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Genetic factors impact behavioral traits relevant to numerous psychiatric disorders and risk-taking behaviors, and different lines of evidence have indicated that discrete neurobiological systems contribute to such individual differences. In this study, we explored the relationship of genetic variants of the prodynorphin (PDYN) gene, which is enriched in the striatonigral/striatomesencephalic pathway, a key neuronal circuit implicated in positive 'Go' behavioral choice and action. Our multidisciplinary approach revealed that the single nucleotide polymorphism (SNP) rs2235749 (in high linkage disequilibrium with rs910080) modifies striatal PDYN expression via impaired binding of miR-365, a microRNA that targets the PDYN 3'-untranslated region (3'UTR), and is significantly associated to novelty- and reward-related behavioral traits in humans and translational animal models. Carriers of the rs2235749G allele exhibited increased levels of PDYN 3'UTR in vitro and had elevated mRNA expression in the medial nucleus accumbens shell (NAcSh) and caudate nucleus in postmortem human brains. There was an association of rs2235749 with novelty-seeking trait and a strong genotype-dose association with positive reinforcement behavior in control subjects, which differed in cannabis-dependent individuals. Using lentiviral miRZip-365 constructs selectively expressed in Pdyn-neurons of the NAcSh, we demonstrated that the Pdyn-miR365 interaction in the NAcSh directly influences novelty-seeking exploratory behavior and facilitates self-administration of natural reward. Overall, this translational study suggests that genetically determined miR-365-mediated epigenetic regulation of PDYN expression in mesolimbic striatonigral/striatomesencephalic circuits possibly contributes to novelty seeking and positive reinforcement traits.

Chronic exposure to drugs of abuse perturbs the endogenous opioid system, which plays a critical role in the development and maintenance of addictive disorders. Opioid genetics may therefore play an important modulatory role in the expression of substance use disorders, but these genes have not been extensively characterized, especially in humans. In the current imaging genetics study, we investigated a single nucleotide polymorphism (SNP) of the protein-coding proenkephalin gene (PENK: rs2609997, recently shown to be associated with cannabis dependence) in 55 individuals with cocaine use disorder and 37 healthy controls. Analyses tested for PENK associations with fMRI response to error (during a classical color-word Stroop task) and gray matter volume (voxel-based morphometry) as a function of Diagnosis (cocaine, control). Results revealed whole-brain Diagnosis×PENK interactions on the neural response to errors (fMRI error>correct contrast) in the right putamen, left rostral anterior cingulate cortex/medial orbitofrontal cortex, and right inferior frontal gyrus; there was also a significant Diagnosis×PENK interaction on right inferior frontal gyrus gray matter volume. These interactions were driven by differences between individuals with cocaine use disorders and controls that were accentuated in individuals carrying the higher-risk PENK C-allele. Taken together, the PENK polymorphism-and potentially opioid neurotransmission more generally-modulates functioning and structural integrity of brain regions previously implicated in error-related processing. PENK could potentially render a subgroup of individuals with cocaine use disorder (i.e., C-allele carriers) more sensitive to mistakes or other related challenges; in future studies, these results could contribute to the development of individualized genetics-informed treatments., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Negative affect is critical for conferring vulnerability to opiate addiction as reflected by the high comorbidity of opiate abuse with major depressive disorder (MDD). Rodent models implicate amygdala prodynorphin (Pdyn) as a mediator of negative affect; however, evidence of PDYN involvement in human negative affect is limited. Here, we found reduced PDYN mRNA expression in the postmortem human amygdala nucleus of the periamygdaloid cortex (PAC) in both heroin abusers and MDD subjects. Similar to humans, rats that chronically self-administered heroin had reduced Pdyn mRNA expression in the PAC at a time point associated with a negative affective state. Using the in vivo functional imaging technology DREAMM (DREADD-assisted metabolic mapping, where DREADD indicates designer receptors exclusively activated by designer drugs), we found that selective inhibition of Pdyn-expressing neurons in the rat PAC increased metabolic activity in the extended amygdala, which is a key substrate of the extrahypothalamic brain stress system. In parallel, PAC-specific Pdyn inhibition provoked negative affect-related physiological and behavioral changes. Altogether, our translational study supports a functional role for impaired Pdyn in the PAC in opiate abuse through activation of the stress and negative affect neurocircuitry implicated in addiction vulnerability.

