The Differential Contribution of Striatonigral and Striatopallidal Neurons in Mediating Responses to Therapeutic Agents and Drugs of Abuse: A Dual Role for DARPP-32

The basal ganglia are a set of subcortical structures which integrate information from diverse brain areas to coordinate vital behaviors including movement, reward, and motivational processes. The striatum is the main input center of the basal ganglia which sends projections to the output nuclei via two pathways, the direct striatonigral pathway and the indirect striatopallidal pathway. These two pathways work together to modulate behavior and imbalance of these pathways can have profound physiological consequences. DARPP-32 is a dual function kinase/phosphatase inhibitor which has been shown to be a key mediator of signaling in both striatonigral and triatopallidal neurons. A variety of therapeutic agents and drugs of abuse can affect the phosphorylation of DARPP-32. Psychostimulants such as cocaine increase DARPP-32 phosphorylation at its main regulatory site, T34. Paradoxically, antipsychotics such as haloperidol also increase T34 phosphorylation to a similar degree. Despite this similar biochemical regulation, psychostimulants and antipsychotics have opposing behavioral and clinical effects. We hypothesized that these drugs act via the same biochemical pathway but in distinct populations of striatal neurons. To directly test this idea, we generated BAC transgenic mice which express epitope tagged DARPP-32 selectively in striatonigral and striatopallidal neurons using the D1 and D2 receptor promoters. We developed a protocol to immunoprecipitate DARPP-32 from drug treated mice and study phosphorylation in a cell-type specific manner. Using this new methodology we demonstrate that the increases in T34 phosphorylation with acute cocaine and haloperidol are restricted to striatonigral and striatopallidal neurons, respectively. Additionally, we show that the changes in DARPP-32 phosphorylation induced by a variety of drugs targeting the striatum have cell-type specific patterns. In a complimentary approach, we generated conditional knock-out mice in which DARPP-32 is selectively deleted in striatonigral or striatopallidal neurons. This allowed us to study the behavioral consequences of alteration in the direct and indirect pathways on psychostimulant and antipsychotic mediated locomotor behavior. These studies provided direct evidence for the theory that the direct and indirect pathways exert opposing influences on locomotor behavior. Additionally, we showed that dopamine can differentially modulate activity in these pathways resulting in a synergistic stimulation of locomotor activity

Corticostriatal Transmission Is Selectively Enhanced in Striatonigral Neurons with Postnatal Loss of Tsc1.

mTORC1 is a central signaling hub that integrates intra- and extracellular signals to regulate a variety of cellular metabolic processes. Mutations in regulators of mTORC1 lead to neurodevelopmental disorders associated with autism, which is characterized by repetitive, inflexible behaviors. These behaviors may result from alterations in striatal circuits that control motor learning and habit formation. However, the consequences of mTORC1 dysregulation on striatal neuron function are largely unknown. To investigate this, we deleted the mTORC1 negative regulator Tsc1 from identified striatonigral and striatopallidal neurons and examined how cell-autonomous upregulation of mTORC1 activity affects their morphology and physiology. We find that loss of Tsc1 increases the excitability of striatonigral, but not striatopallidal, neurons and selectively enhances corticostriatal synaptic transmission. These findings highlight the critical role of mTORC1 in regulating striatal activity in a cell type- and input-specific manner, with implications for striatonigral pathway dysfunction in neuropsychiatric disease

Combinatorial Expression of Grp and Neurod6 Defines Dopamine Neuron Populations with Distinct Projection Patterns and Disease Vulnerability

Midbrain dopamine neurons project to numerous targets throughout the brain to modulate various behaviors and brain states. Within this small population of neurons exists significant heterogeneity based on physiology, circuitry, and disease susceptibility. Recent studies have shown that dopamine neurons can be subdivided based on gene expression; however, the extent to which genetic markers represent functionally relevant dopaminergic subpopulations has not been fully explored. Here we performed single-cell RNA-sequencing of mouse dopamine neurons and validated studies showing that Neurod6 and Grp are selective markers for dopaminergic subpopulations. Using a combination of multiplex fluorescent in situ hybridization, retrograde labeling, and electrophysiology in mice of both sexes, we defined the anatomy, projection targets, physiological properties, and disease vulnerability of dopamine neurons based on Grp and/or Neurod6 expression. We found that the combinatorial expression of Grp and Neurod6 defines dopaminergic subpopulations with unique features. Grp+/Neurod6+ dopamine neurons reside in the ventromedial VTA, send projections to the medial shell of the nucleus accumbens, and have noncanonical physiological properties. Grp+/Neurod6- dopamine neurons are found in the VTA as well as in the ventromedial portion of the SNc, where they project selectively to the dorsomedial striatum. Grp-/Neurod6+ dopamine neurons represent a smaller VTA subpopulation, which is preferentially spared in a 6-OHDA model of Parkinson's disease. Together, our work provides detailed characterization of Neurod6 and Grp expression in the midbrain and generates new insights into how these markers define functionally relevant dopaminergic subpopulations

