Investigation of long interspersed element-1 retrotransposons as potential risk factors for idiopathic temporal lobe epilepsy.

OBJECTIVE: To determine if long interspersed element-1 (L1) retrotransposons convey risk for idiopathic temporal lobe epilepsy (TLE). METHODS: Surgically resected temporal cortex from individuals with TLE (N = 33) and postmortem temporal cortex from individuals with no known neurological disease (N = 33) were analyzed for L1 content by Restriction Enzyme Based Enriched L1Hs sequencing (REBELseq). Expression of three KCNIP4 splice variants was assessed by droplet digital PCR (ddPCR). Protein ANalysis THrough Evolutionary Relationships (PANTHER) was used to determine ontologies and pathways for lists of genes harboring L1 insertions. RESULTS: We identified novel L1 insertions specific to individuals with TLE, and others specific to controls. Although there were no statistically significant differences between cases and controls in the numbers of known and novel L1 insertions, PANTHER analyses of intragenic L1 insertions showed statistically significant enrichments for epilepsy-relevant gene ontologies in both cases and controls. Gene ontologies "neuron projection development" and "calcium ion transmembrane transport" were among those found only in individuals with TLE. We confirmed novel L1 insertions in several genes associated with seizures/epilepsy, including a de novo somatic L1 retrotransposition in KCNIP4 that occurred after neural crest formation in one patient. However, ddPCR results suggest this de novo L1 did not alter KCNIP4 mRNA expression. SIGNIFICANCE: Given current data from this small cohort, we conclude that L1 elements, either rare heritable germline insertions or de novo somatic retrotranspositions, may contribute only minimally to overall genetic risk for idiopathic TLE. We suggest that further studies in additional patients and additional brain regions are warranted.

Differential expression and transcription factor binding associated with genotype at a pharmacogenetic variant in OPRD1.

BACKGROUND: The functional mechanism is unknown for many genetic variants associated with substance use disorder phenotypes. Rs678849, an intronic variant in the delta-opioid receptor gene (OPRD1), has been found to predict regional brain volume, addiction risk, and the efficacy of buprenorphine/naloxone in treating opioid use disorder. The variant has also been implicated as an expression quantitative trait locus (eQTL) for several genes. OBJECTIVES: The objective of this study was to identify functional differences between the two alleles of rs678849 in vitro. We hypothesized that the two alleles of rs678849 would have different effects on transcriptional activity due to differential interactions with transcription factors. METHODS: 15bp regions containing the C or T alleles of rs678849 were cloned into luciferase constructs and transfected into BE(2)C neuroblastoma cells to test the effect on transcription. Electrophoretic mobility shift assays (EMSA) using nuclear lysates from BE(2)C cell or human postmortem medial prefrontal cortex were used to identify proteins that differentially bound the two alleles. RESULTS: At 24 hours post-transfection, the C allele construct had significantly lower luciferase expression than the T allele construct and empty vector control (ANOVA p < .001). Proteomic analysis and supershift assays identified XRCC6 as a transcription factor specifically binding the C allele, whereas hnRNP D0 was found to specifically bind the T allele. CONCLUSION: These functional differences between the C and T alleles may help explain the psychiatric and neurological phenotype differences predicted by rs678849 genotype and the potential role of the variant as an eQTL.

Altered structural and functional synaptic plasticity with motor skill learning in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is the most common inherited intellectual disability. FXS results from a mutation that causes silencing of the FMR1 gene, which encodes the fragile X mental retardation protein. Patients with FXS exhibit a range of neurological deficits, including motor skill deficits. Here, we have investigated motor skill learning and its synaptic correlates in the fmr1 knock-out (KO) mouse. We find that fmr1 KO mice have impaired motor skill learning of a forelimb-reaching task, compared with their wild-type (WT) littermate controls. Electrophysiological recordings from the forelimb region of the primary motor cortex demonstrated reduced, training-induced synaptic strengthening in the trained hemisphere. Moreover, long-term potentiation (LTP) is impaired in the fmr1 KO mouse, and motor skill training does not occlude LTP as it does in the WT mice. Whereas motor skill training induces an increase of synaptic AMPA-type glutamate receptor subunit 1 (GluA1), there is a delay in GluA1 increase in the trained hemisphere of the fmr1 KO mice. Using transcranial in vivo multiphoton microscopy, we find that fmr1 KO mice have similar spine density but increased dendritic spine turnover compared with WT mice. Finally, we report that motor skill training-induced formation of dendritic spines is impaired in fmr1 KO mice. We conclude that FMRP plays a role in motor skill learning and that reduced functional and structural synaptic plasticity might underlie the behavioral deficit in the fmr1 KO mouse.

