie cocaine-induced disorders are unresolved, and effective treatments are lacking. Here, we found that an autophagy-related protein Becn2 is a previously unidentified regulator of cocaine reward behaviors. Becn2 deletion protects mice from cocaine-stimulated locomotion and reward behaviors, as well as cocaine-induced dopamine accumulation and signaling, by increasing presynaptic dopamine receptor 2 (D2R) autoreceptors in dopamine neurons. Becn2 regulates D2R endolysosomal trafficking, degradation, and cocaine-induced behaviors via interacting with a D2R-bound adaptor GASP1. Inactivating Becn2 by upstream autophagy inhibitors stabilizes striatal presynaptic D2R, reduces dopamine release and signaling, and prevents cocaine reward in normal mice. Thus, the autophagy protein Becn2 is essential for cocaine psychomotor stimulation and reward through regulating dopamine neurotransmission, and targeting Becn2 by autophagy inhibitors is a potential strategy to prevent cocaine-induced behaviors.

Neuromodulation mediated by synaptically released endogenous transmitters acting in G protein coupled receptors (GPCRs) is slow primarily because of multistep downstream signaling. What is not known is the spatial and temporal kinetics of transmitter and receptor interaction. The present work used the combination of the dopamine sensor, dLight, to detect the spatial release and diffusion of dopamine and a caged form of a D2-dopamine receptor antagonist, CyHQ-sulpiride, to rapidly block the D2-autoreceptors. Photoactivation of the CyHQ-sulpiride blocked receptors in milliseconds such that the time course of dopamine/receptor interaction was mapped onto the downstream signaling. The results show that highly localized release, but not dopamine diffusion, defines the time course of the functional interaction between dopamine and D2-autoreceptors, which determines downstream inhibition.

Dopamine (DA) signaling affects locomotion, feeding, learning, and memory in C. elegans. Various assays have been developed to study the proteins involved in these behaviors; however, these assays show behavioral output only when there is a drastic change in DA levels. We designed an assay capable of observing behavioral output even with only slight alterations in DA levels. To achieve this, we designed a behavioral paradigm where we combined C. elegans movement with ethanol (EtOH) administration. The behavioral response to alcohol/EtOH and susceptibility to alcohol-use disorders (AUDs) have been linked to DA. Our assay correlates an increase in DA levels due to EtOH and movement obstruction due to a dry surface to a circular sedative behavior, which we designated as EtOH-induced sedative (EIS) behavior. We successfully utilized this assay to assign physiological and behavioral functions to a DA autoreceptor, DOP-2.

Networks of the dorsal-horn of the spinal-cord process nociceptive information from the periphery. In these networks, the excitation/inhibition balance is critical to shape this nociceptive information and to gate it to the brain where it is interpreted as pain. Our aim was to define whether short-term plasticity of inhibitory connections could tune this inhibition/excitation balance by differentially controlling excitatory and inhibitory microcircuits. To this end, we used spinal-cord slices from adult mice expressing enhanced green fluorescent protein (eGFP) under the GAD65 promoter and recorded from both eGFP+ (putative inhibitory) and eGFP- (putative excitatory) neurons of lamina II while stimulating single presynaptic GABAergic interneurons at various frequencies. Our results indicate that GABAergic neurons of lamina II simultaneously contact eGFP- and eGFP+ neurons, but these connections display very different frequency-dependent short-term plasticities. Connections onto eGFP- interneurons displayed limited frequency-dependent changes, and strong time-dependent summation of inhibitory synaptic currents that was however subjected to a tonic activity-dependent inhibition involving A1 adenosine receptors. In contrast, GABAergic connections onto eGFP+ interneurons expressed pronounced frequency-dependent depression, thus favoring disinhibition at these synapses by a mechanism involving the activation of GABAB autoreceptors at low frequency. Interestingly, the balance favors inhibition at frequencies associated with intense pain whether it favors excitation at frequencies associated with low pain. Therefore, these target- and frequency-specific plasticities allow to tune the balance between inhibition and disinhibition while processing frequency-coded information from primary afferents. These short-term plasticities and their modulation by A1 and GABAB receptors might represent an interesting target in pain-alleviating strategies.

