Dopamine cells within the ventral tegmental area of the brain are involved in motivation and reward. Drugs of abuse target these dopamine cells altering their activity and plasticity resulting in addiction. While dopamine cell activity is primarily involved in addiction, the GABA neurons in the VTA have also been shown to have an indirect role. By decreasing the activity of the inhibitory GABA inputs onto dopamine neurons abusive drugs can indirectly increase dopamine cell activity resulting in addictive behaviors. However, although GABA neurons are important in the perception of reward, much less is known about how the excitatory inputs to these cells are regulated and possibly altered by drugs of abuse. Using transgenic mice expressing GFP attached to the GAD promoter, GABA cells were located and patched using whole cell voltage clamp and EPSCs were measured. High frequency stimulation induced LTD of the excitatory inputs to GABA neurons. The endocannabinoid analogue R- methanandamide also induced LTD at these excitatory synapses. These results suggest that endocannabinoids could potentially regulate the activity of GABA cells and as a result the activity of dopamine neurons. The endocannabinoid receptor involved is likely CB1, but not TRPV1 as only the CB1 antagonist AM-251 blocked this high frequency stimulus-induced LTD. Future research could then determine if the pathways involved in this LTD could potentially be altered by drugs of abuse contributing to addiction.

The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system leads to the rewarding properties of alcohol and nicotine (NIC). The mesolimbic DA system consists of DA neurons in the midbrain ventral tegmental area (VTA) that innervate the nucleus accumbens (NAc). DA neurotransmission is regulated by inhibitory VTA GABA neurons, whose excitability is a net effect of glutamate (GLU) and GABA neurotransmission that are modulated by NIC cholinergic receptors (nAChRs) on afferent terminals. We have previously demonstrated that VTA GABA neurons are excited by low-dose ethanol but are inhibited by moderate to high-dose ethanol, and they adapt to chronic ethanol, evincing marked hyperexcitability during withdrawal. The aim of this study was to evaluate the role of α6 nAChRs in ethanol effects on VTA GABA and DA neurons. In order to more conclusively demonstrate the role of α6 nAChRs in alcohol modulation in the VTA, we profiled the pharmacological interactions between ethanol and α6 nAChRs using recombinant α6 nAChRs in human epithelia (SH-EP1) cells and evaluated the effects of α6 nAChR antagonists on ethanol inhibition of GABA-mediated synaptic responses in dissociated GABA neurons of the VTA by recording mIPSCs; and assessed the effects of α6 nAChR antagonists on ethanol inhibition of VTA neurons, via eIPSCs on GABA neurons, sIPSCs on GABA neurons, and firing rate of DA neurons. We found that ethanol enhanced NIC currents in SH-EP1 cells via α6 nAChRs. Electrophysiology studies showed that superfusion of ethanol (5-30 mM) enhanced the frequency and amplitude of mIPSCs recorded in acutely dissociated VTA GABA neurons from GAD-GFP mice. Furthermore, the α6 nAChR antagonist α-conotoxin P1A (10 nM) prevented the ethanol-induced changes in mIPSC. In support, eIPSC experiments demonstrated that low doses of ethanol (1-5 mM) enhanced eIPSC peaks and decreased paired-pulse ratio, suggesting a presynaptic effect with ethanol. Alpha-conotoxin MII (α-CTX MII) blocked ethanol's effects on eIPSCs. This effect on VTA GABA neurons was also seen in sIPSCs, as ethanol decreased GABA firing rate. Similarly, the inhibition caused by ethanol was prevented by α-conotoxin P1A (10 nM). Additionally, CPP studies showed that α6 KO mice and WT mice treated with MEC, a non-competitive, non-α7 antagonist, did not show a preference for EtOH compartments that was found in WT mice. Taken together, these studies indicate that ethanol is acting through α6 nAChRs on GABA terminals to enhance GABA release, suggesting a possible mechanism of action of alcohol and nicotine co-dependence. Through these studies conducted to understand the role of α6 nAChRs in ethanol modulation, we hope to further outline how alcohol alters brain activity so that we can ultimately facilitate the development of therapies/medications for the treatment of alcoholism.

