VTA MC3R neurons control feeding in an activity- and sex-dependent manner in mice.

Increasing evidence indicates that the melanocortin and mesolimbic dopamine (DA) systems interact to regulate feeding and body weight. Because melanocortin-3 receptors (MC3R) are highly expressed in the ventral tegmental area (VTA), we tested whether VTA neurons expressing these receptors (VTA MC3R neurons) control feeding and body weight in vivo. We also tested whether there were sex differences in the ability of VTA MC3R neurons to control feeding, as MC3R -/- mice show sex-dependent alterations in reward feeding and DA levels, and there are clear sex differences in multiple DA-dependent behaviors and disorders. Designer receptors exclusively activated by designer drugs (DREADD) were used to acutely activate and inhibit VTA MC3R neurons and changes in food intake and body weight were measured. Acutely altering the activity of VTA MC3R neurons decreased feeding in an activity- and sex-dependent manner, with acute activation decreasing feeding, but only in females, and acute inhibition decreasing feeding, but only in males. These differences did not appear to be due to sex differences in the number of VTA MC3R neurons, the ability of hM3Dq to activate VTA MC3R neurons, or the proportion of VTA MC3R neurons expressing tyrosine hydroxylase (TH). These studies demonstrate an important role for VTA MC3R neurons in the control of feeding and reveal important sex differences in behavior, whereby opposing changes in neuronal activity in male and female mice cause similar changes in behavior.

Investigating the effects of chronic perinatal alcohol and combined nicotine and alcohol exposure on dopaminergic and non-dopaminergic neurons in the VTA.

The ventral tegmental area (VTA) is the origin of dopaminergic neurons and the dopamine (DA) reward pathway. This pathway has been widely studied in addiction and drug reinforcement studies and is believed to be the central processing component of the reward circuit. In this study, we used a well-established rat model to expose mother dams to alcohol, nicotine-alcohol, and saline perinatally. DA and non-DA neurons collected from the VTA of the rat pups were used to study expression profiles of miRNAs and mRNAs. miRNA pathway interactions, putative miRNA-mRNA target pairs, and downstream modulated biological pathways were analyzed. In the DA neurons, 4607 genes were differentially upregulated and 4682 were differentially downregulated following nicotine-alcohol exposure. However, in the non-DA neurons, only 543 genes were differentially upregulated and 506 were differentially downregulated. Cell proliferation, differentiation, and survival pathways were enriched after the treatments. Specifically, in the PI3K/AKT signaling pathway, there were 41 miRNAs and 136 mRNAs differentially expressed in the DA neurons while only 16 miRNAs and 20 mRNAs were differentially expressed in the non-DA neurons after the nicotine-alcohol exposure. These results depicted that chronic nicotine and alcohol exposures during pregnancy differentially affect both miRNA and gene expression profiles more in DA than the non-DA neurons in the VTA. Understanding how the expression signatures representing specific neuronal subpopulations become enriched in the VTA after addictive substance administration helps us to identify how neuronal functions may be altered in the brain.

Investigating the influence of perinatal nicotine and alcohol exposure on the genetic profiles of dopaminergic neurons in the VTA using miRNA-mRNA analysis.

Nicotine and alcohol are two of the most commonly used and abused recreational drugs, are often used simultaneously, and have been linked to significant health hazards. Furthermore, patients diagnosed with dependence on one drug are highly likely to be dependent on the other. Several studies have shown the effects of each drug independently on gene expression within many brain regions, including the ventral tegmental area (VTA). Dopaminergic (DA) neurons of the dopamine reward pathway originate from the VTA, which is believed to be central to the mechanism of addiction and drug reinforcement. Using a well-established rat model for both nicotine and alcohol perinatal exposure, we investigated miRNA and mRNA expression of dopaminergic (DA) neurons of the VTA in rat pups following perinatal alcohol and joint nicotine-alcohol exposure. Microarray analysis was then used to profile the differential expression of both miRNAs and mRNAs from DA neurons of each treatment group to further explore the altered genes and related biological pathways modulated. Predicted and validated miRNA-gene target pairs were analyzed to further understand the roles of miRNAs within these networks following each treatment, along with their post transcription regulation points affecting gene expression throughout development. This study suggested that glutamatergic synapse and axon guidance pathways were specifically enriched and many miRNAs and genes were significantly altered following alcohol or nicotine-alcohol perinatal exposure when compared to saline control. These results provide more detailed insight into the cell proliferation, neuronal migration, neuronal axon guidance during the infancy in rats in response to perinatal alcohol/ or nicotine-alcohol exposure.