Attention deficit hyperactivity disorder (ADHD) is the most frequently diagnosed neurodevelopmental disorder worldwide. Affected individuals present with hyperactivity, inattention, and cognitive deficits and display a characteristic paradoxical response to drugs affecting the dopaminergic system. However, the underlying pathophysiology of ADHD and how this relates to dopaminergic transmission remains to be fully understood. Sorcs2−/− mice uniquely recapitulate symptoms reminiscent of ADHD in humans. Here, we show that lack of SorCS2 in mice results in lower sucrose intake, indicating general reward deficits. Using in-vivo recordings, we further find that dopaminergic transmission in the ventral tegmental area (VTA) is shifted towards a more regular firing pattern with marked reductions in the relative occurrence of irregular firing in Sorcs2−/− mice. This was paralleled by abnormal acute behavioral responses to dopamine receptor agonists, suggesting fundamental differences in dopaminergic circuits and indicating a perturbation in the balance between the activities of the postsynaptic dopamine receptor DRD1 and the presynaptic inhibitory autoreceptor DRD2. Interestingly, the hyperactivity and drug response of Sorcs2−/− mice were markedly affected by novelty. Taken together, our findings show how loss of a candidate ADHD-risk gene has marked effects on dopaminergic circuit function and the behavioral response to the environment.

Among several serotonin receptors, the 5-HT2B is one of the less studied in the brain. Although this receptor is only weakly expressed in the nervous system, it appears to be involved in several biological mechanisms linked to neuropsychiatric dysfunctions and/or action of several drugs. Indeed, the selective serotonin reuptake inhibitors (SSRIs) act by blocking the serotonin transporter, but their acute and chronic effects are specifically blunted when the 5-HT2B receptor is pharmacologically blocked or when its gene is knocked-out. This lack of effect is also observed in mice submitted to chronic stress paradigms that induce depressive-like states. On the contrary, response to non-serotonergic antidepressants are conserved in mice lacking a functional 5-HT2B receptor. Recent studies have demonstrated that 5-HT2B receptors are expressed by serotonergic neurons, and that they can act as positive autoreceptors, counterbalancing the actions of the negative 5-HT1A autoreceptors. In addition, elegant studies confirm that it is the 5-HT2B autoreceptor that is required for SSRIs effects. All in all, the specific lack of responses to SSRIs exhibited by mice knocked-out for the 5-HT2B receptor make these animals an ideal model for the study of resistance to SSRIs antidepressants, a condition highly reported in clinics. Besides, the impaired response to serotonergic antidepressants, mice constitutively lacking the 5-HT2B receptor have increased basal levels of BDNF in the hippocampus. The “antidepressant-like phenotype” exhibited by these mice offers an interesting tool for the study of new molecules potentially acting as antidepressants.

DYT1-TOR1A dystonia is an inherited dystonia caused by a three base-pair deletion in the TOR1A gene (TOR1AΔE). Although the mechanisms underlying the dystonic movements are largely unknown, abnormalities in striatal dopamine and acetylcholine neurotransmission are consistently implicated whereby dopamine release is reduced while cholinergic tone is increased. Because striatal cholinergic neurotransmission mediates dopamine release, it is not known if the dopamine release deficit is mediated indirectly by abnormal acetylcholine neurotransmission or if Tor1a(ΔE) acts directly within dopaminergic neurons to attenuate release. To dissect the microcircuit that governs the deficit in dopamine release, we conditionally expressed Tor1a(ΔE) in either dopamine neurons or cholinergic interneurons in mice and assessed striatal dopamine release using ex vivo fast scan cyclic voltammetry or dopamine efflux using in vivo microdialysis. Conditional expression of Tor1a(ΔE) in cholinergic neurons did not affect striatal dopamine release. In contrast, conditional expression of Tor1a(ΔE) in dopamine neurons reduced dopamine release to 50% of normal, which is comparable to the deficit in Tor1a+/ΔE knockin mice that express the mutation ubiquitously. Despite the deficit in dopamine release, we found that the Tor1a(ΔE) mutation does not cause obvious nerve terminal dysfunction as other presynaptic mechanisms, including electrical excitability, vesicle recycling/refilling, Ca2+ signaling, D2 dopamine autoreceptor function and GABAB receptor function, are intact. Although the mechanistic link between Tor1a(ΔE) and dopamine release is unclear, these results clearly demonstrate that the defect in dopamine release is caused by the action of the Tor1a(ΔE) mutation within dopamine neurons.

