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Sökning: WFRF:(Wallén Mackenzie Åsa)

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1.
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2.
  • Schweizer, Nadine, et al. (författare)
  • 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
  • 2016
  • Ingår i: eNeuro. - 2373-2822. ; 3:5
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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4.
  • Viereckel, Thomas, et al. (författare)
  • Midbrain Gene Screening Identifies a New Mesoaccumbal Glutamatergic Pathway and a Marker for Dopamine Cells Neuroprotected in Parkinson's Disease
  • 2016
  • Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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5.
  • Viereckel, Thomas, 1987- (författare)
  • United in Diversity : A Physiological and Molecular Characterization of Subpopulations in the Basal Ganglia Circuitry
  • 2017
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • 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.
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6.
  • Viereckel, Thomas, et al. (författare)
  • Validated multi‐step approach for in vivo recording and analysis of optogenetically evoked glutamate in the mouse globus pallidus
  • 2018
  • Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 145:2, s. 125-138
  • Tidskriftsartikel (refereegranskat)abstract
    • Precise quantification of extracellular glutamate concentrations upon neuronal activation is crucial for the understanding of brain function and neurological disorders. While optogenetics is an outstanding method for the correlation between distinct neurons and their role in circuitry and behavior, the electrochemically inactive nature of glutamate has proven challenging for recording upon optogenetic stimulations. This difficulty is due to the necessity for using enzyme-coated microelectrodes and the risk for light-induced artifacts. In this study, we establish a method for the combination of invivo optogenetic stimulation with selective measurement of glutamate concentrations using enzyme-coated multielectrode arrays and amperometry. The glutamatergic subthalamic nucleus (STN), which is the main electrode target site in deep brain stimulation treatment of advanced Parkinsons disease, has recently proven opotogenetically targetable in Pitx2-Cre-transgenic mice and was here used as model system. Upon stereotactic injection of viral Channelrhodopsin2-eYFP constructs into the STN, amperometric recordings were performed at a range of optogenetic stimulation frequencies in the globus pallidus, the main STN target area, in anesthetized mice. Accurate quantification was enabled through a multi-step analysis approach based on self-referencing microelectrodes and repetition of the experimental protocol at two holding potentials, which allowed for the identification, isolation and removal of photoelectric and photoelectrochemical artifacts. This study advances the field of invivo glutamate detection with combined optogenetics and amperometric recordings by providing a validated analysis framework for application in a wide variety of glutamate-based approaches in neuroscience.
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7.
  • Wallén-Mackenzie, Åsa, 1971-, et al. (författare)
  • Spatio-molecular domains identified in the mouse subthalamic nucleus and neighboring glutamatergic and GABAergic brain structures
  • 2020
  • Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
  • Tidskriftsartikel (refereegranskat)abstract
    • The subthalamic nucleus (STN) is crucial for normal motor, limbic and associative function. STN dysregulation is correlated with several brain disorders, including Parkinsons disease and obsessive compulsive disorder (OCD), for which high-frequency stimulation of the STN is increasing as therapy. However, clinical progress is hampered by poor knowledge of the anatomical-functional organization of the STN. Today, experimental mouse genetics provides outstanding capacity for functional decoding, provided selective promoters are available. Here, we implemented single-nuclei RNA sequencing (snRNASeq) of the mouse STN followed through with histological analysis of 16 candidate genes of interest. Our results demonstrate that the mouse STN is composed of at least four spatio-molecularly defined domains, each distinguished by defined sets of promoter activities. Further, molecular profiles dissociate the STN from the adjoining para-STN (PSTN) and neighboring structures of the hypothalamus, mammillary nuclei and zona incerta. Enhanced knowledge of STNs internal organization should prove useful towards genetics-based functional decoding of this clinically relevant brain structure. p id=Par Wallen-Mackenzie et al. investigate anatomical-functional organization of the subthalamic nucleus in mice, using single-nuclei RNA sequencing followed by histological analysis. They identify four domains distinguished by defined sets of promoter activities, providing a valuable resource for functional decoding of the subthalamic nucleus.
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8.
  • Wang, Dong V., et al. (författare)
  • Disrupting Glutamate Co-transmission Does Not Affect Acquisition of Conditioned Behavior Reinforced by Dopamine Neuron Activation
  • 2017
  • Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 18:11, s. 2584-2591
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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9.
  • Alsiö, Johan, et al. (författare)
  • Enhanced Sucrose and Cocaine Self-Administration and Cue-Induced Drug Seeking after Loss of VGLUT2 in Midbrain Dopamine Neurons in Mice
  • 2011
  • Ingår i: Journal of Neuroscience. - 0270-6474 .- 1529-2401. ; 31:35, s. 12593-12603
  • Tidskriftsartikel (refereegranskat)abstract
    • The mesostriatal dopamine (DA) system contributes to several aspects of responses to rewarding substances and is implicated in conditions such as drug addiction and eating disorders. A subset of DA neurons has been shown to express the type 2 Vesicular glutamate transporter (Vglut2) and may therefore corelease glutamate. In the present study, we analyzed mice with a conditional deletion of Vglut2 in DA neurons (Vglut2(f/f;DAT-Cre)) to address the functional significance of the glutamate-DA cophenotype for responses to cocaine and food reinforcement. Biochemical parameters of striatal DA function were also examined by using DA receptor autoradiography, immediate-early gene quantitative in situ hybridization after cocaine challenge, and DA-selective in vivo chronoamperometry. Mice in which Vglut2 expression had been abrogated in DA neurons displayed enhanced operant self-administration of both high-sucrose food and intravenous cocaine. Furthermore, cocaine seeking maintained by drug-paired cues was increased by 76%, showing that reward-dependent plasticity is perturbed in these mice. In addition, several lines of evidence suggest that adaptive changes occurred in both the ventral and dorsal striatum in the absence of VGLUT2: DA receptor binding was increased, and basal mRNA levels of the DA-induced early genes Nur77 and c-fos were elevated as after cocaine induction. Furthermore, in vivo challenge of the DA system by potassium-evoked depolarization revealed less DA release in both striatal areas. This study demonstrates that absence of VGLUT2 in DA neurons leads to perturbations of reward consumption as well as reward-associated memory, features of particular relevance for addictive-like behavior.
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10.
  • Arvidsson, Emma, 1986-, et al. (författare)
  • Age- and Sex-Dependence of Dopamine Release and Capacity for Recovery Identified in the Dorsal Striatum ofC57/Bl6J Mice
  • 2014
  • Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:6, s. e99592-
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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11.
  • Arvidsson, Emma, 1986- (författare)
  • Motion and Emotion : Functional In Vivo Analyses of the Mouse Basal Ganglia
  • 2014
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • 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- and sex-dimorphisms in dopamine kinetics in the dorsal striatum of one of the most commonly used mouse lines worldwide, the C57/Bl6J. Our results point to the importance of taking these dimorphisms into account when utilizing the C57/Bl6J strain as model for neurological and neuropsychiatric disorders.
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12.
  • Bagchi, Sonchita, et al. (författare)
  • In Situ Proximity Ligation Assay (PLA)
  • 2015
  • Ingår i: ELISA. - New York, NY : Springer-Verlag New York. - 9781493927425 - 9781493927418 ; , s. 149-159
  • Bokkapitel (refereegranskat)abstract
    • In situ proximity ligation assay (PLA) is a method to identify physical closeness of proteins, where a signal will only be produced if the two proteins are closer than 40 nm, in tissue section or cell cultures. Modifications of the PLA method can also be used to increase specificity or sensitivity of standard immunohistochemistry protocols.