La queue de l'aire tegmentale ventrale (tVTA) est une région cérébrale GABAergique localisée en arrière de la VTA. Elle projette de façon massive aux neurones dopaminergiques des groupes A9 et A10 du mésencéphale à l'origine des systèmes nigrostrié et mésolimbique. Ces systèmes sont impliqués dans de nombreuses fonctions comme la motricité et les comportements associés aux drogues. L'objectif de ma thèse est d'étudier le rôle de la tVTA via ses projections sur ces systèmes. Pour cela, nous avons utilisé des approches variées comprenant l'immunohistochimie, la pharmacologie in vivo,l'électrophysiologie in vivo et l'étude du comportement moteur. Parmi un ensemble de drogues appartenant à différentes classes, l'activation moléculaire de la tVTA sous forme de l'induction de FosB/ΔFosB est spécifique des psychostimulants et dépend de la dopamine. D'un point de vue physiologique, la tVTA exerce un tonus inhibiteur sur les neurones dopaminergiques de la VTA et joue un rôle crucial dans leur désinhibition par la morphine. Enfin, des lésions de la tVTA influencent des comportements dépendant du système nigrostrié comme le comportement de rotation provoqué par l'amphétamine, les performances motrices et l'apprentissage moteur. / The tail of the ventral tegmenta area (tVTA) is a GABAergic brain region located behind the VTA. It projects massively to dopaminergic neurons in mesencephalic A9 and A10 groups leading to nigrostriatal and mesolimbic systems that play a role in fonctions like motricity and drug-related behaviours. The objective of my thesis is to study the tVTA role through its projections to these systems. For that, we used various approaches including immunohistochemistry, in vivo pharmacology, in vivo electrophysiology, and motor behaviour. Between various drugs belonging to different classes, molecular activation of the tVTA by FosB/ΔFosB induction is only observed following psychostimulant treatment and is mediated by dopamine. From a physiological point of view, the tVTA exerts an inhibitory tone onto VTA dopamine neurons and plays a crucial role in morphine-induced desinhibition. Finally, tVTA lesions modulate nigrostriatal system mediated behaviour like amphetamine-induced rotational behaviour, motor coordination and motor skill learning.

De nombreuses études ont établi que la majorité des neurones libèrent plus qu’une substance chimique. Il est bien connu que les neurones peuvent co-exprimer et co-libérer des neuropeptides en plus de leur neurotransmetteur, mais des évidences de la co-libération de deux petits neurotransmetteurs à action rapide se sont accumulées récemment. Des enregistrements électrophysiologiques ont aussi montré que des neurones sérotoninergiques et dopaminergiques isolés peuvent libérer du glutamate quand ils sont placés en culture. De plus, la présence de glutamate et de glutaminase a été détectée dans des neurones sérotoninergiques, dopaminergiques et noradrénergiques par immunomarquage sur des tranches de cerveau. Malheureusement, en considérant le rôle métabolique du glutamate, sa détection immunologique n’est pas suffisante pour assurer le phénotype glutamatergique d’un neurone. Récemment, la découverte de trois transporteurs vésiculaires du glutamate (VGLUT1-3) a grandement facilité l’identification des neurones glutamatergiques. Ces transporteurs sont nécessaires pour la libération de glutamate et constituent les premiers marqueurs morphologiques du phénotype glutamatergique. Il a été démontré que des neurones noradrénergiques expriment VGLUT2 et que des neurones sérotoninergiques expriment VGLUT3. Mais aucune évidence d’expression d’un des sous-types de VGLUT n’a été reportée pour les neurones dopaminergiques. Le but de notre travail était d’identifier quel sous-type de VGLUT est exprimé par les neurones dopaminergiques mésencéphaliques, et de déterminer si le phénotype glutamatergique de ces neurones peut être modulé dans des conditions particulières. Premièrement, nous avons utilisé des microcultures pour isoler les neurones dopaminergiques et des doubles marquages immunocytochimiques pour observer l’expression de VGLUT dans les neurones positifs pour la tyrosine hydroxylase (TH). Nous avons montré que la majorité (80%) des neurones TH+ isolés exprime spécifiquement VGLUT2. Cette expression est précoce au cours du développement in vitro et limitée aux projections axonales des neurones dopaminergiques. Toutefois, cette forte expression in vitro contraste avec la non-détection de ce transporteur dans les rats adultes in vivo. Nous avons décidé ensuite de regarder si l’expression de VGLUT2 pouvait être régulée pendant le développement cérébral de jeunes rats et sous des conditions traumatiques, par double hybridation in situ. Entre 14 et 16 jours embryonnaires, les