Objective Binge-eating disorder (BED) disrupts dopamine neuron function, in part by altering dopamine transporter (DAT) activity. This study characterized the effects of high-fat bingeing on presynaptic dopamine terminals and tested the hypothesis that acute low-dose amphetamine would restore DAT function. Methods C57BL/6 mice were given limited access (LimA) to a high-fat diet (2 h/d, 3 d/wk) or standard chow (control). After 6 weeks, ex vivo fast-scan cyclic voltammetry was used to characterize dopamine-terminal adaptations in the nucleus accumbens. Prior to undergoing fast-scan cyclic voltammetry, some mice from each group were given amphetamine (0.5 mg/kg intraperitoneally). Results Escalation of high fat intake, termed bingeing, occurred in the LimA group and coincided with increased phasic dopamine release, reduced dopamine uptake rates, and increased dopamine receptor 2 (D2 ) autoreceptor function. Acute amphetamine selectively reversed dopamine uptake changes in the LimA group and restored the potency of amphetamine to inhibit uptake. Conclusions High-fat bingeing enhanced dopaminergic signaling in the nucleus accumbens by promoting phasic dopamine release and reducing clearance. This study's data show that amphetamine was efficacious in restoring impaired DAT function caused by high-fat bingeing but did not reduce dopamine release to normal. These presynaptic changes should be considered if amphetamine-like dopamine releasers are used as treatments for BED.

Severe withdrawal symptoms triggered by cessation of long-term opioid use deter many individuals from seeking treatment. Opioid substitution and α2-adrenergic agonists are the current standard of pharmacotherapy for opioid use disorder in western medicine; however, each is associated with significant complications. Heantos-4 is a non-opioid botanical formulation used to facilitate opioid detoxification in Vietnam. While ongoing clinical use continues to validate its safety and effectiveness, a mechanism of action accounting for these promising effects remains to be specified. Here, we assess the effects of Heantos-4 in a rat model of morphine-dependence and present evidence that alleviation of naloxone-precipitated somatic withdrawal signs is related to an upregulation of mesolimbic dopamine activity and a consequent reversal of a hypodopaminergic state in the nucleus accumbens, a brain region implicated in opioid withdrawal. A central dopaminergic mechanism is further supported by the identification of l-tetrahydropalmatine as a key active ingredient in Heantos-4, which crosses the blood–brain barrier and shows a therapeutic efficacy comparable to its parent formulation in attenuating withdrawal signs. The anti-hypodopaminergic effects of l-tetrahydropalmatine may be related to antagonism of the dopamine autoreceptor, thus constituting a plausible mechanism contributing to the effectiveness of Heantos-4 in facilitating opioid detoxification.

Increased dopamine (DA) signaling in the striatum has been a cornerstone hypothesis about psychosis for over 50 years. Increased dopamine release results in psychotic symptoms, while D2 dopamine receptor (DRD2) antagonists are antipsychotic. Recent schizophrenia GWAS identified risk-associated common variants near the DRD2 gene, but the risk mechanism has been unclear. To gain novel insight into risk mechanisms underlying schizophrenia, we performed a comprehensive analysis of the genetic and transcriptional landscape of schizophrenia in postmortem caudate nucleus from a cohort of 444 individuals. Integrating expression quantitative trait loci (eQTL) analysis, transcriptome wide association study (TWAS), and differential expression analysis, we found many new genes associated with schizophrenia through genetic modulation of gene expression. Using a new approach based on deep neural networks, we construct caudate nucleus gene expression networks that highlight interactions involving schizophrenia risk. Interestingly, we found that genetic risk for schizophrenia is associated with decreased expression of the short isoform of DRD2, which encodes the presynaptic autoreceptor, and not with the long isoform, which encodes the postsynaptic receptor. This association suggests that decreased control of presynaptic DA release is a potential genetic mechanism of schizophrenia risk. Altogether, these analyses provide a new resource for the study of schizophrenia that can bring insight into risk mechanisms and potential novel therapeutic targets.

