Subpopulations of hypocretin/orexin neurons differ in measures of their cell proliferation, dynorphin co-expression, projections, and response to embryonic ethanol exposure

Scientific reports

2023 May 25

Yasmin, N;Collier, AD;Karatayev, O;Abdulai, AR;Yu, B;Fam, M;Khalizova, N;Leibowitz, SF;
PMID: 37231149 | DOI: 10.1038/s41598-023-35432-w

Numerous studies in animals demonstrate that embryonic exposure to ethanol (EtOH) at low-moderate doses stimulates neurogenesis and increases the number of hypothalamic neurons expressing the peptide, hypocretin/orexin (Hcrt). A recent study in zebrafish showed that this effect on the Hcrt neurons in the anterior hypothalamus (AH) is area specific, evident in the anterior (aAH) but not posterior (pAH) part of this region. To understand specific factors that may determine the differential sensitivity to EtOH of these Hcrt subpopulations, we performed additional measures in zebrafish of their cell proliferation, co-expression of the opioid dynorphin (Dyn), and neuronal projections. In association with the increase in Hcrt neurons in the aAH but not pAH, EtOH significantly increased only in the aAH the proliferation of Hcrt neurons and their number lacking Dyn co-expression. The projections of these subpopulations differed markedly in their directionality, with those from the pAH primarily descending to the locus coeruleus and those from the aAH ascending to the subpallium, and they were both stimulated by EtOH, which induced specifically the most anterior subpallium-projecting Hcrt neurons to become ectopically expressed beyond the aAH. These differences between the Hcrt subpopulations suggest they are functionally distinct in their regulation of behavior.

Activation of GABAB receptors in central amygdala attenuates activity of PKCδ + neurons and suppresses punishment-resistant alcohol self-administration in rats

Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology

2023 Feb 04

Domi, E;Xu, L;Toivainen, S;Wiskerke, J;Coppola, A;Holm, L;Augier, E;Petrella, M;Heilig, M;
PMID: 36739350 | DOI: 10.1038/s41386-023-01543-1

Alcohol use despite negative consequences is a core phenomenon of alcohol addiction. We recently used alcohol self-administration that is resistant to footshock punishment as a model of this behavior, and found that activity of PKCδ + GABAergic neurons in the central amygdala (CeA) is a determinant of individual susceptibility for punishment resistance. In the present study, we examined whether activation of GABAB receptors in CeA can attenuate the activity of PKCδ + neurons in this region, and whether this will result in suppression of punishment- resistant alcohol self-administration in the minority of rats that show this behavior. Systemic administration of the clinically approved GABAB agonist baclofen (1 and 3 mg/kg) dose- dependently reduced punishment-resistant alcohol self-administration. Bilateral microinjections of baclofen into CeA (64 ng in 0.3 µl/side) reduced the activity of PKCδ + neurons, as measured by Fos expression. This manipulation also selectively suppressed punished alcohol self-administration in punishment-resistant rats. Expression analysis indicated that virtually all CeA PKCδ + neurons express the GABAB receptor. Using in vitro electrophysiology, we found that baclofen induced hyperpolarization of CeA neurons, reducing their firing rate in response to depolarizing current injections. Together, our findings provide a potential mechanism that contributes to the clinical efficacy of baclofen in alcohol addiction. Therapeutic use of baclofen itself is limited by problems of tolerance and need for dose escalation. Our findings support a mechanistic rationale for developing novel, improved alcohol addiction medications that target GABAB receptors, and that lack these limitations, such as e.g., GABAB positive allosteric modulators (PAM:s).

Low dopamine D2 receptor expression drives gene networks related to GABA, cAMP, growth and neuroinflammation in striatal indirect pathway neurons

Biological Psychiatry Global Open Science

2022 Sep 01

Guerri, L;Dobbs, L;da Silva e Silva, D;Meyers, A;Ge, A;Lecaj, L;Djakuduel, C;Islek, D;Hipolito, D;Martinez, A;Shen, P;Marietta, C;Garamszegi, S;Capobianco, E;Jiang, Z;Schwandt, M;Mash, D;Alvarez, V;Goldman, D;
| DOI: 10.1016/j.bpsgos.2022.08.010

