Spatio-molecular domains identified in the mouse subthalamic nucleus and neighboring glutamatergic and GABAergic brain structures.

The subthalamic nucleus (STN) is crucial for normal motor, limbic and associative function. STN dysregulation is correlated with several brain disorders, including Parkinson's 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 STN´s internal organization should prove useful towards genetics-based functional decoding of this clinically relevant brain structure.

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.

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.

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.

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.

Nurr1 and Retinoid X Receptor Ligands Stimulate Ret Signaling in Dopamine Neurons and Can Alleviate α-Synuclein Disrupted Gene Expression.

α-synuclein, a protein enriched in Lewy bodies and highly implicated in neurotoxicity in Parkinson's disease, is distributed both at nerve terminals and in the cell nucleus. Here we show that a nuclear derivative of α-synuclein induces more pronounced changes at the gene expression level in mouse primary dopamine (DA) neurons compared to a derivative that is excluded from the nucleus. Moreover, by RNA sequencing we analyzed the extent of genome-wide effects on gene expression resulting from expression of human α-synuclein in primary mouse DA neurons. The results implicated the transcription factor Nurr1 as a key dysregulated target of α-synuclein toxicity. Forced Nurr1 expression restored the expression of hundreds of dysregulated genes in primary DA neurons expressing α-synuclein, and therefore prompted us to test the possibility that Nurr1 can be pharmacologically targeted by bexarotene, a ligand for the retinoid X receptor that forms heterodimers with Nurr1. Although our data demonstrated that bexarotene was ineffective in neuroprotection in rats in vivo, the results revealed that bexarotene has the capacity to coregulate subsets of Nurr1 target genes including the receptor tyrosine kinase subunit Ret. Moreover, bexarotene was able to restore dysfunctional Ret-dependent neurotrophic signaling in α-synuclein-overexpressing mouse DA neurons. These data highlight the role of the Nurr1-Ret signaling pathway as a target of α-synuclein toxicity and suggest that retinoid X receptor ligands with appropriate pharmacological properties could have therapeutic potential in Parkinson's disease. SIGNIFICANCE STATEMENT: How α-synuclein, a protein enriched in Lewy bodies in Parkinson's disease, is causing neuropathology in dopamine neurons remains unclear. This study elucidated how α-synuclein is influencing gene expression and how Nurr1, a transcription factor known to protect dopamine neurons against α-synuclein toxicity, can counteract these effects. Moreover, given the protective role of Nurr1, this study also investigated how Nurr1 could be pharmacologically targeted via bexarotene, a ligand of Nurr1's heterodimerization partner retinoid X receptor (RXR). The results showed that RXR ligands could increase neurotrophic signaling, but provided a mixed picture of its potential in a Parkinson's disease rat model in vivo. However, this study clearly emphasized Nurr1's neuroprotective role and indicated that other RXR ligands could have therapeutic potential in Parkinson's disease.

Sox6 and Otx2 control the specification of substantia nigra and ventral tegmental area dopamine neurons.

Distinct midbrain dopamine (mDA) neuron subtypes are found in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA), but it is mainly SNc neurons that degenerate in Parkinson's disease. Interest in how mDA neurons develop has been stimulated by the potential use of stem cells in therapy or disease modeling. However, very little is known about how specific dopaminergic subtypes are generated. Here, we show that the expression profiles of the transcription factors Sox6, Otx2, and Nolz1 define subpopulations of mDA neurons already at the neural progenitor cell stage. After cell-cycle exit, Sox6 selectively localizes to SNc neurons, while Otx2 and Nolz1 are expressed in a subset of VTA neurons. Importantly, Sox6 ablation leads to decreased expression of SNc markers and a corresponding increase in VTA markers, while Otx2 ablation has the opposite effect. Moreover, deletion of Sox6 affects striatal innervation and dopamine levels. We also find reduced Sox6 levels in Parkinson's disease patients. These findings identify Sox6 as a determinant of SNc neuron development and should facilitate the engineering of relevant mDA neurons for cell therapy and disease modeling.

The H3 receptor agonist immepip does not affect l-dopa-induced abnormal involuntary movements in 6-OHDA-lesioned rats.

