Search

Background: Initiation of use/co-use of nicotine and alcohol, commonly occurring in an episodic manner during adolescence, can imprint vulnerability to the developing brain and lead to addiction. The ventral tegmental area (VTA) is a key heterogeneous region of the mesocorticolimbic circuit involved in the binge-drinking and intoxication step of the addiction circuit. Higher human post-mortem VTA expression of vesicular glutamate transporter 2 (VGLUT2), a marker of the glutamatergic phenotype also expressed in dopaminergic [Tyrosine Hydroxylase (Th)-positive] neurons, has been associated with chronic nicotine use and co-use with alcohol. Methods: The present study aimed to map and characterize the Vglut2- and Th-expressing neurons in the VTA of adolescent male rats exposed or not to prolonged (six-weeks) episodic (three consecutive days/week) nicotine and/or alcohol administration. Nicotine (0.35 mg/kg free base) was injected subcutaneously, whereas alcohol (2 g/kg 20%) was administrated via gavage. Vglut2 and Th mRNA was assessed in the anterior and posterior VTA by use of in situ hybridization. Results: The profile of neurons varied with substance-exposure among VTA subregions. Th-only expressing neurons were more abundant in the posterior VTA of the group exposed to nicotine-only, compared to controls. The same neurons were, on the contrary, less present in the anterior VTA of animals exposed to alcohol-only, who also displayed a higher number of Vglut2-expressing neurons in the lateral anterior VTA. Conclusions: VTA Vglut2- and Th-only neurons seem differentially involved in the effects of adolescent episodic nicotine and alcohol exposure in the anterior and posterior VTA.

Background: Initiation of use/co-use of nicotine and alcohol, commonly occurring in an episodic manner during adolescence, can imprint vulnerability to the developing brain and lead to addiction. The ventral tegmental area (VTA) is a key heterogeneous region of the mesocorticolimbic circuit involved in the binge-drinking and intoxication step of the addiction circuit. Higher human post-mortem VTA expression of vesicular glutamate transporter 2 (VGLUT2), a marker of the glutamatergic phenotype also expressed in dopaminergic [Tyrosine Hydroxylase (Th)-positive] neurons, has been associated with chronic nicotine use and co-use with alcohol. Methods: The present study aimed to map and characterize the Vglut2- and Th-expressing neurons in the VTA of adolescent male rats exposed or not to prolonged (six-weeks) episodic (three consecutive days/week) nicotine and/or alcohol administration. Nicotine (0.35 mg/kg free base) was injected subcutaneously, whereas alcohol (2 g/kg 20%) was administrated via gavage. Vglut2 and Th mRNA was assessed in the anterior and posterior VTA by use of in situ hybridization. Results: The profile of neurons varied with substance-exposure among VTA subregions. Th-only expressing neurons were more abundant in the posterior VTA of the group exposed to nicotine-only, compared to controls. The same neurons were, on the contrary, less present in the anterior VTA of animals exposed to alcohol-only, who also displayed a higher number of Vglut2-expressing neurons in the lateral anterior VTA. Conclusions: VTA Vglut2- and Th-only neurons seem differentially involved in the effects of adolescent episodic nicotine and alcohol exposure in the anterior and posterior VTA.

Background: Initiation of use/co-use of nicotine and alcohol, commonly occurring in an episodic manner during adolescence, can imprint vulnerability to the developing brain and lead to addiction. The ventral tegmental area (VTA) is a key heterogeneous region of the mesocorticolimbic circuit involved in the binge-drinking and intoxication step of the addiction circuit. Higher human post-mortem VTA expression of vesicular glutamate transporter 2 (VGLUT2), a marker of the glutamatergic phenotype also expressed in dopaminergic [Tyrosine Hydroxylase (Th)-positive] neurons, has been associated with chronic nicotine use and co-use with alcohol. Methods: The present study aimed to map and characterize the Vglut2- and Th-expressing neurons in the VTA of adolescent male rats exposed or not to prolonged (six-weeks) episodic (three consecutive days/week) nicotine and/or alcohol administration. Nicotine (0.35 mg/kg free base) was injected subcutaneously, whereas alcohol (2 g/kg 20%) was administrated via gavage. Vglut2 and Th mRNA was assessed in the anterior and posterior VTA by use of in situ hybridization. Results: The profile of neurons varied with substance-exposure among VTA subregions. Th-only expressing neurons were more abundant in the posterior VTA of the group exposed to nicotine-only, compared to controls. The same neurons were, on the contrary, less present in the anterior VTA of animals exposed to alcohol-only, who also displayed a higher number of Vglut2-expressing neurons in the lateral anterior VTA. Conclusions: VTA Vglut2- and Th-only neurons seem differentially involved in the effects of adolescent episodic nicotine and alcohol exposure in the anterior and posterior VTA.

Background: Initiation of use/co-use of nicotine and alcohol, commonly occurring in an episodic manner during adolescence, can imprint vulnerability to the developing brain and lead to addiction. The ventral tegmental area (VTA) is a key heterogeneous region of the mesocorticolimbic circuit involved in the binge-drinking and intoxication step of the addiction circuit. Higher human post-mortem VTA expression of vesicular glutamate transporter 2 (VGLUT2), a marker of the glutamatergic phenotype also expressed in dopaminergic [Tyrosine Hydroxylase (Th)-positive] neurons, has been associated with chronic nicotine use and co-use with alcohol. Methods: The present study aimed to map and characterize the Vglut2- and Th-expressing neurons in the VTA of adolescent male rats exposed or not to prolonged (six-weeks) episodic (three consecutive days/week) nicotine and/or alcohol administration. Nicotine (0.35 mg/kg free base) was injected subcutaneously, whereas alcohol (2 g/kg 20%) was administrated via gavage. Vglut2 and Th mRNA was assessed in the anterior and posterior VTA by use of in situ hybridization. Results: The profile of neurons varied with substance-exposure among VTA subregions. Th-only expressing neurons were more abundant in the posterior VTA of the group exposed to nicotine-only, compared to controls. The same neurons were, on the contrary, less present in the anterior VTA of animals exposed to alcohol-only, who also displayed a higher number of Vglut2-expressing neurons in the lateral anterior VTA. Conclusions: VTA Vglut2- and Th-only neurons seem differentially involved in the effects of adolescent episodic nicotine and alcohol exposure in the anterior and posterior VTA.

Background: Initiation of use/co-use of nicotine and alcohol, commonly occurring in an episodic manner during adolescence, can imprint vulnerability to the developing brain and lead to addiction. The ventral tegmental area (VTA) is a key heterogeneous region of the mesocorticolimbic circuit involved in the binge-drinking and intoxication step of the addiction circuit. Higher human post-mortem VTA expression of vesicular glutamate transporter 2 (VGLUT2), a marker of the glutamatergic phenotype also expressed in dopaminergic [Tyrosine Hydroxylase (Th)-positive] neurons, has been associated with chronic nicotine use and co-use with alcohol. Methods: The present study aimed to map and characterize the Vglut2- and Th-expressing neurons in the VTA of adolescent male rats exposed or not to prolonged (six-weeks) episodic (three consecutive days/week) nicotine and/or alcohol administration. Nicotine (0.35 mg/kg free base) was injected subcutaneously, whereas alcohol (2 g/kg 20%) was administrated via gavage. Vglut2 and Th mRNA was assessed in the anterior and posterior VTA by use of in situ hybridization. Results: The profile of neurons varied with substance-exposure among VTA subregions. Th-only expressing neurons were more abundant in the posterior VTA of the group exposed to nicotine-only, compared to controls. The same neurons were, on the contrary, less present in the anterior VTA of animals exposed to alcohol-only, who also displayed a higher number of Vglut2-expressing neurons in the lateral anterior VTA. Conclusions: VTA Vglut2- and Th-only neurons seem differentially involved in the effects of adolescent episodic nicotine and alcohol exposure in the anterior and posterior VTA.

A key function of the mesencephalic locomotor region (MLR) is to control the speed of forward symmetrical locomotor movements. However, the ability of freely moving mammals to integrate environmental cues to brake and turn during MLR stimulation is poorly documented. Here, we investigated whether freely behaving mice could brake or turn, based on environmental cues during MLR stimulation. We photostimulated the cuneiform nucleus (part of the MLR) in mice expressing channelrhodopsin in Vglut2-positive neurons in a Cre-dependent manner (Vglut2-ChR2-EYFP) using optogenetics. We detected locomotor movements using deep learning. We used patch-clamp recordings to validate the functional expression of channelrhodopsin and neuroanatomy to visualize the stimulation sites. In the linear corridor, gait diagram and limb kinematics were similar during spontaneous and optogenetic-evoked locomotion. In the open-field arena, optogenetic stimulation of the MLR evoked locomotion, and increasing laser power increased locomotor speed. Mice could brake and make sharp turns (~90°) when approaching a corner during MLR stimulation in the open-field arena. The speed during the turn was scaled with the speed before the turn, and with the turn angle. Patch-clamp recordings in Vglut2-ChR2-EYFP mice show that blue light evoked short-latency spiking in MLR neurons. Our results strengthen the idea that different brainstem neurons convey braking/turning and MLR speed commands in mammals. Our study also shows that Vglut2-positive neurons of the cuneiform nucleus are a relevant target to increase locomotor activity without impeding the ability to brake and turn when approaching obstacles, thus ensuring smooth and adaptable navigation. Our observations may have clinical relevance since cuneiform nucleus stimulation is increasingly considered to improve locomotion function in pathological states such as Parkinson’s disease, spinal cord injury, or stroke., Peer Reviewed