Estrogen Receptor α Regulates Ethanol Excitation of Ventral Tegmental Area Neurons and Binge Drinking in Female Mice.

Elevations in estrogen (17ß-estradiol, E2) are associated with increased alcohol drinking by women and experimentally in rodents. E2 alters the activity of the dopamine system, including the VTA and its projection targets, which plays an important role in binge drinking. A previous study demonstrated that, during high E2 states, VTA neurons in female mice are more sensitive to ethanol excitation. However, the mechanisms responsible for the ability of E2 to enhance ethanol sensitivity of VTA neurons have not been investigated. In this study, we used selective agonists and antagonists to examine the role of ER subtypes (ERα and ERß) in regulating the ethanol sensitivity of VTA neurons in female mice and found that ERα promotes the enhanced ethanol response of VTA neurons. We also demonstrated that enhancement of ethanol excitation requires the activity of the metabotropic glutamate receptor, mGluR1, which is known to couple with ERα at the plasma membrane. To investigate the behavioral relevance of these findings, we administered lentivirus-expressing short hairpin RNAs targeting either ERα or ERß into the VTA and found that knockdown of each receptor in the VTA reduced binge-like ethanol drinking in female, but not male, mice. Reducing ERα in the VTA had a more dramatic effect on binge-like drinking than reducing ERß, consistent with the ability of ERα to alter ethanol sensitivity of VTA neurons. These results provide important insight into sex-specific mechanisms that drive excessive alcohol drinking.SIGNIFICANCE STATEMENT Estrogen has potent effects on the dopamine system and increases the vulnerability of females to develop addiction to substances, such as alcohol. We investigated the mechanisms by which estrogen increases the response of neurons in the VTA to ethanol. We found that activation of the ERα increased the ethanol-induced excitation of VTA neurons. 17ß-Estradiol-mediated enhancement of ethanol-induced excitation required the metabotropic glutamate receptor mGluR1. We also demonstrated that ERs in the VTA regulate binge-like alcohol drinking by female, but not male, mice. The influence of ERs on binge drinking in female mice suggests that treatments for alcohol use disorder in women may need to account for this sex difference.

Investigating the influence of perinatal nicotine exposure on genetic profiles of neurons in the sub-regions of the VTA.

Chronic nicotine exposure during pregnancy has been shown to induce physiological and anatomical alterations in offspring. Previously, we investigated the complexity of dopamine (DA) neuron firing in the sub-regions of the ventral tegmental area (VTA) following perinatal nicotine exposure. Using approximate entropy, we found that within the middle sub-region, the parainterfascicular nucleus (PIF), there was higher complexity indicating more random neural firing and a less homogeneous neuron population. Therefore, we sought to investigate the neuron populations within the sub-regions of the VTA following perinatal nicotine exposure. We used real time PCR in order to find the relative quantity of glutamate to γ-aminobutyric acid (GABA), DA, and glutamate neurons within three sub-regions: the parabrachial pigmented nucleus (PBP), parainterfascicular nucleus (PIF), and paranigral nucleus (PN). Our results showed that the PIF region of the VTA contained a more diverse population of neurons resulting in a more complex system. In addition, we found that DA neurons are more activated in PN sub-region of the VTA, which mediates the rewarding effects of drugs including nicotine. Lastly, using immunohistochemistry, we observed an overall decrease in DA neurons following perinatal nicotine exposure.

Neural connectivity between the hypothalamic supramammillary nucleus and appetite- and motivation-related regions of the rat brain.