marquages de VGLUT2 et de TH montraient une superposition significative qui n’était pas retrouvée à des stades ultérieurs. Dans le mésencéphale de jeunes rats postnataux, nous avons détecté l’ARNm de VGLUT2 dans environs 1-2% des neurones exprimant l’ARNm de TH dans la substance noire et l’aire tegmentaire ventrale (ATV). Pour explorer la régulation de l’expression de VGLUT2 dans des conditions traumatiques, nous avons utilisé la 6-hydroxydopamine (6-OHDA) pour léser les neurones dopaminergiques dans les jeunes rats. Dix jours après la chirurgie, nous avons trouvé que 27% des neurones dopaminergiques survivants dans l’ATV exprimaient l’ARNm de VGLUT2 dans les rats 6-OHDA. Finalement, nous avons observé la colocalisation de la protéine VGLUT2 dans les terminaisons TH positives par microscopie électronique. Dans les rats normaux, la protéine VGLUT2 est retrouvée dans 28% des terminaisons axonales TH dans le noyau accumbens. Dans les rats lésés à la 6-OHDA, nous avons observé une diminution considérable des terminaisons TH positives, et une augmentation dans la proportion (37%) des terminaisons dopaminergiques présentant du VGLUT2. Nos résultats suggèrent que le phénotype glutamatergique des neurones dopaminergiques est régulé au cours du développement, peut être réactivé dans des états pathologiques, et que ces neurones peuvent libérer du glutamate dans conditions spécifiques. / Numerous studies have established that the majority of neurons release more than one chemical substance. It is well known that neurons can co-express and co-release neuropeptides in addition to their neurotransmitter, but evidence of co-release of two small and fast-acting neurotransmitters has been accumulated recently. Electrophysiological recordings have also shown that isolated serotonine and dopamine neurons can release glutamate as a co-transmitter when they are placed in culture. Furthermore, the presence of glutamate and glutaminase has been detected in serotonine, dopamine and noradrenaline neurons by immunolabelling in brain slices. Unfortunately, considering the metabolic role of glutamate, its immunodetection is not sufficient to assert the glutamatergic phenotype of a neuron. Recently, the discovery of three vesicular glutamate transporters (VGLUT1-3) has greatly facilitated the identification of glutamate neurons. These transporters are necessary for the glutamate release by neurons and constitute the first molecular markers of a glutamatergic phenotype. Interestingly, it was demonstrated that some noradrenaline neurons express VGLUT2 and that some serotonin neurons express VGLUT3. But no evidence for expression of any VGLUT subtypes was initially reported for dopamine neurons. The goal of our work was to identify which VGLUT subtype is expressed by mesencephalic dopamine neurons, and to determine if the glutamatergic phenotype of these neurons can be modulated under specific conditions. First, we used microcultures to isolate dopamine neurons and double immunocytochemistry to visualize VGLUT expression in tyrosine hydroxylase (TH) positive neurons. We showed that the majority (80%) of isolated TH+ neurons express specifically VGLUT2. This expression occurred early during in vitro development and was limited to axonal projections of dopamine neurons. However, this strong expression in vitro contrasted with the lack of detection of this transporter in adult rats in vivo. We next decided to investigate if VGLUT2 expression could be regulated during brain development of young rats and under traumatic conditions, using double in situ hybridization. At embryonic days 14 to 16, VGLUT2 and TH labelling displayed significant overlap which was no longer found at later stages. In postnatal mesencephalon of young rats, we detected VGLUT2 mRNA in approximately 1-2% of neurons expressing TH mRNA in the substantia nigra and in ventral tegmental area (VTA). To explore the regulation of VGLUT2 expression under traumatic condition, we used 6-hydroxydopamine (6-OHDA) to damage dopamine neurons in young rats. Ten days post-surgery, we found that 27% of surviving dopamine neurons in the VTA expressed VGLUT2 mRNA in 6-OHDA animals. Finally, we observed the colocalisation of VGLUT2 protein in TH positive terminals by electron microscopy. In normal rats, VGLUT2 protein was found in 28% of TH positive axon terminals in nucleus accumbens. In 6 OHDA-lesioned rats, we observed a considerable reduction of TH positive terminals, and an increase in the proportion (37%) of dopamine terminals displaying VGLUT2. Our results suggest that the glutamatergic phenotype of dopamine neurons is developmentally regulated, can be reactivated under pathological states, and that these neurons are able to release glutamate under specific conditions.