Central histaminergic H3 receptor (H3R) has been extensively investigated as a potential therapeutic target for various neurological and neurodegenerative disorders. Despite promising results in preclinical rodent models, clinical trials have not provided conclusive evidence for the benefit of H3R antagonists to alleviate cognitive and behavioral symptoms of these disorders. Inconsistent pharmacological efficacies may arise from aberrant changes in H3R over time during disease development. Because H3R is involved in feedback inhibition of histamine synthesis and secretion, the expression of the autoreceptor may also be reciprocally regulated by altered histamine levels in a pathological condition. Thus, we investigated H3R expression in a mouse model of cow’s milk allergy, a condition associated with increased histamine levels. Mice were sensitized to bovine whey proteins (WP) over 5 weeks and H3R protein and transcript levels were examined in the brain. Substantially increased H3R immunoreactivity was observed in various brain regions of WP-sensitized mice compared to sham mice. Elevated H3R expression was also found in the thalamic/hypothalamic region. The expression of histaminergic H1, but not H2, receptor subtype was also increased in this and the midbrain regions. Unlike the brain, all three histaminergic receptors were increased in the small intestine. These results indicated that the central histaminergic receptors were altered in WP-sensitized mice in a subtype- and region-specific manner, which likely contributed to behavioral changes we observed in these mice. Our study also suggests that altered levels of H3R could be considered during a pharmacological intervention of a neurological disease.

The mesolimbic dopamine system-which originates in the ventral tegmental area and projects to the striatum-has been shown to be involved in the expression of sex-specific behavior and is thought to be a critical mediator of many psychiatric diseases. While substantial work has focused on sex differences in the anatomy of dopamine neurons and relative dopamine levels between males and females, an important characteristic of dopamine release from axon terminals in the striatum is that it is rapidly modulated by local regulatory mechanisms independent of somatic activity. These processes can occur via homosynaptic mechanisms-such as presynaptic dopamine autoreceptors and dopamine transporters-as well as heterosynaptic mechanisms, such as retrograde signaling from postsynaptic cholinergic and GABAergic systems, among others. These regulators serve as potential targets for the expression of sex differences in dopamine regulation in both ovarian hormone-dependent and independent fashions. This review describes how sex differences in microcircuit regulatory mechanisms can alter dopamine dynamics between males and females. We then describe what is known about the hormonal mechanisms controlling/regulating these processes. Finally, we highlight the missing gaps in our knowledge of these systems in females. Together, a more comprehensive and mechanistic understanding of how sex differences in dopamine function manifest will be particularly important in developing evidence-based therapeutics that target this system and show efficacy in both sexes.

The secretion of prolactin from the pituitary is negatively controlled by tuberoinfundibular dopamine (TIDA) neurones. The electrical properties of TIDA cells have recently been identified as a modulatory target of neurotransmitters and hormones in the lactotrophic axis. The role of the GABAB receptor in this control has received little attention, yet is of particular interest because it may act as a TIDA neurone autoreceptor. Here, this issue was explored in a spontaneously active rat TIDA in vitro slice preparation using whole-cell recordings. Application of the GABAB receptor agonist, baclofen, dose-dependently slowed down or abolished the network oscillations typical of this preparation. Pharmacological manipulations identify the underlying mechanism as an outward current mediated by G-protein-coupled inwardly rectifying K+ -like channels. In addition to this postsynaptic modulation, we describe a presynaptic modulation where GABAB receptors restrain the release of glutamate and GABA onto TIDA neurones. Our data identify both pre- and postsynaptic modulation of TIDA neurones by GABAB receptors that may play a role in the neuronal network control of pituitary prolactin secretion and lactation.