The supramammillary nucleus (SuM) has an emerging role in appetite control. We have shown that the rat SuM is activated during hunger or food anticipation, or by ghrelin administration. In the present study, we characterised the connectivity between the SuM and key appetite- and motivation-related nuclei in the rat. In adult wild-type rats, or rats expressing Cre recombinase under the control of the tyrosine hydroxylase (TH) promoter (TH-Cre rats), we used c-Fos immunohistochemistry to visualise and correlate the activation of medial SuM (SuMM) with activation in the lateral hypothalamic area (LH), the dorsomedial hypothalamus (DMH) or the ventral tegmental area (VTA) after voluntary consumption of a high-sugar, high-fat food. To determine neuroanatomical connectivity, we used retrograde and anterograde tracing methods to specifically investigate the neuronal inputs and outputs of the SuMM. After consumption of the food there were positive correlations between c-Fos expression in the SuMM and the LH, DMH and VTA (P = 0.0001, 0.01 and 0.004). Using Fluoro-Ruby as a retrograde tracer, we demonstrate the existence of inputs from the LH, DMH, VTA and ventromedial hypothalamus (VMH) to the SuMM. The SuMM showed reciprocal inputs to the LH and DMH, and we identified a TH-positive output from SuMM to DMH. We co-labelled retrogradely-labelled sections for TH in the VMH, or for TH, orexin and melanin-concentrating hormone in the LH and DMH. However, we did not observe any colocalisation of immunoreactivity with any retrogradely-labelled cells. Viral mapping in TH-Cre rats confirms the existence of a reciprocal SuMM-DMH connection and shows that TH-positive cells project from the SuMM and VTA to the lateral septal area and cingulate cortex, respectively. These data provide evidence for the connectivity of the SuMM to brain regions involved in appetite control, and form the foundation for functional and behavioural studies aiming to further characterise the brain circuitry controlling eating behaviours.

Synergy of Distinct Dopamine Projection Populations in Behavioral Reinforcement.

Dopamine neurons of the ventral tegmental area (VTA) regulate reward association and motivation. It remains unclear whether there are distinct dopamine populations to mediate these functions. Using mouse genetics, we isolated two populations of dopamine-producing VTA neurons with divergent projections to the nucleus accumbens (NAc) core and shell. Inhibition of VTA-core-projecting neurons disrupted Pavlovian reward learning, and activation of these cells promoted the acquisition of an instrumental response. VTA-shell-projecting neurons did not regulate Pavlovian reward learning and could not facilitate acquisition of an instrumental response, but their activation could drive robust responding in a previously learned instrumental task. Both populations are activated simultaneously by cues, actions, and rewards, and this co-activation is required for robust reinforcement of behavior. Thus, there are functionally distinct dopamine populations in the VTA for promoting motivation and reward association, which operate on the same timescale to optimize behavioral reinforcement.

Why flavored vape products may be attractive: Green apple tobacco flavor elicits reward-related behavior, upregulates nAChRs on VTA dopamine neurons, and alters midbrain dopamine and GABA neuron function.

While nicotine is the primary addictive component in tobacco products, additional flavors have become a concern with the growing popularity of electronic nicotine delivery systems (ENDS). For this reason, we have begun to investigate popular tobacco and ENDS flavors. Here, we examined farnesol, a chemical flavorant used in green apple and fruit flavors in ENDS e-liquids, for its ability to produce reward-related behavior. Using male and female 3-6 month old C57BL/6 J mice and farnesol doses of 0.1, 1, and 10 mg/kg we identified a sex-dependent effect in a conditioned place preference assay: farnesol-alone produces reward-related behavior in only male mice. Despite this sex-dependent effect, 1.0 mg/kg farnesol enhances locomotor activity in both male and female mice. To understand farnesol's effect on reward-related behavior, we used whole-cell patch-clamp electrophysiology and confocal microscopy to investigate changes in putative dopamine and GABA neurons. For these approaches, we utilized genetically modified mice that contain fluorescent nicotinic acetylcholine receptors (nAChRs). Our electrophysiological assays with male mice revealed that farnesol treatment increases ventral tegmental area (VTA) dopamine neuron firing frequency and this may be due to a decrease in inhibitory tone from GABA neurons. Our microscopy assays revealed that farnesol treatment produces a significant upregulation of α6* nAChRs in male mice but not female mice. This was supported by an observed increase in α6* nAChR function in additional electrophysiology assays. These data provide evidence that popular tobacco flavorants may alter smoking-related behavior and promote the need to examine additional ENDS flavors.

