United in Diversity : A Physiological and Molecular Characterization of Subpopulations in the Basal Ganglia Circuitry

The Basal Ganglia consist of a number of different nuclei that form a diverse circuitry of GABAergic, dopaminergic and glutamatergic neurons. This complex network is further organized in subcircuits that govern limbic and motor functions in humans and other vertebrates. Due to the interconnection of the individual structures, dysfunction in one area or cell population can affect the entire network, leading to synaptic and molecular alterations in the circuitry as a whole. The studies in this doctoral thesis aimed at characterizing restricted subpopulations of neurons in the Basal Ganglia circuitry and their importance in the wider function of the network. To this end, we identified subpopulations of neurons in the subthalamic nucleus (STN), substantia nigra (SN) and ventral tegmental area (VTA), characterized their molecular profile and investigated their physiological role in the circuitry. Within the mouse STN, reduction of glutamatergic neurotransmission in a subpopulation expressing Paired-like homeodomain transcription factor 2 (Pitx2) led to structural alterations in the nucleus as well as biochemical alterations of the dopaminergic system in the Nucleus accumbens (NAc) and changes in reward-related behavior. In the ventral midbrain, we identified and characterized novel marker genes selective to the VTA or SN. Of these, transient receptor potential cation channel subfamily V member 1 (TrpV1) marks a population of mainly glutamatergic neurons in the VTA which project to the NAc, while gastrin releasing peptide (Grp) is expressed in a population of dopaminergic neurons neuroprotected in Parkinson's disease. Furthermore, we discovered that disruption of glutamatergic co-release of dopaminergic neurons expressing dopamine transporter (DAT), diminishes fast EPSCs and glutamate release but does not affect the acquisition of reward-related behavioral tasks. To selectively quantify glutamate release from specific subpopulations, we devised a technique combining glutamate-amperometry and optogenetics. This was used to measure glutamate released from Pitx2-expressing synaptic terminals in the Globus pallidus as well as DAT- or TrpV1-expressing terminals in the NAc. In summary, this doctoral thesis has furthered understanding of the function and importance of specific subpopulations within the Basal Ganglia circuitry and provides a novel means to investigate glutamate in the intact rodent brain within clearly defined, restricted cell populations

United in Diversity : A Physiological and Molecular Characterization of Subpopulations in the Basal Ganglia Circuitry

The Basal Ganglia consist of a number of different nuclei that form a diverse circuitry of GABAergic, dopaminergic and glutamatergic neurons. This complex network is further organized in subcircuits that govern limbic and motor functions in humans and other vertebrates. Due to the interconnection of the individual structures, dysfunction in one area or cell population can affect the entire network, leading to synaptic and molecular alterations in the circuitry as a whole. The studies in this doctoral thesis aimed at characterizing restricted subpopulations of neurons in the Basal Ganglia circuitry and their importance in the wider function of the network. To this end, we identified subpopulations of neurons in the subthalamic nucleus (STN), substantia nigra (SN) and ventral tegmental area (VTA), characterized their molecular profile and investigated their physiological role in the circuitry. Within the mouse STN, reduction of glutamatergic neurotransmission in a subpopulation expressing Paired-like homeodomain transcription factor 2 (Pitx2) led to structural alterations in the nucleus as well as biochemical alterations of the dopaminergic system in the Nucleus accumbens (NAc) and changes in reward-related behavior. In the ventral midbrain, we identified and characterized novel marker genes selective to the VTA or SN. Of these, transient receptor potential cation channel subfamily V member 1 (TrpV1) marks a population of mainly glutamatergic neurons in the VTA which project to the NAc, while gastrin releasing peptide (Grp) is expressed in a population of dopaminergic neurons neuroprotected in Parkinson's disease. Furthermore, we discovered that disruption of glutamatergic co-release of dopaminergic neurons expressing dopamine transporter (DAT), diminishes fast EPSCs and glutamate release but does not affect the acquisition of reward-related behavioral tasks. To selectively quantify glutamate release from specific subpopulations, we devised a technique combining glutamate-amperometry and optogenetics. This was used to measure glutamate released from Pitx2-expressing synaptic terminals in the Globus pallidus as well as DAT- or TrpV1-expressing terminals in the NAc. In summary, this doctoral thesis has furthered understanding of the function and importance of specific subpopulations within the Basal Ganglia circuitry and provides a novel means to investigate glutamate in the intact rodent brain within clearly defined, restricted cell populations

Age- and Sex-Dependence of Dopamine Release and Capacity for Recovery Identified in the Dorsal Striatum ofC57/Bl6J Mice

