Paternal nicotine taking elicits heritable sex-specific phenotypes that are mediated by hippocampal Satb2.

Nicotine intake, whether through tobacco smoking or e-cigarettes, remains a global health concern. An emerging preclinical literature indicates that parental nicotine exposure produces behavioral, physiological, and molecular changes in subsequent generations. However, the heritable effects of voluntary parental nicotine taking are unknown. Here, we show increased acquisition of nicotine taking in male and female offspring of sires that self-administered nicotine. In contrast, self-administration of sucrose and cocaine were unaltered in male and female offspring suggesting that the intergenerational effects of paternal nicotine taking may be reinforcer specific. Further characterization revealed memory deficits and increased anxiety-like behaviors in drug-naive male, but not female, offspring of nicotine-experienced sires. Using an unbiased, genome-wide approach, we discovered that these phenotypes were associated with decreased expression of Satb2, a transcription factor known to play important roles in synaptic plasticity and memory formation, in the hippocampus of nicotine-sired male offspring. This effect was sex-specific as no changes in Satb2 expression were found in nicotine-sired female offspring. Finally, increasing Satb2 levels in the hippocampus prevented the escalation of nicotine intake and rescued the memory deficits associated with paternal nicotine taking in male offspring. Collectively, these findings indicate that paternal nicotine taking produces heritable sex-specific molecular changes that promote addiction-like phenotypes and memory impairments in male offspring.

Post-treatment effects of topiramate on alcohol-related outcomes: A combined analysis of two placebo-controlled trials.

Topiramate reduces drinking and alcohol-related problems and is increasingly being used to treat alcohol use disorder (AUD). In a randomized controlled trial (RCT) of topiramate, rs2832407, a single nucleotide polymorphism (SNP) in the GRIK1 gene moderated topiramate's effects (Study 1). However, a second RCT (Study 2) did not replicate the SNP's moderating effect during treatment. The current analysis combines data from these two studies to examine topiramate's effects on alcohol-related outcomes and on its pharmacogenetic moderation during a 6-month post-treatment period. This analysis includes 308 individuals with problematic alcohol use (67% male; mean age = 51.1; topiramate: 49%, placebo: 51%). It uses generalized linear mixed models to examine changes in self-reported alcohol consumption and alcohol-related problems and concentrations of the liver enzyme γ-glutamyltransferase. The report combines published 3- and 6-month follow-up data from Study 1 with similar, unpublished data from Study 2. Despite robust effects of topiramate on drinking during treatment, the overall multivariate medication effects on outcomes during 3- and 6-month follow-up were not significant (p = 0.08 and p = 0.26, respectively). The moderating effect of the SNP on primary treatment outcomes was also not significant during either follow-up period (p = 0.13 and p = 0.16, respectively). However, during the 3-month post-treatment period, drinks per day was significantly lower in the topiramate group than the placebo group in the rs2832407*CC-genotype group. The robust effects of topiramate on alcohol-related outcomes during treatment diminish substantially once the medication is discontinued. Research is needed both to determine the optimal treatment duration and to identify clinically useful pharmacogenetic moderators of topiramate for treating AUD.

Placental DNA methylation profiles in opioid-exposed pregnancies and associations with the neonatal opioid withdrawal syndrome.

Opioid abuse during pregnancy can result in Neonatal Opioid Withdrawal Syndrome (NOWS). We investigated genome-wide methylation analyses of 96 placental tissue samples, including 32 prenatally opioid-exposed infants with NOWS who needed therapy (+Opioids/+NOWS), 32 prenatally opioid-exposed infants with NOWS who did not require treatment (+Opioids/-NOWS), and 32 prenatally unexposed controls (-Opioids/-NOWS, control). Statistics, bioinformatics, Artificial Intelligence (AI), including Deep Learning (DL), and Ingenuity Pathway Analyses (IPA) were performed. We identified 17 dysregulated pathways thought to be important in the pathophysiology of NOWS and reported accurate AI prediction of NOWS diagnoses. The DL had an AUC (95% CI) =0.98 (0.95-1.0) with a sensitivity and specificity of 100% for distinguishing NOWS from the +Opioids/-NOWS group and AUCs (95% CI) =1.00 (1.0-1.0) with a sensitivity and specificity of 100% for distinguishing NOWS versus control and + Opioids/-NOWS group versus controls. This study provides strong evidence of methylation dysregulation of placental tissue in NOWS development.