Comparison between dopaminergic and non-dopaminergic neurons in the VTA following chronic nicotine exposure during pregnancy.

Exposure to nicotine during pregnancy through maternal smoking or nicotine replacement therapy is associated with adverse birth outcomes as well as several cognitive and neurobehavioral deficits. Several studies have shown that nicotine produces long-lasting effects on gene expression within many brain regions, including the ventral tegmental area (VTA), which is the origin of dopaminergic neurons and the dopamine reward pathway. Using a well-established rat model for perinatal nicotine exposure, we sought to investigate altered biological pathways using mRNA and miRNA expression profiles of dopaminergic (DA) and non-dopaminergic (non-DA) neurons in this highly-valuable area. Putative miRNA-gene target interactions were assessed as well as miRNA-pathway interactions. Our results indicate that extracellular matrix (ECM) receptor interactions were significantly altered in DA and non-DA neurons due to chronic nicotine exposure during pregnancy. They also show that the PI3K/AKT signaling pathway was enriched in DA neurons with multiple significant miRNA-gene targets, but the same changes were not seen in non-DA neurons. We speculate that nicotine exposure during pregnancy could differentially affect the gene expression of DA and non-DA neurons in the VTA.

Gene expression and neurochemical characterization of the rostromedial tegmental nucleus (RMTg) in rats and mice.

The rostromedial tegmental nucleus (RMTg), also known as the tail of the ventral tegmental area (tVTA), is a GABAergic structure identified in 2009 that receives strong inputs from the lateral habenula and other sources, sends dense inhibitory projections to midbrain dopamine (DA) neurons, and plays increasingly recognized roles in aversive learning, addiction, and other motivated behaviors. In general, little is known about the genetic identity of these neurons. However, recent work has identified the transcription factor FoxP1 as enhanced in the mouse RMTg (Lahti et al. in Development 143(3):516-529, 2016). Hence, in the current study, we used RNA sequencing to identify genes significantly enhanced in the rat RMTg as compared to adjacent VTA, and then examined the detailed distribution of two genes in particular, prepronociceptin (Pnoc) and FoxP1. In rats and mice, both Pnoc and FoxP1 were expressed at high levels in the RMTg and colocalized strongly with previously established RMTg markers. FoxP1 was particularly selective for RMTg neurons, as it was absent in most adjacent brain regions. We used these gene expression patterns to refine the anatomic characterization of RMTg in rats, extend this characterization to mice, and show that optogenetic manipulation of RMTg in mice bidirectionally modulates real-time place preference. Hence, RMTg neurons in both rats and mice exhibit distinct genetic profiles that correlate with their distinct connectivity and function.

Investigating the genetic profile of dopaminergic neurons in the VTA in response to perinatal nicotine exposure using mRNA-miRNA analyses.

Maternal smoking during pregnancy is associated with an increased risk of developmental, behavioral, and cognitive deficits. Nicotine, the primary addictive component in tobacco, has been shown to modulate changes in gene expression when exposure occurs during neurodevelopment. The ventral tegmental area (VTA) is believed to be central to the mechanism of addiction because of its involvement in the reward pathway. The purpose of this study was to build a genetic profile for dopamine (DA) neurons in the VTA and investigate the disruptions to the molecular pathways after perinatal nicotine exposure. Initially, we isolated the VTA from rat pups treated perinatally with either nicotine or saline (control) and collected DA neurons using fluorescent-activated cell sorting. Using microarray analysis, we profiled the differential expression of mRNAs and microRNAs from DA neurons in the VTA in order to explore potential points of regulation and enriched pathways following perinatal nicotine exposure. Furthermore, mechanisms of miRNA-mediated post-transcriptional regulation were investigated using predicted and validated miRNA-gene targets in order to demonstrate the role of miRNAs in the mesocorticolimbic DA pathway. This study provides insight into the genetic profile as well as biological pathways of DA neurons in the VTA of rats following perinatal nicotine exposure.

c-Fos marking of identified midbrain neurons coactive after nicotine administration in-vivo.