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13.
  • Berube-Carriere, Noemie, et al. (författare)
  • Ultrastructural characterization of the mesostriatal dopamine innervation in mice, including two mouse lines of conditional VGLUT2 knockout in dopamine neurons
  • 2012
  • Ingår i: European Journal of Neuroscience. - : Wiley. - 0953-816X .- 1460-9568. ; 35:4, s. 527-538
  • Tidskriftsartikel (refereegranskat)abstract
    • Despite the increasing use of genetically modified mice to investigate the dopamine (DA) system, little is known about the ultrastructural features of the striatal DA innervation in the mouse. This issue is particularly relevant in view of recent evidence for expression of the vesicular glutamate transporter 2 (VGLUT2) by a subset of mesencephalic DA neurons in mouse as well as rat. We used immuno-electron microscopy to characterize tyrosine hydroxylase (TH)-labeled terminals in the core and shell of nucleus accumbens and the neostriatum of two mouse lines in which the Vglut2 gene was selectively disrupted in DA neurons (cKO), their control littermates, and C57BL/6/J wild-type mice, aged P15 or adult. The three regions were also examined in cKO mice and their controls of both ages after dual THVGLUT2 immunolabeling. Irrespective of the region, age and genotype, the TH-immunoreactive varicosities appeared similar in size, vesicular content, percentage with mitochondria, and exceedingly low frequency of synaptic membrane specialization. No dually labeled axon terminals were found at either age in control or in cKO mice. Unless TH and VGLUT2 are segregated in different axon terminals of the same neurons, these results favor the view that the glutamatergic cophenotype of mesencephalic DA neurons is more important during the early development of these neurons than for the establishment of their scarce synaptic connectivity. They also suggest that, in mouse even more than rat, the mesostriatal DA system operates mainly through non-targeted release of DA, diffuse transmission and the maintenance of an ambient DA level.
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14.
  • Bimpisidis, Zisis, et al. (författare)
  • Increased sucrose consumption in mice gene-targeted for Vmat2 selectively in NeuroD6-positive neurons of the ventral tegmental area
  • 2023
  • Ingår i: Frontiers in Molecular Neuroscience. - : Frontiers Media S.A.. - 1662-5099. ; 16
  • Tidskriftsartikel (refereegranskat)abstract
    • Ventral tegmental area (VTA) dopamine (DA) neurons are implicated in reward processing, motivation, reward prediction error, and in substance use disorder. Recent studies have identified distinct neuronal subpopulations within the VTA that can be clustered based on their molecular identity, neurotransmitter profile, physiology, projections and behavioral role. One such subpopulation is characterized by expression of the NeuroD6 gene, and projects primarily to the nucleus accumbens medial shell. We recently showed that optogenetic stimulation of these neurons induces real-time place preference while their targeted deletion of the Vmat2 gene caused altered response to rewarding substances, including ethanol and psychostimulants. Based on these recent findings, we wanted to further investigate the involvement of the NeuroD6-positive VTA subpopulation in reward processing. Using the same NeuroD6(Cre+/wt);Vmat2(flox/flox) mice as in our prior study, we now addressed the ability of the mice to process sucrose reward. In order to assess appetitive behavior and motivation to obtain sucrose reward, we tested conditional knockout (cKO) and control littermate mice in an operant sucrose self-administration paradigm. We observed that cKO mice demonstrate higher response rates to the operant task and consume more sucrose rewards than control mice. However, their motivation to obtain sucrose is identical to that of control mice. Our results highlight previous observations that appetitive behavior and motivation to obtain rewards can be served by distinct neuronal circuits, and demonstrate that the NeuroD6 VTA subpopulation is involved in mediating the former, but not the latter. Together with previous studies on the NeuroD6 subpopulation, our findings pinpoint the importance of unraveling the molecular and functional role of VTA subpopulations in order to better understand normal behavior and psychiatric disease.
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15.
  • Bimpisidis, Zisis, et al. (författare)
  • Neurocircuitry of Reward and Addiction : Potential Impact of Dopamine-Glutamate Co-release as Future Target in Substance Use Disorder
  • 2019
  • Ingår i: Journal of Clinical Medicine. - : MDPI. - 2077-0383. ; 8:11
  • Forskningsöversikt (refereegranskat)abstract
    • Dopamine-glutamate co-release is a unique property of midbrain neurons primarily located in the ventral tegmental area (VTA). Dopamine neurons of the VTA are important for behavioral regulation in response to rewarding substances, including natural rewards and addictive drugs. The impact of glutamate co-release on behaviors regulated by VTA dopamine neurons has been challenging to probe due to lack of selective methodology. However, several studies implementing conditional knockout and optogenetics technologies in transgenic mice have during the past decade pointed towards a role for glutamate co-release in multiple physiological and behavioral processes of importance to substance use and abuse. In this review, we discuss these studies to highlight findings that may be critical when considering mechanisms of importance for prevention and treatment of substance abuse.
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16.
  • Bimpisidis, Zisis, et al. (författare)
  • The NeuroD6 Subtype of VTA Neurons Contributes to Psychostimulant Sensitization and Behavioral Reinforcement
  • 2019
  • Ingår i: eNeuro. - : SOC NEUROSCIENCE. - 2373-2822. ; 6:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Reward-related behavior is complex and its dysfunction correlated with neuropsychiatric illness. Dopamine (DA) neurons of the ventral tegmental area (VTA) have long been associated with different aspects of reward function, but it remains to be disentangled how distinct VTA DA neurons contribute to the full range of behaviors ascribed to the VTA. Here, a recently identified subtype of VTA neurons molecularly defined by NeuroD6 (NEX1M) was addressed. Among all VTA DA neurons, less than 15% were identified as positive for NeuroD6. In addition to dopaminergic markers, sparse NeuroD6 neurons expressed the vesicular glutamate transporter 2 (Vglut2) gene. To achieve manipulation of NeuroD6 VTA neurons, NeuroD6(NEX)-Cre-driven mouse genetics and optogenetics were implemented. First, expression of vesicular monoamine transporter 2 (VMAT2) was ablated to disrupt dopaminergic function in NeuroD6 VTA neurons. Comparing Vmat2(Cre)(lox/lox;NEX-) conditional knock-out (cKO) mice with littermate controls, it was evident that baseline locomotion, preference for sugar and ethanol, and place preference upon amphetamine-induced and cocaine-induced conditioning were similar between genotypes. However, locomotion upon repeated psychostimulant administration was significantly elevated above control levels in cKO mice. Second, optogenetic activation of NEX-Cre VTA neurons was shown to induce DA release and glutamatergic postsynaptic currents within the nucleus accumbens. Third, optogenetic stimulation of NEX-Cre VTA neurons in vivo induced significant place preference behavior, while stimulation of VTA neurons defined by Calretinin failed to cause a similar response. The results show that NeuroD6 VTA neurons exert distinct regulation over specific aspects of reward-related behavior, findings that contribute to the current understanding of VTA neurocircuitry.
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17.