Dopamine (DA) neurons of the ventral tegmental area (VTA) continue to gain attention as far more heterogeneous than previously realized. Within the medial aspect of the VTA, the unexpected presence of TrpV1 mRNA has been identified. TrpV1 encodes the Transient Receptor Potential cation channel subfamily V member 1, TRPV1, also known as the capsaicin receptor, well recognized for its role in heat and pain processing by peripheral neurons. In contrast, the brain distribution of TrpV1 has been debated. Here, we hypothesized that the TrpV1+ identity defines a distinct subpopulation of VTA DA neurons. To explore these brain TrpV1+ neurons, histological analyses and Cre-driven mouse genetics were employed. TrpV1 mRNA was most strongly detected at the perinatal stage forming a band of scattered neurons throughout the medial VTA, reaching into the posterior hypothalamus. Within the VTA, the majority of TrpV1 co-localized with both Tyrosine hydroxylase (Th) and Vesicular monoamine transporter 2 (Vmat2), confirming a DA phenotype. However, TrpV1 also co-localized substantially with Vesicular glutamate transporter 2 (Vglut2), representing the capacity for glutamate (GLU) release. These TrpV1+/Th+/Vglut2+/Vmat2+ neurons thus constitute a molecularly and anatomically distinct subpopulation of DA-GLU co-releasing neurons. To assess behavioral impact, a TrpV1Cre-driven strategy targeting the Vmat2 gene in mice was implemented. This manipulation was sufficient to alter psychomotor behavior induced by amphetamine. The acute effect of the drug was accentuated above control levels, suggesting super-sensitivity in the drug-na ve state resembling a “pre-sensitized” phenotype. However, no progressive increase with repeated injections was observed. This study identifies a distinct TrpV1+ VTA subpopulation as a critical modulatory component in responsiveness to amphetamine. Moreover, expression of the gene encoding TRPV1 in selected VTA neurons opens up for new possibilities in pharmacolog

Dopamine (DA) neurons of the ventral tegmental area (VTA) continue to gain attention as far more heterogeneous than previously realized. Within the medial aspect of the VTA, the unexpected presence of TrpV1 mRNA has been identified. TrpV1 encodes the Transient Receptor Potential cation channel subfamily V member 1, TRPV1, also known as the capsaicin receptor, well recognized for its role in heat and pain processing by peripheral neurons. In contrast, the brain distribution of TrpV1 has been debated. Here, we hypothesized that the TrpV1+ identity defines a distinct subpopulation of VTA DA neurons. To explore these brain TrpV1+ neurons, histological analyses and Cre-driven mouse genetics were employed. TrpV1 mRNA was most strongly detected at the perinatal stage forming a band of scattered neurons throughout the medial VTA, reaching into the posterior hypothalamus. Within the VTA, the majority of TrpV1 co-localized with both Tyrosine hydroxylase (Th) and Vesicular monoamine transporter 2 (Vmat2), confirming a DA phenotype. However, TrpV1 also co-localized substantially with Vesicular glutamate transporter 2 (Vglut2), representing the capacity for glutamate (GLU) release. These TrpV1+/Th+/Vglut2+/Vmat2+ neurons thus constitute a molecularly and anatomically distinct subpopulation of DA-GLU co-releasing neurons. To assess behavioral impact, a TrpV1Cre-driven strategy targeting the Vmat2 gene in mice was implemented. This manipulation was sufficient to alter psychomotor behavior induced by amphetamine. The acute effect of the drug was accentuated above control levels, suggesting super-sensitivity in the drug-na ve state resembling a “pre-sensitized” phenotype. However, no progressive increase with repeated injections was observed. This study identifies a distinct TrpV1+ VTA subpopulation as a critical modulatory component in responsiveness to amphetamine. Moreover, expression of the gene encoding TRPV1 in selected VTA neurons opens up for new possibilities in pharmacolog

Dopamine (DA) neurons of the ventral tegmental area (VTA) continue to gain attention as far more heterogeneous than previously realized. Within the medial aspect of the VTA, the unexpected presence of TrpV1 mRNA has been identified. TrpV1 encodes the Transient Receptor Potential cation channel subfamily V member 1, TRPV1, also known as the capsaicin receptor, well recognized for its role in heat and pain processing by peripheral neurons. In contrast, the brain distribution of TrpV1 has been debated. Here, we hypothesized that the TrpV1+ identity defines a distinct subpopulation of VTA DA neurons. To explore these brain TrpV1+ neurons, histological analyses and Cre-driven mouse genetics were employed. TrpV1 mRNA was most strongly detected at the perinatal stage forming a band of scattered neurons throughout the medial VTA, reaching into the posterior hypothalamus. Within the VTA, the majority of TrpV1 co-localized with both Tyrosine hydroxylase (Th) and Vesicular monoamine transporter 2 (Vmat2), confirming a DA phenotype. However, TrpV1 also co-localized substantially with Vesicular glutamate transporter 2 (Vglut2), representing the capacity for glutamate (GLU) release. These TrpV1+/Th+/Vglut2+/Vmat2+ neurons thus constitute a molecularly and anatomically distinct subpopulation of DA-GLU co-releasing neurons. To assess behavioral impact, a TrpV1Cre-driven strategy targeting the Vmat2 gene in mice was implemented. This manipulation was sufficient to alter psychomotor behavior induced by amphetamine. The acute effect of the drug was accentuated above control levels, suggesting super-sensitivity in the drug-na ve state resembling a “pre-sensitized” phenotype. However, no progressive increase with repeated injections was observed. This study identifies a distinct TrpV1+ VTA subpopulation as a critical modulatory component in responsiveness to amphetamine. Moreover, expression of the gene encoding TRPV1 in selected VTA neurons opens up for new possibilities in pharmacolog

Dopamine (DA) neurons of the ventral tegmental area (VTA) continue to gain attention as far more heterogeneous than previously realized. Within the medial aspect of the VTA, the unexpected presence of TrpV1 mRNA has been identified. TrpV1 encodes the Transient Receptor Potential cation channel subfamily V member 1, TRPV1, also known as the capsaicin receptor, well recognized for its role in heat and pain processing by peripheral neurons. In contrast, the brain distribution of TrpV1 has been debated. Here, we hypothesized that the TrpV1+ identity defines a distinct subpopulation of VTA DA neurons. To explore these brain TrpV1+ neurons, histological analyses and Cre-driven mouse genetics were employed. TrpV1 mRNA was most strongly detected at the perinatal stage forming a band of scattered neurons throughout the medial VTA, reaching into the posterior hypothalamus. Within the VTA, the majority of TrpV1 co-localized with both Tyrosine hydroxylase (Th) and Vesicular monoamine transporter 2 (Vmat2), confirming a DA phenotype. However, TrpV1 also co-localized substantially with Vesicular glutamate transporter 2 (Vglut2), representing the capacity for glutamate (GLU) release. These TrpV1+/Th+/Vglut2+/Vmat2+ neurons thus constitute a molecularly and anatomically distinct subpopulation of DA-GLU co-releasing neurons. To assess behavioral impact, a TrpV1Cre-driven strategy targeting the Vmat2 gene in mice was implemented. This manipulation was sufficient to alter psychomotor behavior induced by amphetamine. The acute effect of the drug was accentuated above control levels, suggesting super-sensitivity in the drug-na ve state resembling a “pre-sensitized” phenotype. However, no progressive increase with repeated injections was observed. This study identifies a distinct TrpV1+ VTA subpopulation as a critical modulatory component in responsiveness to amphetamine. Moreover, expression of the gene encoding TRPV1 in selected VTA neurons opens up for new possibilities in pharmacolog

A key function of the mesencephalic locomotor region (MLR) is to control the speed of forward symmetrical locomotor movements. However, the ability of freely moving mammals to integrate environmental cues to brake and turn during MLR stimulation is poorly documented. Here, we investigated whether freely behaving mice could brake or turn, based on environmental cues during MLR stimulation. We photostimulated the cuneiform nucleus (part of the MLR) in mice expressing channelrhodopsin in Vglut2-positive neurons in a Cre-dependent manner (Vglut2-ChR2-EYFP) using optogenetics. We detected locomotor movements using deep learning. We used patch-clamp recordings to validate the functional expression of channelrhodopsin and neuroanatomy to visualize the stimulation sites. In the linear corridor, gait diagram and limb kinematics were similar during spontaneous and optogenetic-evoked locomotion. In the open-field arena, optogenetic stimulation of the MLR evoked locomotion, and increasing laser power increased locomotor speed. Mice could brake and make sharp turns (~90°) when approaching a corner during MLR stimulation in the open-field arena. The speed during the turn was scaled with the speed before the turn, and with the turn angle. Patch-clamp recordings in Vglut2-ChR2-EYFP mice show that blue light evoked short-latency spiking in MLR neurons. Our results strengthen the idea that different brainstem neurons convey braking/turning and MLR speed commands in mammals. Our study also shows that Vglut2-positive neurons of the cuneiform nucleus are a relevant target to increase locomotor activity without impeding the ability to brake and turn when approaching obstacles, thus ensuring smooth and adaptable navigation. Our observations may have clinical relevance since cuneiform nucleus stimulation is increasingly considered to improve locomotion function in pathological states such as Parkinson’s disease, spinal cord injury, or stroke., Peer Reviewed