Despite the reduced life expectancy and staggering financial burden of medical treatment associated with tobacco smoking, the molecular, cellular, and ensemble adaptations associated with chronic nicotine consumption remain poorly understood. Complex circuitry interconnecting dopaminergic and cholinergic regions of the midbrain and mesopontine tegmentum are critical for nicotine associated reward. Yet our knowledge of the nicotine activation of these regions is incomplete, in part due to their cell type diversity. We performed double immunohistochemistry for the immediate early gene and surrogate activity sensor, c-Fos, and markers for either cholinergic, dopaminergic or GABAergic cell types in mice treated with nicotine. Both acute (0.5 mg/kg) and chronic (0.5 mg/kg/day for 7 days) nicotine strongly activated GABAergic neurons of the interpeduncular nucleus and medial terminal nucleus of the accessory optic tract (MT). Acute but not chronic nicotine also activated small percentages of dopaminergic and other neurons in the ventral tegmental area (VTA) as well as noncholinergic neurons in the pedunculotegmental and laterodorsal tegmental nuclei (PTg/LDTg). Twenty four hours of nicotine withdrawal after chronic nicotine treatment suppressed c-Fos activation in the MT. In comparison to nicotine, a single dose of cocaine caused a similar activation in the PTg/LDTg but not the VTA where GABAergic cells were strongly activated but dopaminergic neurons were not affected. These results indicate the existence of drug of abuse specific ensembles. The loss of ensemble activation in the VTA and PTg/LDTg after chronic nicotine represents a molecular and cellular tolerance which may have implications for the mechanisms underlying nicotine dependence.

Role of the mesolimbic dopamine system in relief learning.

The relief from an aversive event is rewarding. Since organisms are able to learn which environmental cues can cease an aversive event, relief learning helps to better cope with future aversive events. Literature data suggest that relief learning is affected in various psychopathological conditions, such as anxiety disorders. Here, we investigated the role of the mesolimbic dopamine system in relief learning. Using a relief learning procedure in Sprague Dawley rats, we applied a combination of behavioral experiments with anatomical tracing, c-Fos immunohistochemistry, and local chemogenetic and pharmacological interventions to broadly characterize the role of the mesolimbic dopamine system. The present study shows that a specific part of the mesolimbic dopamine system, the projection from the posterior medial ventral tegmental area (pmVTA) to the nucleus accumbens shell (AcbSh), is activated by aversive electric stimuli. 6-OHDA lesions of the pmVTA blocked relief learning but fear learning and safety learning were not affected. Chemogenetic silencing of the pmVTA-AcbSh projection using the DREADD approach, as well as intra-AcbSh injections of the dopamine D2/3 receptor antagonist raclopride inhibited relief learning. Taken together, the present data demonstrate that the dopaminergic pmVTA-AcbSh projection is critical for relief learning but not for similar learning phenomena. This novel finding may have clinical implications since the processing of signals predicting relief and safety is often impaired in patients suffering from anxiety disorders. Furthermore, it may help to better understand psychological conditions like non-suicidal self-injury, which are associated with pain offset relief.

Lateral septum stimulation disinhibits dopaminergic neurons in the antero-ventral region of the ventral tegmental area: Role of GABA-A alpha 1 receptors.

The mechanisms commanding the activity of dopaminergic neurons of the ventral tegmental area (VTA) and the location of these neurons are relevant for the coding and expression of motivated behavior associated to reward-related signals. Anatomical evidence shows that several brain regions modulate VTA dopaminergic neurons activity via multiple mechanisms. However, there is still scarce knowledge of how the lateral septum (LS) modulates VTA activity. We performed in-vivo dual-probe microdialysis to measure VTA dopamine, glutamate and GABA extracellular levels after LS stimulation in the presence or absence of GABAergic antagonists. Anterograde tracing and immunohistochemical analysis was used to reveal the anatomical relationship between LS and VTA. LS stimulation significantly increased dopamine and GABA, but not glutamate, VTA extracellular levels. Intra VTA infusion of bicuculline, GABA-A receptor antagonist, inhibited the increase of dopamine but not of GABA VTA levels induced by LS stimulation. Intra VTA infusion of indiplon, selective positive allosteric modulator of GABA-A receptors containing alpha1 subunit, significantly increases VTA dopamine extracellular levels induced by LS. Combined c-Fos and tyrosine hydroxylase immunohistochemistry, revealed that LS stimulation increases the activity of dopaminergic neurons in the antero-ventral region of the VTA. Consistently, anterograde tracing with biotinylated dextran amine revealed the existence of fibers arising from the LS to the antero-ventral region of the VTA. Taken together, our results suggest that LS modulates dopaminergic activity in the antero-ventral region of VTA by inhibiting GABAergic interneurons bearing GABA-A receptors containing alpha1 subunit.

Addiction-related interactions of pregabalin with morphine in mice and humans: reinforcing and inhibiting effects.

The gabapentinoid pregabalin is a rapid-acting anxiolytic and analgesic, possibly suitable in supervised opioid detoxification. However, clinicians have been cautious in using it because of its unknown addictive risk and rising number of mortalities after pregabalin self-medication in opioid abusers. Here, we studied interactions of pregabalin and morphine on reward functions of the dopamine system in mice and the efficacy of pregabalin on withdrawal in opioid addicts. After the treatment of mice with pregabalin and morphine, we used electrophysiology to study neuroplasticity in midbrain slices, self-administration and conditioned place preference tests to investigate the rewarding potential of pregabalin and naloxone-precipitated morphine withdrawal to evaluate opioid withdrawal symptoms. Further, we ran a pilot single-blind, randomized, controlled trial (34 heroin addicts) to evaluate the efficacy and safety of pregabalin in the treatment of opioid withdrawal syndrome. Pregabalin alone did not induce glutamate receptor neuroplasticity of dopamine neurons in the ventral tegmental area, but pre-treatment with pregabalin suppressed morphine-induced neuroplasticity, hyperlocomotion and morphine self-administration. Pregabalin administration after chronic morphine exposure failed to induce any rewarding effects. Instead, pregabalin suppressed withdrawal symptoms in both morphine-treated mice and opioid addicts and was well tolerated. Intriguingly, pregabalin administration after a low dose of morphine strongly facilitated ventral tegmental area neuroplasticity and led to increased conditioned place preference. Pregabalin appears to have the efficacy to counteract both reinforcing and withdrawal effects of opioids, but it also has a potentiating effect when given to mice with existing opioid levels.

Estradiol increases the sensitivity of ventral tegmental area dopamine neurons to dopamine and ethanol.

Gender differences in psychiatric disorders such as addiction may be modulated by the steroid hormone estrogen. For instance, 17ß-estradiol (E2), the predominant form of circulating estrogen in pre-menopausal females, increases ethanol consumption, suggesting that E2 may affect the rewarding properties of ethanol and thus the development of alcohol use disorder in females. The ventral tegmental area (VTA) is critically involved in the rewarding and reinforcing effects of ethanol. In order to determine the role of E2 in VTA physiology, gonadally intact female mice were sacrificed during diestrus II (high E2) or estrus (low E2) for electrophysiology recordings. We measured the excitation by ethanol and inhibition by dopamine (DA) of VTA DA neurons and found that both excitation by ethanol and inhibition by dopamine were greater in diestrus II compared with estrus. Treatment of VTA slices from mice in diestrus II with an estrogen receptor antagonist (ICI 182,780) reduced ethanol-stimulated neuronal firing, but had no effect on ethanol-stimulated firing of neurons in slices from mice in estrus. Surprisingly, ICI 182,780 did not affect the inhibition by DA, indicating different mechanisms of action of estrogen receptors in altering ethanol and DA responses. We also examined the responses of VTA DA neurons to ethanol and DA in ovariectomized mice treated with E2 and found that E2 treatment enhanced the responses to ethanol and DA in a manner similar to what we observed in mice in diestrus II. Our data indicate that E2 modulates VTA neuron physiology, which may contribute to both the enhanced reinforcing and rewarding effects of alcohol and the development of other psychiatric disorders in females that involve alterations in DA neurotransmission.