Background: Marijuana use by teenagers often predates the use of harder drugs, but the neurobiological underpinnings of such vulnerability are unknown. Animal studies suggest enhanced heroin self-administration (SA) and dysregulation of the endogenous opioid system in the nucleus accumbens shell (NAcsh) of adults following adolescent Δ(9)-tetrahydrocannabinol (THC) exposure. However, a causal link between proenkephalin (Penk) expression and vulnerability to heroin has yet to be established., Methods: To investigate the functional significance of NAcsh Penk tone, selective viral-mediated knockdown and overexpression of Penk was performed, followed by analysis of subsequent heroin SA behavior. To determine whether adolescent THC exposure was associated with chromatin alteration, we analyzed levels of histone H3 methylation in the NAcsh via chromatin immunoprecipitation at five sites flanking the Penk gene transcription start site., Results: Here we show that regulation of the Penk opioid neuropeptide gene in NAcsh directly regulates heroin SA behavior. Selective viral-mediated knockdown of Penk in striatopallidal neurons attenuates heroin SA in adolescent THC-exposed rats, whereas Penk overexpression potentiates heroin SA in THC-naïve rats. Furthermore, we report that adolescent THC exposure mediates Penk upregulation through reduction of histone H3 lysine 9 (H3K9) methylation in the NAcsh, thereby disrupting the normal developmental pattern of H3K9 methylation., Conclusions: These data establish a direct association between THC-induced NAcsh Penk upregulation and heroin SA and indicate that epigenetic dysregulation of Penk underlies the long-term effects of THC., (Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Background: Many young people experiment with cannabis, yet only a subgroup progress to dependence suggesting individual differences that could relate to factors such as genetics and behavioral traits. Dopamine receptor D2 (DRD2) and proenkephalin (PENK) genes have been implicated in animal studies with cannabis exposure. Whether polymorphisms of these genes are associated with cannabis dependence and related behavioral traits is unknown., Methodology/principal Findings: Healthy young adults (18-27 years) with cannabis dependence and without a dependence diagnosis were studied (N = 50/group) in relation to a priori-determined single nucleotide polymorphisms (SNPs) of the DRD2 and PENK genes. Negative affect, Impulsive Risk Taking and Neuroticism-Anxiety temperamental traits, positive and negative reward-learning performance and stop-signal reaction times were examined. The findings replicated the known association between the rs6277 DRD2 SNP and decisions associated with negative reinforcement outcomes. Moreover, PENK variants (rs2576573 and rs2609997) significantly related to Neuroticism and cannabis dependence. Cigarette smoking is common in cannabis users, but it was not associated to PENK SNPs as also validated in another cohort (N = 247 smokers, N = 312 non-smokers). Neuroticism mediated (15.3%-19.5%) the genetic risk to cannabis dependence and interacted with risk SNPs, resulting in a 9-fold increase risk for cannabis dependence. Molecular characterization of the postmortem human brain in a different population revealed an association between PENK SNPs and PENK mRNA expression in the central amygdala nucleus emphasizing the functional relevance of the SNPs in a brain region strongly linked to negative affect., Conclusions/significance: Overall, the findings suggest an important role for Neuroticism as an endophenotype linking PENK polymorphisms to cannabis-dependence vulnerability synergistically amplifying the apparent genetic risk.

Striatal enkephalin and dynorphin opioid systems mediate reward and negative affect, respectively, relevant to addiction disorders. We examined polymorphisms of proenkephalin (PENK) and prodynorphin (PDYN) genes in relation to heroin abuse and gene expression in the human striatum and the relevance of genetic dopaminergic tone, critical for drug reward and striatal function. Heroin abuse was significantly associated with PENK polymorphic 3' UTR dinucleotide (CA) repeats; 79% of subjects homozygous for the 79-bp allele were heroin abusers. Such individuals tended to express higher PENK mRNA than the 81-bp homozygotes, but PENK levels within the nucleus accumbens (NAc) shell were most strongly correlated to catecholamine-O-methyltransferase (COMT) genotype. Control Met/Met individuals expressed lower PENK mRNA than Val carriers, a pattern reversed in heroin users. Up-regulation of NAc PENK in Met/Met heroin abusers was accompanied by impaired tyrosine hydroxylase (TH) mRNA expression in mesolimbic dopamine neurons. In contrast to PENK, no association was detected between PDYN genotype (68-bp repeat element containing one to four copies of AP-1 binding sites in the promoter region) and heroin abuse, although there was a clear functional association with striatal PDYN mRNA expression: an increased number of inducible repeats (three and four) correlated with higher PDYN levels than adult or fetal subjects with noninducible (one and two) alleles. Moreover, PDYN expression was not related to COMT genotype. Altogether, the data suggest that dysfunction of the opioid reward system is significantly linked to opiate abuse vulnerability and that heroin use alters the apparent influence of heritable dopamine tone on mesolimbic PENK and TH function.