  • Bimpisidis, Zisis, et al. (författare)
  • Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse
  • 2020
  • Ingår i: Journal of Visualized Experiments. - : JOURNAL OF VISUALIZED EXPERIMENTS. - 1940-087X. ; :156
  • Tidskriftsartikel (refereegranskat)abstract
    • Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with behavioral testing in validated paradigms allows the measurement of behavioral consequences upon stimulation of distinct neurons in real-time with high spatial and temporal selectivity, and thus the establishment of causal relationships between neuronal activation and behavior. Here, we describe a step-by-step protocol fora real-time place preference (RT-PP) paradigm, a modified version of the classical conditioned place preference (CPP) test. The RT-PP is performed in a three-compartment apparatus and can be utilized to answer if optogenetic stimulation of a specific neuronal population is rewarding or aversive. We also describe an alternative version of the RT-PP protocol, the socalled neutral compartment preference (NCP) protocol, which can be used to confirm aversion. The two approaches are based on extensions of classical methodology originating from behavioral pharmacology and recent implementation of optogenetics within the neuroscience field. Apart from measuring place preference in real time, these setups can also give information regarding conditioned behavior. We provide easyto-follow step-by-step protocols alongside examples of our own data and discuss important aspects to consider when applying these types of experiments.
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18.
  • Birgner, Carolina, et al. (författare)
  • VGLUT2 in dopamine neurons is required for psychostimulant-induced behavioural activation
  • 2010
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:1, s. 389-394
  • Tidskriftsartikel (refereegranskat)abstract
    • The “One neuron-one neurotransmitter” concept has been challenged frequently during the last three decades, and the coexistence of neurotransmitters in individual neurons is now regarded as a common phenomenon. The functional significance of neurotransmitter coexistence is, however, less well understood. Several studies have shown that a subpopulation of dopamine (DA) neurons in the ventral tegmental area (VTA) expresses the vesicular glutamate transporter 2 (VGLUT2) and has been suggested to use glutamate as a cotransmitter. The VTA dopamine neurons project to limbic structures including the nucleus accumbens, and are involved in mediating the motivational and locomotor activating effects of psychostimulants. To determine the functional role of glutamate cotransmission by these neurons, we deleted VGLUT2 in DA neurons by using a conditional gene-targeting approach in mice. A DAT-Cre/Vglut2Lox mouse line (Vglut2f/f;DAT-Cre mice) was produced and analyzed by in vivo amperometry as well as by several behavioral paradigms. Although basal motor function was normal in the Vglut2f/f;DAT-Cre mice, their risk-taking behavior was altered. Interestingly, in both home-cage and novel environments, the gene targeted mice showed a greatly blunted locomotor response to the psychostimulant amphetamine, which acts via the midbrain DA system. Our results show that VGLUT2 expression in DA neurons is required for normal emotional reactivity as well as for psychostimulant-mediated behavioral activation.
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19.
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20.
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21.
  • Dumas, Sylvie, et al. (författare)
  • Developmental Co-expression of Vglut2 and Nurr1 in a Mes-Di-Encephalic Continuum Preceeds Dopamine and Glutamate Neuron Specification
  • 2019
  • Ingår i: Frontiers in Cell and Developmental Biology. - : FRONTIERS MEDIA SA. - 2296-634X. ; 7
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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22.
  • El Mestikawy, Salah, et al. (författare)
  • From glutamate co-release to vesicular synergy : vesicular glutamate transporters
  • 2011
  • Ingår i: Nature Reviews Neuroscience. - : Springer Science and Business Media LLC. - 1471-003X .- 1471-0048. ; 12:4, s. 204-216
  • Forskningsöversikt (refereegranskat)abstract
    • Recent data indicate that 'classical' neurotransmitters can also act as co-transmitters. This notion has been strengthened by the demonstration that three vesicular glutamate transporters (vesicular glutamate transporter 1 (VGLUT1), VGLUT2 and VGLUT3) are present in central monoamine, acetylcholine and GABA neurons, as well as in primarily glutamatergic neurons. Thus, intriguing questions are raised about the morphological and functional organization of neuronal systems endowed with such a dual signalling capacity. In addition to glutamate co-release, vesicular synergy - a process leading to enhanced packaging of the 'primary' transmitter - is increasingly recognized as a major property of the glutamatergic co-phenotype. The behavioural relevance of this co-phenotype is presently the focus of considerable interest.
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23.
  • Fortin, G. M., et al. (författare)
  • Glutamate corelease promotes growth and survival of midbrain dopamine neurons
  • 2012
  • Ingår i: Journal of Neuroscience. - 0270-6474 .- 1529-2401. ; 32:48, s. 17477-17491
  • Tidskriftsartikel (refereegranskat)abstract
    • Recent studies have proposed that glutamate corelease by mesostriatal dopamine (DA) neurons regulates behavioral activation by psychostimulants.How and when glutamate release by DA neurons might play this role remains unclear. Considering evidence for early expression of the type 2 vesicular glutamate transporter in mesencephalic DA neurons, we hypothesized that this cophenotype is particularly important during development. Using a conditional gene knock-out approach to selectively disrupt the Vglut2 gene in mouse DA neurons, we obtained in vitro and in vivo evidence for reduced growth and survival of mesencephalic DA neurons, associated with a decrease in the density of DA innervation in the nucleus accumbens, reduced activity-dependent DA release, and impaired motor behavior. These findings provide strong evidence for a functional role of the glutamatergic cophenotype in the development of mesencephalic DA neurons, opening new perspectives into the pathophysiology of neurodegenerative disorders involving the mesostriatal DA system.
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24.
  • Gezelius, Henrik, et al. (författare)
  • Role of glutamate in locomotor rhythm generating neuronal circuitry
  • 2006
  • Ingår i: Journal of Physiology - Paris. - : Elsevier BV. - 0928-4257 .- 1769-7115. ; 100:5-6, s. 297-303
  • Tidskriftsartikel (refereegranskat)abstract
    • Central pattern generators (CPGs) are defined as neuronal circuits capable of producing a rhythmic and coordinated output without the influence of sensory input. The locomotor and respiratory neuronal circuits are two of the better-characterized CPGs, although much work remains to fully understand how these networks operate. Glutamatergic neurons are involved in most neuronal circuits of the nervous system and considerable efforts have been made to study glutamate receptors in nervous system signaling using a variety of approaches. Because of the complexity of glutamate-mediated signaling and the variety of receptors triggered by glutamate, it has been difficult to pinpoint the role of glutamatergic neurons in neuronal circuits. In addition, glutamate is an amino acid used by every cell, which has hampered identification of glutamatergic neurons. Glutamatergic excitatory neurotransmission is dependent on the release from glutamate-filled presynaptic vesicles loaded by three members of the solute carrier family, Slc17a6-8, which function as vesicular glutamate transporters (VGLUTs). Recent data describe that Vglut2 (Slc17a6) null mutant mice die immediately after birth due to a complete loss of the stable autonomous respiratory rhythm generated by the pre-Bötzinger complex. Surprisingly, we found that basal rhythmic locomotor activity is not affected in Vglut2 null mutant embryos. With this perspective, we discuss data regarding presence of VGLUT1, VGLUT2 and VGLUT3 positive neuronal populations in the spinal cord.
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25.