Eff ect of a single asenapine treatment on Fos expression in the brain catecholamine-synthesizing neurons: impact of a chronic mild stress preconditioning.

OBJECTIVE: Fos protein expression in catecholamine-synthesizing neurons of the substantia nigra (SN) pars compacta (SNC, A8), pars reticulata (SNR, A9), and pars lateralis (SNL), the ventral tegmental area (VTA, A10), the locus coeruleus (LC, A6) and subcoeruleus (sLC), the ventrolateral pons (PON-A5), the nucleus of the solitary tract (NTS-A2), the area postrema (AP), and the ventrolateral medulla (VLM-A1) was quantitatively evaluated aft er a single administration of asenapine (ASE) (designated for schizophrenia treatment) in male Wistar rats preconditioned with a chronic unpredictable variable mild stress (CMS) for 21 days. Th e aim of the present study was to reveal whether a single ASE treatment may 1) activate Fos expression in the brain areas selected; 2) activate tyrosine hydroxylase (TH)-synthesizing cells displaying Fos presence; and 3) be modulated by CMS preconditioning. METHODS: Control (CON), ASE, CMS, and CMS+ASE groups were used. CMS included restraint, social isolation, crowding, swimming, and cold. Th e ASE and CMS+ASE groups received a single dose of ASE (0.3 mg/kg, s.c.) and CON and CMS saline (300 µl/rat, s.c.). The animals were sacrificed 90 min aft er the treatments. Fos protein and TH-labeled immunoreactive perikarya were analyzed on double labeled histological sections and enumerated on captured pictures using combined light and fluorescence microscope illumination. RESULTS: Saline or CMS alone did not promote Fos expression in any of the structures investigated. ASE alone or in combination with CMS elicited Fos expression in two parts of the SN (SNC, SNR) and the VTA. Aside from some cells in the central gray tegmental nuclei adjacent to LC, where a small number of Fos profiles occurred, none or negligible Fos occurrence was detected in the other structures investigated including the LC and sLC, PON-A5, NTS-A2, AP, and VLM-A1. CMS preconditioning did not infl uence the level of Fos induction in the SN and VTA elicited by ASE administration. Similarly, the ratio between the amount of free Fos and Fos colocalized with TH was not aff ected by stress preconditioning in the SNC, SNR, and the VTA. CONCLUSIONS: Th e present study provides an anatomical/functional knowledge about the nature of the acute ASE treatment on the catecholamine-synthesizing neurons activity in certain brain structures and their missing interplay with the CMS preconditioning.

The Epac-Phospholipase Cε Pathway Regulates Endocannabinoid Signaling and Cocaine-Induced Disinhibition of Ventral Tegmental Area Dopamine Neurons.