The involvement of dopamine (DA) in the emotional and psychiatric disturbances associated with schizophrenia and psychomotor stimulant abuse is well known; however, the mechanism by which DA mediates fear expression and anxiety is not well defined. Accordingly, the objective of the present thesis was to determine the fear-motivational functions of DA neurons in the ventral tegmental area (VTA) and to examine the role of DA in fear extinction using the potentiated startle paradigm. In Experiment 1, it was observed that electrical stimulation of the VTA produced a pronounced increase in the amplitude of the acoustic startle reflex. In subsequent experiments fear-potentiated startle was assessed following axon-sparing N-methyl-D-aspartic acid (NMDA) lesions of the VTA and after bilateral intra-VTA infusion of the DA D_2/3 receptor agonist quinpirole (Experiments 2-4). The NMDA lesions resulted in substantial cell loss in the medial ventral tegmentum and blocked fear-potentiated startle. Similarly, inhibition of DA neuronal activity associated with locally-administered quinpirole suppressed the expression of the conditioned fear-induced increase in startle amplitudes. The quinpirole results implicate DA neuronal functioning in fear motivation. To explore further the involvement of DA in aversive emotional behavior, pharmacological experiments were conducted in which the effects of peripherally-administered DA agonist drugs on fear extinction were assessed. Subjects in Experiment 5 received an acute injection of either cocaine hydrochloride (40.0 mg/kg), d-amphetamine sulphate (5.0 mg/kg), the D_2/3 agonist quinpirole hydrochloride (5.0 mg/kg), or the D₁-type agonist SKF 38393 (5.0 mg/kg) during a single extinction session following fear acquisition. Animals treated with cocaine, d-amphetamine, and SKF 38393 exhibited fear-potentiated startle, whereas quinpirole treatment failed to alter fear extinction to the nonreinforced conditioned stimulus (CS). Also, it was revealed using a within-subjects design in Experiment 6 that cocaine administration reinstated fear-potentiated startle following extinction. Taken together, the results of the present experiments suggests fundamental role for DA and DA D₁ receptors in fear expression. It was proposed that VTA DA neurons gate levels of aversive emotional arousal within the amygdala-based fear system.

<p>Dopamine regulates movement, cognition and the rewarding effects of addictive drugs. Sex differences mediated by gonadal hormones affect each of these processes. An extensive literature suggests that estrogen augments dopaminergic function. Our laboratory found that female rats exhibit increased locomotor stimulation in response to cocaine and greater cocaine-induced dopamine overflow compared to males, sex differences that emerge in early adulthood. Currently, the underlying mechanisms for these differences are poorly understood. I hypothesized that female rats would have more dopamine neurons in midbrain regions and that ovarian hormones would exert trophic effects on dopamine neurons. Immunohistochemical and stereological techniques were used to quantitate the number of cells in the SNpc and VTA of male and female rats and mice to assess: (1) if sex differences in dopamine neuron number exist and when they emerge, (2) how gonadal hormones influence dopaminergic cell number and dopamine-mediated behaviors (3) the role of specific hormone receptors in the effects on cell number (4) the possibility that dopamine neuron number is directly linked to cocaine-stimulated behavior and electrically-stimulated dopamine release and that these responses to cocaine are mediated through gonadal hormone modulation of midbrain dopamine neuron number. I discovered sex differences in midbrain dopamine neuron number; adult female rodents have more neurons in the SNpc and VTA. We also found that gonadectomy in adulthood reduced midbrain dopamine neuron number in females and increased neuron number in males, establishing the trophic effects of estrogen in the intact midbrain and possible suppressive effects of androgens. Treatment with agonists for estrogen receptor subtypes alpha and beta and androgen receptor reversed the effects of gonadectomy on cell number in females and males, respectively. In an effort to bridge cocaine-stimulated behavior and cell number in sham ovariectomized and ovariectomized females, we discovered cocaine-stimulated behavior, dopamine release and SNpc cell density were positively correlated in intact female rats, an effect that is lost with ovariectomy. This dissertation demonstrates that estrogen is critical for the maintenance of dopaminergic cell populations that enhance behavioral responses to psychostimulants in females, thereby contributing to the observed sex differences.</p> / Dissertation