La maladie de Parkinson (MP) est une pathologie neurodégénérative caractérisée par de sévères déficits locomoteurs dus à la dégénérescence des neurones dopaminergiques méso-diencéphaliques. Ces neurones modulent plusieurs structures cérébrales impliquées dans la programmation du mouvement, dont la Région Locomotrice Mésencéphalique (MLR). Cette région du tronc cérébral contrôle l’initiation de la locomotion et la vitesse locomotrice chez les vertébrés. Lorsque les neurones dopaminergiques meurent, l’activité de la MLR diminue, ce qui pourrait contribuer aux déficits locomoteurs. À ce jour, les facteurs de vulnérabilité des neurones dopaminergiques dans la MP ne sont pas encore bien définis, mais des études suggèrent que les neurones utilisant le glutamate comme co-transmetteur lors de leur développement pourraient être plus vulnérables. Toutefois, aucune comparaison n’a été faite avec les neurones ne dégénérant pas dans la MP. Au niveau des traitements de la MP, les approches basées sur l’administration de L-DOPA ou la stimulation cérébrale profonde des ganglions de la base suggèrent que les effets bénéfiques sur la fonction locomotrice mettraient en jeu une augmentation de l’activité de la MLR. Or, la stimulation de la MLR n’a encore jamais été étudiée dans un modèle animal de la MP. Les traitements actuels perdent de leur efficacité à mesure que la pathologie progresse, soulignant l’importance de trouver de nouvelles cibles thérapeutiques afin d’améliorer la fonction locomotrice dans la MP. Le premier objectif de mes travaux doctoraux a été de comparer l’expression d’un gène caractéristique du phénotype glutamatergique, le transporteur vésiculaire au glutamate de type 2 (Vglut2), dans les noyaux dopaminergiques A8-A13 en utilisant une technique de cartographie du destin cellulaire chez la souris. Mes travaux montrent que les noyaux dopaminergiques connus pour dégénérer dans la MP expriment à un moment dans leur vie Vglut2, contrairement à ceux qui ne dégénèrent pa, Parkinson's disease (PD) is a neurodegenerative pathology characterized by severe locomotor deficits due to the degeneration of meso-diencephalic dopaminergic neurons. These neurons modulate several brain structures involved in movement programming, including the Mesencephalic Locomotor Region (MLR). This brainstem region controls the initiation of locomotion and locomotor speed in vertebrates. When dopaminergic neurons are lost, MLR activity decreases, which could contribute to locomotor deficits. To date, the vulnerability factors of dopaminergic neurons in PD are not yet well defined, but studies suggest that neurons using glutamate as a co-transmitter during their development may be more vulnerable. However, no comparison has been made with neurons that do not degenerate in PD. Regarding the treatment of PD, approaches based on the administration of L-DOPA or deep brain stimulation of the basal ganglia suggest that the beneficial effects on locomotor function would involve an increased activity in the MLR. However, the effects of MLR stimulation have never been studied in an animal model of PD. Current treatments lose their effectiveness as the disease progresses, underlining the importance of finding new therapeutic targets to improve locomotor function in PD. The first objective of my doctoral work was to compare the expression of a gene specifically associated with the glutamatergic phenotype, the vesicular glutamate transporter type 2 (Vglut2), in A8-A13 dopaminergic nuclei using a cell fate mapping technique in mice. My work shows that the dopaminergic nuclei known to degenerate in PD express at some point in their life Vglut2, unlike those which do not degenerate. The second objective was to study in vivo the effects of MLR stimulation on the locomotor deficits induced in a mouse model of PD based on intracerebral injection of a neurotoxin. My work shows that optogenetic stimulation of glutamatergic neurons (Vglut2+) in the cuneiform nucleus, the

Dopamine (DA) neurons of the ventral tegmental area (VTA) continue to gain attention as far more heterogeneous than previously realized. Within the medial aspect of the VTA, the unexpected presence of TrpV1 mRNA has been identified. TrpV1 encodes the Transient Receptor Potential cation channel subfamily V member 1, TRPV1, also known as the capsaicin receptor, well recognized for its role in heat and pain processing by peripheral neurons. In contrast, the brain distribution of TrpV1 has been debated. Here, we hypothesized that the TrpV1+ identity defines a distinct subpopulation of VTA DA neurons. To explore these brain TrpV1+ neurons, histological analyses and Cre-driven mouse genetics were employed. TrpV1 mRNA was most strongly detected at the perinatal stage forming a band of scattered neurons throughout the medial VTA, reaching into the posterior hypothalamus. Within the VTA, the majority of TrpV1 co-localized with both Tyrosine hydroxylase (Th) and Vesicular monoamine transporter 2 (Vmat2), confirming a DA phenotype. However, TrpV1 also co-localized substantially with Vesicular glutamate transporter 2 (Vglut2), representing the capacity for glutamate (GLU) release. These TrpV1+/Th+/Vglut2+/Vmat2+ neurons thus constitute a molecularly and anatomically distinct subpopulation of DA-GLU co-releasing neurons. To assess behavioral impact, a TrpV1Cre-driven strategy targeting the Vmat2 gene in mice was implemented. This manipulation was sufficient to alter psychomotor behavior induced by amphetamine. The acute effect of the drug was accentuated above control levels, suggesting super-sensitivity in the drug-na ve state resembling a “pre-sensitized” phenotype. However, no progressive increase with repeated injections was observed. This study identifies a distinct TrpV1+ VTA subpopulation as a critical modulatory component in responsiveness to amphetamine. Moreover, expression of the gene encoding TRPV1 in selected VTA neurons opens up for new possibilities in pharmacolog

Like ventral tegmental area (VTA) dopamine (DA) neurons, VTA glutamate neuron activity can support positive reinforcement. However, a subset of VTA neurons co-release DA and glutamate, and DA release might be responsible for behavioral reinforcement induced by VTA glutamate neuron activity. To test this, we used optogenetics to stimulate VTA glutamate neurons in which tyrosine hydroxylase (TH), and thus DA biosynthesis, was conditionally ablated using either floxed Th mice or viral-based CRISPR/Cas9. Both approaches led to loss of TH expression in VTA glutamate neurons and loss of DA release from their distal terminals in nucleus accumbens (NAc). Despite loss of the DA signal, optogenetic activation of VTA glutamate cell bodies or axon terminals in NAc was sufficient to support reinforcement. These results suggest that glutamate release from VTA is sufficient to promote reinforcement independent of concomitant DA co-release, establishing a non-DA mechanism by which VTA activity can support reward-seeking behaviors.

The importance of Vesicular Glutamate Transporter 2 (VGLUT2)-mediated neurotransmission has been highlighted in studies on addiction-related phenotypes. The single nucleotide polymorphism rs2290045 in VGLUT2 has been associated with alcohol dependence, but it is unknown whether or how this association is affected by environmental factors. The present study determined whether the association of alcohol-related problems with the rs2290045 in the VGLUT2 gene was modified by negative and positive environmental factors. Three samples were included: a clinical sample of 131 adolescents followed from age 17 to 22; a general population sample of 1794 young adults; and a general population sample of 1687 adolescents followed from age 14 to 17. DNA was extracted from saliva and the rs2290045 (T/C) was genotyped. Alcohol-related problems were assessed using the Alcohol Use Disorders Identification Test. Stressful life events (SLE) and parenting were assessed by questionnaires. Gene-environment interactions were investigated using a dual statistical approach. In all samples (effect sizes 0.6-6.2%), and consistent with the differential susceptibility framework, T carriers exposed to SLE reported more alcohol-related problems if they had experienced poor parenting, and lower alcohol-related problems if they had received supportive parenting. T carriers not exposed to SLE reported higher alcohol-related problems if they had received supportive parenting and lower alcohol-related problems if they had received poor parenting. Among CC carriers, alcohol-related problems did not vary as a function of negative and positive environmental factors. In conclusion, in three samples of youths, alcohol-related problems were associated with an interaction of VGLUT2 rs2290045, SLE, and parenting.

Transgenic mouse lines are instrumental in our attempt to understand brain function. Promoters driving transgenic expression of the gene encoding Cre recombinase are crucial to ensure selectivity in Cre-mediated targeting of floxed alleles using the Cre-Lox system. For the study of dopamine (DA) neurons, promoter sequences driving expression of the Dopamine transporter (Dat) gene are often implemented and several DAT-Cre transgenic mouse lines have been found to faithfully direct Cre activity to DA neurons. While evaluating an established DAT-Cre mouse line, reporter gene expression was unexpectedly identified in cell somas within the amygdala. To indiscriminately explore Cre activity in DAT-Cre transgenic lines, systematic whole-brain analysis of two DAT-Cre mouse lines was performed upon recombination with different types of floxed reporter alleles. Results were compared with data available from the Allen Institute for Brain Science. The results identified restricted DAT-Cre-driven reporter gene expression in cell clusters within several limbic areas, including amygdaloid and mammillary subnuclei, septum and habenula, areas classically associated with glutamatergic and GABAergic neurotransmission. While no Dat gene expression was detected, ample co-localization between DAT-Cre-driven reporter and markers for glutamatergic and GABAergic neurons was found. Upon viral injection of a fluorescent reporter into the amygdala and habenula, distinct projections from non-dopaminergic DAT-Cre neurons could be distinguished. The study demonstrates that DAT-Cre transgenic mice, beyond their usefulness in recombination of floxed alleles in DA neurons, could be implemented as tools to achieve selective targeting in restricted excitatory and inhibitory neuronal populations within the limbic neurocircuitry.