Single-nucleus RNA-seq identifies one galanin neuronal subtype in mouse preoptic hypothalamus activated during recovery from sleep deprivation.

The preoptic area of the hypothalamus (POA) is essential for sleep regulation. However, the cellular makeup of the POA is heterogeneous, and the molecular identities of the sleep-promoting cells remain elusive. To address this question, this study compares mice during recovery sleep following sleep deprivation to mice allowed extended sleep. Single-nucleus RNA sequencing (single-nucleus RNA-seq) identifies one galanin inhibitory neuronal subtype that shows upregulation of rapid and delayed activity-regulated genes during recovery sleep. This cell type expresses higher levels of growth hormone receptor and lower levels of estrogen receptor compared to other galanin subtypes. single-nucleus RNA-seq also reveals cell-type-specific upregulation of purinergic receptor (P2ry14) and serotonin receptor (Htr2a) during recovery sleep in this neuronal subtype, suggesting possible mechanisms for sleep regulation. Studies with RNAscope validate the single-nucleus RNA-seq findings. Thus, the combined use of single-nucleus RNA-seq and activity-regulated genes identifies a neuronal subtype functionally involved in sleep regulation.

An endogenous GLP-1 circuit engages VTA GABA neurons to regulate mesolimbic dopamine neurons and attenuate cocaine seeking.

Recent studies show that systemic administration of a glucagon-like peptide-1 receptor (GLP-1R) agonist is sufficient to attenuate the reinstatement of cocaine-seeking behavior, an animal model of relapse. However, the neural mechanisms mediating these effects and the role of endogenous central GLP-1 signaling in cocaine seeking remain unknown. Here, we show that voluntary cocaine taking decreased plasma GLP-1 levels in rats and that chemogenetic activation of GLP-1-producing neurons in the nucleus tractus solitarius (NTS) that project to the ventral tegmental area (VTA) decreased cocaine reinstatement. Single nuclei transcriptomics and FISH studies revealed GLP-1Rs are expressed primarily on GABA neurons in the VTA. Using in vivo fiber photometry, we found that the efficacy of a systemic GLP-1R agonist to attenuate cocaine seeking was associated with increased activity of VTA GABA neurons and decreased activity of VTA dopamine neurons. Together, these findings suggest that targeting central GLP-1 circuits may be an effective strategy toward reducing cocaine relapse and highlight a novel functional role of GABAergic GLP-1R-expressing midbrain neurons in drug seeking.

Enhancing translation: A need to leverage complex preclinical models of addictive drugs to accelerate substance use treatment options.

Preclinical models of addictive drugs have been developed for decades to model aspects of the clinical experience in substance use disorders (SUDs). These include passive exposure as well as volitional intake models across addictive drugs and have been utilized to also measure withdrawal symptomatology and potential neurobehavioral mechanisms underlying relapse to drug seeking or taking. There are a number of Food and Drug Administration (FDA)-approved medications for SUDs, however, many demonstrate low clinical efficacy as well as potential sex differences, and we also note gaps in the continuum of care for certain aspects of clinical experiences in individuals who use drugs. In this review, we provide a comprehensive update on both frequently utilized and novel behavioral models of addiction with a focus on translational value to the clinical experience and highlight the need for preclinical research to follow epidemiological trends in drug use patterns to stay abreast of clinical treatment needs. We then note areas in which models could be improved to enhance the medications development pipeline through efforts to enhance translation of preclinical models. Next, we describe neuroscience efforts that can be leveraged to identify novel biological mechanisms to enhance medications development efforts for SUDs, focusing specifically on advances in brain transcriptomics approaches that can provide comprehensive screening and identification of novel targets. Together, the confluence of this review demonstrates the need for careful selection of behavioral models and methodological parameters that better approximate the clinical experience combined with cutting edge neuroscience techniques to advance the medications development pipeline for SUDs.