Adolescence is characterized by changes in behavior, such as increases in sensation seeking and risk taking, and increased vulnerability to developing a range of psychiatric disorders, including substance abuse disorders (SUD) and mood disorders. The mesolimbic dopamine system plays an essential role in mediating these behaviors and disorders. Therefore, it is imperative to understand how the dopamine system and its regulation are changing during this period of development. Here, we used ex vivo fast scan cyclic voltammetry to compare stimulated dopamine release and its local circuitry regulation between early adolescent and adult male and female Sprague-Dawley rats. We found that, compared to adults, adolescent males have decreased stimulated dopamine release in the NAc core, while adolescent females have increased dopamine release in the NAc shell, NAc core, and DMS. We also found sex- and region-specific differences in other dopamine dynamics, including maximal dopamine uptake (Vmax), release across a range of stimulation frequencies, and autoreceptor regulation of dopamine release. Better understanding how the dopamine system develops during adolescence will be imperative for understanding what mediates adolescent vulnerability to developing psychiatric disorders and how disruptions during this period of reorganization could alter behaviors and vulnerability into adulthood.

Prenatal exposure to bisphenol A (BPA) influences the development of sex differences neurologically and behaviorally across many species of vertebrates. These effects are a consequence of BPA&rsquo, s estrogenic activity and its ability to act as an endocrine disrupter even, at very low doses. When exposure to BPA occurs during critical periods of development, it can interfere with the normal activity of sex steroids, impacting the fate of neurons, neural connectivity and the development of brain regions sensitive to steroid activity. Among the most sensitive behavioral targets of BPA action are behaviors that are characterized by a sexual dimorphism, especially emotion and anxiety related behaviors, such as the amount of time spent investigating a novel environment, locomotive activity and arousal. Moreover, in some species of rodents, BPA exposure affected males&rsquo, sexual behaviors. Interestingly, these behaviors are at least in part modulated by the catecholaminergic system, which has been reported to be a target of BPA action. In the present study we investigated the influence of prenatal exposure of mice to a very low single dose of BPA on emotional and sexual behaviors and on the density and binding characteristics of alpha2 adrenergic receptors. Alpha2 adrenergic receptors are widespread in the central nervous system and they can act as autoreceptors, inhibiting the release of noradrenaline and other neurotransmitters from presynaptic terminals. BPA exposure disrupted sex differences in behavioral responses to a novel environment, but did not affect male mice sexual behavior. Importantly, BPA exposure caused a change in the binding affinity of alpha2 adrenergic receptors in the locus coeruleus and medial preoptic area (mPOA) and it eliminated the sexual dimorphism in the density of the receptors in the mPOA.

RationaleIn rodents, exposure to novel environments elicits initial anxiety-like behavior (neophobia) followed by intense exploration (neophilia) that gradually subsides as the environment becomes familiar. Thus, innate novelty-induced behaviors are useful indices of anxiety and motivation in animal models of psychiatric disease. Noradrenergic neurons are activated by novelty and implicated in exploratory and anxiety-like responses, but the role of norepinephrine (NE) in neophobia has not been clearly delineated.ObjectiveWe sought to define the role of central NE transmission in neophilic and neophobic behaviors.MethodsWe assessed dopamine β-hydroxylase knockout (Dbh -/-) mice lacking NE and their NE-competent (Dbh +/-) littermate controls in neophilic (novelty-induced locomotion; NIL) and neophobic (novelty-suppressed feeding; NSF) behavioral tests with subsequent quantification of brain-wide c-fos induction. We complimented the gene knockout approach with pharmacological interventions.ResultsDbh -/- mice exhibited blunted locomotor responses in the NIL task and completely lacked neophobia in the NSF test. Neophobia was rescued in Dbh -/- mice by acute pharmacological restoration of central NE with the synthetic precursor L-3,4-dihydroxyphenylserine (DOPS), and attenuated in control mice by the inhibitory α2-adrenergic autoreceptor agonist guanfacine. Following either NSF or NIL, Dbh -/- mice demonstrated reduced c-fos in the anterior cingulate cortex, medial septum, ventral hippocampus, bed nucleus of the stria terminalis, and basolateral amygdala.ConclusionThese findings indicate that central NE signaling is required for the expression of both neophilic and neophobic behaviors. Further, we describe a putative noradrenergic novelty network as a potential therapeutic target for treating anxiety and substance abuse disorders.