Midbrain dopamine (DA) neurons exist as several subtypes and are found in a heterogeneous environment including GABAergic and glutamatergic neurons as well as various types of co-releasing neurons. Developmental programs underlying this heterogeneity have remained elusive. In this study, combinatorial mRNA analysis was performed at stages when neuronal phenotypes are first specified. Vesicular transporters for dopamine and other monoamines (VMAT2), GABA (VIAAT), and glutamate (VGLUT2) were assessed by systematically applying fluorescent in situ hybridization through the mes-di-encephalon of the mouse embryo at embryonal days (E) 9.5-14.5. The results show that early differentiating dopamine neurons express the gene encoding VGLUT2 before onset of any dopaminergic markers. Prior to its down-regulation in maturing dopamine neurons, Vglut2 mRNA co-localizes extensively with Tyrosine hydroxylase (Th) and Nurr1, commonly used as markers for DA neurons. Further, Vglut2 and Nurr1 mRNAs are shown to overlap substantially in diencephalic neurons that maintain a glutamatergic phenotype. The results suggest that Vglut2/Nurr1-double positive cells give rise both to dopaminergic and glutamatergic neurons within the mes-di-encephalic area. Finally, analysis of markers representing subtypes of dopamine neurons, including the newly described NeuroD6 subtype, shows that certain subtype specifications arise early. Histological findings are outlined in the context of neuroanatomical concepts and the prosomeric model of brain development. The study contributes to the current decoding of the recently discovered heterogeneity among neurons residing along the cephalic flexure.

The importance of Vesicular Glutamate Transporter 2 (VGLUT2)-mediated neurotransmission has been highlighted in studies on addiction-related phenotypes. The single nucleotide polymorphism rs2290045 in VGLUT2 has been associated with alcohol dependence, but it is unknown whether or how this association is affected by environmental factors. The present study determined whether the association of alcohol-related problems with the rs2290045 in the VGLUT2 gene was modified by negative and positive environmental factors. Three samples were included: a clinical sample of 131 adolescents followed from age 17 to 22; a general population sample of 1794 young adults; and a general population sample of 1687 adolescents followed from age 14 to 17. DNA was extracted from saliva and the rs2290045 (T/C) was genotyped. Alcohol-related problems were assessed using the Alcohol Use Disorders Identification Test. Stressful life events (SLE) and parenting were assessed by questionnaires. Gene-environment interactions were investigated using a dual statistical approach. In all samples (effect sizes 0.6-6.2%), and consistent with the differential susceptibility framework, T carriers exposed to SLE reported more alcohol-related problems if they had experienced poor parenting, and lower alcohol-related problems if they had received supportive parenting. T carriers not exposed to SLE reported higher alcohol-related problems if they had received supportive parenting and lower alcohol-related problems if they had received poor parenting. Among CC carriers, alcohol-related problems did not vary as a function of negative and positive environmental factors. In conclusion, in three samples of youths, alcohol-related problems were associated with an interaction of VGLUT2 rs2290045, SLE, and parenting.

The importance of Vesicular Glutamate Transporter 2 (VGLUT2)-mediated neurotransmission has been highlighted in studies on addiction-related phenotypes. The single nucleotide polymorphism rs2290045 in VGLUT2 has been associated with alcohol dependence, but it is unknown whether or how this association is affected by environmental factors. The present study determined whether the association of alcohol-related problems with the rs2290045 in the VGLUT2 gene was modified by negative and positive environmental factors. Three samples were included: a clinical sample of 131 adolescents followed from age 17 to 22; a general population sample of 1794 young adults; and a general population sample of 1687 adolescents followed from age 14 to 17. DNA was extracted from saliva and the rs2290045 (T/C) was genotyped. Alcohol-related problems were assessed using the Alcohol Use Disorders Identification Test. Stressful life events (SLE) and parenting were assessed by questionnaires. Gene-environment interactions were investigated using a dual statistical approach. In all samples (effect sizes 0.6-6.2%), and consistent with the differential susceptibility framework, T carriers exposed to SLE reported more alcohol-related problems if they had experienced poor parenting, and lower alcohol-related problems if they had received supportive parenting. T carriers not exposed to SLE reported higher alcohol-related problems if they had received supportive parenting and lower alcohol-related problems if they had received poor parenting. Among CC carriers, alcohol-related problems did not vary as a function of negative and positive environmental factors. In conclusion, in three samples of youths, alcohol-related problems were associated with an interaction of VGLUT2 rs2290045, SLE, and parenting.

Midbrain dopamine (DA) neurons exist as several subtypes and are found in a heterogeneous environment including GABAergic and glutamatergic neurons as well as various types of co-releasing neurons. Developmental programs underlying this heterogeneity have remained elusive. In this study, combinatorial mRNA analysis was performed at stages when neuronal phenotypes are first specified. Vesicular transporters for dopamine and other monoamines (VMAT2), GABA (VIAAT), and glutamate (VGLUT2) were assessed by systematically applying fluorescent in situ hybridization through the mes-di-encephalon of the mouse embryo at embryonal days (E) 9.5-14.5. The results show that early differentiating dopamine neurons express the gene encoding VGLUT2 before onset of any dopaminergic markers. Prior to its down-regulation in maturing dopamine neurons, Vglut2 mRNA co-localizes extensively with Tyrosine hydroxylase (Th) and Nurr1, commonly used as markers for DA neurons. Further, Vglut2 and Nurr1 mRNAs are shown to overlap substantially in diencephalic neurons that maintain a glutamatergic phenotype. The results suggest that Vglut2/Nurr1-double positive cells give rise both to dopaminergic and glutamatergic neurons within the mes-di-encephalic area. Finally, analysis of markers representing subtypes of dopamine neurons, including the newly described NeuroD6 subtype, shows that certain subtype specifications arise early. Histological findings are outlined in the context of neuroanatomical concepts and the prosomeric model of brain development. The study contributes to the current decoding of the recently discovered heterogeneity among neurons residing along the cephalic flexure.

Midbrain dopamine (DA) neurons exist as several subtypes and are found in a heterogeneous environment including GABAergic and glutamatergic neurons as well as various types of co-releasing neurons. Developmental programs underlying this heterogeneity have remained elusive. In this study, combinatorial mRNA analysis was performed at stages when neuronal phenotypes are first specified. Vesicular transporters for dopamine and other monoamines (VMAT2), GABA (VIAAT), and glutamate (VGLUT2) were assessed by systematically applying fluorescent in situ hybridization through the mes-di-encephalon of the mouse embryo at embryonal days (E) 9.5-14.5. The results show that early differentiating dopamine neurons express the gene encoding VGLUT2 before onset of any dopaminergic markers. Prior to its down-regulation in maturing dopamine neurons, Vglut2 mRNA co-localizes extensively with Tyrosine hydroxylase (Th) and Nurr1, commonly used as markers for DA neurons. Further, Vglut2 and Nurr1 mRNAs are shown to overlap substantially in diencephalic neurons that maintain a glutamatergic phenotype. The results suggest that Vglut2/Nurr1-double positive cells give rise both to dopaminergic and glutamatergic neurons within the mes-di-encephalic area. Finally, analysis of markers representing subtypes of dopamine neurons, including the newly described NeuroD6 subtype, shows that certain subtype specifications arise early. Histological findings are outlined in the context of neuroanatomical concepts and the prosomeric model of brain development. The study contributes to the current decoding of the recently discovered heterogeneity among neurons residing along the cephalic flexure.

Transgenic mouse lines are instrumental in our attempt to understand brain function. Promoters driving transgenic expression of the gene encoding Cre recombinase are crucial to ensure selectivity in Cre-mediated targeting of floxed alleles using the Cre-Lox system. For the study of dopamine (DA) neurons, promoter sequences driving expression of the Dopamine transporter (Dat) gene are often implemented and several DAT-Cre transgenic mouse lines have been found to faithfully direct Cre activity to DA neurons. While evaluating an established DAT-Cre mouse line, reporter gene expression was unexpectedly identified in cell somas within the amygdala. To indiscriminately explore Cre activity in DAT-Cre transgenic lines, systematic whole-brain analysis of two DAT-Cre mouse lines was performed upon recombination with different types of floxed reporter alleles. Results were compared with data available from the Allen Institute for Brain Science. The results identified restricted DAT-Cre-driven reporter gene expression in cell clusters within several limbic areas, including amygdaloid and mammillary subnuclei, septum and habenula, areas classically associated with glutamatergic and GABAergic neurotransmission. While no Dat gene expression was detected, ample co-localization between DAT-Cre-driven reporter and markers for glutamatergic and GABAergic neurons was found. Upon viral injection of a fluorescent reporter into the amygdala and habenula, distinct projections from non-dopaminergic DAT-Cre neurons could be distinguished. The study demonstrates that DAT-Cre transgenic mice, beyond their usefulness in recombination of floxed alleles in DA neurons, could be implemented as tools to achieve selective targeting in restricted excitatory and inhibitory neuronal populations within the limbic neurocircuitry.