Environmental experiences play a critical role in an individualʼs risk of becoming addicted. Positive experiences may mitigate addiction vulnerability, whereas adverse experiences, particularly during adolescence, have been shown to increase addiction risk. Social isolation in rodents is a model system used to study the effects of such experiences, yet its impact on the learning and memory processes that underlie addiction remains elusive. Although social isolation is known to alter the functioning of the dopaminergic system, as well as reward processing and learning, its effect on dopamine (DA) neurons of the ventral tegmental area (VTA) is unknown. The data presented in this dissertation demonstrate that social isolation of rats during a critical period in adolescence (postnatal days 21-42) enhances long-term potentiation (LTP) of N-methyl D-aspartate receptor (NMDAR)-mediated glutamatergic transmission in the VTA. Activation of NMDARs is critical to the generation of DA neuron bursts that encode rewards and reward-predictive cues, and NMDARs are necessary for associative reward learning. The isolation-induced enhancement of NMDAR LTP results from augmentation of metabotropic glutamate receptor (mGluR)-dependent calcium (Ca²⁺) signaling via an increase in inositol 1,4,5-trisphosphate(IP3) sensitivity. Isolation-mediated effects on Ca²⁺ signaling and NMDAR plasticity were not reversed by a subsequent period of resocialization. Furthermore, social isolation during this critical period occludes the effect of repeated amphetamine exposure on mGluR/IP₃-mediated Ca²⁺ signaling and synaptic plasticity. Although corticotropin releasing factor (CRF) further facilitates mGluR/IP3-mediated Ca²⁺ signaling in DA neurons, alterations in CRF receptors are not responsible for the effects of isolation on Ca²⁺ signaling and synaptic plasticity. In addition, the learning of associations between environmental stimuli and drug rewards is acquired more quickly and is more resistant to extinction in isolated animals. Data presented in this dissertation lend support to the theory that enhanced mGluR/IP₃-mediated Ca²⁺ signaling and NMDAR plasticity facilitate the learning and memory of drug-associated stimuli. This dissertation provides the first demonstration of a cellular basis for the critical time window of social isolation during adolescence. NMDAR plasticity in the VTA may thus represent a neural substrate by which early life experiences regulate addiction vulnerability. (Note: Behavioral data were acquired by Mickael Degoulet) / text

De nombreuses études ont établi que la majorité des neurones libèrent plus qu’une substance chimique. Il est bien connu que les neurones peuvent co-exprimer et co-libérer des neuropeptides en plus de leur neurotransmetteur, mais des évidences de la co-libération de deux petits neurotransmetteurs à action rapide se sont accumulées récemment. Des enregistrements électrophysiologiques ont aussi montré que des neurones sérotoninergiques et dopaminergiques isolés peuvent libérer du glutamate quand ils sont placés en culture. De plus, la présence de glutamate et de glutaminase a été détectée dans des neurones sérotoninergiques, dopaminergiques et noradrénergiques par immunomarquage sur des tranches de cerveau. Malheureusement, en considérant le rôle métabolique du glutamate, sa détection immunologique n’est pas suffisante pour assurer le phénotype glutamatergique d’un neurone. Récemment, la découverte de trois transporteurs vésiculaires du glutamate (VGLUT1-3) a grandement facilité l’identification des neurones glutamatergiques. Ces transporteurs sont nécessaires pour la libération de glutamate et constituent les premiers marqueurs morphologiques du phénotype glutamatergique. Il a été démontré que des neurones noradrénergiques expriment VGLUT2 et que des neurones sérotoninergiques expriment VGLUT3. Mais aucune évidence d’expression d’un des sous-types de VGLUT n’a été reportée pour les neurones dopaminergiques. Le but de notre travail était d’identifier quel sous-type de VGLUT est exprimé par les neurones dopaminergiques mésencéphaliques, et de déterminer si le phénotype glutamatergique de ces neurones peut être modulé dans des conditions particulières. Premièrement, nous avons utilisé des microcultures pour isoler les neurones dopaminergiques et des doubles marquages immunocytochimiques pour observer l’expression de VGLUT dans les neurones positifs pour la tyrosine hydroxylase (TH). Nous avons montré que la majorité (80%) des neurones TH+ isolés exprime spécifiquement VGLUT2. Cette expression est précoce au cours du développement in vitro et limitée aux projections axonales des neurones dopaminergiques. Toutefois, cette forte expression in vitro contraste avec la non-détection de ce transporteur dans les rats adultes in vivo. Nous avons décidé ensuite de regarder si l’expression de VGLUT2 pouvait être régulée pendant le développement cérébral de jeunes