Maternal opioid use disorder: Placental transcriptome analysis for neonatal opioid withdrawal syndrome.

Excessive prenatal opioid exposure may lead to the development of Neonatal Opioid Withdrawal Syndrome (NOWS). RNA-seq was done on 64 formalin-fixed paraffin-embedded placental tissue samples from 32 mothers with opioid use disorder, with newborns with NOWS that required treatment, and 32 prenatally unexposed controls. We identified 93 differentially expressed genes in the placentas of infants with NOWS compared to unexposed controls. There were 4 up- and 89 downregulated genes. Among these, 7 genes CYP1A1, APOB, RPH3A, NRXN1, LINC01206, AL157396.1, UNC80 achieved an FDR p-value of <0.01. The remaining 87 genes were significant with FDR p-value <0.05. The 4 upregulated, CYP1A1, FP671120.3, RAD1, RN7SL856P, and the 10 most significantly downregulated genes were RNA5SP364, GRIN2A, UNC5D, DMBT1P1, MIR3976HG, LINC02199, LINC02822, PANTR1, AC012178.1, CTNNA2. Ingenuity Pathway Analysis identified the 7 most likely to play an important role in the etiology of NOWS. Our study expands insights into the genetic mechanisms of NOWS development.

A Delta-Opioid Receptor Gene Polymorphism Moderates the Therapeutic Response to Extended-Release Buprenorphine in Opioid Use Disorder.

BACKGROUND: Buprenorphine treatment is not equally effective in all patients with opioid use disorder (OUD). Two retrospective studies showed that, among African Americans (AAs), rs678849, a polymorphism in the delta-opioid receptor gene, moderated the therapeutic effect of sublingual buprenorphine. METHODS: We examined rs678849 as a moderator of the response to an extended-release subcutaneous buprenorphine formulation (BUP-XR) in a 24-week OUD treatment study of 127 AAs and 327 European Americans (EAs). Participants were randomly assigned to receive: (1) BUP-XR as 2 monthly injections of 300 mg followed by either 300 mg monthly or 100 mg monthly for 4 months, or (2) monthly volume-matched placebo injections. Generalized estimating equations logistic regression analyses tested, per population group, the main and interaction effects of treatment (BUP-XR vs placebo) and genotype group (rs678849*CC vs CT/TT) on weekly urine drug screens (UDS). RESULTS: Among AAs, the placebo group had higher rates of opioid-positive UDS than the BUP-XR group (log odds ratio = 1.67, 95% CI = 0.36, 2.98), but no genotype by treatment effect (P = .80). Among EAs, the placebo group also showed higher rates of opioid-positive UDS than the BUP-XR group (log odds ratio = 1.97, 95% CI = 1.14, 2.79) but a significant genotype by treatment interaction (χ 2(1) = 4.33, P = .04). CONCLUSION: We found a moderating effect of rs678849 on the response to buprenorphine treatment of OUD in EAs, but not AAs. These findings require replication in well-powered, prospective studies of both AA and EA OUD patients treated with BUP-XR and stratified on rs678849 genotype.

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.

The importance of buprenorphine research in the opioid crisis.

With the urgency to treat patients more effectively for opioid use disorder in the midst of the opioid epidemic, a key area for precision medicine is to improve individualized medication-assisted treatment for opioid use disorder. The expansion of medication-assisted treatment is a key to reducing illicit opioid use, preventing opioid overdose deaths, and reducing the comorbidities and societal impacts of opioid use disorder. The most common medication for opioid use disorder will soon be buprenorphine. Research to date shows the successful impact of buprenorphine treatment, including the pharmacogenomics of buprenorphine response and treatment efficacy. Buprenorphine is also a promising treatment for depression and anxiety, and neonatal opioid withdrawal syndrome (NOWS). However, the rates of success with medication-assisted treatment for opioid use disorder, particularly at the beginning of treatment, still show many individuals relapsing to illicit opioid use. With the scope of the opioid crisis, there is an urgent need for expansion of buprenorphine treatment research to provide critical information for improving outcomes of opioid use disorder. Implementing the best strategies for opioid use disorder treatment is of dire urgency and will save lives.