Analysis of single-cell transcriptome data from a mouse model implicates protein synthesis dysfunction in schizophrenia.

BACKGROUND: Schizophrenia is a mental disorder that causes considerable morbidity, whose risk largely results from genetic factors. Setd1a is a gene implicated in schizophrenia. OBJECTIVE: To study the gene expression changes found in heterozygous Setd1a± knockout mice in order to gain useful insight into schizophrenia pathogenesis. METHODS: We mined a single-cell RNA sequencing (scRNAseq) dataset from the prefrontal cortex (PFC) and striatum of Setd1a± mice and identified cell type-specific differentially expressed genes (DEGs) and differential transcript usage (DTU). DEGs and genes containing DTU found in each cell type were used to identify affected biological pathways using Ingenuity Pathway Analysis (IPA). RESULTS: We identified 273 unique DEGs across all cell types in PFC and 675 unique gene peaks containing DTU. In striatum, we identified 327 unique DEGs across all cell types and 8 unique gene peaks containing DTU. Key IPA findings from the analysis of DEGs found in PFC and striatum implicate processes involved in protein synthesis, mitochondrial function, cell metabolism, and inflammation. IPA analysis of genes containing DTU in PFC points to protein synthesis, as well as cellular activities involving intracellular signaling and neurotransmission. One canonical pathway, 'EIF2 Signaling', which is involved in the regulation of protein synthesis, was detected in PFC DEGs, striatum DEGs, and PFC genes containing DTU, drawing attention to its importance in schizophrenia pathophysiology. CONCLUSION: Processes involving protein synthesis in general and the 'EIF2 Signaling' pathway in particular could be targets for the development of new research strategies and biomarkers in schizophrenia.

A nociceptive amygdala-striatal pathway for chronic pain aversion.

The basolateral amygdala (BLA) is essential for assigning positive or negative valence to sensory stimuli. Noxious stimuli that cause pain are encoded by an ensemble of nociceptive BLA projection neurons (BLAnoci ensemble). However, the role of the BLAnoci ensemble in mediating behavior changes and the molecular signatures and downstream targets distinguishing this ensemble remain poorly understood. Here, we show that the same BLAnoci ensemble neurons are required for both acute and chronic neuropathic pain behavior. Using single nucleus RNA-sequencing, we characterized the effect of acute and chronic pain on the BLA and identified enrichment for genes with known functions in axonal and synaptic organization and pain perception. We thus examined the brain-wide targets of the BLAnoci ensemble and uncovered a previously undescribed nociceptive hotspot of the nucleus accumbens shell (NAcSh) that mirrors the stability and specificity of the BLAnoci ensemble and is recruited in chronic pain. Notably, BLAnoci ensemble axons transmit acute and neuropathic nociceptive information to the NAcSh, highlighting this nociceptive amygdala-striatal circuit as a unique pathway for affective-motivational responses across pain states.

A single-nucleus transcriptomic atlas of medium spiny neurons in the rat nucleus accumbens.

Neural processing of rewarding stimuli involves several distinct regions, including the nucleus accumbens (NAc). The majority of NAc neurons are GABAergic projection neurons known as medium spiny neurons (MSNs). MSNs are broadly defined by dopamine receptor expression, but evidence suggests that a wider array of subtypes exist. To study MSN heterogeneity, we analyzed single-nucleus RNA sequencing data from the largest available rat NAc dataset. Analysis of 48,040 NAc MSN nuclei identified major populations belonging to the striosome and matrix compartments. Integration with mouse and human data indicated consistency across species and disease-relevance scoring using genome-wide association study results revealed potentially differential roles for MSN populations in substance use disorders. Additional high-resolution clustering identified 34 transcriptomically distinct subtypes of MSNs definable by a limited number of marker genes. Together, these data demonstrate the diversity of MSNs in the NAc and provide a basis for more targeted genetic manipulation of specific populations.

A single-nucleus transcriptomic atlas of medium spiny neurons in the rat nucleus accumbens.