Brain serotonin (5-HT) neurotransmission plays an important role in male sexual behavior and it is well established that activating 5-HT1A receptors in rats facilitate ejaculatory behavior. However, the relative contribution of 5-HT1A somatodendritic autoreceptors and heteroreceptors in this pro-sexual behavior is unclear. Moreover, it is unclear whether the contribution of somatodendritic 5-HT1A autoreceptors and postsynaptic 5-HT1A heteroreceptors alter when extracellular 5-HT levels are chronically increased. Serotonin transporter knockout (SERT-/-) rats exhibit enhanced extracellular 5-HT levels and desensitized 5-HT1A receptors. These rats model neurochemical changes underlying chronic SSRI-induced sexual dysfunction. We want to determine the role of presynaptic versus postsynaptic 5-HT1A receptors in the pro-sexual effects of 5-HT1A receptor agonists in SERT+/+ and in SERT-/- rats. Therefore, acute effects of the biased 5-HT1A receptor agonists F-13714, a preferential 5-HT1A autoreceptor agonist, or F-15599, a preferential 5-HT1A heteroreceptor agonist, and S15535 a mixed 5-HT1A autoreceptor agonist/heteroreceptor antagonist, on male sexual behavior were assessed. A clear and stable genotype effect was found after training where SERT+/+ performed sexual behavior at a higher level than SERT-/- rats. Both F-15599 and F-13714 induced pro-sexual activity in SERT+/+ and SERT-/- animals. Compared to SERT+/+, the F13714-dose-response curve in SERT-/- rats was shifted to the right. SERT+/+ and SERT-/- rats responded similar to F15599. Within both SERT+/+ and SERT-/- rats the potency of F-13714 was much stronger compared to F-15599. S15535 had no effect on sexual behavior in either genotype. In SERT+/+ and SERT-/- rats that were selected on comparable low sexual activity (SERT+/+ 3 or less ejaculations and SERT-/- 5 or less ejaculations in 10 weeks) S15535 also did not influence sexual behavior. The two biased compounds with differential effects on 5-HT1A auto- and hetero-receptors, exerted pro-sexual activity in both SERT+/+ and SERT-/- rats. Applying these specific pharmacological tools has not solved whether pre- or post-synaptic 5-HT1A receptors are involved in pro-sexual activity. Moreover, the inactivity of S15535 in male sexual behavior in either genotype was unexpected. The question is whether the in vivo pharmacological profile of the different 5-HT1A receptor ligands used, is sufficient to differentiate pre- and/or post-synaptic 5-HT1A receptor contributions in male rat sexual behavior.

The neurotransmitter dopamine is implicated in diverse functions, including reward processing, reinforcement learning and cognitive control. The tendency to discount future rewards in value over time has long been discussed in the context of potential dopaminergic modulation. Here we examined the effect of a single dose of the D2 receptor antagonist Haloperidol (2mg) on temporal discounting. Our approach extends previous human pharmacological studies in two ways. First, we applied state-of-the-art computational modeling based on the drift diffusion model to comprehensively examine choice dynamics. Second, we examined dopaminergic modulation of reward magnitude effects on temporal discounting. Drift diffusion modeling revealed reduced temporal discounting and substantially faster non-decision times under Haloperidol. Temporal discounting was substantially increased for low vs. high reward magnitudes, but this magnitude effect was largely unaffected by Haloperidol. These results were corroborated by model-free analyses as well as modeling via more standard approaches using softmax action selection. We previously reported elevated caudate activation under Haloperidol in this sample of participants, supporting the idea that Haloperidol elevated dopamine neurotransmission, e.g. by blocking inhibitory feedback via presynaptic D2 autoreceptors. The present modeling results show that during inter-temporal choice, this leads to attenuated temporal discounting and increased response vigor (shorter non-decision times).