  • Guillaumin, Adriane, et al. (författare)
  • Anatomical-functional analysis of the spatially restricted Transient receptor vanilloid-1 (Trpv1)-positive domain within the medial hypothalamic-mesencephalic area
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • The distribution pattern and functional role of brain neurons expressing the Transient receptor vanilloid-1 (Trpv1) gene, which in the sensory nervous system regulates body temperature, heat and pain, have remained obscure. Two studies using both in situ hybridization to detect endogenous Trpv1 mRNA and a floxed reporter allel in Trpv1-Cre mice, have detected sparse expression in the caudal aspect of the hypothalamus and the ventral tegmental area of the midbrain. This area has during recent years been associated with aversion processing via glutamatergic neurons expressing the Vesicular glutamate transporter 2 (Vglut2) gene. Glutamatergic neurons of this area thus seem to be selectively correlated with aversion, while dopaminergic neurons in the ventral midbrain have long been associated with reward processing. A previous study from our laboratory found that the Trpv1-positive neuronal population consisted of both glutamatergic and dopaminergic neurons. It was therefore of particular interest to study this population further. Would this small but distinct hypothalamic-mesencephalic neuronal group of neurons regulate any specific type of behaviour if selectively activated? The results presented show that Trpv1 mRNA is primarily detected at the perinatal stage forming a band of neurons stretching from the posterior hypothalamus through to, and including, the ventral tegmental area. In the mature mouse, application of optogenetic constructs to the Trpv1-Cre-positive population enabled the analysis of both projection pattern and behavioural role of the Trpv1 neurons. These rare neurons, forming a hypothalamic-mesenecphalic continuum, project to several areas of the limbic system including the hippocampus, septum, nucleus accumbens and the preoptic area. When optogenetically activated, Trpv1-Cre mice show normal movements in the open field test, but display progressive avoidance behaviour in an optogenetic real-time place preference test. The results identify a new neuronal population tentatively involved in the complex network of aversive behaviour.
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26.
  • Guillaumin, Adriane, et al. (författare)
  • Experimental investigation into the role of the subthalamic nucleus (STN) in motor control using optogenetics in mice
  • 2021
  • Ingår i: Brain Research. - : Elsevier. - 0006-8993 .- 1872-6240. ; 1755
  • Tidskriftsartikel (refereegranskat)abstract
    • The subthalamic nucleus (STN) is critical for the execution of intended movements. Loss of its normal function is strongly associated with several movement disorders, including Parkinson's disease for which the STN is an important target area in deep brain stimulation (DBS) therapy. Classical basal ganglia models postulate that two parallel pathways, the direct and indirect pathways, exert opposing control over movement, with the STN acting within the indirect pathway. The STN is regulated by both inhibitory and excitatory input, and is itself excitatory. While most functional knowledge of this clinically relevant brain structure has been gained from pathological conditions and models, primarily parkinsonian, experimental evidence for its role in normal motor control has remained more sparse. The objective here was to tease out the selective impact of the STN on several motor parameters required to achieve intended movement, including locomotion, balance and motor coordination. Optogenetic excitation and inhibition using both bilateral and unilateral stimulations of the STN were implemented in freely-moving mice. The results demonstrate that selective optogenetic inhibition of the STN enhances locomotion while its excitation reduces locomotion. These findings lend experimental support to basal ganglia models of the STN in terms of locomotion. In addition, optogenetic excitation in freely-exploring mice induced self-grooming, disturbed gait and a jumping/escaping behavior, while causing reduced motor coordination in advanced motor tasks, independent of grooming and jumping. This study contributes experimentally validated evidence for a regulatory role of the STN in several aspects of motor control.
  •  
27.
  • Guillaumin, Adriane, et al. (författare)
  • Improving well-being and survival in the 6-OHDA lesion model of Parkinson´s disease in mice : Literature review and step-by-step protocol
  • 2022
  • Ingår i: Scandinavian Journal of Laboratory Animal Science. - : Scandinavian Society for Laboratory Animal Science. - 0901-3393. ; 48:1, s. 1-21
  • Forskningsöversikt (refereegranskat)abstract
    • Parkinson's disease (PD) is the most common neurodegenerative motor disorder and primarily affects movement control but also a range of non-motor functions. With unknown etiology and lack of cure, much research is dedicated to unravel pathological mechanisms and improve clinical prospects for symptom alleviation, prevention and treatment. To achieve these goals, animal models intended to represent symptoms similar to those observed in the complex clinical display of PD play a key role. It is important to bear in mind that, in any studies with laboratory animals, it is crucial to take the 3Rs principle (Refine, Reduce, Replace) into account. The main pathology of PD includes degeneration of dopamine neurons in the substantia nigra pars compacta (SNc). The 6-hydroxydopamine (6-OHDA) lesion model, in which dopaminergic neurons are chemically destroyed, is often favored as a laboratory model of PD in both rodents and primates. However, while reproducing several features of clinical PD, mice exposed to 6-OHDA frequently experience systemic dysfunction causing premature death. To avoid suffering and unnecessary deaths of laboratory mice, there is a need for improved experimental protocols in accordance with the 3Rs principle. Based on current literature and our own previous experiments, we decided to test the effect of three parameters: 1) reduced dose of the 6-OHDA toxin; 2) daily post-operative care to avoid hypothermia and energy loss; 3) shortened interval from surgical injection of toxin to time of sacrifice. By implementing a 6-OHDA lesion protocol using a lower dose of toxin than commonly seen in the literature alongside careful post-operative care and decreased time post-injection, a fully recovered weight post-surgery and high survival rate was obtained. This was achieved despite full expression of the 6-OHDA-induced locomotor phenotype. A step-by-step protocol was formulated. Validation using histological analysis confirmed toxin-induced degeneration of midbrain dopamine neurons with concomitant loss of dopaminergic projections in the lesioned hemisphere. Notably, while SNc dopamine neurons were drastically reduced, those located in the ventral tegmental area (VTA) were less affected in a medialhigh survival to laterallow survival manner. The Refine and Reduce parameters of the 3Rs principle in experimental animal welfare were specifically addressed which allowed us to improve well-being and survival of mice while maintaining characteristic parkinsonian features in the 6-OHDA lesion model. A table summarizing current literature on the 6-OHDA model in rodents and our validated step-by-step experimental protocol are provided.
  •  
28.
  • Guillaumin, Adriane, et al. (författare)
  • Optimization protocol for the 6-OHDA model of Parkinson´s disease in wildtype mice
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Parkinson’s disease is a severe progressive neurodegenerative disease which usually appears in patients above 60 years old. The main motor symptoms are bradykinesia, akinesia, rigidity and tremor. The cause of the disease is still unknown but the consequence is a degeneration of the dopaminergic neurons of the substantia nigra pars compacta (SNc). Current treatments for PD focus on replacing the loss of dopamine or to stimulate at high frequency the subthalamic nucleus. In order to improve treatments for PD, researchers use parkinsonian animal models. Among those, the neurotoxin-based models are commonly used, in particular the 6-hydroxydopamine (6-OHDA) model. The 6-OHDA is a neurotoxin which, when injected intracerebrally, selectively destroys catecholaminergic neurons. The 6-OHDA model reproduces several features of PD including motor impairments and loss of tyrosine hydroxylase (TH) in dopaminergic neurons of the SNc and partially in dopaminergic neurons of the ventral tegmental area (VTA). In this study we optimized the 6-OHDA model in mice to increase the survival rate and well-being of the mice while inducing parkinsonian symptoms.