Transgenic mouse lines are instrumental in our attempt to understand brain function. Promoters driving transgenic expression of the gene encoding Cre recombinase are crucial to ensure selectivity in Cre-mediated targeting of floxed alleles using the Cre-Lox system. For the study of dopamine (DA) neurons, promoter sequences driving expression of the Dopamine transporter (Dat) gene are often implemented and several DAT-Cre transgenic mouse lines have been found to faithfully direct Cre activity to DA neurons. While evaluating an established DAT-Cre mouse line, reporter gene expression was unexpectedly identified in cell somas within the amygdala. To indiscriminately explore Cre activity in DAT-Cre transgenic lines, systematic whole-brain analysis of two DAT-Cre mouse lines was performed upon recombination with different types of floxed reporter alleles. Results were compared with data available from the Allen Institute for Brain Science. The results identified restricted DAT-Cre-driven reporter gene expression in cell clusters within several limbic areas, including amygdaloid and mammillary subnuclei, septum and habenula, areas classically associated with glutamatergic and GABAergic neurotransmission. While no Dat gene expression was detected, ample co-localization between DAT-Cre-driven reporter and markers for glutamatergic and GABAergic neurons was found. Upon viral injection of a fluorescent reporter into the amygdala and habenula, distinct projections from non-dopaminergic DAT-Cre neurons could be distinguished. The study demonstrates that DAT-Cre transgenic mice, beyond their usefulness in recombination of floxed alleles in DA neurons, could be implemented as tools to achieve selective targeting in restricted excitatory and inhibitory neuronal populations within the limbic neurocircuitry.

The importance of Vesicular Glutamate Transporter 2 (VGLUT2)-mediated neurotransmission has been highlighted in studies on addiction-related phenotypes. The single nucleotide polymorphism rs2290045 in VGLUT2 has been associated with alcohol dependence, but it is unknown whether or how this association is affected by environmental factors. The present study determined whether the association of alcohol-related problems with the rs2290045 in the VGLUT2 gene was modified by negative and positive environmental factors. Three samples were included: a clinical sample of 131 adolescents followed from age 17 to 22; a general population sample of 1794 young adults; and a general population sample of 1687 adolescents followed from age 14 to 17. DNA was extracted from saliva and the rs2290045 (T/C) was genotyped. Alcohol-related problems were assessed using the Alcohol Use Disorders Identification Test. Stressful life events (SLE) and parenting were assessed by questionnaires. Gene-environment interactions were investigated using a dual statistical approach. In all samples (effect sizes 0.6-6.2%), and consistent with the differential susceptibility framework, T carriers exposed to SLE reported more alcohol-related problems if they had experienced poor parenting, and lower alcohol-related problems if they had received supportive parenting. T carriers not exposed to SLE reported higher alcohol-related problems if they had received supportive parenting and lower alcohol-related problems if they had received poor parenting. Among CC carriers, alcohol-related problems did not vary as a function of negative and positive environmental factors. In conclusion, in three samples of youths, alcohol-related problems were associated with an interaction of VGLUT2 rs2290045, SLE, and parenting.

Midbrain dopamine (DA) neurons exist as several subtypes and are found in a heterogeneous environment including GABAergic and glutamatergic neurons as well as various types of co-releasing neurons. Developmental programs underlying this heterogeneity have remained elusive. In this study, combinatorial mRNA analysis was performed at stages when neuronal phenotypes are first specified. Vesicular transporters for dopamine and other monoamines (VMAT2), GABA (VIAAT), and glutamate (VGLUT2) were assessed by systematically applying fluorescent in situ hybridization through the mes-di-encephalon of the mouse embryo at embryonal days (E) 9.5-14.5. The results show that early differentiating dopamine neurons express the gene encoding VGLUT2 before onset of any dopaminergic markers. Prior to its down-regulation in maturing dopamine neurons, Vglut2 mRNA co-localizes extensively with Tyrosine hydroxylase (Th) and Nurr1, commonly used as markers for DA neurons. Further, Vglut2 and Nurr1 mRNAs are shown to overlap substantially in diencephalic neurons that maintain a glutamatergic phenotype. The results suggest that Vglut2/Nurr1-double positive cells give rise both to dopaminergic and glutamatergic neurons within the mes-di-encephalic area. Finally, analysis of markers representing subtypes of dopamine neurons, including the newly described NeuroD6 subtype, shows that certain subtype specifications arise early. Histological findings are outlined in the context of neuroanatomical concepts and the prosomeric model of brain development. The study contributes to the current decoding of the recently discovered heterogeneity among neurons residing along the cephalic flexure.

Transgenic mouse lines are instrumental in our attempt to understand brain function. Promoters driving transgenic expression of the gene encoding Cre recombinase are crucial to ensure selectivity in Cre-mediated targeting of floxed alleles using the Cre-Lox system. For the study of dopamine (DA) neurons, promoter sequences driving expression of the Dopamine transporter (Dat) gene are often implemented and several DAT-Cre transgenic mouse lines have been found to faithfully direct Cre activity to DA neurons. While evaluating an established DAT-Cre mouse line, reporter gene expression was unexpectedly identified in cell somas within the amygdala. To indiscriminately explore Cre activity in DAT-Cre transgenic lines, systematic whole-brain analysis of two DAT-Cre mouse lines was performed upon recombination with different types of floxed reporter alleles. Results were compared with data available from the Allen Institute for Brain Science. The results identified restricted DAT-Cre-driven reporter gene expression in cell clusters within several limbic areas, including amygdaloid and mammillary subnuclei, septum and habenula, areas classically associated with glutamatergic and GABAergic neurotransmission. While no Dat gene expression was detected, ample co-localization between DAT-Cre-driven reporter and markers for glutamatergic and GABAergic neurons was found. Upon viral injection of a fluorescent reporter into the amygdala and habenula, distinct projections from non-dopaminergic DAT-Cre neurons could be distinguished. The study demonstrates that DAT-Cre transgenic mice, beyond their usefulness in recombination of floxed alleles in DA neurons, could be implemented as tools to achieve selective targeting in restricted excitatory and inhibitory neuronal populations within the limbic neurocircuitry.

The importance of Vesicular Glutamate Transporter 2 (VGLUT2)-mediated neurotransmission has been highlighted in studies on addiction-related phenotypes. The single nucleotide polymorphism rs2290045 in VGLUT2 has been associated with alcohol dependence, but it is unknown whether or how this association is affected by environmental factors. The present study determined whether the association of alcohol-related problems with the rs2290045 in the VGLUT2 gene was modified by negative and positive environmental factors. Three samples were included: a clinical sample of 131 adolescents followed from age 17 to 22; a general population sample of 1794 young adults; and a general population sample of 1687 adolescents followed from age 14 to 17. DNA was extracted from saliva and the rs2290045 (T/C) was genotyped. Alcohol-related problems were assessed using the Alcohol Use Disorders Identification Test. Stressful life events (SLE) and parenting were assessed by questionnaires. Gene-environment interactions were investigated using a dual statistical approach. In all samples (effect sizes 0.6-6.2%), and consistent with the differential susceptibility framework, T carriers exposed to SLE reported more alcohol-related problems if they had experienced poor parenting, and lower alcohol-related problems if they had received supportive parenting. T carriers not exposed to SLE reported higher alcohol-related problems if they had received supportive parenting and lower alcohol-related problems if they had received poor parenting. Among CC carriers, alcohol-related problems did not vary as a function of negative and positive environmental factors. In conclusion, in three samples of youths, alcohol-related problems were associated with an interaction of VGLUT2 rs2290045, SLE, and parenting.

The importance of Vesicular Glutamate Transporter 2 (VGLUT2)-mediated neurotransmission has been highlighted in studies on addiction-related phenotypes. The single nucleotide polymorphism rs2290045 in VGLUT2 has been associated with alcohol dependence, but it is unknown whether or how this association is affected by environmental factors. The present study determined whether the association of alcohol-related problems with the rs2290045 in the VGLUT2 gene was modified by negative and positive environmental factors. Three samples were included: a clinical sample of 131 adolescents followed from age 17 to 22; a general population sample of 1794 young adults; and a general population sample of 1687 adolescents followed from age 14 to 17. DNA was extracted from saliva and the rs2290045 (T/C) was genotyped. Alcohol-related problems were assessed using the Alcohol Use Disorders Identification Test. Stressful life events (SLE) and parenting were assessed by questionnaires. Gene-environment interactions were investigated using a dual statistical approach. In all samples (effect sizes 0.6-6.2%), and consistent with the differential susceptibility framework, T carriers exposed to SLE reported more alcohol-related problems if they had experienced poor parenting, and lower alcohol-related problems if they had received supportive parenting. T carriers not exposed to SLE reported higher alcohol-related problems if they had received supportive parenting and lower alcohol-related problems if they had received poor parenting. Among CC carriers, alcohol-related problems did not vary as a function of negative and positive environmental factors. In conclusion, in three samples of youths, alcohol-related problems were associated with an interaction of VGLUT2 rs2290045, SLE, and parenting.

Midbrain dopamine (DA) neurons exist as several subtypes and are found in a heterogeneous environment including GABAergic and glutamatergic neurons as well as various types of co-releasing neurons. Developmental programs underlying this heterogeneity have remained elusive. In this study, combinatorial mRNA analysis was performed at stages when neuronal phenotypes are first specified. Vesicular transporters for dopamine and other monoamines (VMAT2), GABA (VIAAT), and glutamate (VGLUT2) were assessed by systematically applying fluorescent in situ hybridization through the mes-di-encephalon of the mouse embryo at embryonal days (E) 9.5-14.5. The results show that early differentiating dopamine neurons express the gene encoding VGLUT2 before onset of any dopaminergic markers. Prior to its down-regulation in maturing dopamine neurons, Vglut2 mRNA co-localizes extensively with Tyrosine hydroxylase (Th) and Nurr1, commonly used as markers for DA neurons. Further, Vglut2 and Nurr1 mRNAs are shown to overlap substantially in diencephalic neurons that maintain a glutamatergic phenotype. The results suggest that Vglut2/Nurr1-double positive cells give rise both to dopaminergic and glutamatergic neurons within the mes-di-encephalic area. Finally, analysis of markers representing subtypes of dopamine neurons, including the newly described NeuroD6 subtype, shows that certain subtype specifications arise early. Histological findings are outlined in the context of neuroanatomical concepts and the prosomeric model of brain development. The study contributes to the current decoding of the recently discovered heterogeneity among neurons residing along the cephalic flexure.

