Figure - available from: Molecular Psychiatry
This content is subject to copyright. Terms and conditions apply.
Behavioral and pharmacological characterization of D1-Cre-flTrkB mice a Rotational behavior in D1-Cre-flTrkB-S, D1-Cre-flTrkB-NS, and D1-Cre control mice from juvenile to young-adult ages (*p < 0.05); b Only D1-Cre-flTrkB-S mice display head tics between the age of 3 and 8 weeks (**p < 0.01); (a–b) n = 13 D1-Cre-flTrkB-S, 7 D1-Cre-flTrkB-NS, and 12 D1-Cre; c Rotational behavior following vehicle (veh) and 0.02 mg/kg (sc.) ecopipam (SCH-39166) in 8-week-old mice (***p < 0.001); d Rotational behavior following vehicle (veh) and 0.05 mg/kg (sc.) haloperidol in 8-week-old mice (***p < 0.001); (c–d) n = 5 in each group; e Representative image of AAV-DIO-hM4d(Gi)-mCherry virus injection in the dorsal striatum (ac anterior commissure, NAc nucleus accumbens); f Rotational behavior following vehicle (veh) and 0.5 mg/kg (ip.) clozapine-N-oxide (CNO) in 4-week-old mice (**p < 0.01), n = 8 in each group; g Rotational behavior following vehicle and 0.5 mg/kg (ip.) CNO in 8-week-old mice (***p < 0.001), n = 8, 8, 7 respectively. For ‘Rotations’, please note the scale of the y-axis.
Source publication
Motor stereotypies occurring in early-onset neuropsychiatric diseases are associated with dysregulated basal ganglia direct-pathway activity. Disruptions in network connectivity through impaired neuronal structure have been implicated in both rodents and humans. However, the neurobiological mechanisms leading to direct-pathway neuron disconnectivit...
Citations
... In addition to social behavior, SPN ablation increased motor stereotypies. Insufficient D1R-SPN output (72), excessive D2R-SPN activity, and conversely, insufficient D2R-SPN activity (73) were all reported to cause stereotypies. In contrast, complete ablation of striatal CINs did not produce such an outcome (69), suggesting a limited contribution of this neuronal population. ...
Background:
Deficient social interactions are a hallmark of major neuropsychiatric disorders, and accumulating evidence points to altered social reward and motivation as key underlying mechanisms of these pathologies. In the present study, we further explored the role of the balance of activity between D1 and D2 dopamine receptor-expressing striatal projection neurons (D1R- and D2R-SPNs) in the control of social behavior, challenging the hypothesis that excessive D2R-SPN activity, rather than deficient D1R-SPN activity, compromises social behavior.
Methods:
We selectively ablated D1R- and D2R-SPNs using an inducible diphtheria toxin receptor (DTR)-mediated cell targeting strategy and assessed social behavior as well as repetitive/perseverative behavior, motor function and anxiety levels. We tested the effects of optogenetic stimulation of D2R-SPNs in the nucleus accumbens (NAc) and pharmacological compounds repressing D2R-SPN.
Results:
Targeted deletion of D1R-SPNs in the NAc blunted social behavior in mice, facilitated motor skill learning and increased anxiety levels. These behaviors were normalized by pharmacological inhibition of D2R-SPN, which also repressed transcription in the efferent nucleus, the ventral pallidum. Ablation of D1R-SPNs in the dorsal striatum had no impact on social behavior but impaired motor skill learning and decreased anxiety levels. Deletion of D2R-SPNs in the NAc produced motor stereotypies but facilitated social behavior and impaired motor skill learning. We mimicked excessive D2R-SPN activity by optically stimulating D2R-SPNs in the NAc and observed a severe deficit in social interaction that was prevented by D2R-SPN pharmacological inhibition.
Conclusions:
Repressing D2R-SPN activity may represent a promising therapeutic strategy to relieve social deficits in neuropsychiatric disorders.