The objective of this study was to determine if prenatal cocaine affects the levels of prodynorphin and proenkephalin mRNA in male and female adolescent rats. Pregnant dams received cocaine or vehicle from gestational days 8-22 and upon delivery, the pups were fostered. At postnatal days 42-44, pups were killed and brains removed and frozen. Sections of striatum and nucleus accumbens were processed for prodynorphin and proenkephalin mRNA expression. Prenatal cocaine did not affect the expression of proenkephalin mRNA, but males showed higher expression than females. However, prodynorphin mRNA was lower in female rats exposed to cocaine compared to controls. Prenatal cocaine appears to have unique effects on neuropeptides during adolescence.

The classical view postulates that neuropeptide precursors in neurons are processed into mature neuropeptides in the somatic trans-Golgi network (TGN) and in secretory vesicles during axonal transport. Here we show that prodynorphin (PDYN), precursor to dynorphin opioid peptides, is predominantly located in axon terminals and dendrites in hippocampal and striatal neurons. The molar content of unprocessed PDYN was much greater than that of dynorphin peptides in axon terminals of PDYN-containing neurons projecting to the CA3 region of the hippocampus and in the striatal projections to the ventral tegmental area. Electron microscopy showed coexistence of PDYN and dynorphins in the same axon terminals with occasional codistribution in individual dense core vesicles. Thus, the precursor protein is apparently stored at presynaptic sites. In comparison with the hippocampus and striatum, PDYN and dynorphins were more equally distributed between neuronal somata and processes in the amygdala and cerebral cortex, suggesting regional differences in the regulation of trafficking and processing of the precursor protein. Potassium-induced depolarization activated PDYN processing and secretion of opioid peptides in neuronal cultures and in a model cell line. Regulation of PDYN storage and processing at synapses by neuronal activity or extracellular stimuli may provide a local mechanism for regulation of synaptic transmission.

Fetal development is a period sensitive to environmental influences such as maternal drug use. The most commonly used illicit drug by pregnant women is marijuana. The present study investigated the effects of in utero marijuana exposure on expression levels of opioid-related genes in the human fetal forebrain in light of the strong interaction between the cannabinoid and opioid systems. The study group consisted of 42 midgestation fetuses from saline-induced voluntary abortions. The opioid peptide precursors (preprodynorphin and preproenkephalin (PENK)) and receptor (mu, kappa and delta) mRNA expression were assessed in distinct brain regions. The effect of prenatal cannabis exposure was analyzed by multiple regression controlling for confounding variables (maternal alcohol and cigarette use, fetal age, sex, growth measure and post-mortem interval). Prenatal cannabis exposure was significantly associated with increased mu receptor expression in the amygdala, reduced kappa receptor mRNA in mediodorsal thalamic nucleus and reduced preproenkephalin expression in the caudal putamen. Prenatal alcohol exposure primarily influenced the kappa receptor mRNA with reduced levels in the amygdala, claustrum, putamen and insula cortex. No significant effect of prenatal nicotine exposure could be discerned in the present study group. These results indicate that maternal cannabis and alcohol exposure during pregnancy differentially impair opioid-related genes in distinct brain circuits that may have long-term effects on cognitive and emotional behaviors.

Transcription from multiple promoters along with alternative mRNA splicing constitutes the basis for cell-specific gene expression and mRNA and protein diversity. The prodynorphin gene (PDYN) gives rise to prodynorphin (PDYN), precursor to dynorphin opioid peptides that regulate diverse physiological functions and are implicated in various neuropsychiatric disorders. Here, we characterized PDYN transcripts and proteins in the adult human brain and studied PDYN processing and intracellular localization in model cell lines. Seven PDYN mRNAs were identified in the human brain; two of the transcripts, FL1 and FL2, encode the full-length PDYN. The dominant, FL1 transcript shows high expression in limbic-related structures such as the nucleus accumbens and amygdala. The second, FL2 transcript is only expressed in few brain structures such as the claustrum and hypothalamus. FL-PDYN was identified for the first time in the brain as the dominant PDYN protein product. Three novel PDYNs expressed from spliced or truncated PDYN transcripts either lack a central segment but are still processed into dynorphins, or are translated into N-terminally truncated proteins. One truncated PDYN is located in the cell nucleus, suggesting a novel nonopioid function for this protein. The complexity of PDYN expression and diversity of its protein products may be relevant for diverse levels of plasticity in adaptive responses for the dynorphin system.

Several human and rat studies suggest that the striatal dynorphin system is important for neuroadaptation following cocaine exposure. In the current study, prodynorphin (PDYN) mRNA expression was examined in monkeys at initial and chronic phases of cocaine self-administration. Adult Rhesus monkeys were trained to self-administer food (banana flavoured pellets) or cocaine (0.03 or 0.3 mg/kg per injection) on a fixed interval 3-min schedule for 5 or 100 sessions. Each session ended after 30 reinforcers were delivered. The PDYN mRNA expression was analysed in the precommissural striatum using in situ hybridization histochemistry. We found a specific activation of PDYN mRNA expression in the limbic-innervated patch/striosome compartment of the dorsal caudate and dorsal putamen during the initial (i.e. 5 day) phase of the high dose cocaine self-administration. After 100 days of the high dose exposure, the patch/striosome compartment remained activated, but an increase in PDYN mRNA levels was also evident in the sensorimotor-connected matrix compartment of the caudate. Neither self-administration phase resulted in significant changes in the corresponding striatal regions of the low dose cocaine-exposed primates. Moreover, cocaine self-administration failed to alter the PDYN mRNA expression in high- or low-expressing PDYN cell populations in the nucleus accumbens during any condition studied. These results demonstrate the vulnerability of the dorsal striatum (in particular the caudate) to neuroadaptations following long-term high dose cocaine self-administration. In addition, the temporal nature of the changes in PDYN gene expression within the striatal compartments could reflect a change in drug responsivity that occurs during the transition to drug dependence.

The dynorphin system has been associated with the regulation of mood. The expression of the prodynorphin mRNA was currently studied in the amygdaloid complex, a brain region critical for emotional processing, in subjects (14-15 per group) diagnosed with major depression, bipolar disorder, or schizophrenia and compared to normal controls. In situ hybridization histochemistry was used to characterize the anatomical distribution and expression levels of the prodynorphin mRNA within the amygdaloid complex. High prodynorphin mRNA levels were expressed in the parvicellular division of the accessory basal, posterior cortical, periamygdaloid cortex, and amygdalohippocampal area in normal subjects. Individuals with major depression had significantly reduced (41-68%) expression of the prodynorphin mRNA in the accessory basal (both parvicellular and magnocellular divisions; P < 0.01) and amygdalohippocampal area (P < 0.001) as compared to controls. The bipolar disorder group also showed a significant reduction (37-38%, P < 0.01) of the mRNA expression levels in the amygdalohippocampal area and in the parvicellular division of the accessory basal. No other amygdala nuclei studied showed any significant differences for the prodynorphin mRNA levels measured in the major depression and bipolar disorder subjects. Additionally, the prodynorphin mRNA expression levels did not differ significantly between the schizophrenic and normal control subjects in any of the amygdala areas examined. These findings indicate specific prodynorphin amygdala impairment in association with mood disorder.

The present study examined the prodynorphin and kappa opioid receptor mRNA expression levels in the anterior cingulate and dorsolateral prefrontal cortices of subjects diagnosed with schizophrenia, bipolar disorder, or major depression as compared with normal controls without a psychiatric diagnosis. Multivariate analyses failed to reveal any differences in the mRNA expression levels between the four diagnostic groups, though a group trend (non-significant) was evident for the expression of the kappa opioid receptor and prodynorphin mRNAs in the prefrontal cortex. The mRNA expression levels were not associated with lifetime history of antipsychotic treatment or with suicide as a cause of death. The results, however, suggested an influence of certain drugs of abuse on the prodynorphin cortical mRNA expression. Prodynorphin mRNA expression levels were found to be elevated in individuals with a history of marihuana or stimulant use, but not alcohol. Overall, our data do not provide strong evidence for impaired prodynorphin or kappa opioid receptor mRNA levels in the dorsolateral prefrontal or cingulate cortices of schizophrenic, bipolar disorder, or major depressed subjects.