The dorsal striatum is the main input structure of the basal ganglia and the major target area of dopaminergic projections originating in the substantia nigra pars compacta. Heavily involved in the regulation of voluntary movement and habit formation, this structure is of strong importance in Parkinson's disease, obsessive-compulsive disorder, Tourette's syndrome and addiction. The C57/Bl6J mouse strain, the most commonly used strain in preclinical research today, is frequently used as a model organism for analysis of dopaminergic parameters implicated in human pathophysiology. Several components of the dopamine system have been shown to vary with age and sex, however knowledge of the contribution of these factors for dopamine release kinetics in the C57/Bl6J mouse strain is lacking. In the present study, we used an intracranial KCl-stimulation challenge paradigm to provoke release from dopaminergic terminals in the dorsal striatum of anaesthetized C57/Bl6J mice. By high-speed in vivo chronoamperometric recordings, we analyzed DA release parameters in male and female mice of two different ages. Our experiments demonstrate elevated DA amplitudes in adult compared to young mice of both sexes and higher DA amplitudes in females compared to males at both ages. Adult mice exhibited higher recovery capabilities after repeated stimulation than did young mice and also showed a lower variability in the kinetic parameters trise and t80 between stimulations. These results identified age- and sex- dimorphisms in DA release parameters and point to the importance of taking these dimorphisms into account when utilizing the C57/Bl6J mouse strain as model for neurological and neuropsychiatric disorders

High-speed <i>in vivo</i> chronoamperometry recording setup and parameters.

<p><b>A</b>: Experimental setup: 6 consecutive KCl-stimulations (1–6) in 2-minute intervals were followed by a 15-minute stimulation-free period upon which followed stimulation 7. Basal release capacity was defined by stimulation 1; recovery capacity was defined as a significant increase in DA amplitude upon stimulation 7 compared to stimulation 6; regain of basal release capability was defined as the absence of a significant difference in DA amplitude between stimulation 1 and stimulation 7. <b>B</b>: Left: Schematic illustration of a coronal brain section at Bregma 1.1 with recording electrode implicated by green dotted line. Right: Close-up of representative photomicrograph depicting the dorsal striatum (DStr) shown to the left as indicated by gray dotted lines; histological verification of the position of the DiO-coated carbon-fiber recording electrode (green) implanted in the DStr of a C57/BL6J mouse. <b>C</b>: Representative traces of DA release kinetics obtained by the high-speed chronoamperometry system following KCl-stimulation 1 for all four experimental groups: Green - adult females; Red - adult males; Black - young females; Blue - young males (shown in inset). Illustration of parameters analysed (green trace used as example): Amplitude, defined as the peak DA concentration (µM) from baseline; <i>t</i>_rise, the time (seconds) between injection and maximum peak concentration; and t₈₀, the time (seconds) from maximum peak concentration until 80% decrease of the maximum amplitude as a measure of DA clearance. CC; corpus callosum, LV; lateral ventricle.</p

Comparison of DA release and reuptake parameters between adult and young groups of mice of both sexes.

<p>Scatter plots of DA amplitude vs. t_rise (A–C) and DA amplitude vs. t₈₀ (D–F) as logarithmic values (natural logarithm) for each individual mouse after stimulation 1 (A, D), 6 (B, E) and 7 (C, F), respectively. Same color-coding as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099592#pone-0099592-g001" target="_blank">Fig. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099592#pone-0099592-g002" target="_blank">2</a>: Green - adult females; Red - adult males; Black - young females; Blue - young males. Bars on the side of each plot depict mean values of the groups upon each specific stimulation (1, 6 and 7) and stars indicate statistically significant differences between indicated groups. p<0.05: *, p<0.01: **, p<0.001: *** as detected by 3-way repeated measures ANOVA and post-hoc test with Bonferroni correction.</p

DA release and reuptake parameters within adult and young groups of mice of both sexes.

<p>Mean values obtained for each stimulation (1–7) within each group (Green - adult females (A–C); Red - adult males (D–F); Black - young females (G–I); Blue - young males (J–L) for parameters amplitude (A,D, G, J), <i>t</i>_rise (B, E, H, K) and t₈₀ (C, F, I, L); Recovery capacity (stimulation 7 vs. 6) and regain of basal release capability (stimulation 7 vs. 1) shown as bar graph inserts for all groups and parameters (adult females (A′–C′); adult males (D′–F′); young females (G′–I′); young males (J′–L′). p<0.05: #, p<0.01: ##, p<0.001: ### as detected by 1-way repeated measure ANOVA, p<0.05: *, p<0.01: **, p<0.001: *** as detected by post-hoc test with Bonferroni correction (A-L) and paired student's t-test (A′-L′)</p

Reduced Vglut2/Slc17a6 Gene Expression Levels throughout the Mouse Subthalamic Nucleus Cause Cell Loss and Structural Disorganization Followed by Increased Motor Activity and Decreased Sugar Consumption.