A deficient serotonergic system is associated with psychopathological behaviors in various species, among which, feather pecking (FP) in chickens. Deficiency in the serotonergic system can predispose birds to develop FP, while the serotonergic system is affected in birds that feather peck. Serotonin (5-HT) can further influence dopamine (DA) activity. Lines with high FP tendency generally have low central 5-HT and DA turnovers at a young age, but high turnovers at an adult age in brain areas involved in somato-motor regulation and goal-directed behavior. Agonizing 5-HT1A and 5-HT1B receptors increases FP, while antagonizing D2 receptor reduces FP. Genetic associations exist between FP, 5-HT1A and 5-HT1B receptor functioning and metabolism of 5-HT and DA. Birds with deficient functioning of the somatodendritic 5-HT1A autoreceptor and 5-HT metabolism appear predisposed to develop FP. Birds which feather peck often eat feathers, have low whole-blood 5-HT, different gut-microbiota composition and immune competence compared to non-peckers. FP and feather eating likely affect the interaction between gut microbiota, immune system and serotonergic system, but this needs further investigation.

Cocaine is a highly abused drug, and cocaine addiction affects millions of individuals worldwide. Cocaine blocks normal uptake function at the dopamine transporter (DAT), thus increasing extracellular dopamine. Currently, no chemical therapies are available to treat cocaine abuse. Previous works showed that the selective inhibitors of protein kinase Cβ (PKCβ), enzastaurin and ruboxistaurin, attenuate dopamine overflow and locomotion stimulated by another psychostimulant drug, amphetamine. We now test if ruboxistaurin similarly affects cocaine action. Perfusion of 1 μM ruboxistaurin directly into the core of the nucleus accumbens via retrodialysis reduced cocaine-stimulated increases in dopamine overflow, measured using microdialysis sampling, with simultaneous reductions in locomotor behavior. Because cocaine activity is highly regulated by dopamine autoreceptors, we examined whether ruboxistaurin was acting at the level of the D2 autoreceptor. Perfusion of 5 μM raclopride, a selective D2-like receptor antagonist, before addition of ruboxistaurin, abrogated the effect of ruboxistaurin on cocaine-stimulated dopamine overflow and hyperlocomotion. Further, ruboxistaurin was inactive against cocaine-stimulated locomotor activity in mice with a genetic deletion in D2 receptors as compared to wild-type mice. In contrast, blockade or deletion of dopamine D2 receptors did not abolish the attenuating effect of ruboxistaurin on amphetamine-stimulated activities. Therefore, the inhibition of PKCβ reduces dopamine overflow and locomotor activity stimulated by both cocaine and amphetamine, but the mechanism of action differs for each stimulant. These data suggest that inhibition of PKCβ would serve as a target to reduce the abuse of either amphetamine or cocaine.

Cariprazine, an orally active and potent dopamine D3–preferring D3/D2 receptor partial agonist, is approved to treat adults with schizophrenia (in the United States and Europe) and manic or mixed episodes associated with bipolar I disorder (in the United States). Cariprazine also displays partial agonism at serotonin [5-hydroxytryptamine (5-HT)] 5-HT1A receptors and antagonism at 5-HT2A and 5-HT2B receptors in vitro. The study objective was to determine whether cariprazine leads to functional alterations of monoamine systems in vivo via electrophysiological recordings from anesthetized rats. Dorsal raphe nucleus (DRN), locus coeruleus (LC), and hippocampus pyramidal neurons were recorded, and cariprazine was administered systemically or locally through iontophoresis. In the DRN, cariprazine completely inhibited the firing activity of 5-HT neurons, which was fully reversed by the 5-HT1A receptor antagonist, WAY100635. In the LC, cariprazine reversed the inhibitory effect of the preferential 5-HT2A receptor agonist, 2,5-dimethoxy-4-iodoamphetamine, on norepinephrine (NE) neurons (ED50 = 66 µg/kg) but did not block the inhibitory effect of the α2-adrenergic receptor agonist, clonidine. Cariprazine, iontophorized into the hippocampus, diminished pyramidal neuronal firing through activation of 5-HT1A receptors, while its concomitant administration did not dampen the suppressant effect of 5-HT. These results indicate that, in vivo, cariprazine acted as a 5-HT1A autoreceptor agonist in the DRN, a 5-HT2A receptor antagonist in modulating the firing activity of LC NE neurons, and a full agonist at 5-HT1A receptors mediating the electrophysiological effect of 5-HT on pyramidal neurons. The modulatory actions of cariprazine on these monoaminergic systems may contribute to its therapeutic effectiveness in patients with depressive episodes.