  •  
29.
  • Guillaumin, Adriane, 1991-, et al. (författare)
  • Optogenetic investigation into the role of the subthalamic nucleus in motor control
  • 2024
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • The subthalamic nucleus is important achieve intended movements. Loss of its normal function is strongly associated with several movement disorders. Classical basal ganglia models postulate that two parallel pathways, the direct and indirect pathways, exert opposing control over movement, with the subthalamic nucleus part of the indirect pathway through which competing motor programs are prevented. The subthalamic nucleus is regulated by both inhibitory and excitatory projections but experimental evidence for its role in motor control has remained sparse. The objective here was to tease out the selective impact of the subthalamic nucleus on several motor parameters required to achieve intended movement, including locomotion, balance and motor coordination. Optogenetic excitation and inhibition using both bilateral and unilateral stimulations of the subthalamic nucleus were implemented in freely-moving mice. The results demonstrate that selective optogenetic inhibition of the subthalamic nucleus enhances locomotion while its excitation reduces locomotion. These findings lend experimental support to basal ganglia models in terms of locomotion. However, further analysis of subthalamic nucleus excitation revealed grooming and disturbed gait. Selective excitation also caused reduced motor coordination, independent of grooming, in advanced motor tasks.  This study contributes experimental evidence for a regulatory role of the subthalamic nucleus in motor control.
  •  
30.
  • Guillaumin, Adriane (författare)
  • The subthalamic nucleus in motor and affective functions : An optogenetic in vivo-investigation
  • 2020
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • The basal ganglia form a group of subcortical interconnected nuclei involved in motor, limbic and cognitive functions. According to the classical model of the basal ganglia, two main pathways exert opposing control over movement, one facilitating movement and the other suppressing movement. The subthalamic nucleus (STN) plays a critical role in this function, and has also been implicated in reward processing. Despite ample knowledge of the role of the STN in motor dysfunctions in relation to Parkinson’s disease, less is known about STN’s natural role in healthy subjects.The studies described in this thesis aimed to address the functional role of the STN in its natural neurocircuitry by using a transgenic mouse line which expresses Cre recombinase under the Pitx2 promoter. The Pitx2 gene is restricted to the STN and the use of Pitx2-Cre mice thereby allows selective manipulation of STN neurons by using optogenetics. By expressing Channelrhodopsin (ChR2) or Archaerhodopsin (Arch) in Pitx2-Cre neurons, we could optogenetically excite or inhibit STN Pitx2-Cre neurons and investigate the role of the STN in motor and affective functions. We showed that optogenetic inhibition and excitation of the STN induce opposite effects on motor activity. STN excitation reduced locomotion while STN inhibition enhanced locomotion, thereby providing experimental evidence to classical motor models postulating this role. We also showed that optogenetic excitation of the STN induces potent place avoidance, a behaviour relevant to aversion. Projections from the STN to the ventral pallidum (VP) exist that when excited induced the same behaviour. The VP projects to the lateral habenula (LHb), a structure known for its role in aversion. A glutamatergic multi-synaptic connection between the STN and the LHb was confirmed.Aversive behaviour is also mediated by the hypothalamic-mesencephalic area. The Trpv1 gene is expressed within the posterior hypothalamus. By applying optogenetics in a Trpv1-Cre mouse line, projection patterns to limbic brain areas were identified, and optogenetic excitation of Trpv1-Cre neurons was found to induce place avoidance.The STN and posterior hypothalamus are thereby demonstrated as new players in the aversion neurocircuitry, while the long-assumed role of the STN in motor behaviour is confirmed. To enable future analyses of how STN manipulation might rescue motor and affective deficiency relevant to human disorders, a neuronal degeneration mouse model was generated.To conclude, the results presented in this thesis contribute to enhanced neurobiological understanding of the role played by the STN in motor and affective functions.
  •  
31.
  • König, Niclas, 1986-, et al. (författare)
  • Selective Knockout of the Vesicular Monoamine Transporter 2 (Vmat2) Gene in Calbindin2/Calretinin-Positive Neurons Results in Profound Changes in Behavior and Response to Drugs of Abuse
  • 2020
  • Ingår i: Frontiers in Behavioral Neuroscience. - : FRONTIERS MEDIA SA. - 1662-5153. ; 14
  • Tidskriftsartikel (refereegranskat)abstract
    • The vesicular monoamine transporter 2 (VMAT2) has a range of functions in the central nervous system, from sequestering toxins to providing conditions for the quantal release of monoaminergic neurotransmitters. Monoamine signaling regulates diverse functions from arousal to mood, movement, and motivation, and dysregulation of VMAT2 function is implicated in various neuropsychiatric diseases. While all monoamine-releasing neurons express the Vmat2 gene, only a subset is positive for the calcium-binding protein Calbindin 2 (Calb2; aka Calretinin, 29 kDa Calbindin). We recently showed that about half of the dopamine neurons in the mouse midbrain are positive for Calb2 and that Calb2 is an early developmental marker of midbrain dopamine cells. Calb2-positive neurons have also been identified in other monoaminergic areas, yet the role of Calb2-positive monoaminergic neurons is poorly understood. To selectively address the impact of Calb2-positive monoaminergic neurons in behavioral regulation, we took advantage of the Cre-LoxP system to create a new conditional knockout (cKO) mouse line in which Vmat2 expression is deleted selectively in Calb2-Cre-positive neurons. In this Vmat2(lox/lox;Calb2-Cre) cKO mouse line, gene targeting of Vmat2 was observed in several distinct monoaminergic areas. By comparing control and cKO mice in a series of behavioral tests, specific dissimilarities were identified. In particular, cKO mice were smaller than control mice and showed heightened sensitivity to the stereotypy-inducing effects of amphetamine and slight reductions in preference toward sucrose and ethanol, as well as a blunted response in the elevated plus maze test. These data uncover new knowledge about the role of genetically defined subtypes of neurons in the brain's monoaminergic systems.
  •  
32.
  • Lagerström, Malin C., et al. (författare)
  • A sensory subpopulation depends on vesicular glutamate transporter 2 for mechanical pain, and together with substance P, inflammatory pain
  • 2011
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:14, s. 5789-5794
  • Tidskriftsartikel (refereegranskat)abstract
    • Ablating or functionally compromising sets of sensory neurons has provided important insights into peripheral modality-specific wiring in the somatosensory system. Inflammatory hyperalgesia, cold pain, and noxious mechanosensation have all been shown to depend upon Na(v)1.8-positive sensory neurons. The release of fast-acting neurotransmitters, such as glutamate, and more slowly released neuropeptides, such as substance P (SP), contribute to the diversified responses to external stimuli. Here we show that deleting Vglut2 in Na(v)1.8(Cre)-positive neurons compromised mechanical pain and NGF-induced thermal hyperalgesia, whereas tactile-evoked sensation, thermal, formalin-evoked, and chronic neuropathic pain were normal. However, when Vglut2(f/f); Na(v)1.8(Cre) mice were injected with a SP antagonist before the formalin test, the second phase pain response was nearly completely abolished, whereas in control mice, the pain response was unaffected. Our results suggest that VGLUT2-dependent signaling originating from Na(v)1.8-positive neurons is a principal sensing mechanism for mechanical pain and, together with SP, inflammatory pain. These data define sets of primary afferents associated with specific modalities and provide useful genetic tools with which to analyze the pathways that are activated by functionally distinct neuronal populations and transmitters.