Exchange protein directly activated by cAMP (Epac) is a direct effector for the ubiquitous second messenger cAMP. Epac activates the phospholipase Cε (PLCε) pathway. PLCß has been linked to the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Here, we report that Epac facilitates endocannabinoid-mediated retrograde synaptic depression through activation of PLCε. Intracellular loading of a selective Epac agonist 8-CPT-2Me-cAMP into ventral tegmental area (VTA) dopamine neurons enabled previously ineffective stimuli to induce depolarization-induced suppression of inhibition (DSI) and long-term depression of IPSCs (I-LTD) in the VTA. DSI and I-LTD are mediated by 2-AG since they were blocked by a diacylglycerol lipase inhibitor. The effects of 8-CPT-2Me-cAMP on DSI and I-LTD were absent in Epac2 and PLCε knock-out mice, but remained intact in Epac1 knock-out mice. These results identify a novel mechanism for on-demand synthesis of retrograde signaling 2-AG by the Epac2-PLCε pathway. We investigated the functional significance of Epac2-PLCε-2-AG signaling in regulating inhibitory synaptic plasticity in VTA dopamine neurons induced by in vivo cocaine exposure. We showed that cocaine place conditioning led to a decrease in the frequency and amplitude of spontaneous IPSCs and an increase in action potential firing in wild-type mice, but not in Epac2 or PLCε knock-out mice. Together, these results indicate that the Epac2-PLCε-2-AG signaling cascade contributes to cocaine-induced disinhibition of VTA dopamine neurons.SIGNIFICANCE STATEMENT 2-arachidonoylglycerol (2-AG) is an endogenous cannabinoid that depresses synaptic transmission through stimulation of CB1 receptors. Among the six isoforms of phospholipase C (PLC; PLCß, PLCγ, PLCδ, PLCε, PLCζ, PLCη), only PLCß has been linked to 2-AG synthesis. Here we demonstrate that 8-CPT-2Me-cAMP, a selective agonist of the cAMP sensor protein Epac, enhances 2-AG-mediated synaptic depression in ventral tegmental area (VTA) dopamine neurons via activation of PLCε. These results identify a novel mechanism for 2-AG synthesis via activation of the Epac-PLCε pathway. Furthermore, we show that cocaine-induced conditioned place preference and disinhibition of VTA dopamine neurons were impaired in mice lacking Epac or PLCε. Thus, the Epac-PLCε signaling pathway contributes to cocaine-induced disinhibition of VTA dopamine neurons and formation of drug-associated memories.

Interactions between cannabidiol and Δ9-THC following acute and repeated dosing: Rebound hyperactivity, sensorimotor gating and epigenetic and neuroadaptive changes in the mesolimbic pathway.

The evidence base for the use of medical cannabis preparations containing specific ratios of cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) is limited. While there is abundant data on acute interactions between CBD and THC, few studies have assessed the impact of their repeated co-administration. We previously reported that CBD inhibited or potentiated the acute effects of THC dependent on the measure being examined at a 1:1 CBD:THC dose ratio. Further, CBD decreased THC effects on brain regions involved in memory, anxiety and body temperature regulation. Here we extend on these finding by examining over 15 days of treatment whether CBD modulated the repeated effects of THC on behaviour and neuroadaption markers in the mesolimbic dopamine pathway. After acute locomotor suppression, repeated THC caused rebound locomotor hyperactivity that was modestly inhibited by CBD. CBD also slightly reduced the acute effects of THC on sensorimotor gating. These subtle effects were found at a 1:1 CBD:THC dose ratio but were not accentuated by a 5:1 dose ratio. CBD did not alter the trajectory of enduring THC-induced anxiety nor tolerance to the pharmacological effects of THC. There was no evidence of CBD potentiating the behavioural effects of THC. However we demonstrated for the first time that repeated co-administration of CBD and THC increased histone 3 acetylation (H3K9/14ac) in the VTA and ΔFosB expression in the nucleus accumbens. These changes suggest that while CBD may have protective effects acutely, its long-term molecular actions on the brain are more complex and may be supradditive.

Investigating the Effect of Perinatal Nicotine Exposure on Dopaminergic Neurons in the VTA Using miRNA Expression Profiles.

Maternal smoking during pregnancy is associated with developmental, cognitive, and behavioral disorders, including low birth weight, attention deficit hyperactivity disorder, learning disabilities, and drug abuse later in life. Nicotine activates the reward-driven behavior characteristic of drug abuse. Dopaminergic (DA) neurons originating from the ventral tegmental area (VTA) of the brain, which are stimulated by nicotine and other stimuli, are widely implicated in the natural reward pathway that is known to contribute to addiction. In recent years, microRNAs have been implicated in disrupting regulatory mechanisms due to their capability of targeting multiple genes and thus inducing downstream effects along many pathways. In order to investigate miRNA expression of dopaminergic neurons from the VTA, we employed patch clamping to identify and harvest both DA and non-DA neurons from rats perinatally exposed to nicotine for use in single-cell RT-qPCR. Our data indicated that miR-140-5p and miR-140-3p were upregulated in DA neurons; while miR-140-3p and miR-212 were differentially expressed in non-DA neurons. A functional enrichment analysis was also performed on our miRNA-gene prediction network and predicted that our miRNAs target genes involved in drug response and neuroplasticity.