Background A salient effect of addictive drugs is to hijack the dopamine reward system, an evolutionarily conserved driver of goal-directed behavior and learning. Reduced dopamine type-II receptor (D2R) availability in the striatum is an important pathophysiological mechanism for addiction that is both consequential and causal for other molecular, cellular, and neuronal network differences etiologic for this disorder. Here, we sought to identify gene expression changes attributable to innate low expression of the Drd2 gene in the striatum and specific to striatal indirect medium spiny neurons (iMSNs). Methods Cre-conditional, Translating Ribosome Affinity Purification (TRAP) was used to purify and analyze the translatome (ribosome-bound mRNA) of iMSNs from mice with low/heterozygous or wild-type Drd2 expression in iMSNs. Complementary electrophysiological recordings and gene expression analysis of postmortem brain tissue from human cocaine users were performed. Results Innate low expression of Drd2 in iMSNs led to differential expression of genes involved in GABA and cAMP signaling, neural growth, lipid metabolism, neural excitability, and inflammation. Creb1 was identified as a likely upstream regulator, among others. In human brain, expression of FXYD2, a modulatory subunit of the Na/K pump, was negatively correlated with DRD2 mRNA expression. In iMSN-TRAP-Drd2HET mice, increased Cartpt and reduced S100a10 (p11) expression recapitulated previous observations in cocaine paradigms. Electrophysiology experiments supported a higher GABA tone in iMSN-Drd2HET mice. Conclusion This study provides strong molecular evidence that in addiction inhibition by the indirect pathway is constitutively enhanced through neural growth and increased GABA signaling.

Ventral tegmental area astrocytes modulate cocaine reward by tonically releasing GABA

Neuron

2023 Jan 10

Yang, J;Chen, J;Liu, Y;Chen, KH;Baraban, JM;Qiu, Z;
PMID: 36681074 | DOI: 10.1016/j.neuron.2022.12.033

Addictive drugs increase ventral tegmental area (VTA) dopamine (DA) neuron activity through distinct cellular mechanisms, one of which involves disinhibition of DA neurons by inhibiting local GABA neurons. How drugs regulate VTA GABA neuron activity and drive addictive behaviors remains poorly understood. Here, we show that astrocytes control VTA GABA neuron activity in cocaine reward via tonic inhibition in mice. Repeated cocaine exposure potentiates astrocytic tonic GABA release through volume-regulated anion channels (VRACs) and augments tonic inhibition of VTA GABA neurons, thus downregulating their activities and disinhibiting nucleus accumbens (NAc) projecting DA neurons. Attenuation of tonic inhibition by either deleting Swell1 (Lrrc8a), the obligatory subunit of VRACs, in VTA astrocytes or disrupting δ subunit of GABAA receptors in VTA GABA neurons reduces cocaine-evoked changes in neuron activity, locomotion, and reward behaviors in mice. Together, our findings reveal the critical role of astrocytes in regulating the VTA local circuit and cocaine reward.

CREB binding at the Zfp189 promoter within medium spiny neuron subtypes differentially regulates behavioral and physiological adaptations over the course of cocaine use

Biological Psychiatry

2022 Aug 01

Teague, C;Picone, J;Wright, W;Browne, C;Silva, G;Futamura, R;Minier-Toribio, A;Estill, M;Ramakrishnan, A;Martinez-Rivera, F;Godino, A;Parise, E;Schmidt, K;Pulido, N;Lorsch, Z;Kim, J;Shen, L;Neve, R;Dong, Y;Nestler, E;Hamilton, P;
| DOI: 10.1016/j.biopsych.2022.07.022

Background Over the course of chronic drug use, brain transcriptional neuroadaptation are thought to contribute to a change in drug use behavior over time. The function of the transcription factor CREB within the nucleus accumbens (NAc) has been well documented in opposing the rewarding properties of many classes of drugs, yet the gene targets through which CREB causally manifests these lasting neuroadaptations remain unknown. Here, we identify zinc finger protein 189 (Zfp189) as a CREB target gene that is transcriptionally responsive to acute and chronic cocaine use within mouse NAc. Methods To query the role of the CREB-Zfp189 interaction in cocaine use, we virally delivered modified CRISPR/dCas9 constructs, capable of selectively localizing CREB to the Zfp189 gene promoter in the NAc of mice. Results We observe that CREB binding to the Zfp189 promoter increases Zfp189 expression and diminishes the reinforcing responses to cocaine. We show further that NAc Zfp189 expression is increased within D1 medium spiny neurons (MSNs) in response to acute cocaine, but increased in both D1 and D2 MSNs in response to chronic cocaine. CREB-mediated induction of Zfp189 potentiates electrophysiological activity of D1 and D2 MSNs - recapitulating the known effect of CREB on these neurons. Lastly, targeting CREB to the Zfp189 promoter within NAc Drd2-expressing neurons, but not Drd1-expressing neurons, was sufficient to diminish cocaine-conditioned behaviors. Conclusions Together, these findings point to the CREB-Zfp189 interaction within NAc Drd2+ neurons as a molecular signature of chronic cocaine use that is causal in counteracting the reinforcing effects of cocaine.