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33.
  • Lagerström, Malin C., et al. (författare)
  • VGLUT2-Dependent Sensory Neurons in the TRPV1 Population Regulate Pain and Itch
  • 2010
  • Ingår i: Neuron. - : Elsevier BV. - 0896-6273 .- 1097-4199. ; 68:3, s. 529-542
  • Tidskriftsartikel (refereegranskat)abstract
    • The natural response to itch sensation is to scratch, which relieves the itch through an unknown mechanism. Interaction between pain and itch has been frequently demonstrated, and the selectivity hypothesis of itch, based on data from electrophysiological and behavioral experiments, postulates the existence of primary pain afferents capable of repressing itch. Here, we demonstrate that deletion of vesicular glutamate transporter (VGLUT) 2 in a subpopulation of neurons partly overlapping with the vanilloid receptor (TRPV1) primary afferents resulted in a dramatic increase in itch behavior accompanied by a reduced responsiveness to thermal pain. The increased itch behavior was reduced by administration of antihistaminergic drugs and by genetic deletion of the gastrin-releasing peptide receptor, demonstrating a dependence on VGLUT2 to maintain normal levels of both histaminergic and nonhistaminergic itch. This study establishes that VGLUT2 is a major player in TRPV1 thermal nociception and also serves to regulate a normal itch response.
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34.
  • Larhammar, Martin, 1985- (författare)
  • Neuronal Networks of Movement : Slc10a4 as a Modulator & Dmrt3 as a Gait-keeper
  • 2014
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Nerve cells are organized into complex networks that comprise the building blocks of our nervous system. Neurons communicate by transmitting messenger molecules released from synaptic vesicles. Alterations in neuronal circuitry and synaptic signaling contribute to a wide range of neurological conditions, often with consequences for movement. Intrinsic neuronal networks in the spinal cord serve to coordinate vital rhythmic motor functions. In spite of extensive efforts to address the organization of these neural circuits, much remains to be revealed regarding the identity and function of specific interneuron cell types and how neuromodulation tune network activity. In this thesis, two novel genes initially identified as markers for spinal neuronal populations were investigated: Slc10a4 and Dmrt3.The orphan transporter SLC10A4 was found to be expressed on synaptic vesicles of the cholinergic system, including motor neurons, as well as in the monoaminergic system, including dopaminergic, serotonergic and noradrenergic nuclei. Thus, it constitutes a novel molecular denominator shared by these classic neuromodulatory systems. SLC10A4 was found to influence vesicular transport of dopamine and affect neuronal release and reuptake efficiency in the striatum. Mice lacking Slc10a4 displayed impaired monoamine homeostasis and were hypersensitive to the drugs amphetamine and tranylcypromine. These findings demonstrate that SLC10A4 is capable of modulating the modulatory systems of the brain with potential clinical relevance for neurological and mental disorders.The transcription factor encoded by Dmrt3 was found to be expressed in a population of inhibitory commissural interneurons originating from the dorsal interneuron 6 (dI6) domain in the spinal cord. In parallel, a genome-wide association study revealed that a non-sense mutation in horse DMRT3 is permissive for the ability to perform pace among other alternate gaits. Further analysis of Dmrt3 null mutant mice showed that Dmrt3 has a central role for spinal neuronal network development with consequences for locomotor behavior. The dI6 class has been suggested to take part in motor circuits but remains one of the least studied classes due to lack of molecular markers. To further investigate the Dmrt3-derived neurons, and the dI6 population in general, a Dmrt3Cre mouse line was generated which allowed for characterization on the molecular, cellular and  behavioral level. It was found that Dmrt3 neurons synapse onto motor neurons, receive extensive synaptic inputs from various neuronal sources and are rhythmically active during fictive locomotion. Furthermore, silencing of Dmrt3 neurons in Dmrt3Cre;Viaatlx/lx mice led to impaired motor coordination and alterations in gait, together demonstrating the importance of this neuronal population in the control of movement.
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35.
  • Larhammar, Martin, 1985-, et al. (författare)
  • SLC10A4 Is a Vesicular Amine-Associated Transporter Modulating Dopamine Homeostasis
  • 2015
  • Ingår i: Biological Psychiatry. - : Elsevier BV. - 0006-3223 .- 1873-2402. ; 77:6, s. 526-536
  • Tidskriftsartikel (refereegranskat)abstract
    • BackgroundThe neuromodulatory transmitters, biogenic amines, have profound effects on multiple neurons and are essential for normal behavior and mental health. Here we report that the orphan transporter SLC10A4, which in the brain is exclusively expressed in presynaptic vesicles of monoaminergic and cholinergic neurons, has a regulatory role in dopamine homeostasis.MethodsWe used a combination of molecular and behavioral analyses, pharmacology, and in vivo amperometry to assess the role of SLC10A4 in dopamine-regulated behaviors.ResultsWe show that SLC10A4 is localized on the same synaptic vesicles as either vesicular acetylcholine transporter or vesicular monoamine transporter 2. We did not find evidence for direct transport of dopamine by SLC10A4; however, synaptic vesicle preparations lacking SLC10A4 showed decreased dopamine vesicular uptake efficiency. Furthermore, we observed an increased acidification in synaptic vesicles isolated from mice overexpressing SLC10A4. Loss of SLC10A4 in mice resulted in reduced striatal serotonin, noradrenaline, and dopamine concentrations and a significantly higher dopamine turnover ratio. Absence of SLC10A4 led to slower dopamine clearance rates in vivo, which resulted in accumulation of extracellular dopamine. Finally, whereas SLC10A4 null mutant mice were slightly hypoactive, they displayed hypersensitivity to administration of amphetamine and tranylcypromine.ConclusionsOur results demonstrate that SLC10A4 is a vesicular monoaminergic and cholinergic associated transporter that is important for dopamine homeostasis and neuromodulation in vivo. The discovery of SLC10A4 and its role in dopaminergic signaling reveals a novel mechanism for neuromodulation and represents an unexplored target for the treatment of neurological and mental disorders.
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36.
  •  
37.
  • Nordenankar, Karin, 1981- (författare)
  • Functional Analysis of the Vesicular Glutamate Transporter 2 in Specific Neuronal Circuits of the Brain
  • 2012
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • A key issue in neuroscience is to determine the connection between neuronal circuits and behaviour. In the adult brain, all neuronal circuits include a glutamatergic component. Three proteins designated Vesicular glutamate transporter 1-3 (VGLUT1-3) possess the capability of packaging glutamate into presynaptic vesicles for release of glutamate at the nerve terminal. The present study aimed at determining the role of VGLUT2 in neuronal circuits of higher brain function, emotion, and reward-pocessing. A conditional knockout (cKO) strategy was utilised, and three different mouse lines were produced to delete VGLUT2 in specific neuronal circuits in a temporally and spatially controlled manner. First, we produced a cKO mouse in which Vglut2 was deleted in specific subpopulations of the cortex, amygdala and hippocampus from preadolescence. This resulted in blunted aspects in cognitive, emotional and social behaviour in a schizophrenia-related phenotype. Furthermore, we showed a downstream effect of the targeted deletion on the dopaminergic system. In a subsequent analysis of the same cKO mice, we showed that female cKO mice were more affected their male counterparts, and we also found that female schizophrenia patients, but not male patients, had increased Vglut2 levels in the cortex.  Second, we produced and analysed cKO mice in which Vglut2 was deleted in the cortex, amygdala and hippocampus already from midgestation, and could show that this deletion affected emotional, but not cognitive, function. Third, we addressed the role of VGLUT2 in midbrain dopamine neurons by targeting Vglut2 specifically in these neurons. These cKO mice showed a blunted activational response to the psychostimulant amphetamine and increased operant self-administration of both sugar and cocaine reinforcers. Further, the cKO mice displayed strongly enhanced cocaine-seeking in response to cocaine-associated cues, a behaviour of relevance for addiction in humans. In summary, this thesis work has addressed the role of the presynaptic glutamatergic neuron in different neuronal circuits and shown that the temporal and spatial distribution of VGLUT2 is of great significance for normal brain function.
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38.
  • Nordenankar, Karin, et al. (författare)
  • Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity
  • 2015
  • Ingår i: Brain Structure and Function. - : Springer Science and Business Media LLC. - 1863-2653 .- 1863-2661. ; 220:4, s. 2171-2190
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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39.
  • Nordenankar, Karin, et al. (författare)
  • Targeted deletion of Vglut2 expression in the embryonal telencephalon promotes an anxiolytic phenotype of the adult mouse
  • 2015
  • Ingår i: Upsala Journal of Medical Sciences. - : Uppsala Medical Society. - 0300-9734 .- 2000-1967. ; 120:3
  • Tidskriftsartikel (refereegranskat)abstract
    • BACKGROUND: Anxiety is a natural emotion experienced by all individuals. However, when anxiety becomes excessive, it contributes to the substantial group of anxiety disorders that affect one in three people and thus are among the most common psychiatric disorders. Anxiolysis, the reduction of anxiety, is mediated via several large groups of therapeutical compounds, but the relief is often only temporary, and increased knowledge of the neurobiology underlying anxiety is needed in order to improve future therapies.AIM: We previously demonstrated that mice lacking forebrain expression of the Vesicular glutamate transporter 2 (Vglut2) from adolescence showed a strong anxiolytic behaviour as adults. In the current study, we wished to analyse if removal of Vglut2 expression already from mid-gestation of the mouse embryo would give rise to similar anxiolysis in the adult mouse.METHODS: We produced transgenic mice lacking Vglut2 from mid-gestation and analysed their affective behaviour, including anxiety, when they had reached adulthood.RESULTS: The transgenic mice lacking Vglut2 expression from mid-gestation showed certain signs of anxiolytic behaviour, but this phenotype was not as prominent as when Vglut2 was removed during adolescence.CONCLUSION: Our results suggest that both embryonal and adolescent forebrain expression of Vglut2 normally contributes to balancing the level of anxiety. As the neurobiological basis for anxiety is similar across species, our results in mice may help improve the current understanding of the neurocircuitry of anxiety, and hence anxiolysis, also in humans.
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40.
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41.
  • Papathanou, Maria, 1984-, et al. (författare)
  • Off-Target Effects in Transgenic Mice : Characterization of Dopamine Transporter (DAT)-Cre Transgenic Mouse Lines Exposes Multiple Non-Dopaminergic Neuronal Clusters Available for Selective Targeting within Limbic Neurocircuitry
  • 2019
  • Ingår i: eNeuro. - 2373-2822. ; 6:5
  • Tidskriftsartikel (refereegranskat)abstract
    • 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.
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42.
  • Papathanou, Maria, 1984-, et al. (författare)
  • Targeting VGLUT2 in Mature Dopamine Neurons Decreases Mesoaccumbal Glutamatergic Transmission and Identifies a Role for Glutamate Co-release in Synaptic Plasticity by Increasing Baseline AMPA/NMDA Ratio
  • 2018
  • Ingår i: Frontiers in Neural Circuits. - : Frontiers Media SA. - 1662-5110. ; 12, s. 1-20
  • Tidskriftsartikel (refereegranskat)abstract
    • Expression of the Vglut2/Slc17a6 gene encoding the Vesicular glutamate transporter 2 (VGLUT2) in midbrain dopamine (DA) neurons enables these neurons to co-release glutamate in the nucleus accumbens (NAc), a feature of putative importance to drug addiction. For example, it has been shown that conditional deletion of Vglut2 gene expression within developing DA neurons in mice causes altered locomotor sensitization to addictive drugs, such as amphetamine and cocaine, in adulthood. Alterations in DA neurotransmission in the mesoaccumbal pathway has been proposed to contribute to these behavioral alterations but the underlying molecular mechanism remains largely elusive. Repeated exposure to cocaine is known to cause lasting adaptations of excitatory synaptic transmission onto medium spiny neurons (MSNs) in the NAc, but the putative contribution of VGLUT2-mediated glutamate co-release from the mesoaccumbal projection has never been investigated. In this study, we implemented a tamoxifen-inducible Cre-LoxP strategy to selectively probe VGLUT2 in mature DA neurons of adult mice. Optogenetics-coupled patch clamp analysis in the NAc demonstrated a significant reduction of glutamatergic neurotransmission, whilst behavioral analysis revealed a normal locomotor sensitization to amphetamine and cocaine. When investigating if the reduced level of glutamate co-release from DA neurons caused a detectable post-synaptic effect on MSNs, patch clamp analysis identified an enhanced baseline AMPA/NMDA ratio in DA receptor subtype 1 (DRD1)-expressing accumbal MSNs which occluded the effect of cocaine on synaptic transmission. We conclude that VGLUT2 in mature DA neurons actively contributes to glutamatergic neurotransmission in the NAc, a finding which for the first time highlights VGLUT2-mediated glutamate co-release in the complex mechanisms of synaptic plasticity in drug addiction.
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43.
  • Prasad, Asheeta A., et al. (författare)
  • Architecture of the subthalamic nucleus
  • 2024
  • Ingår i: Communications Biology. - : Springer Nature. - 2399-3642. ; 7:1
  • Forskningsöversikt (refereegranskat)abstract
    • The subthalamic nucleus (STN) is a major neuromodulation target for the alleviation of neurological and neuropsychiatric symptoms using deep brain stimulation (DBS). STN-DBS is today applied as treatment in Parkinson ' s disease, dystonia, essential tremor, and obsessive-compulsive disorder (OCD). STN-DBS also shows promise as a treatment for refractory Tourette syndrome. However, the internal organization of the STN has remained elusive and challenges researchers and clinicians: How can this small brain structure engage in the multitude of functions that renders it a key hub for therapeutic intervention of a variety of brain disorders ranging from motor to affective to cognitive? Based on recent gene expression studies of the STN, a comprehensive view of the anatomical and cellular organization, including revelations of spatio-molecular heterogeneity, is now possible to outline. In this review, we focus attention to the neurobiological architecture of the STN with specific emphasis on molecular patterns discovered within this complex brain area. Studies from human, non-human primate, and rodent brains now reveal anatomically defined distribution of specific molecular markers. Together their spatial patterns indicate a heterogeneous molecular architecture within the STN. Considering the translational capacity of targeting the STN in severe brain disorders, the addition of molecular profiling of the STN will allow for advancement in precision of clinical STN-based interventions. The subthalamic nucleus is a highly evolutionary conserved brain region. Emerging studies reveal spatio-molecular heterogeneity leading to improved understanding of the organization of the STN and targeting for specific neurological disorders.
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44.
  • Pupe, Stefano, et al. (författare)
  • Cre-driven optogenetics in the heterogeneous genetic panorama of the VTA
  • 2015
  • Ingår i: TINS - Trends in Neurosciences. - : Elsevier BV. - 0166-2236 .- 1878-108X. ; 38:6, s. 375-386
  • Forskningsöversikt (refereegranskat)abstract
    • The selectivity of optogenetics commonly relies on genetic promoters to manipulate specific populations of neurons through the use of Cre-driver lines. All studies performed in the ventral tegmental area (VTA) so far have utilized promoters present in groups of cells that release dopamine (DA), GABA, or glutamate. However, neurons that co-release neurotransmitters and variabilities within groups of neurons that release the same neurotransmitter present challenges when evaluating the results. Further complexity is introduced by ectopic expression patterns often occurring in transgenic Cre-drivers. New perspectives could be unfolded by identifying and selecting different types of promoter for driving the Cre recombinase. Here, we discuss some promising candidates and highlight the advantages or disadvantages of different methods for creating novel transgenic lines.
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45.
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46.
  • Rajagopalan, Aparna, et al. (författare)
  • Reduced Gene Expression Levels of Munc13-1 and Additional Components of the Presynaptic Exocytosis Machinery Upon Conditional Targeting of Vglut2 in the Adolescent Mouse
  • 2014
  • Ingår i: Synapse. - : Wiley. - 0887-4476 .- 1098-2396. ; 68:12, s. 624-633
  • Tidskriftsartikel (refereegranskat)abstract
    • Presynaptic proteins orchestrate an intricate interplay of dynamic interactions in order to regulate quantal exocytosis of transmitter-filled vesicles, and their dysregulation might cause neurological and neuropsychiatric dysfunction. Mice carrying a spatiotemporal restriction in the expression of the Vesicular glutamate transporter 2 (Vglut2; aka Slc17a6) in the cortex, amygdala and hippocampal subiculum from the third postnatal week show a strong anxiolytic phenotype and certain behavioral correlates of schizophrenia. To further understand the molecular consequences of this targeted deletion of Vglut2, we performed an unbiased microarray analysis comparing gene expression levels in the subiculum of these conditional Vglut2 knockout mice (Vglut2(f/f;CamKII) cKO) to those in control littermates. Expression of Unc13C (Munc13-3), a member of the Unc/Munc family, previously shown to be important for glutamatergic transmission, was identified to be significantly down-regulated. Subsequent analysis by quantitative RT-PCR revealed a 50% down-regulation of Munc 13-1, the gene encoding the Unc/Munc subtype described as an essential component in the majority of glutamtergic synapses in the hippocampus. Genes encoding additional components of the presynaptic machinery were also found regulated, including Rab3A, RIM1, as well as Syntaxin1 and Synaptobrevin. Altered expression levels of these genes were further found in the amygdala and in the retrosplenial group of the cortex, additional regions in which Vglut2 was conditionally targeted. These findings suggest that expression levels of Vglut2 might be important for the maintenance of gene expression in the presynaptic machinery in the adult mouse brain. Synapse 68:624-633, 2014.
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47.
  • Santin, Marie des Neiges, et al. (författare)
  • Anatomical characterisation of three different psychosurgical targets in the subthalamic area : from the basal ganglia to the limbic system
  • 2023
  • Ingår i: Brain Structure and Function. - : Springer. - 1863-2653 .- 1863-2661. ; 228, s. 1977-1992
  • Tidskriftsartikel (refereegranskat)abstract
    • Effective neural stimulation for the treatment of severe psychiatric disorders needs accurate characterisation of surgical targets. This is especially true for the medial subthalamic region (MSR) which contains three targets: the anteromedial STN for obsessive compulsive disorder (OCD), the medial forebrain bundle (MFB) for depression and OCD, and the "Sano triangle" for pathological aggressiveness. Blocks containing the subthalamic area were obtained from two human brains. After obtaining 11.7-Tesla MRI, blocks were cut in regular sections for immunohistochemistry. Fluorescent in situ hybridisation was performed on the macaque MSR. Electron microscopic observation for synaptic specialisation was performed on human and macaque subthalamic fresh samples. Images of human brain sections were reconstructed in a cryoblock which was registered on the MRI and histological slices were then registered. The STN contains glutamatergic and fewer GABAergic neurons and has no strict boundary with the adjacent MSR. The anteromedial STN has abundant dopaminergic and serotoninergic innervation with very sparse dopaminergic neurons. The MFB is composed of dense anterior dopaminergic and posterior serotoninergic fibres, and fewer cholinergic and glutamatergic fibres. Medially, the Sano triangle presumably contains orexinergic terminals from the hypothalamus, and neurons with strong nuclear oestrogen receptor-alpha staining with a decreased anteroposterior and mediolateral gradient of staining. These findings provide new insight regarding MSR cells and their fibre specialisation, forming a transition zone between the basal ganglia and the limbic systems. Our 3D reconstruction enabled us to visualize the main histological features of the three targets which should enable better targeting and understanding of neuromodulatory stimulation results in severe psychiatric conditions.
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48.
  • Schweizer, Nadine, 1985- (författare)
  • Across Borders : A Histological and Physiological Study of the Subthalamic Nucleus in Reward and Movement
  • 2016
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • 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 STN development, function in the adult central nervous system and defects caused by specific deletion. Eventually identifying and characterizing subpopulations of the STN can contribute to the optimization of deep brain stimulation and help to reduce its side-effects, or even open up possibilities for genetic or optogenetic therapy approaches.
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49.
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50.
  • Schweizer, Nadine, et al. (författare)
  • Limiting glutamate transmission in a Vglut2-expressing subpopulation of the subthalamic nucleus is sufficient to cause hyperlocomotion
  • 2014
  • Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 111:21, s. 7837-7842
  • Tidskriftsartikel (refereegranskat)abstract
    • The subthalamic nucleus (STN) is a key area of the basal ganglia circuitry regulating movement. We identified a subpopulation of neurons within this structure that coexpresses Vglut2 and Pitx2, and by conditional targeting of this subpopulation we reduced Vglut2 expression levels in the STN by 40%, leaving Pitx2 expression intact. This reduction diminished, yet did not eliminate, glutamatergic transmission in the substantia nigra pars reticulata and entopeduncular nucleus, two major targets of the STN. The knockout mice displayed hyperlocomotion and decreased latency in the initiation of movement while preserving normal gait and balance. Spatial cognition, social function, and level of impulsive choice also remained undisturbed. Furthermore, these mice showed reduced dopamine transporter binding and slower dopamine clearance in vivo, suggesting that Vglut2-expressing cells in the STN regulate dopaminergic transmission. Our results demonstrate that altering the contribution of a limited population within the STN is sufficient to achieve results similar to STN lesions and high-frequency stimulation, but with fewer side effects.
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