rats et sous des conditions traumatiques, par double hybridation in situ. Entre 14 et 16 jours embryonnaires, les marquages de VGLUT2 et de TH montraient une superposition significative qui n’était pas retrouvée à des stades ultérieurs. Dans le mésencéphale de jeunes rats postnataux, nous avons détecté l’ARNm de VGLUT2 dans environs 1-2% des neurones exprimant l’ARNm de TH dans la substance noire et l’aire tegmentaire ventrale (ATV). Pour explorer la régulation de l’expression de VGLUT2 dans des conditions traumatiques, nous avons utilisé la 6-hydroxydopamine (6-OHDA) pour léser les neurones dopaminergiques dans les jeunes rats. Dix jours après la chirurgie, nous avons trouvé que 27% des neurones dopaminergiques survivants dans l’ATV exprimaient l’ARNm de VGLUT2 dans les rats 6-OHDA. Finalement, nous avons observé la colocalisation de la protéine VGLUT2 dans les terminaisons TH positives par microscopie électronique. Dans les rats normaux, la protéine VGLUT2 est retrouvée dans 28% des terminaisons axonales TH dans le noyau accumbens. Dans les rats lésés à la 6-OHDA, nous avons observé une diminution considérable des terminaisons TH positives, et une augmentation dans la proportion (37%) des terminaisons dopaminergiques présentant du VGLUT2. Nos résultats suggèrent que le phénotype glutamatergique des neurones dopaminergiques est régulé au cours du développement, peut être réactivé dans des états pathologiques, et que ces neurones peuvent libérer du glutamate dans conditions spécifiques. / Numerous studies have established that the majority of neurons release more than one chemical substance. It is well known that neurons can co-express and co-release neuropeptides in addition to their neurotransmitter, but evidence of co-release of two small and fast-acting neurotransmitters has been accumulated recently. Electrophysiological recordings have also shown that isolated serotonine and dopamine neurons can release glutamate as a co-transmitter when they are placed in culture. Furthermore, the presence of glutamate and glutaminase has been detected in serotonine, dopamine and noradrenaline neurons by immunolabelling in brain slices. Unfortunately, considering the metabolic role of glutamate, its immunodetection is not sufficient to assert the glutamatergic phenotype of a neuron. Recently, the discovery of three vesicular glutamate transporters (VGLUT1-3) has greatly facilitated the identification of glutamate neurons. These transporters are necessary for the glutamate release by neurons and constitute the first molecular markers of a glutamatergic phenotype. Interestingly, it was demonstrated that some noradrenaline neurons express VGLUT2 and that some serotonin neurons express VGLUT3. But no evidence for expression of any VGLUT subtypes was initially reported for dopamine neurons. The goal of our work was to identify which VGLUT subtype is expressed by mesencephalic dopamine neurons, and to determine if the glutamatergic phenotype of these neurons can be modulated under specific conditions. First, we used microcultures to isolate dopamine neurons and double immunocytochemistry to visualize VGLUT expression in tyrosine hydroxylase (TH) positive neurons. We showed that the majority (80%) of isolated TH+ neurons express specifically VGLUT2. This expression occurred early during in vitro development and was limited to axonal projections of dopamine neurons. However, this strong expression in vitro contrasted with the lack of detection of this transporter in adult rats in vivo. We next decided to investigate if VGLUT2 expression could be regulated during brain development of young rats and under traumatic conditions, using double in situ hybridization. At embryonic days 14 to 16, VGLUT2 and TH labelling displayed significant overlap which was no longer found at later stages. In postnatal mesencephalon of young rats, we detected VGLUT2 mRNA in approximately 1-2% of neurons expressing TH mRNA in the substantia nigra and in ventral tegmental area (VTA). To explore the regulation of VGLUT2 expression under traumatic condition, we used 6-hydroxydopamine (6-OHDA) to damage dopamine neurons in young rats. Ten days post-surgery, we found that 27% of surviving dopamine neurons in the VTA expressed VGLUT2 mRNA in 6-OHDA animals. Finally, we observed the colocalisation of VGLUT2 protein in TH positive terminals by electron microscopy. In normal rats, VGLUT2 protein was found in 28% of TH positive axon terminals in nucleus accumbens. In 6 OHDA-lesioned rats, we observed a considerable reduction of TH positive terminals, and an increase in the proportion (37%) of dopamine terminals displaying VGLUT2. Our results suggest that the glutamatergic phenotype of dopamine neurons is developmentally regulated, can be reactivated under pathological states, and that these neurons are able to release glutamate under specific conditions.