Neural processing of rewarding stimuli involves several distinct regions, including the nucleus accumbens (NAc). The majority of NAc neurons are GABAergic projection neurons known as medium spiny neurons (MSNs). MSNs are broadly defined by dopamine receptor expression, but evidence suggests that a wider array of subtypes exist. To study MSN heterogeneity, we analyzed single-nucleus RNA sequencing data from the largest available rat NAc dataset. Analysis of 48,040 NAc MSN nuclei identified major populations belonging to the striosome and matrix compartments. Integration with mouse and human data indicated consistency across species and disease-relevance scoring using genome-wide association study results revealed potentially differential roles for MSN populations in substance use disorders. Additional high-resolution clustering identified 34 transcriptomically distinct subtypes of MSNs definable by a limited number of marker genes. Together, these data demonstrate the diversity of MSNs in the NAc and provide a basis for more targeted genetic manipulation of specific populations.

Mimicking opioid analgesia in cortical pain circuits.

The anterior cingulate cortex plays a pivotal role in the cognitive and affective aspects of pain perception. Both endogenous and exogenous opioid signaling within the cingulate mitigate cortical nociception, reducing pain unpleasantness. However, the specific functional and molecular identities of cells mediating opioid analgesia in the cingulate remain elusive. Given the complexity of pain as a sensory and emotional experience, and the richness of ethological pain-related behaviors, we developed a standardized, deep-learning platform for deconstructing the behavior dynamics associated with the affective component of pain in mice-LUPE (Light aUtomated Pain Evaluator). LUPE removes human bias in behavior quantification and accelerated analysis from weeks to hours, which we leveraged to discover that morphine altered attentional and motivational pain behaviors akin to affective analgesia in humans. Through activity-dependent genetics and single-nuclei RNA sequencing, we identified specific ensembles of nociceptive cingulate neuron-types expressing mu-opioid receptors. Tuning receptor expression in these cells bidirectionally modulated morphine analgesia. Moreover, we employed a synthetic opioid receptor promoter-driven approach for cell-type specific optical and chemical genetic viral therapies to mimic morphine's pain-relieving effects in the cingulate, without reinforcement. This approach offers a novel strategy for precision pain management by targeting a key nociceptive cortical circuit with on-demand, non-addictive, and effective analgesia.

Unraveling Psychiatric Disorders through Neural Single-Cell Transcriptomics Approaches.

The development of single-cell and single-nucleus transcriptome technologies is enabling the unraveling of the molecular and cellular heterogeneity of psychiatric disorders. The complexity of the brain and the relationships between different brain regions can be better understood through the classification of individual cell populations based on their molecular markers and transcriptomic features. Analysis of these unique cell types can explain their involvement in the pathology of psychiatric disorders. Recent studies in both human and animal models have emphasized the importance of transcriptome analysis of neuronal cells in psychiatric disorders but also revealed critical roles for non-neuronal cells, such as oligodendrocytes and microglia. In this review, we update current findings on the brain transcriptome and explore molecular studies addressing transcriptomic alterations identified in human and animal models in depression and stress, neurodegenerative disorders (Parkinson's and Alzheimer's disease), schizophrenia, opioid use disorder, and alcohol and psychostimulant abuse. We also comment on potential future directions in single-cell and single-nucleus studies.

Calcitonin receptor signaling in nucleus accumbens D1R- and D2R-expressing medium spiny neurons bidirectionally alters opioid taking in male rats.

The high rates of relapse associated with current medications used to treat opioid use disorder (OUD) necessitate research that expands our understanding of the neural mechanisms regulating opioid taking to identify molecular substrates that could be targeted by novel pharmacotherapies to treat OUD. Recent studies show that activation of calcitonin receptors (CTRs) is sufficient to reduce the rewarding effects of addictive drugs in rodents. However, the role of central CTR signaling in opioid-mediated behaviors has not been studied. Here, we used single nuclei RNA sequencing (snRNA-seq), fluorescent in situ hybridization (FISH), and immunohistochemistry (IHC) to characterize cell type-specific patterns of CTR expression in the nucleus accumbens (NAc), a brain region that plays a critical role in voluntary drug taking. Using these approaches, we identified CTRs expressed on D1R- and D2R-expressing medium spiny neurons (MSNs) in the medial shell subregion of the NAc. Interestingly, Calcr transcripts were expressed at higher levels in D2R- versus D1R-expressing MSNs. Cre-dependent viral-mediated miRNA knockdown of CTRs in transgenic male rats was then used to determine the functional significance of endogenous CTR signaling in opioid taking. We discovered that reduced CTR expression specifically in D1R-expressing MSNs potentiated/augmented opioid self-administration. In contrast, reduced CTR expression specifically in D2R-expressing MSNs attenuated opioid self-administration. These findings highlight a novel cell type-specific mechanism by which CTR signaling in the ventral striatum bidirectionally modulates voluntary opioid taking and support future studies aimed at targeting central CTR-expressing circuits to treat OUD.