... The sex differences in our behavioral data align with the previous finding from germline Nrxn2 KO [17] suggesting a sex-dependent role for Nrxn2 in social and repetitive behaviors. The lack of social reward experience and the repetitive shredding behavior in Nrxn2 cKO mice could be a consequence of a disrupted connection from the prefrontal cortex to the dorsal striatum, part of the basal ganglia circuitry which underlies behavioral flexibility and reward processing [58][59][60]. Future investigations on the synaptic function and behaviors mediated by the corticostriatal circuits would expand our current understanding of the role of Nrxn2. ...
Neurexins (Nrxns) have been extensively studied for their role in synapse organization and have been linked to many neuropsychiatric disorders, including autism spectrum disorder (ASD), and epilepsy. However, no studies have provided direct evidence that Nrxns may be the key regulator in the shared pathogenesis of these conditions largely due to complexities among Nrxns and their non-canonical functions in different synapses. Recent studies identified NRXN2 mutations in ASD and epilepsy, but little is known about Nrxn2’s role in a circuit-specific manner. Here, we report that conditional deletion of Nrxn2 from the hippocampus and cortex (Nrxn2 cKO) results in behavioral abnormalities, including reduced social preference and increased nestlet shredding behavior. Electrophysiological recordings identified an overall increase in hippocampal CA3→CA1 network activity in Nrxn2 cKO mice. Using intracranial electroencephalogram recordings, we observed unprovoked spontaneous reoccurring electrographic and behavioral seizures in Nrxn2 cKO mice. This study provides the first evidence that conditional deletion of Nrxn2 induces increased network activity that manifests into spontaneous recurrent seizures and behavioral impairments.
... For RNA sequencing, only samples with RNA integrity numbers >8 were used. Samples were submitted in biological quadruplicates for RNA sequencing at the UMSOM Institute for Genome Sciences (IGS) and processed as described previously (15). Libraries were prepared from 10 ng of RNA from each sample using the SMART-Seq . ...
BACKGROUND: Use of the synthetic opioid fentanyl increased ~300% in the last decade, including among women of reproductive ages. Adverse neonatal outcomes and long-term behavioral disruptions are associated with perinatal opioid exposure. Our previous work demonstrated that perinatal fentanyl exposed (PFE) mice displayed enhanced negative affect and somatosensory circuit and behavioral disruptions during adolescence. However, little is known about molecular adaptations across brain regions that underlie these outcomes. We performed RNA-sequencing across three reward and two sensory brain areas to study transcriptional programs in PFE juvenile mice.
METHODS: C57BL/6 pregnant dams received 10ug/ml fentanyl in the drinking water from embryonic day 0 (E0) through gestational periods until weaning at postnatal day 21 (P21). RNA was extracted from nucleus accumbens (NAc), prelimbic cortex (PrL), ventral tegmental area (VTA), somatosensory cortex (S1) and ventrobasal thalamus (VBT) from PFE mice of both sexes at P35. RNA-sequencing was performed, followed by analysis of differentially expressed genes (DEGs) and gene co-expression networks.
RESULTS: Transcriptome analysis revealed sex-specific DEGs and gene modules significantly associated with PFE. The VTA had the most DEGs, while pronounced gene enrichment occurred in NAc. Genes enriched in mitochondrial respiration were pronounced in NAc and VTA of PFE males, extracellular matrix (ECM) and neuronal migration enrichment were pronounced in NAc and VTA of PFE males, while genes associated with vesicular cycling and synaptic signaling were markedly altered in NAc of PFE female mice. In sensory areas from PFE females, we found alterations in mitochondrial respiration, synaptic and ciliary organization processes.
CONCLUSION: Our findings demonstrate sex-specific neuroadaptations in PFE mice. Our studies identify distinct transcriptomes across reward and sensory brain regions that may underlie disrupted behavioral and circuit states in PFE mice.
... The sex differences in our behavioral data are in alignment with the previous nding from germline Nrxn2 KO (17) suggesting a sexdependent role for Nrxn2 in social and repetitive behaviors. The lack of social reward experience and the repetitive shredding behavior in Nrxn2 cKO mice could be a consequence of a disrupted connection from the prefrontal cortex to the dorsal striatum, part of the basal ganglia circuitry which underlies behavioral exibility and reward processing (57)(58)(59). Future investigations on the synaptic function and behaviors mediated by the corticostriatal circuits would expand our current understanding of the role of Nrxn2. ...