Hyperexcitability of the local cortical circuit in mouse models of tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a neurogenetic disorder associated with epilepsy, intellectual disabilities, and autistic behaviors. These neurological symptoms result from synaptic dysregulations, which shift a balance between excitation and inhibition. To decipher the synaptic substrate of hyperexcitability, we examined pan-neuronal Tsc1 knockout mouse and found a reduction in surface expression of a GABA receptor (GABAR) subunit but not AMPA receptor (AMPAR) subunit. Using electrophysiological recordings, we found a significant reduction in the frequency of GABAR-mediated miniature inhibitory postsynaptic currents (GABAR-mIPSCs) but not AMPAR-mediated miniature excitatory postsynaptic currents (AMPAR-mEPSCs) in layer 2/3 pyramidal neurons. To determine a subpopulation of interneurons that are especially vulnerable to the absence of TSC1 function, we also analyzed two strains of conditional knockout mice targeting two of the prominent interneuron subtypes that express parvalbumin (PV) or somatostatin (SST). Unlike pan-neuronal knockout mice, both interneuron-specific Tsc-1 knockout mice did not develop spontaneous seizures and grew into adults. Further, the properties of AMPAR-mEPSCs and GABAR-mIPSCs were normal in both Pv-Cre and Sst-Cre x Tsc1fl/fl knockout mice. These results indicate that removal of TSC1 from all neurons in a local cortical circuit results in hyperexcitability while connections between pyramidal neurons and interneurons expressing PV and SST are preserved in the layer 2/3 visual cortex. Our study suggests that another inhibitory cell type or a combination of multiple subtypes may be accountable for hyperexcitability in TSC. Keywords: Tuberous sclerosis complex; E/I balance; AMPA receptor; GABA receptor; Autism; Epilepsy; mTOR pathwa

Bupropion Increases Selection of High Effort Activity in Rats Tested on a Progressive Ratio/Chow Feeding Choice Procedure: Implications for Treatment of Effort-Related Motivational Symptoms

Background: Depression and related disorders are characterized by deficits in behavioral activation, exertion of effort, and other psychomotor/motivational dysfunctions. Depressed patients show alterations in effort-related decision making and a bias towards selection of low effort activities. It has been suggested that animal tests of effort-related decision making could be useful as models of motivational dysfunctions seen in psychopathology. Methods: Because clinical studies have suggested that inhibition of catecholamine uptake may be a useful strategy for treatment of effort-related motivational symptoms, the present research assessed the ability of bupropion to increase work output in rats responding on a test of effort-related decision-making (ie, a progressive ratio/chow feeding choice task). With this task, rats can choose between working for a preferred food (high-carbohydrate pellets) by lever pressing on a progressive ratio schedule vs obtaining a less preferred laboratory chow that is freely available in the chamber. Results: Bupropion (10.0–40.0 mg/kg intraperitoneal) significantly increased all measures of progressive ratio lever pressing, but decreased chow intake. These effects were greatest in animals with low baseline levels of work output on the progressive ratio schedule. Because accumbens dopamine is implicated in effort-related processes, the effects of bupropion on markers of accumbens dopamine transmission were examined. Bupropion elevated extracellular dopamine levels in accumbens core as measured by microdialysis and increased phosphorylated dopamine and cyclic-AMP related phosphoprotein 32 kDaltons (pDARPP-32) immunoreactivity in a manner consistent with D1 and D2 receptor stimulation. Conclusion: The ability of bupropion to increase exertion of effort in instrumental behavior may have implications for the pathophysiology and treatment of effort-related motivational symptoms in humans

Perturbed proteostasis in autism spectrum disorders

Dynamic changes in synaptic strength rely on de novo protein synthesis and protein degradation by the ubiquitin proteasome system (UPS). Disruption of either of these cellular processes will result in significant impairments in synaptic plasticity and memory formation. Mutations in several genes encoding regulators of mRNA translation and members of the UPS have been associated with an increased risk for the development of autism spectrum disorders. It is possible that these mutations result in a similar imbalance in protein homeostasis (proteostasis) at the synapse. This review will summarize recent work investigating the role of the UPS in synaptic plasticity at glutamatergic synapses, and propose that dysfunctional proteostasis is a common consequence of several genetic mutations linked to autism spectrum disorders. [Image: see text] Dynamic changes in synaptic strength rely on de novo protein synthesis and protein degradation by the ubiquitin proteasome system (UPS). Disruption of either of these cellular processes will result in significant impairments in synaptic plasticity and memory formation. Mutations in several genes encoding regulators of mRNA translation (i.e. FMR1) and protein degradation (i.e. UBE3A) have been associated with an increased risk for autism spectrum disorders and intellectual disability (ASD/ID). These mutations similarly disrupt protein homeostasis (proteostasis). Compensatory changes that reset the rate of proteostasis may contribute to the neurological symptoms of ASD/ID. This review summarizes recent work investigating the role of the UPS in synaptic plasticity at glutamatergic synapses, and proposes that dysfunctional proteostasis is a common consequence of several genetic mutations linked to ASD. This article is part of a mini review series: “Synaptic Function and Dysfunction in Brain Diseases”

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties