Biology and Bias in Cell Type-Specific RNAseq of Nucleus Accumbens Medium Spiny Neurons
Hope KronmanFelix RichterBenoît LabontéRamesh ChandraShan ZhaoGabriel E. HoffmanMary Kay LoboEric E. SchadtEric J. Nestler
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Abstract Isolation of cell populations is untangling complex biological interactions, but studies comparing methodologies lack in vivo complexity and draw limited conclusions about the types of transcripts identified by each technique. Furthermore, few studies compare FACS-based techniques to ribosomal affinity purification, and none do so genome-wide. We addressed this gap by systematically comparing nuclear-FACS, whole cell-FACS, and RiboTag affinity purification in the context of D1 or D2 dopamine receptor-expressing medium spiny neuron (MSN) subtypes of the nucleus accumbens (NAc), a key brain reward region. We find that nuclear-FACS-seq generates a substantially longer list of differentially expressed genes between these cell types, and a significantly larger number of neuropsychiatric GWAS hits than the other two methods. RiboTag-seq has much lower coverage of the transcriptome than the other methods, but very efficiently distinguishes D1- and D2-MSNs. We also demonstrate differences between D1- and D2-MSNs with respect to RNA localization, suggesting fundamental cell type differences in mechanisms of transcriptional regulation and subcellular transport of RNAs. Together, these findings guide the field in selecting the RNAseq method that best suits the scientific questions under investigation.Keywords:
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Abstract Substance use disorders (SUDs) induce widespread molecular dysregulation in the nucleus accumbens (NAc), a brain region pivotal for coordinating motivation and reward. These molecular changes are thought to support lasting neural and behavioral disturbances that promote drug-seeking in addiction. However, different drug classes exert unique influences on neural circuits, cell types, physiology, and gene expression despite the overlapping symptomatology of SUDs. To better understand common and divergent molecular mechanisms governing SUD pathology, our goal was to survey cell-type-specific restructuring of the NAc transcriptional landscape in after psychostimulant or opioid exposure. We combined fluorescence-activated nuclei sorting and RNA sequencing to profile NAc D1 and D2 medium spiny neurons (MSNs) across cocaine and morphine exposure paradigms, including initial exposure, prolonged withdrawal after repeated exposure, and re-exposure post-withdrawal. Our analyses reveal that D1 MSNs display many convergent transcriptional responses across drug classes during exposure, whereas D2 MSNs manifest mostly divergent responses between cocaine and morphine, with morphine causing more adaptations in this cell type. Utilizing multiscale embedded gene co-expression network analysis (MEGENA), we discerned transcriptional regulatory networks subserving biological functions shared between cocaine and morphine. We observed largely integrative engagement of overlapping gene networks across drug classes in D1 MSNs, but opposite regulation of key D2 networks, highlighting potential therapeutic gene network targets within MSNs. These studies establish a landmark, cell-type-specific atlas of transcriptional regulation induced by cocaine and by morphine that can serve as a foundation for future studies towards mechanistic understanding of SUDs. Our findings, and future work leveraging this dataset, will pave the way for the development of targeted therapeutic interventions, addressing the urgent need for more effective treatments for cocaine use disorder and enhancing the existing strategies for opioid use disorder.
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R. Christopher Pierce1 and Marina E. Wolf2 1Center for Neurobiology and Behavior, Department of Psychiatry, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania 19104 2Department of Neuroscience, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064 Correspondence: marina.wolf{at}rosalindfranklin.edu
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The present study examined the effect of deep brain stimulation (DBS) in the nucleus accumbens shell on cocaine seeking and neuronal plasticity in rats. Electrical DBS of the accumbens shell attenuated cocaine primed reinstatement across a range of frequencies as low as 12 Hz in male rats. Nucleus accumbens medium spiny neurons (MSNs) can be differentiated by expression of dopamine D1 receptors (D1DRs) or D2DRs. Low-frequency optogenetic-DBS in D1DR- or D2DR-containing neurons attenuated cocaine seeking in male but not female rats. In slice electrophysiology experiments, 12 Hz electrical stimulation evoked long term potentiation (LTP) in D1DR-MSNs and D2DR-MSNs from cocaine naive male and female rats. However, in cocaine-experienced rats, electrical and optical DBS only elicited LTP in D2DR-MSNs from male rats. These results suggest that low frequency DBS in the nucleus accumbens shell effectively, but sex-specifically, suppresses cocaine seeking, which may be associated with the reversal of synaptic plasticity deficits in D2DR-MSNs.
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The nucleus accumbens septi of the normal adult rat was investigated by means of the rapid Golgi impregnation technique according to Valverde (1970). 5 neuron types could be differentiated: type 1: spiny neurons; type 2: few spiny neurons; type 3: aspiny giant neurons; type 4: spiny spindle shaped neurons; type 5: aspiny spidery neurons. The types 1, 4 and 5, resp., are considered, with respect to morphological criteria, to represent interneurons, whereas the types 2 and 3 are considered to be accumbens output neurons. The cytoarchitecture of the nc. accumbens septi thus displays striking similarities with the caudate-putamen-complex. An hypothetical correlation of these neurons in a functional -- biochemical sense as well as their position in the nc. accumbens which is suggested to be a transition area between limbic and extrapyramidal motor systems, is discussed.
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Stress alters the structure and function of brain reward circuitry and is an important risk factor for developing depression. In the nucleus accumbens (NAc), structural and physiological plasticity of medium spiny neurons (MSNs) have been linked to increased stress-related and depression-like behaviors. NAc MSNs have opposing roles in driving stress-related behaviors that is dependent on their dopamine receptor expression. After chronic social defeat stress, NAc MSNs exhibit increased dendritic spine density. However, it remains unclear if the dendritic spine plasticity is MSN subtype specific. Here we use viral labeling to characterize dendritic spine morphology specifically in dopamine D2 receptor expressing MSNs (D2-MSNs). After chronic social defeat, D2-MSNs exhibit increased spine density that is correlated with enhanced social avoidance behavior. Together, our data indicate dendritic spine plasticity is MSN subtype specific, improving our understanding of structural plasticity after chronic stress.
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The nucleus accumbens (nAc) is involved in addiction-related behaviour caused by several drugs of abuse, including alcohol. Glycine receptors (GlyRs) are potentiated by ethanol and they have been implicated in the regulation of accumbal dopamine levels. We investigated the presence of GlyR subunits in nAc and their modulation by ethanol in medium spiny neurons (MSNs) of the mouse nAc. We found that the GlyR α1 subunit is preferentially expressed in nAc and is potentiated by ethanol. Our study shows that GlyR α1 in nAc is a new target for development of novel pharmacological tools for behavioural intervention in drug abuse.Alcohol abuse causes major social, economic and health-related problems worldwide. Alcohol, like other drugs of abuse, increases levels of dopamine in the nucleus accumbens (nAc), facilitating behavioural reinforcement and substance abuse. Previous studies suggested that glycine receptors (GlyRs) are involved in the regulation of accumbal dopamine levels. Here, we investigated the presence of GlyRs in accumbal dopamine receptor medium spiny neurons (MSNs) of C57BL/6J mice, analysing mRNA expression levels and immunoreactivity of GlyR subunits, as well as ethanol sensitivity. We found that GlyR α1 subunits are expressed at higher levels than α2, α3 and β in the mouse nAc and were located preferentially in dopamine receptor 1 (DRD1)-positive MSNs. Interestingly, the glycine-evoked currents in dissociated DRD1-positive MSNs were potentiated by ethanol. Also, the potentiation of the GlyR-mediated tonic current by ethanol suggests that they modulate the excitability of DRD1-positive MSNs in nAc. This study should contribute to understanding the role of GlyR α1 in the reward system and might help to develop novel pharmacological therapies to treat alcoholism and other addiction-related and compulsive behaviours.
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The nucleus accumbens (NAc) is clearly implicated in reward processing and drug addiction, as well as in numerous neurological and psychiatric disorders; nevertheless, the circuit mechanisms underlying the diverse functions of the NAc remain poorly understood. Here, we characterized the whole-brain and monosynaptic inputs to two main projection cell types - D1 dopamine receptor expressing medium spiny neurons (D1R-MSNs) and D2 dopamine receptor expressing medium spiny neurons (D2R-MSNs) - within the NAc core and NAc shell by rabies-mediated trans-synaptic tracing. We discovered that D1R-MSNs and D2R-MSNs in both NAc subregions receive similar inputs from diverse sources. Inputs to the NAc core are broadly scattered, whereas inputs to the NAc shell are relatively concentrated. Furthermore, we identified numerous brain areas providing important contrasting inputs to different NAc subregions. The anterior cortex preferentially innervates the NAc core for both D1R-MSNs and D2R-MSNs, whereas the lateral hypothalamic area (LH) preferentially targets D1R-MSNs in the NAc shell. Characterizing the cell-type-specific connectivity of different NAc subregions lays a foundation for studying how diverse functions of the NAc are mediated by specific pathways.
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