Neurexins (Nrxns) have been extensively studied for their role in synapse organization and have been linked to many neuropsychiatric disorders, including autism spectrum disorder (ASD), and epilepsy. However, no studies have provided direct evidence that Nrxns may be the key regulator in the shared pathogenesis of these conditions largely due to complexities among Nrxns and their non-canonical functions in different synapses. Recent studies identified NRXN2 mutations in ASD and epilepsy, but little is known about Nrxn2’s role in a circuit-specific manner. Here, we report that conditional deletion of Nrxn2 from the hippocampus and cortex ( Nrxn2 cKO) results in behavioral abnormalities, including reduced social preference and increased nestlet shredding behavior. Electrophysiological recordings identified an overall increase in hippocampal CA3◊CA1 network activity in Nrxn2 cKO mice. Using intracranial electroencephalogram recordings, we observed unprovoked spontaneous reoccurring electrographic and behavioral seizures in Nrxn2 cKO mice. This study provides the first evidence that conditional deletion of Nrxn2 induces increased network activity that manifests into spontaneous recurrent seizures and behavioral impairments.
... To determine potential molecular mechanisms underlying D1-MSN-specific atrophy and physiology changes, we extracted RNA from immunoprecipitated polyribosomes in D1-and D2-MSNs as in our previous work (56,62). We sequenced D1-and D2-MSN translatomes ( Figure 3A, B; drinking in Figure S3A in Supplement 1) and found little overlap in the differentially expressed genes between the MSN subtypes after fentanyl abstinence ( Figure 3C). ...
Background
Opioid discontinuation generates a withdrawal syndrome marked by increased negative affect. Increased symptoms of anxiety and dysphoria during opioid discontinuation are a significant barrier to achieving long-term abstinence in opioid-dependent individuals. While adaptations in the nucleus accumbens are implicated in the opioid abstinence syndrome, the precise neural mechanisms are poorly understood. Additionally, our current knowledge is limited to changes following natural and semi-synthetic opioids, despite recent increases in synthetic opioid use and overdose.
Methods
We used a combination of cell subtype specific viral-labeling and electrophysiology in male and female mice to investigate structural and functional plasticity in nucleus accumbens medium spiny neuron (MSNs) subtypes after fentanyl abstinence. We characterized molecular adaptations after fentanyl abstinence with subtype specific RNAseq and Weighted Gene Co-expression Network Analysis. We used viral-mediated gene transfer to manipulate the molecular signature of fentanyl abstinence in D1-MSNs.
Results
Here we show fentanyl abstinence increases anxiety-like behavior, decreases social interaction, and engenders MSN subtype-specific plasticity in both sexes. D1, but not D2-MSNs exhibit dendritic atrophy and an increase in excitatory drive. We identified a cluster of co-expressed dendritic morphology genes downregulated selectively in D1-MSNs that are transcriptionally co-regulated by E2F1. E2f1 expression in D1-MSNs protects against loss of dendritic complexity, altered physiology, and negative affect-like behaviors caused by fentanyl abstinence.
Conclusion
Our findings indicate fentanyl abstinence causes unique structural, functional, and molecular changes in nucleus accumbens D1-MSNs that can be targeted to alleviate negative affective symptoms during abstinence.
... For RNA sequencing, only samples with RNA integrity numbers >8 were used. Samples were submitted in biological quadruplicates for RNA sequencing at the UMSOM Institute for Genome Sciences (IGS) and processed as in 14 . ...
... Polyribosome immunoprecipitation was achieved as described in 14,33 . Briefly, pooled tissue from RiboTag (RT) +/+ mice with virally mediated Cre expression in VPàMDT neurons (n= 4-5 mice per sample) was homogenized and 800-μL of the supernatant was incubated in HA-coupled magnetic beads (Invitrogen: 100.03D; ...
... No more than 3 animals per group were excluded. Sample sizes were determined from previous studies using self-administration in mice, RNA sequencing, cell-type-specific RNA isolation and neuronal morphology 14,15,24 . In figure legends: *p≤ 0.05, **p< 0.01, ***p< 0.001. ...
Psychostimulant exposure alters the activity of ventral pallidum (VP) projection neurons. However, the molecular underpinnings of these circuit dysfunctions are unclear. We used RNA-sequencing to reveal alterations in the transcriptional landscape of the VP that are induced by cocaine self-administration in mice. We then probed gene expression in select VP neuronal subpopulations to isolate a circuit associated with cocaine intake. Finally, we used both overexpression and CRISPR-mediated knockdown to test the role of a gene target on cocaine-mediated behaviors as well as dendritic spine density. Our results showed that a large proportion (55%) of genes associated with structural plasticity were changed 24 h following cocaine intake. Among them, the transcription factor Nr4a1 (Nuclear receptor subfamily 4, group A, member 1, or Nur77) showed high expression levels. We found that the VP to mediodorsal thalamus (VP → MDT) projection neurons specifically were recapitulating this increase in Nr4a1 expression. Overexpressing Nr4a1 in VP → MDT neurons enhanced drug-seeking and drug-induced reinstatement, while Nr4a1 knockdown prevented self-administration acquisition and subsequent cocaine-mediated behaviors. Moreover, we showed that Nr4a1 negatively regulated spine dynamics in this specific cell subpopulation. Together, our study identifies for the first time the transcriptional mechanisms occurring in VP in drug exposure. Our study provides further understanding on the role of Nr4a1 in cocaine-related behaviors and identifies the crucial role of the VP → MDT circuit in drug intake and relapse-like behaviors.
... To determine the molecular mechanisms underlying D1-MSN specific atrophy and increased excitability, we extracted RNA from immunoprecipitated polyribosomes in D1-and D2 MSNs as in our previous work (Engeln et al., 2020;Fox et al., 2020a). We performed RNA sequencing of D1-and D2-MSN translatomes after fentanyl abstinence (Fig 3A-B, fentanyl drinking data in Fig S3A), and found very little overlap in the differentially expressed genes between MSN subtypes after fentanyl abstinence (Fig 3C). ...
... To further refine the fentanyl modules, we performed Gene Ontology (GO) Analysis on the genes from each module using BiNGO (Maere et al., 2005) similar to our previous work (Engeln et al., 2020) (Supplemental File 2). Given the changes to dendritic complexity and excitability, we looked for overrepresentation of synaptic and dendritic complexity GO terms (Fig 4A), and found enrichment in both Green and Magenta modules. ...
... Sections containing NAc were sampled from bregma AP:1.5-1.0 mm and Z-stack images were acquired at 0.6 µm increments using a 40x objective. MSNs were 3D reconstructed using Imaris 8.3 software (Bitplane, Oxford Instruments) as previously (Engeln et al., 2020;Fox et al., 2020a). Surfaces were masked to was not certified by peer review) is the author/funder. ...
Opioid discontinuation generates a withdrawal syndrome marked by a negative emotional state. Increased anxiety and dysphoria during opioid discontinuation are a significant barrier to achieving long-term abstinence in opioid-dependent individuals. Adaptations in brain-reward circuitry are implicated in the opioid abstinence syndrome, but current knowledge is limited to changes following natural and semi-synthetic opioids. Here we report abstinence from the synthetic opioid fentanyl engenders structural, functional, and molecular plasticity in nucleus accumbens neuron subtypes (MSNs) that mediate negative emotional behaviors. We show fentanyl abstinence causes dendritic atrophy and increased excitatory drive exclusive to D1-receptor containing MSNs. Using subtype specific RNAseq and Weighted Gene Co-Expression Network Analysis, we identified molecular signatures of fentanyl abstinence in MSN subtypes. We found a network of co-expressed genes downregulated selectively in D1-MSNs, and transcriptionally co-regulated by E2F1. We show targeting abstinence-induced molecular changes protects D1-MSNs from maladaptive plasticity and alleviates negative emotional behaviors after fentanyl abstinence.
... Similarly, optogenetic activation of dMSNs also resulted in sustained and chronic repetitive behaviors (Bouchekioua et al., 2018). Engeln et al. (2021) found chemogenetic inhibition of dMSN can reduce repetitive rotations. In addition, the increased RRBs related to aberrant dMSNs have been found in Neuroligin 1 and Neuroligin-3 mutant mice (Rothwell et al., 2014;Espinosa et al., 2015). ...
Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by deficits in social communication, social interaction, and repetitive restricted behaviors (RRBs). It is usually detected in early childhood. RRBs are behavioral patterns characterized by repetition, inflexibility, invariance, inappropriateness, and frequent lack of obvious function or specific purpose. To date, the classification of RRBs is contentious. Understanding the potential mechanisms of RRBs in children with ASD, such as neural connectivity disorders and abnormal immune functions, will contribute to finding new therapeutic targets. Although behavioral intervention remains the most effective and safe strategy for RRBs treatment, some promising drugs and new treatment options (e.g., supplementary and cell therapy) have shown positive effects on RRBs in recent studies. In this review, we summarize the latest advances of RRBs from mechanistic to therapeutic approaches and propose potential future directions in research on RRBs.
... Projection-specific RNA isolation was performed using polyribosome immunoprecipitation as described previously 12,13 . Briefly, pooled tissue from RiboTag (RT)+/+ mice with virally mediated Cre expression in VP projection neurons (n= 4-5 mice per sample) was homogenized and 800 μL of the supernatant was incubated in HAcoupled magnetic beads (Invitrogen: 100.03D; ...
The ventral pallidum (VP), a major component of the basal ganglia, plays a critical role in motivational disorders. It sends projections to many different brain regions but it is not yet known whether and how these projections differ in their cellular properties, gene expression patterns, connectivity and role in reward seeking. In this study, we focus on four major outputs of the VP - to the lateral hypothalamus (LH), ventral tegmental area (VTA), mediodorsal thalamus (MDT), and lateral habenula (LHb) - and examine the differences between them in 1) baseline gene expression profiles using projection-specific RNA-sequencing; 2) physiological parameters using whole-cell patch clamp; and 3) their influence on cocaine reward using chemogenetic tools. We show that these four VP efferents differ in all three aspects and highlight specifically differences between the projections to the LH and the VTA. These two projections originate largely from separate populations of neurons, express distinct sets of genes related to neurobiological functions, and show opposite physiological and behavioral properties. Collectively, our data demonstrates for the first time that VP neurons exhibit distinct molecular and cellular profiles in a projection-specific manner, suggesting that they represent different cell types.
... For RNA sequencing, only samples with RNA integrity numbers >8 were used. Samples were submitted in biological quadruplicates for RNA sequencing at the UMSOM Institute for Genome Sciences (IGS) and processed as in 14 . ...
... Polyribosome immunoprecipitation was achieved as described in 14,33 . Briefly, pooled tissue from RiboTag (RT) +/+ mice with virally mediated Cre expression in VPàMDT neurons (n= 4-5 mice per sample) was homogenized and 800-μL of the supernatant was incubated in HA-coupled magnetic beads (Invitrogen: 100.03D; ...
... No more than 3 animals per group were excluded. Sample sizes were determined from previous studies using self-administration in mice, RNA sequencing, cell-type-specific RNA isolation and neuronal morphology 14,15,24 . In figure legends: *p≤ 0.05, **p< 0.01, ***p< 0.001. ...
Psychostimulant exposure alters the activity of ventral pallidum (VP) projection-neurons. However, the molecular underpinnings of these circuit dysfunctions are unclear. Using RNA-sequencing followed by circuit-specific gene expression assays, we revealed a key role for the VP to mediodorsal thalamus (VP-MDT) projection neurons in cocaine-related behaviors in mice. Our analyses demonstrated that the transcription factor Nr4a1 bidirectionally modulated dendritic spine dynamics in VP-MDT neurons and positively regulated pathological drug use.