The treatment of dyskinesia in Parkinson׳s disease remains poor but H3 receptor agonists have been suggested as a novel pharmacological approach. We examined the effects of the H3 agonist, immepip, in 6-OHDA-lesioned rats exhibiting AIMs (abnormal involuntary movements), a rat analogue of dyskinesia, in response to l-dopa compared to the known anti-dyskinetic agents amantadine, MK-801 and 8-OHDPAT. We then attempted to extend these studies in to dyskinetic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated common marmosets. Amantadine, MK-801 and 8-OHDPAT all dose-dependently reduced l-dopa-induced axial, lingual and oral (ALO) AIMs in 6-OHDA-lesioned animals accompanied by a reduction in contralateral rotation with higher doses of amantadine and MK-801. By contrast, immepip had no effect on AIMs expression or contralateral rotation. In the MPTP-treated common marmoset exhibiting dyskinesia to l-dopa, immepip alone induced retching and in combination with l-dopa administered subcutaneously or orally induced the rapid onset of retching and vomiting which was not controlled by pretreatment with domperidone. Administration of the unrelated H3 agonist, imetit had the same effect. Despite causing negative side-effects, it appears that both agonists reduced the antiparkinsonian response to l-dopa resulting in reduced dyskinesia. H3 agonists appear unlikely candidates for the treatment of dyskinesia in PD based on lack of evidence of efficacy and potential adverse effects.

Levodopa infusion does not decrease the onset of abnormal involuntary movements in parkinsonian rats.

The short duration of effect of levodopa is linked to pulsatile stimulation of striatal dopamine receptors and dyskinesia induction. However, the recent introduction of intraduodenal (i.d.) infusions and novel oral controlled release formulations of l-dopa may prevent dyskinesia induction and reduce the severity of established involuntary movements. We have compared the effects of twice-daily intraperitoneal (i.p.) administration and daily i.d. infusion of l-dopa on the induction and expression of abnormal involuntary movements in 6-hydroxydopamine (6-OHDA)-lesioned rats. Animals were treated with either twice-daily i.p. administration of l-dopa/carbidopa (7.85/12.5 mg/kg) or an 8-hour i.d. infusion of l-dopa/carbidopa (20/5 mg/mL; infusion rate: 0.04 mL/h) for 14 days, after which treatments were switched between groups and continued for a further 14 days. Pulsatile i.p. administration of l-dopa induced moderate to severe abnormal involuntary movements, which gradually increased in severity over the 14 days, but i.d. infusion of l-dopa induced abnormal involuntary movements of a similar severity. Switching from continuous i.d. to pulsatile i.p. administration of l-dopa continued to provoke severe abnormal involuntary movements expression. Switching from pulsatile i.p. to continuous i.d. l-dopa administration did not alter the peak abnormal involuntary movement severity but tended to reduce their duration. Treatment with less pulsatile l-dopa administration using i.d. infusion does not reduce the risk of the appearance of dyskinesia. By contrast, the duration of established dyskinesia can be reduced by more continuous l-dopa delivery in agreement with clinical experience.

Induction and expression of abnormal involuntary movements is related to the duration of dopaminergic stimulation in 6-OHDA-lesioned rats.

Dyskinesia induction in Parkinson's disease (PD) appears less marked with long-acting dopamine agonists than with short-acting L-Dopa, but the relationship to duration of drug action is unknown. It is also unclear whether the duration of drug action affects the expression of established dyskinesia. This study compared the ability of L-Dopa and four dopamine agonists of different duration of action to induce abnormal involuntary movements (AIMs) in 6-hydroxydopamine (6-OHDA)-lesioned rats, and their ability to express established AIMs following prior exposure to L-Dopa. 6-OHDA-lesioned rats were treated with saline, L-Dopa/benserazide, apomorphine, ropinirole, pramipexole or pergolide once daily for 15 days. Repeated administration of the short-acting dopamine agonists, apomorphine (duration 80 min) and ropinirole (duration 90 min) induced marked axial, limb and orolingual AIMs at peak effect. L-Dopa (duration 100 min) produced moderate AIMs at peak effect, while administration of the long-acting dopamine agonists, pramipexole (duration 150 min) and pergolide (duration 240 min) resulted in mild AIMs. In rats primed to exhibit severe AIMs following repeated L-Dopa administration, acute administration of apomorphine, ropinirole and L-Dopa induced severe AIMs. By contrast, pramipexole and pergolide evoked only mild-moderate AIMs. Again, there was a negative correlation between duration of effect and the severity of AIMs expressed. These studies show that both the induction and expression of AIMs in 6-OHDA-lesioned rats are related to the duration of action of dopaminergic drugs. These findings suggest that continuous dopaminergic stimulation could be used both to avoid dyskinesia induction and to improve motor function in late-stage PD when troublesome dyskinesia is evident.