GPR-160 Receptor Signaling in the Dorsal Vagal Complex of Male Rats Modulates Meal Microstructure and CART-Mediated Hypophagia.

The g-protein coupled receptor GPR-160, recently identified as a putative receptor for the cocaine and amphetamine-regulated transcript (CART) peptide, shows abundant expression in the energy-balance control nuclei, including the dorsal vagal complex (DVC). However, its physiological role in the control of food intake has yet to be fully explored. Here, we performed a virally mediated, targeted knockdown (KD) of Gpr160 in the DVC of male rats to evaluate its physiological role in control of feeding. Our results indicate that DVC Gpr160 KD affects meal microstructure. Specifically, DVC Gpr160 KD animals consumed more frequent, but shorter meals during the dark phase and showed decreased caloric intake and duration of meals during the light phase. Cumulatively, however, these bidirectional effects on feeding resulted in no difference in body weight gain. We next tested the role of DVC GPR-160 in mediating the anorexigenic effects of exogenous CART. Our results show that DVC Gpr160 KD partially attenuates CART's anorexigenic effects. To further characterize Gpr160+ cells in the DVC, we utilized single-nucleus RNA sequencing data to uncover abundant GPR-160 expression in DVC microglia and only minimal expression in neurons. Altogether, our results suggest that DVC CART signaling may be mediated by Gpr160+ microglia, which in turn may be modulating DVC neuronal activity to control food intake.

Type I interferon response in astrocytes promotes brain metastasis by enhancing monocytic myeloid cell recruitment.

Cancer metastasis to the brain is a significant clinical problem. Metastasis is the consequence of favorable interactions between invaded cancer cells and the microenvironment. Here, we demonstrate that cancer-activated astrocytes create a sustained low-level activated type I interferon (IFN) microenvironment in brain metastatic lesions. We further confirm that the IFN response in astrocytes facilitates brain metastasis. Mechanistically, IFN signaling in astrocytes activates C-C Motif Chemokine Ligand 2 (CCL2) production, which further increases the recruitment of monocytic myeloid cells. The correlation between CCL2 and monocytic myeloid cells is confirmed in clinical brain metastasis samples. Lastly, genetically or pharmacologically inhibiting C-C Motif Chemokine Receptor 2 (CCR2) reduces brain metastases. Our study clarifies a pro-metastatic effect of type I IFN in the brain even though IFN response has been considered to have anti-tumor effects. Moreover, this work expands our understandings on the interactions between cancer-activated astrocytes and immune cells in brain metastasis.

GIP receptor agonism blocks chemotherapy-induced nausea and vomiting.

OBJECTIVE: Nausea and vomiting remain life-threatening obstacles to successful treatment of chronic diseases, despite a cadre of available antiemetic medications. Our inability to effectively control chemotherapy-induced nausea and vomiting (CINV) highlights the need to anatomically, molecularly, and functionally characterize novel neural substrates that block CINV. METHODS: Behavioral pharmacology assays of nausea and emesis in 3 different mammalian species were combined with histological and unbiased transcriptomic analyses to investigate the beneficial effects of glucose-dependent insulinotropic polypeptide receptor (GIPR) agonism on CINV. RESULTS: Single-nuclei transcriptomics and histological approaches in rats revealed a topographical, molecularly distinct, GABA-ergic neuronal population in the dorsal vagal complex (DVC) that is modulated by chemotherapy but rescued by GIPR agonism. Activation of DVCGIPR neurons substantially decreased behaviors indicative of malaise in cisplatin-treated rats. Strikingly, GIPR agonism blocks cisplatin-induced emesis in both ferrets and shrews. CONCLUSION: Our multispecies study defines a peptidergic system that represents a novel therapeutic target for the management of CINV, and potentially other drivers of nausea/emesis.