Transgenic mouse lines are instrumental in our attempt to understand brain function. Promoters driving transgenic expression of the gene encoding Cre recombinase are crucial to ensure selectivity in Cre-mediated targeting of floxed alleles using the Cre-Lox system. For the study of dopamine (DA) neurons, promoter sequences driving expression of the Dopamine transporter (Dat) gene are often implemented and several DAT-Cre transgenic mouse lines have been found to faithfully direct Cre activity to DA neurons. While evaluating an established DAT-Cre mouse line, reporter gene expression was unexpectedly identified in cell somas within the amygdala. To indiscriminately explore Cre activity in DAT-Cre transgenic lines, systematic whole-brain analysis of two DAT-Cre mouse lines was performed upon recombination with different types of floxed reporter alleles. Results were compared with data available from the Allen Institute for Brain Science. The results identified restricted DAT-Cre-driven reporter gene expression in cell clusters within several limbic areas, including amygdaloid and mammillary subnuclei, septum and habenula, areas classically associated with glutamatergic and GABAergic neurotransmission. While no Dat gene expression was detected, ample co-localization between DAT-Cre-driven reporter and markers for glutamatergic and GABAergic neurons was found. Upon viral injection of a fluorescent reporter into the amygdala and habenula, distinct projections from non-dopaminergic DAT-Cre neurons could be distinguished. The study demonstrates that DAT-Cre transgenic mice, beyond their usefulness in recombination of floxed alleles in DA neurons, could be implemented as tools to achieve selective targeting in restricted excitatory and inhibitory neuronal populations within the limbic neurocircuitry.

The importance of Vesicular Glutamate Transporter 2 (VGLUT2)-mediated neurotransmission has been highlighted in studies on addiction-related phenotypes. The single nucleotide polymorphism rs2290045 in VGLUT2 has been associated with alcohol dependence, but it is unknown whether or how this association is affected by environmental factors. The present study determined whether the association of alcohol-related problems with the rs2290045 in the VGLUT2 gene was modified by negative and positive environmental factors. Three samples were included: a clinical sample of 131 adolescents followed from age 17 to 22; a general population sample of 1794 young adults; and a general population sample of 1687 adolescents followed from age 14 to 17. DNA was extracted from saliva and the rs2290045 (T/C) was genotyped. Alcohol-related problems were assessed using the Alcohol Use Disorders Identification Test. Stressful life events (SLE) and parenting were assessed by questionnaires. Gene-environment interactions were investigated using a dual statistical approach. In all samples (effect sizes 0.6-6.2%), and consistent with the differential susceptibility framework, T carriers exposed to SLE reported more alcohol-related problems if they had experienced poor parenting, and lower alcohol-related problems if they had received supportive parenting. T carriers not exposed to SLE reported higher alcohol-related problems if they had received supportive parenting and lower alcohol-related problems if they had received poor parenting. Among CC carriers, alcohol-related problems did not vary as a function of negative and positive environmental factors. In conclusion, in three samples of youths, alcohol-related problems were associated with an interaction of VGLUT2 rs2290045, SLE, and parenting., Funding Agencies:Alcohol Research Council of the Swedish Alcohol Retailing MonopolyLars Hierta's Minne foundationLundberg's and Karlsson's foundationUppsala and Örebro Regional Research CouncilCounty Council of VästmanlandSwedish Psychiatric FoundationResearch Foundations of Bertil Hallsten and AhlenEuropean Union (EU)INCA 600398 SciLifeLab

The subthalamic nucleus (STN) is a small excitatory nucleus with a significant role in response inhibition. STN activity has been previously shown to be correlated with behavioral stopping or switching, but no studies have yet shown a causal role. This dissertation provides the first evidence that STN activation interrupts behavior, and blocking the STN blunts the interruptive effect of surprise. Thus, the STN is both necessary and sufficient for such forms of behavioral response suppression. Further, STN activation also is sufficient to interrupt cognition, decreasing performance on a working memory task. Finally, viral tracing evidence demonstrates a topological organization of cortical “hyperdirect pathway” inputs onto STN, with motor cortex projecting dominantly to lateral STN and limbic and associative-related cortical regions projecting dominantly to medial STN.

The subthalamic nucleus (STN) is a small excitatory nucleus with a significant role in response inhibition. STN activity has been previously shown to be correlated with behavioral stopping or switching, but no studies have yet shown a causal role. This dissertation provides the first evidence that STN activation interrupts behavior, and blocking the STN blunts the interruptive effect of surprise. Thus, the STN is both necessary and sufficient for such forms of behavioral response suppression. Further, STN activation also is sufficient to interrupt cognition, decreasing performance on a working memory task. Finally, viral tracing evidence demonstrates a topological organization of cortical “hyperdirect pathway” inputs onto STN, with motor cortex projecting dominantly to lateral STN and limbic and associative-related cortical regions projecting dominantly to medial STN.

Itch is a sensation that promotes the desire to scratch, which can be evoked by mechanical and chemical stimuli. In the spinal cord, neurons expressing the gastrin-releasing peptide receptor (GRPR) have been identified as specific mediators of itch. However, our understanding of the GRPR population in the spinal cord, and thus how these neurons exercise their functions, is limited. For this purpose, we constructed a Cre line designed to target the GRPR population of neurons (Grpr-Cre). Our analysis revealed that Grpr-Cre cells in the spinal cord are predominantly excitatory interneurons that are found in the dorsal lamina, especially in laminae II-IV. Application of the specific agonist gastrin-releasing peptide induced spike responses in 43.3% of the patched Grpr-Cre neurons, where the majority of the cells displayed a tonic firing property. Additionally, our analysis showed that the Grpr-Cre population expresses Vglut2 mRNA, and mice ablated of Vglut2 in Grpr-Cre cells (Vglut2-lox; Grpr-Cre mice) displayed less spontaneous itch and attenuated responses to both histaminergic and nonhistaminergic agents. We could also show that application of the itch-inducing peptide, natriuretic polypeptide B, induces calcium influx in a subpopulation of Grpr-Cre neurons. To summarize, our data indicate that the Grpr-Cre spinal cord neural population is composed of interneurons that use VGLUT2-mediated signaling for transmitting chemical and spontaneous itch stimuli to the next, currently unknown, neurons in the labeled line of itch., Title in thesis list of papers: Spinal Cord Interneurons Expressing the Gastrin Releasing Peptide Receptor Convey Itch through VGLUT2-mediated Signaling

Itch is a sensation that promotes the desire to scratch, which can be evoked by mechanical and chemical stimuli. In the spinal cord, neurons expressing the gastrin-releasing peptide receptor (GRPR) have been identified as specific mediators of itch. However, our understanding of the GRPR population in the spinal cord, and thus how these neurons exercise their functions, is limited. For this purpose, we constructed a Cre line designed to target the GRPR population of neurons (Grpr-Cre). Our analysis revealed that Grpr-Cre cells in the spinal cord are predominantly excitatory interneurons that are found in the dorsal lamina, especially in laminae II-IV. Application of the specific agonist gastrin-releasing peptide induced spike responses in 43.3% of the patched Grpr-Cre neurons, where the majority of the cells displayed a tonic firing property. Additionally, our analysis showed that the Grpr-Cre population expresses Vglut2 mRNA, and mice ablated of Vglut2 in Grpr-Cre cells (Vglut2-lox; Grpr-Cre mice) displayed less spontaneous itch and attenuated responses to both histaminergic and nonhistaminergic agents. We could also show that application of the itch-inducing peptide, natriuretic polypeptide B, induces calcium influx in a subpopulation of Grpr-Cre neurons. To summarize, our data indicate that the Grpr-Cre spinal cord neural population is composed of interneurons that use VGLUT2-mediated signaling for transmitting chemical and spontaneous itch stimuli to the next, currently unknown, neurons in the labeled line of itch., Title in thesis list of papers: Spinal Cord Interneurons Expressing the Gastrin Releasing Peptide Receptor Convey Itch through VGLUT2-mediated Signaling

Itch is a sensation that promotes the desire to scratch, which can be evoked by mechanical and chemical stimuli. In the spinal cord, neurons expressing the gastrin-releasing peptide receptor (GRPR) have been identified as specific mediators of itch. However, our understanding of the GRPR population in the spinal cord, and thus how these neurons exercise their functions, is limited. For this purpose, we constructed a Cre line designed to target the GRPR population of neurons (Grpr-Cre). Our analysis revealed that Grpr-Cre cells in the spinal cord are predominantly excitatory interneurons that are found in the dorsal lamina, especially in laminae II-IV. Application of the specific agonist gastrin-releasing peptide induced spike responses in 43.3% of the patched Grpr-Cre neurons, where the majority of the cells displayed a tonic firing property. Additionally, our analysis showed that the Grpr-Cre population expresses Vglut2 mRNA, and mice ablated of Vglut2 in Grpr-Cre cells (Vglut2-lox; Grpr-Cre mice) displayed less spontaneous itch and attenuated responses to both histaminergic and nonhistaminergic agents. We could also show that application of the itch-inducing peptide, natriuretic polypeptide B, induces calcium influx in a subpopulation of Grpr-Cre neurons. To summarize, our data indicate that the Grpr-Cre spinal cord neural population is composed of interneurons that use VGLUT2-mediated signaling for transmitting chemical and spontaneous itch stimuli to the next, currently unknown, neurons in the labeled line of itch., Title in thesis list of papers: Spinal Cord Interneurons Expressing the Gastrin Releasing Peptide Receptor Convey Itch through VGLUT2-mediated Signaling

The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content.