Replication of the pharmacogenetic effect of rs678849 on buprenorphine efficacy in African-Americans with opioid use disorder.

Many patients with opioid use disorder do not have successful outcomes during treatment but the underlying reasons are not well understood. An OPRD1 variant (rs678849) was previously associated with methadone and buprenorphine efficacy in African-Americans with opioid use disorder. The objective of this study was to determine if the effect of rs678849 on opioid use disorder treatment outcome could be replicated in an independent population. Participants were recruited from African-American patients who had participated in previous studies of methadone or buprenorphine treatment at the outpatient treatment research clinic of the NIDA Intramural Research Program in Baltimore, MD, USA between 2000 and 2017. Rs678849 was genotyped retrospectively, and genotypes were compared with urine drug screen results from the previous studies for opioids other than the one prescribed for treatment. Genotypes were available for 24 methadone patients and 55 buprenorphine patients. After controlling for demographics, the effect of rs678849 genotype was significant in the buprenorphine treatment group (RR = 1.69, 95% confidence interval (CI) 1.59-1.79, p = 0.021). Buprenorphine patients with the C/C genotype were more likely to have opioid-positive drug screens than individuals with the C/T or T/T genotypes, replicating the original pharmacogenetic finding. The effect of genotype was not significant in the methadone group (p = 0.087). Thus, the genotype at rs678849 is associated with buprenorphine efficacy in African-Americans being treated for opioid use disorder. This replication suggests that rs678849 genotype may be a valuable pharmacogenetic marker for deciding which opioid use disorder medication to prescribe in this population.

OPRD1 Genetic Variation and Human Disease.

The OPRD1 gene encodes the delta-opioid receptor, which has multiple functions including regulating reward pathways. The gene contains more than 2,000 verified genetic variants but only 2 currently have evidence for specific functions: rs1042114 disrupts maturation of the receptor and rs569356 affects OPRD1 expression. These polymorphisms and others in the gene have been found to be associated with human diseases. The most reproducible data are associations between opioid addiction and three variants in intron 1 (rs2236861, rs2236857, and rs3766951), which have been described in a number of independent populations. Several publications also point toward an association between anorexia and a haplotype block containing rs569356 and rs533123. Unfortunately the mechanisms underlying these two effects are currently unknown. In contrast, rs1042114 has been linked to Alzheimer's disease through an increasingly well-defined mechanism by which the variant allele reduces production of the beta-amyloid plaques associated with the disease. Additional studies of OPRD1 variants are necessary to replicate current findings and to delineate the functional roles of relevant polymorphisms.

Pharmacogenetic analysis of opioid dependence treatment dose and dropout rate.

BACKGROUND: Currently, no pharmacogenetic tests for selecting an opioid-dependence pharmacotherapy have been approved by the US Food and Drug Administration. OBJECTIVES: Determine the effects of variants in 11 genes on dropout rate and dose in patients receiving methadone or buprenorphine/naloxone (ClinicalTrials.gov Identifier: NCT00315341). METHODS: Variants in six pharmacokinetic genes (CYP1A2, CYP2B6, CYP2C19, CYP2C9, CYP2D6, CYP3A4) and five pharmacodynamic genes (HTR2A, OPRM1, ADRA2A, COMT, SLC6A4) were genotyped in samples from a 24-week, randomized, open-label trial of methadone and buprenorphine/naloxone for the treatment of opioid dependence (n = 764; 68.7% male). Genotypes were then used to determine the metabolism phenotype for each pharmacokinetic gene. Phenotypes or genotypes for each gene were analyzed for association with dropout rate and mean dose. RESULTS: Genotype for 5-HTTLPR in the SLC6A4 gene was nominally associated with dropout rate when the methadone and buprenorphine/naloxone groups were combined. When the most significant variants associated with dropout rate were analyzed using pairwise analyses, SLC6A4 (5-HTTLPR) and COMT (Val158Met; rs4860) had nominally significant associations with dropout rate in methadone patients. None of the genes analyzed in the study was associated with mean dose of methadone or buprenorphine/naloxone. CONCLUSIONS: This study suggests that functional polymorphisms related to synaptic dopamine or serotonin levels may predict dropout rates during methadone treatment. Patients with the S/S genotype at 5-HTTLPR in SLC6A4 or the Val/Val genotype at Val158Met in COMT may require additional treatment to improve their chances of completing addiction treatment. Replication in other methadone patient populations will be necessary to ensure the validity of these findings.