Neuron Subtype Specific Molecular Mechanisms in Fentanyl Abstinence

Biological Psychiatry

2022 May 01

Fox, M;Wulff, A;Franco, D;Choi, E;Engeln, M;Calarco, C;Turner, M;Chandra, R;Thompson, S;Ament, S;Lobo, M;
| DOI: 10.1016/j.biopsych.2022.02.052

Background Opioid withdrawal or abstinence generates a negative affective state and causes dendritic atrophy in nucleus accumbens (NAc) medium spiny neurons (MSNs). Based on our previous work, we hypothesized that opioid abstinence-induced atrophy is MSN subtype-specific, and blocking the subtype-specific molecular mediators can reverse behavioral changes caused by opioid abstinence. Methods All experiments were conducted in 8-10-week-old mice using sex as a biological variable. Cohorts of n=8-12 per condition were used in behavioral experiments; n=4-5 per condition for morphology or electrophysiology (3-7 cells/mouse); n=24 per condition (4 mice pooled/sample) for molecular biology. We used Cre-dependent viruses to label D1- and D2-MSNs in D1- and A2A-Cre mice for morphology or patch-clamp electrophysiology. We used D1- or A2A-Cre x RiboTag mice for subtype-specific RNAseq. We analyzed RNAseq data with weighted correlation network analysis (WGCNA), then further validated identified genes with Nanostring and RNAscope. Results Mice exhibit increased social-withdrawal and stress-like behaviors after fentanyl abstinence. (2-way ANOVA P=0.018). Stress-like behaviors after abstinence were associated with reduced dendritic complexity of NAc D1-, but not D2-MSNs (D1: 2-way ANOVA P=0.0129, D2:P=0.97). Using WGCNA, we identified 11 MSN subtype-specific gene networks altered by fentanyl abstinence (P0.05). Conclusions Our findings indicate fentanyl abstinence causes unique structural, functional, and molecular changes in NAc D1-MSNs that can be targeted to alleviate abstinence-induced dendritic atrophy and stress-like behaviors.

Lack of Causal Roles of Cannabinoid and Dopamine Neurotransmitter Systems in Orbitofrontal and Piriform Cortex in Fentanyl Relapse in Rats

eNeuro

2022 Jul 20

Claypool, SM;Behdin, S;Applebey, SV;Orihuel, J;Ma, Z;Reiner, DJ;
PMID: 35768212 | DOI: 10.1523/ENEURO.0496-21.2022

The orbitofrontal cortex (OFC) and piriform cortex (Pir) play a role in fentanyl relapse after food choice-induced voluntary abstinence, a procedure mimicking abstinence because of availability of alternative nondrug rewards. We used in situ hybridization and pharmacology to determine the role of OFC and Pir cannabinoid and dopamine receptors in fentanyl relapse. We trained male and female rats to self-administer food pellets for 6 d (6 h/d) and intravenous fentanyl (2.5 µg/kg/infusion) for 12 d (6 h/d). We assessed fentanyl relapse after 12 discrete choice sessions between fentanyl and food (20 trials/d), in which rats voluntarily reduced fentanyl self-administration. We used RNAscope to determine whether fentanyl relapse is associated with activity (indicated by Fos) in OFC and Pir cells expressing Cnr1 [which encodes cannabinoid 1 (CB1) receptors] or Drd1 and Drd2 (which encode dopamine D1 and D2 receptors). We injected a CB1 receptor antagonist or agonist (0.3 or 1.0 µg AM251 or WIN55,212-2/hemisphere) into OFC or a dopamine D1 receptor antagonist (1.0 or 3.0 µg SCH39166/hemisphere) into Pir to determine the effect on fentanyl relapse. Fentanyl relapse was associated with OFC cells co-expressing Fos and Cnr1 and Pir cells co-expressing Fos and Drd1 However, injections of the CB1 receptor antagonist AM251 or agonist WIN55,212-2 into OFC or the dopamine D1 receptor antagonist SCH39166 into Pir had no effect on fentanyl relapse. Fentanyl relapse is associated with activation of Cnr1-expressing OFC cells and Drd1-expressing Pir cells, but pharmacological manipulations do not support causal roles of OFC CB1 receptors or Pir dopamine D1 receptors in fentanyl relapse.

A red nucleus-VTA glutamate pathway underlies exercise reward and the therapeutic effect of exercise on cocaine use

Science advances

2022 Sep 02

He, Y;Madeo, G;Liang, Y;Zhang, C;Hempel, B;Liu, X;Mu, L;Liu, S;Bi, GH;Galaj, E;Zhang, HY;Shen, H;McDevitt, RA;Gardner, EL;Liu, QS;Xi, ZX;
PMID: 36054363 | DOI: 10.1126/sciadv.abo1440

Physical exercise is rewarding and protective against drug abuse and addiction. However, the neural mechanisms underlying these actions remain unclear. Here, we report that long-term wheel-running produced a more robust increase in c-fos expression in the red nucleus (RN) than in other brain regions. Anatomic and functional assays demonstrated that most RN magnocellular portion (RNm) neurons are glutamatergic. Wheel-running activates a subset of RNm glutamate neurons that project to ventral tegmental area (VTA) dopamine neurons. Optogenetic stimulation of this pathway was rewarding, as assessed by intracranial self-stimulation and conditioned place preference, whereas optical inhibition blocked wheel-running behavior. Running wheel access decreased cocaine self-administration and cocaine seeking during extinction. Last, optogenetic stimulation of the RNm-to-VTA glutamate pathway inhibited responding to cocaine. Together, these findings indicate that physical exercise activates a specific RNm-to-VTA glutamatergic pathway, producing exercise reward and reducing cocaine intake.

Ventral pallidum DRD3 potentiates a pallido-habenular circuit driving accumbal dopamine release and cocaine seeking

Neuron

2021 May 21

Pribiag, H;Shin, S;Wang, EH;Sun, F;Datta, P;Okamoto, A;Guss, H;Jain, A;Wang, XY;De Freitas, B;Honma, P;Pate, S;Lilascharoen, V;Li, Y;Lim, BK;
PMID: 34048697 | DOI: 10.1016/j.neuron.2021.05.002

Drugs of abuse induce persistent remodeling of reward circuit function, a process thought to underlie the emergence of drug craving and relapse to drug use. However, how circuit-specific, drug-induced molecular and cellular plasticity can have distributed effects on the mesolimbic dopamine reward system to facilitate relapse to drug use is not fully elucidated. Here, we demonstrate that dopamine receptor D3 (DRD3)-dependent plasticity in the ventral pallidum (VP) drives potentiation of dopamine release in the nucleus accumbens during relapse to cocaine seeking after abstinence. We show that two distinct VP DRD3+ neuronal populations projecting to either the lateral habenula (LHb) or the ventral tegmental area (VTA) display different patterns of activity during drug seeking following abstinence from cocaine self-administration and that selective suppression of elevated activity or DRD3 signaling in the LHb-projecting population reduces drug seeking. Together, our results uncover how circuit-specific DRD3-mediated plasticity contributes to the process of drug relapse.

Leveraging circuits to understand addiction

Neurocircuitry of Addiction

2023 Jan 19

Salling, M;
| DOI: 10.1016/B978-0-12-823453-2.00002-3

Advancements in neuroscientific methods often drive new waves of insight into our understanding of addiction. While addiction research questions persist, technical improvements can augment our observational sensitivity, allowing us to update and extend existing addiction models through method development, creative application, and scientific discovery. As a result of this iterative process, we have reached the point where neuroscientists can now readily identify, monitor, and control specific neural circuits during behavior, thereby opening new windows of inquiry into the neurobiology of addiction. The objective of this chapter is to familiarize the reader with standard and emerging techniques used to observe and interrogate neural circuitry that are prevalent in contemporary clinical and preclinical addiction neuroscience labs and that are presented throughout the book. This chapter will further discuss the historical context, benefits, and limitations of these techniques with a look forward into how they can be applied to questions of addiction neurocircuitry.

Cell-type specific molecular architecture for mu opioid receptor function in pain and addiction circuits

Neuropharmacology

2023 Jun 02

Ochandarena, NE;Niehaus, J;Tassou, A;Scherrer, G;
PMID: 37271281 | DOI: 10.1016/j.neuropharm.2023.109597

Opioids are potent analgesics broadly used for pain management; however, they can produce dangerous side effects including addiction and respiratory depression. These harmful effects have led to an epidemic of opioid abuse and overdose deaths, creating an urgent need for the development of both safer pain medications and treatments for opioid use disorders. Both the analgesic and addictive properties of opioids are mediated by the mu opioid receptor (MOR), making resolution of the cell types and neural circuits responsible for each of the effects of opioids a critical research goal. Single-cell RNA sequencing (scRNA-seq) technology is enabling the identification of MOR-expressing cell types throughout the nervous system, creating new opportunities for mapping distinct opioid effects onto newly discovered cell types. Here, we describe molecularly defined MOR-expressing neuronal cell types throughout the peripheral and central nervous systems and their potential contributions to opioid analgesia and addiction.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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