Itch is a sensation that promotes the desire to scratch, which can be evoked by mechanical and chemical stimuli. In the spinal cord, neurons expressing the gastrin-releasing peptide receptor (GRPR) have been identified as specific mediators of itch. However, our understanding of the GRPR population in the spinal cord, and thus how these neurons exercise their functions, is limited. For this purpose, we constructed a Cre line designed to target the GRPR population of neurons (Grpr-Cre). Our analysis revealed that Grpr-Cre cells in the spinal cord are predominantly excitatory interneurons that are found in the dorsal lamina, especially in laminae II-IV. Application of the specific agonist gastrin-releasing peptide induced spike responses in 43.3% of the patched Grpr-Cre neurons, where the majority of the cells displayed a tonic firing property. Additionally, our analysis showed that the Grpr-Cre population expresses Vglut2 mRNA, and mice ablated of Vglut2 in Grpr-Cre cells (Vglut2-lox; Grpr-Cre mice) displayed less spontaneous itch and attenuated responses to both histaminergic and nonhistaminergic agents. We could also show that application of the itch-inducing peptide, natriuretic polypeptide B, induces calcium influx in a subpopulation of Grpr-Cre neurons. To summarize, our data indicate that the Grpr-Cre spinal cord neural population is composed of interneurons that use VGLUT2-mediated signaling for transmitting chemical and spontaneous itch stimuli to the next, currently unknown, neurons in the labeled line of itch., Title in thesis list of papers: Spinal Cord Interneurons Expressing the Gastrin Releasing Peptide Receptor Convey Itch through VGLUT2-mediated Signaling

Itch is a sensation that promotes the desire to scratch, which can be evoked by mechanical and chemical stimuli. In the spinal cord, neurons expressing the gastrin-releasing peptide receptor (GRPR) have been identified as specific mediators of itch. However, our understanding of the GRPR population in the spinal cord, and thus how these neurons exercise their functions, is limited. For this purpose, we constructed a Cre line designed to target the GRPR population of neurons (Grpr-Cre). Our analysis revealed that Grpr-Cre cells in the spinal cord are predominantly excitatory interneurons that are found in the dorsal lamina, especially in laminae II-IV. Application of the specific agonist gastrin-releasing peptide induced spike responses in 43.3% of the patched Grpr-Cre neurons, where the majority of the cells displayed a tonic firing property. Additionally, our analysis showed that the Grpr-Cre population expresses Vglut2 mRNA, and mice ablated of Vglut2 in Grpr-Cre cells (Vglut2-lox; Grpr-Cre mice) displayed less spontaneous itch and attenuated responses to both histaminergic and nonhistaminergic agents. We could also show that application of the itch-inducing peptide, natriuretic polypeptide B, induces calcium influx in a subpopulation of Grpr-Cre neurons. To summarize, our data indicate that the Grpr-Cre spinal cord neural population is composed of interneurons that use VGLUT2-mediated signaling for transmitting chemical and spontaneous itch stimuli to the next, currently unknown, neurons in the labeled line of itch., Title in thesis list of papers: Spinal Cord Interneurons Expressing the Gastrin Releasing Peptide Receptor Convey Itch through VGLUT2-mediated Signaling

The basal ganglia are the key circuitry controlling movement and reward behavior. Both locomotion and reward-related behavior are also modified by dopaminergic input from the substantia nigra and the ventral tegmental area (VTA). If the basal ganglia are severed by lesion or in disease, such as in Parkinson’s disease, the affected individuals suffer from severe motor impairments and often of affective and reward-related symptoms. The subthalamic nucleus (STN) is a glutamatergic key area of the basal ganglia and a common target for deep brain stimulation in Parkinson’s disease to alleviate motor symptoms. The STN serves not only motoric, but also limbic and cognitive functions, which is often attributed to a tripartite anatomical subdivision. However, the functional output of both VTA and STN may rely more on intermingled subpopulations than on a strictly anatomical subdivision. In this doctoral thesis, the role of subpopulations within and associated with the basal ganglia is addressed from both a genetic and a behavioral angle. The identification of a genetically defined subpopulation within the STN, co-expressing Paired-like homeodomain transcription factor 2 (Pitx2) and Vesicular glutamate transport 2 (Vglut2), made it possible to conditionally reduce glutamatergic transmission from this subgroup of neurons and to investigate its influence on locomotion and motivational behavior, giving interesting insights into the mechanisms possibly underlying deep brain stimulation therapy and its side-effects. We address the strong influence of the Pitx2-Vglut2 subpopulation on movement, as well as the more subtle changes in reward-related behavior and the impact of the alterations on the reward-related dopaminergic circuitry. We also further elucidate the genetic composition of the STN by finding new markers for putative STN subpopulations, thereby opening up new possibilities to target those cells genetically and optogenetically. This will help in future to examine both ST

Neuronal calcium-binding protein 1 and -2 (NECAB1/2) localize to multiple excitatory neuron populations in the mouse spinal cord. Here, we analyzed rat and human spinal cord, combining in situ hybridization and immunohistochemistry, complementing newly collated data on mouse spinal cord for direct comparisons. Necab1/2 mRNA transcripts showed complementary distribution in rodent’s spinal cord. Multiple-labeling fluorescence histochemistry with neuronal phenotypic markers localized NECAB1 to a dense fiber plexus in the dorsal horn, to neurons mainly in superficial layers and to commissural interneurons in both rodent species. NECAB1-positive (+) motor neurons were only found in mice. NECAB1 distribution in the human spinal cord was similar with the addition of NECAB1-like immunoreactivity surrounding myelinated axons. NECAB2 was mainly present in excitatory synaptic boutons in the dorsal horn of all three species, and often in calbindin-D28k+ neuronal somata. Rodent ependymal cells expressed calbindin-D28k. In humans, they instead were NECAB2+ and/or calretinin+. Our results reveal that the association of NECAB2 to excitatory neuronal circuits in the spinal cord is evolutionarily conserved across the mammalian species investigated so far. In contrast, NECAB1 expression is more heterogeneous. Thus, our study suggests that the phenotypic segregation of NECAB1 and -2 to respective excitatory and inhibitory spinal systems can underpin functional modalities in determining the fidelity of synaptic neurotransmission and neuronal responsiveness, and might bear translational relevance to humans., QC 20160610

The basal ganglia are the key circuitry controlling movement and reward behavior. Both locomotion and reward-related behavior are also modified by dopaminergic input from the substantia nigra and the ventral tegmental area (VTA). If the basal ganglia are severed by lesion or in disease, such as in Parkinson’s disease, the affected individuals suffer from severe motor impairments and often of affective and reward-related symptoms. The subthalamic nucleus (STN) is a glutamatergic key area of the basal ganglia and a common target for deep brain stimulation in Parkinson’s disease to alleviate motor symptoms. The STN serves not only motoric, but also limbic and cognitive functions, which is often attributed to a tripartite anatomical subdivision. However, the functional output of both VTA and STN may rely more on intermingled subpopulations than on a strictly anatomical subdivision. In this doctoral thesis, the role of subpopulations within and associated with the basal ganglia is addressed from both a genetic and a behavioral angle. The identification of a genetically defined subpopulation within the STN, co-expressing Paired-like homeodomain transcription factor 2 (Pitx2) and Vesicular glutamate transport 2 (Vglut2), made it possible to conditionally reduce glutamatergic transmission from this subgroup of neurons and to investigate its influence on locomotion and motivational behavior, giving interesting insights into the mechanisms possibly underlying deep brain stimulation therapy and its side-effects. We address the strong influence of the Pitx2-Vglut2 subpopulation on movement, as well as the more subtle changes in reward-related behavior and the impact of the alterations on the reward-related dopaminergic circuitry. We also further elucidate the genetic composition of the STN by finding new markers for putative STN subpopulations, thereby opening up new possibilities to target those cells genetically and optogenetically. This will help in future to examine both ST

The basal ganglia are the key circuitry controlling movement and reward behavior. Both locomotion and reward-related behavior are also modified by dopaminergic input from the substantia nigra and the ventral tegmental area (VTA). If the basal ganglia are severed by lesion or in disease, such as in Parkinson’s disease, the affected individuals suffer from severe motor impairments and often of affective and reward-related symptoms. The subthalamic nucleus (STN) is a glutamatergic key area of the basal ganglia and a common target for deep brain stimulation in Parkinson’s disease to alleviate motor symptoms. The STN serves not only motoric, but also limbic and cognitive functions, which is often attributed to a tripartite anatomical subdivision. However, the functional output of both VTA and STN may rely more on intermingled subpopulations than on a strictly anatomical subdivision. In this doctoral thesis, the role of subpopulations within and associated with the basal ganglia is addressed from both a genetic and a behavioral angle. The identification of a genetically defined subpopulation within the STN, co-expressing Paired-like homeodomain transcription factor 2 (Pitx2) and Vesicular glutamate transport 2 (Vglut2), made it possible to conditionally reduce glutamatergic transmission from this subgroup of neurons and to investigate its influence on locomotion and motivational behavior, giving interesting insights into the mechanisms possibly underlying deep brain stimulation therapy and its side-effects. We address the strong influence of the Pitx2-Vglut2 subpopulation on movement, as well as the more subtle changes in reward-related behavior and the impact of the alterations on the reward-related dopaminergic circuitry. We also further elucidate the genetic composition of the STN by finding new markers for putative STN subpopulations, thereby opening up new possibilities to target those cells genetically and optogenetically. This will help in future to examine both ST