Ventral hippocampus neurons encode meal-related memory.

The ability to encode and retrieve meal-related information is critical to efficiently guide energy acquisition and consumption, yet the underlying neural processes remain elusive. Here we reveal that ventral hippocampus (HPCv) neuronal activity dynamically elevates during meal consumption and this response is highly predictive of subsequent performance in a foraging-related spatial memory task. Targeted recombination-mediated ablation of HPCv meal-responsive neurons impairs foraging-related spatial memory without influencing food motivation, anxiety-like behavior, or escape-mediated spatial memory. These HPCv meal-responsive neurons project to the lateral hypothalamic area (LHA) and single-nucleus RNA sequencing and in situ hybridization analyses indicate they are enriched in serotonin 2a receptors (5HT2aR). Either chemogenetic silencing of HPCv-to-LHA projections or intra-HPCv 5HT2aR antagonist yielded foraging-related spatial memory deficits, as well as alterations in caloric intake and the temporal sequence of spontaneous meal consumption. Collective results identify a population of HPCv neurons that dynamically respond to eating to encode meal-related memories.

Analysis of differential gene expression and transcript usage in hippocampus of Apoe null mutant mice: Implications for Alzheimer's disease.

A dataset of single-nucleus RNA sequencing (snRNAseq) data was analyzed using Seurat, Sierra, and Ingenuity Pathway Analysis (IPA) programs to assess differentially expressed genes (DEGs) and differential transcript usage (DTU) in mouse hippocampal cell types. Seurat identified DEGs between the wild type (WT) and Apoe knockout (EKO) mice. IPA identified 11 statistically significant canonical pathways in >1 cell type. Sierra identified Sipa1l1 with DTU between WT and EKO samples. Analysis of the Sipa1l1 peak region identified an alternative non-canonical polyadenylation signal and a putative cytoplasmic polyadenylation element. APOE regulation of gene transcription and co-transcriptional RNA processing may underlie Alzheimer's disease.

Single Nucleus Transcriptome Data from Alzheimer's Disease Mouse Models Yield New Insight into Pathophysiology.

BACKGROUND: 5XFAD humanized mutant mice and Trem2 knockout (T2KO) mice are two mouse models relevant to the study of Alzheimer's disease (AD)-related pathology. OBJECTIVE: To determine hippocampal transcriptomic and polyadenylation site usage alterations caused by genetic mutations engineered in 5XFAD and T2KO mice. METHODS: Employing a publicly available single-nucleus RNA sequencing dataset, we used Seurat and Sierra analytic programs to identify differentially expressed genes (DEGs) and differential transcript usage (DTU), respectively, in hippocampal cell types from each of the two mouse models. We analyzed cell type-specific DEGs further using Ingenuity Pathway Analysis (IPA). RESULTS: We identified several DEGs in both neuronal and glial cell subtypes in comparisons of wild type (WT) versus 5XFAD and WT versus T2KO mice, including Ttr, Fth1, Pcsk1n, Malat1, Rpl37, Rtn1, Sepw1, Uba52, Mbp, Arl6ip5, Gm26917, Vwa1, and Pgrmc1. We also observed DTU in common between the two comparisons in neuronal and glial subtypes, specifically in the genes Prnp, Rbm4b, Pnisr, Opcml, Cpne7, Adgrb1, Gabarapl2, Ubb, Ndfip1, Car11, and Stmn4. IPA identified three statistically significant canonical pathways that appeared in multiple cell types and that overlapped between 5XFAD and T2KO comparisons to WT, including 'FXR/RXR Activation', 'LXR/RXR Activation', and 'Acute Phase Response Signaling'. CONCLUSION: DEG, DTU, and IPA findings, derived from two different mouse models of AD, highlight the importance of energy imbalance and inflammatory processes in specific hippocampal cell types, including subtypes of neurons and glial cells, in the development of AD-related pathology. Additional studies are needed to further characterize these findings.