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.

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.

Candidate Genes from an FDA-Approved Algorithm Fail to Predict Opioid Use Disorder Risk in Over 450,000 Veterans.

Importance: Recently, the Food and Drug Administration gave pre-marketing approval to algorithm based on its purported ability to identify genetic risk for opioid use disorder. However, the clinical utility of the candidate genes comprising the algorithm has not been independently demonstrated. Objective: To assess the utility of 15 variants in candidate genes from an algorithm intended to predict opioid use disorder risk. Design: This case-control study examined the association of 15 candidate genetic variants with risk of opioid use disorder using available electronic health record data from December 20, 1992 to September 30, 2022. Setting: Electronic health record data, including pharmacy records, from Million Veteran Program participants across the United States. Participants: Participants were opioid-exposed individuals enrolled in the Million Veteran Program (n = 452,664). Opioid use disorder cases were identified using International Classification of Disease diagnostic codes, and controls were individuals with no opioid use disorder diagnosis. Exposures: Number of risk alleles present across 15 candidate genetic variants. Main Outcome and Measures: Predictive performance of 15 genetic variants for opioid use disorder risk assessed via logistic regression and machine learning models. Results: Opioid exposed individuals (n=33,669 cases) were on average 61.15 (SD = 13.37) years old, 90.46% male, and had varied genetic similarity to global reference panels. Collectively, the 15 candidate genetic variants accounted for 0.4% of variation in opioid use disorder risk. The accuracy of the ensemble machine learning model using the 15 genes as predictors was 52.8% (95% CI = 52.1 - 53.6%) in an independent testing sample. Conclusions and Relevance: Candidate genes that comprise the approved algorithm do not meet reasonable standards of efficacy in predicting opioid use disorder risk. Given the algorithm's limited predictive accuracy, its use in clinical care would lead to high rates of false positive and negative findings. More clinically useful models are needed to identify individuals at risk of developing opioid use disorder.

Clinical Pharmacogenetics Implementation Consortium Guideline for CYP2B6 Genotype and Methadone Therapy.

Methadone is a mu (µ) opioid receptor agonist used clinically in adults and children to manage opioid use disorder, neonatal abstinence syndrome, and acute and chronic pain. It is typically marketed as a racemic mixture of R- and S-enantiomers. R-methadone has 30-to 50-fold higher analgesic potency than S-methadone, and S-methadone has a greater adverse effect (prolongation) on the cardiac QTc interval. Methadone undergoes stereoselective metabolism. CYP2B6 is the primary enzyme responsible for catalyzing the metabolism of both enantiomers to the inactive metabolites, S- and R-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (S- and R-EDDP). Genetic variation in the CYP2B6 gene has been investigated in the context of implications for methadone pharmacokinetics, dose, and clinical outcomes. Most CYP2B6 variants result in diminished or loss of CYP2B6 enzyme activity, which can lead to higher plasma methadone concentrations (affecting S- more than R-methadone). However, the data do not consistently indicate that CYP2B6-based metabolic variability has a clinically significant effect on methadone dose, efficacy, or QTc prolongation. Expert analysis of the published literature does not support a change from standard methadone prescribing based on CYP2B6 genotype (updates at www.cpicpgx.org).

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.