The basal ganglia are the key circuitry controlling movement and reward behavior. Both locomotion and reward-related behavior are also modified by dopaminergic input from the substantia nigra and the ventral tegmental area (VTA). If the basal ganglia are severed by lesion or in disease, such as in Parkinson’s disease, the affected individuals suffer from severe motor impairments and often of affective and reward-related symptoms. The subthalamic nucleus (STN) is a glutamatergic key area of the basal ganglia and a common target for deep brain stimulation in Parkinson’s disease to alleviate motor symptoms. The STN serves not only motoric, but also limbic and cognitive functions, which is often attributed to a tripartite anatomical subdivision. However, the functional output of both VTA and STN may rely more on intermingled subpopulations than on a strictly anatomical subdivision. In this doctoral thesis, the role of subpopulations within and associated with the basal ganglia is addressed from both a genetic and a behavioral angle. The identification of a genetically defined subpopulation within the STN, co-expressing Paired-like homeodomain transcription factor 2 (Pitx2) and Vesicular glutamate transport 2 (Vglut2), made it possible to conditionally reduce glutamatergic transmission from this subgroup of neurons and to investigate its influence on locomotion and motivational behavior, giving interesting insights into the mechanisms possibly underlying deep brain stimulation therapy and its side-effects. We address the strong influence of the Pitx2-Vglut2 subpopulation on movement, as well as the more subtle changes in reward-related behavior and the impact of the alterations on the reward-related dopaminergic circuitry. We also further elucidate the genetic composition of the STN by finding new markers for putative STN subpopulations, thereby opening up new possibilities to target those cells genetically and optogenetically. This will help in future to examine both ST

The basal ganglia are the key circuitry controlling movement and reward behavior. Both locomotion and reward-related behavior are also modified by dopaminergic input from the substantia nigra and the ventral tegmental area (VTA). If the basal ganglia are severed by lesion or in disease, such as in Parkinson’s disease, the affected individuals suffer from severe motor impairments and often of affective and reward-related symptoms. The subthalamic nucleus (STN) is a glutamatergic key area of the basal ganglia and a common target for deep brain stimulation in Parkinson’s disease to alleviate motor symptoms. The STN serves not only motoric, but also limbic and cognitive functions, which is often attributed to a tripartite anatomical subdivision. However, the functional output of both VTA and STN may rely more on intermingled subpopulations than on a strictly anatomical subdivision. In this doctoral thesis, the role of subpopulations within and associated with the basal ganglia is addressed from both a genetic and a behavioral angle. The identification of a genetically defined subpopulation within the STN, co-expressing Paired-like homeodomain transcription factor 2 (Pitx2) and Vesicular glutamate transport 2 (Vglut2), made it possible to conditionally reduce glutamatergic transmission from this subgroup of neurons and to investigate its influence on locomotion and motivational behavior, giving interesting insights into the mechanisms possibly underlying deep brain stimulation therapy and its side-effects. We address the strong influence of the Pitx2-Vglut2 subpopulation on movement, as well as the more subtle changes in reward-related behavior and the impact of the alterations on the reward-related dopaminergic circuitry. We also further elucidate the genetic composition of the STN by finding new markers for putative STN subpopulations, thereby opening up new possibilities to target those cells genetically and optogenetically. This will help in future to examine both ST

ObjectivesThe ventromedial hypothalamic nucleus (VMH) regulates energy homeostasis as well as social and emotional behaviors. Nearly all VMH neurons, including those in the sexually dimorphic ventrolateral VMH (VMHvl) subregion, release the excitatory neurotransmitter glutamate and use the vesicular glutamate transporter 2 (Vglut2). Here, we asked how glutamatergic signaling contributes to the collective metabolic and behavioral responses attributed to the VMH and VMHvl.MethodsUsing Sf1-Cre and a Vglut2 floxed allele, Vglut2 was knocked-out in SF-1 VMH neurons (Vglut2 (Sf1-Cre) ). Metabolic and neurobehavioral assays were carried out initially on Vglut2 (fl/fl) and Vglut2 (Sf1-Cre) mice in a mixed, and then in the C57BL/6 genetic background, which is prone to hyperglycemia and diet induced obesity (DIO).ResultsSeveral phenotypes observed in Vglut2 (Sf1-Cre) mice were largely unexpected based on prior studies that have perturbed VMH development or VMH glutamate signaling. In our hands, Vglut2 (Sf1-Cre) mice failed to exhibit the anticipated increase in body weight after high fat diet (HFD) or the impaired glucose homeostasis after fasting. Instead, there was a significant sex-dependent attenuation of DIO in Vglut2 (Sf1-Cre) females. Vglut2 (Sf1-Cre) males also display a sex-specific loss of conditioned-fear responses and aggression accompanied by more novelty-associated locomotion. Finally, unlike the higher anxiety noted in Sf1 (Nestin-Cre) mice that lack a fully formed VMH, both male and female Vglut2 (Sf1-Cre) mice were less anxious.ConclusionsLoss of VMH glutamatergic signaling sharply decreased DIO in females, attenuated aggression and learned fear in males, and was anxiolytic in males and females. Collectively, our findings demonstrate that while glutamatergic output from the VMH appears largely dispensable for counter regulatory responses to hypoglycemia, it drives sex-dependent differences in metabolism and social behaviors and is essential for adaptive respo

ObjectivesThe ventromedial hypothalamic nucleus (VMH) regulates energy homeostasis as well as social and emotional behaviors. Nearly all VMH neurons, including those in the sexually dimorphic ventrolateral VMH (VMHvl) subregion, release the excitatory neurotransmitter glutamate and use the vesicular glutamate transporter 2 (Vglut2). Here, we asked how glutamatergic signaling contributes to the collective metabolic and behavioral responses attributed to the VMH and VMHvl.MethodsUsing Sf1-Cre and a Vglut2 floxed allele, Vglut2 was knocked-out in SF-1 VMH neurons (Vglut2 (Sf1-Cre) ). Metabolic and neurobehavioral assays were carried out initially on Vglut2 (fl/fl) and Vglut2 (Sf1-Cre) mice in a mixed, and then in the C57BL/6 genetic background, which is prone to hyperglycemia and diet induced obesity (DIO).ResultsSeveral phenotypes observed in Vglut2 (Sf1-Cre) mice were largely unexpected based on prior studies that have perturbed VMH development or VMH glutamate signaling. In our hands, Vglut2 (Sf1-Cre) mice failed to exhibit the anticipated increase in body weight after high fat diet (HFD) or the impaired glucose homeostasis after fasting. Instead, there was a significant sex-dependent attenuation of DIO in Vglut2 (Sf1-Cre) females. Vglut2 (Sf1-Cre) males also display a sex-specific loss of conditioned-fear responses and aggression accompanied by more novelty-associated locomotion. Finally, unlike the higher anxiety noted in Sf1 (Nestin-Cre) mice that lack a fully formed VMH, both male and female Vglut2 (Sf1-Cre) mice were less anxious.ConclusionsLoss of VMH glutamatergic signaling sharply decreased DIO in females, attenuated aggression and learned fear in males, and was anxiolytic in males and females. Collectively, our findings demonstrate that while glutamatergic output from the VMH appears largely dispensable for counter regulatory responses to hypoglycemia, it drives sex-dependent differences in metabolism and social behaviors and is essential for adaptive respo

A major challenge in the field of neuroscience is to link behavior with specific neuronal circuitries and cellular events. One way of facing this challenge is to identify unique cellular markers and thus have the ability to, through various mouse genetics tools, mimic, manipulate and control various aspects of neuronal activity to decipher their correlation to behavior. The Vesicular Glutamate Transporter 2 (VGLUT2) packages glutamate into presynaptic vesicles for axonal terminal release. In this thesis, VGLUT2 was used to specifically target cell populations within the basal ganglia of mice with the purpose of investigating its connectivity, function and involvement in behavior. The motor and limbic loops of the basal ganglia are important for processing of voluntary movement and emotions. During such physiological events, dopamine plays a central role in modulating the activity of these systems. The brain reward system is mainly formed by dopamine projections from the ventral tegmental area (VTA) to the ventral striatum. Certain dopamine neurons within the VTA exhibit the ability to co-release dopamine and glutamate. In paper I, glutamate and dopamine co-release was targeted and our results demonstrate that the absence of VGLUT2 in dopamine neurons leads to perturbations of reward consumption and reward-associated memory, probably due to reduced DA release observed in the striatum as detected by in vivo chronoamperometry. In papers II and IV, VGLUT2 in a specific subpopulation within the subthalamic nucleus (STN) was identified and targeted. Based on the described role of the STN in movement control, we hypothesized that the mice would be hyperlocomotive. As shown in paper II, this was indeed the case. In paper IV, a putative reward-related phenotype was approached and we could show reduced operant-self administration of sugar and altered dopamine release levels suggesting a role for the STN in reward processes. In paper III, we investigated and identified age- an

Neuronal calcium (Ca2+)-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca2+-binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small-and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca2+-binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca2+-binding proteins in pain-related DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord., QC 20140505