abstract: Intermittent social defeat stress induces cross-sensitization to psychostimulants and escalation of drug self-administration. These behaviors could result from the stress-induced neuroadaptation in the mesocorticolimbic dopamine circuit. Brain-derived neurotrophic factor (BDNF) in the ventral tegmental area (VTA) is persistently elevated after social defeat stress, and may contribute to the stress-induced neuroadaptation in the mesocorticolimbic dopamine circuit. BDNF modulates synaptic plasticity, and facilitates stress- and drug-induced neuroadaptations in the mesocorticolimbic system. The present research examined the role of mesolimbic BDNF signaling in social defeat stress-induced cross-sensitization to psychostimulants and the escalation of cocaine self-administration in rats. We measured drug taking behavior with the acquisition, progressive ratio, and binge paradigms during self-administration. With BDNF overexpression in the ventral tegmental area (VTA), single social defeat stress-induced cross-sensitization to amphetamine (AMPH) was significantly potentiated. VTA-BDNF overexpression also facilitates acquisition of cocaine self-administration, and a positive correlation between the level of VTA BDNF and drug intake during 12 hour binge was observed. We also found significant increase of DeltaFosB expression in the nucleus accumbens (NAc), the projection area of the VTA, in rats received intra-VTA BDNF overexpression. We therefore examined whether BDNF signaling in the NAc is important for social defeat stress-induced cross-sensitization by knockdown of the receptor of BDNF (neurotrophin tyrosine kinase receptor type 2, TrkB) there. NAc TrkB knockdown prevented social defeat stress-induced cross-sensitization to psychostimulant. Also social defeat stress-induced increase of DeltaFosB in the NAc was prevented by TrkB knockdown. Several other factors up-regulated by stress, such as the GluA1 subunit of Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and BDNF in the VTA were also prevented. We conclude that BDNF signaling in the VTA increases social defeat stress-induced vulnerability to psychostimulants, manifested as potentiated cross-sensitization/sensitization to AMPH and escalation of cocaine self-administration. Also BDNF signaling in the NAc is necessary for the stress-induced neuroadaptation and behavioral sensitization to psychostimulants. Therefore, TrkB in the NAc could be a therapeutic target to prevent stress-induced vulnerability to drugs of abuse in the future. DeltaFosB in the NAc shell could be a neural substrate underlying persistent cross-sensitization and augmented cocaine self-administration induced by social defeat stress. / Dissertation/Thesis / Ph.D. Neuroscience 2013

Maternal behavior is the complex array of caregiving behaviors females display towards offspring. In rats, the transition to motherhood depends on the action of various hormones, especially estradiol near parturition, which primes the maternal circuitry to respond to pups upon first encounter at parturition with appropriate maternal behavior. Although virgin rats avoid pups, new mothers are highly motivated to interact with pups, and their maternal behavior depends on the functional interaction between the medial preoptic area (mPOA) and the ventral tegmental area (VTA). However, a precise mapping of the VTA-projecting mPOA neurons remains to be elucidated. To determine whether pup-responsive neurons in the mPOA project to the VTA, we injected the retrograde tracer Fluorogold (FG) into the VTA of new mother and virgin female rats. Six days later, females were exposed to 3 pups for 5 minutes, and their brains processed to visualize FG and c-Fos immunostaining. In addition, we further characterized the molecular phenotype of these neurons by performing immunohistochemistry against estrogen receptor alpha (Esr1). As expected, the behavior of postpartum and virgin females toward pups was different. Mothers readily approached pups and displayed maternal behavior, whereas virgins avoided interaction with pups. Despite these disparate responses to pups, no differences were found in the number and distribution of mPOAc-Fos→VTA neurons. In addition, in both postpartum and virgin females, a significant proportion of these pup-responsive mPOA→VTA projecting neurons also express Esr1. Further functional interrogation of these c-Fos+/Esr1+ mPOA→VTA neurons in virgins and mothers might elucidate distinct circuit dynamics potentially underlying their behavioral differences towards pups.