The subthalamic nucleus (STN) plays a central role in motor, cognitive, and affective behavior. Deep brain stimulation (DBS) of the STN is the most common surgical intervention for advanced Parkinson's disease (PD), and STN has lately gained attention as target for DBS in neuropsychiatric disorders, including obsessive compulsive disorder, eating disorders, and addiction. Animal studies using STN-DBS, lesioning, or inactivation of STN neurons have been used extensively alongside clinical studies to unravel the structural organization, circuitry, and function of the STN. Recent studies in rodent STN models have exposed different roles for STN neurons in reward-related functions. We have previously shown that the majority of STN neurons express the vesicular glutamate transporter 2 gene (Vglut2/Slc17a6) and that reduction of Vglut2 mRNA levels within the STN of mice [conditional knockout (cKO)] causes reduced postsynaptic activity and behavioral hyperlocomotion. The cKO mice showed less interest in fatty rewards, which motivated analysis of reward-response. The current results demonstrate decreased sugar consumption and strong rearing behavior, whereas biochemical analyses show altered dopaminergic and peptidergic activity in the striatum. The behavioral alterations were in fact correlated with opposite effects in the dorsal versus the ventral striatum. Significant cell loss and disorganization of the STN structure was identified, which likely accounts for the observed alterations. Rare genetic variants of the human VGLUT2 gene exist, and this study shows that reduced Vglut2/Slc17a6 gene expression levels exclusively within the STN of mice is sufficient to cause strong modifications in both the STN and the mesostriatal dopamine system

Midbrain Gene Screening Identifies a New Mesoaccumbal Glutamatergic Pathway and a Marker for Dopamine Cells Neuroprotected in Parkinson's Disease

The ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) of the midbrain are associated with Parkinson's disease (PD), schizophrenia, mood disorders and addiction. Based on the recently unraveled heterogeneity within the VTA and SNc, where glutamate, GABA and co-releasing neurons have been found to co-exist with the classical dopamine neurons, there is a compelling need for identification of gene expression patterns that represent this heterogeneity and that are of value for development of human therapies. Here, several unique gene expression patterns were identified in the mouse midbrain of which NeuroD6 and Grp were expressed within different dopaminergic subpopulations of the VTA, and TrpV1 within a small heterogeneous population. Optogenetics-coupled in vivo amperometry revealed a previously unknown glutamatergic mesoaccumbal pathway characterized by TrpV1-Cre-expression. Human GRP was strongly detected in non-melanized dopaminergic neurons within the SNc of both control and PD brains, suggesting GRP as a marker for neuroprotected neurons in PD. This study thus unravels markers for distinct subpopulations of neurons within the mouse and human midbrain, defines unique anatomical subregions within the VTA and exposes an entirely new glutamatergic pathway. Finally, both TRPV1 and GRP are implied in midbrain physiology of importance to neurological and neuropsychiatric disorders

Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity

Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations ("TH-Vglut2 Class1") also expressed the dopamine transporter (DAT) gene while one did not ("TH-Vglut2 Class2"), and the remaining population did not express TH at all ("Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area

Disrupting Glutamate Co-transmission Does Not Affect Acquisition of Conditioned Behavior Reinforced by Dopamine Neuron Activation.

Dopamine neurons in the ventral tegmental area (VTA) were previously found to express vesicular glutamate transporter 2 (VGLUT2) and to co-transmit glutamate in the ventral striatum (VStr). This capacity may play an important role in reinforcement learning. Although it is known that activation of the VTA-VStr dopamine system readily reinforces behavior, little is known about the role of glutamate co-transmission in such reinforcement. By combining electrode recording and optogenetics, we found that stimulation of VTA dopamine neurons in vivo evoked fast excitatory responses in many VStr neurons of adult mice. Whereas conditional knockout of the gene encoding VGLUT2 in dopamine neurons largely eliminated fast excitatory responses, it had little effect on the acquisition of conditioned responses reinforced by dopamine neuron activation. Therefore, glutamate co-transmission appears dispensable for acquisition of conditioned responding reinforced by DA neuron activation