The substituted phenylpiperidine (-)-OSU6162 is a novel modulator of the dopaminergic systems with low affinity for dopamine D(2) receptors and potent normalizing effects on l-DOPA-induced dyskinesias. We studied the effects of coadministration of (-)-OSU6162 with l-DOPA on the regulation of striatal preproenkephalin (PPE) and prodynorphin (PDyn) mRNA expression in the primate brain by in situ hybridization histochemistry. Common marmoset monkeys sustaining unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway received l-DOPA/carbidopa, l-DOPA/carbidopa plus (-)-OSU6162, or vehicle over 14 days. In vehicle-treated animals, PPE mRNA levels were markedly increased in the sensorimotor territory of the lesioned striatum. By contrast, a rather uniform lesion-induced reduction of PDyn mRNA levels was found in the vehicle group. Subchronic l-DOPA treatment induced a further increase in PPE mRNA expression in a number of sensorimotor and associative subregions of the denervated striatum. Coadministration of (-)-OSU6162 with l-DOPA partially reversed the lesion- and l-DOPA-induced elevation of PPE expression and, by affecting PPE mRNA expression differentially on the intact and lesioned striatum, markedly reduced the side-to-side difference in PPE mRNA expression. The effects on PPE mRNA expression were apparent throughout the rostrocaudal extent of the putamen and the dorsal portions of the caudate nucleus. l-DOPA treatment resulted in an enhancement in PDyn mRNA expression in all functional compartments of the striatum. Coadministration of (-)-OSU6162 had no apparent influence on these l-DOPA-induced changes in PDyn mRNA expression. The present results suggest that (-)-OSU6162 acts primarily by modifying striatal output via the indirect pathway., (Copyright 2001 Academic Press.)

The mRNA expression for preprotachykinin-A (PPT-A) was studied throughout the human and cynomolgus monkey brain to assess the neuroanatomical expression pattern of the PPT-A gene in primates. In situ hybridization showed that the PPT-A mRNA is expressed highly in specific regions of the postmortem human brain, including the striatum, islands of Calleja, hypothalamus (posterior, premammillary, medial mammillary, and ventromedial nuclei), superior and inferior colliculi, periaqueductal gray, and oculomotor nuclear complex. PPT-A mRNA-expressing neurons also were present in the paranigralis (ventral tegmental area) and were scattered in the bed nucleus stria terminalis throughout the sublenticular substantia innominata region, including the diagonal band of Broca and the nucleus basalis of Meynert. In the hippocampus, high PPT-A mRNA expression was localized predominantly to the polymorphic layer of the dentate gyrus; no labeled cells were present in the granular layer. Positively labeled cells also were found scattered in the CA regions as well as in the amygdaloid complex. Neocortical expression of PPT-A mRNA was localized mainly to the deep laminae (layers V/VI), except for the striate cortex (labeling was seen also in superficial layers). The subiculum, thalamus, globus pallidus, ventral pallidum, substantia nigra pars compacta, red nucleus, pontine nuclei, and cerebellum were characterized by very weak to undetectable expression of PPT-A mRNA. An expression pattern was evident in the monkey forebrain similar to that observed in the human, except for the absence of PPT mRNA-expressing cells in the medial mammillary nucleus despite intense expression in supramammillary, lateral mammillary, and premammillary nuclei. Overall, more similarities than differences are apparent between primate species in the expression pattern of the PPT-A gene. J. Comp. Neurol. 411;56-72, 1999., (Copyright 1999 Wiley-Liss, Inc.)

Preprodynorphin mRNA expression in the striatal-olfactory tubercle complex was studied in adult rats exposed to cocaine (50 mg/kg) during postnatal days (PnD) 11-20. While multiple regions of the striatum and olfactory tubercle were examined, alterations were only found in the nucleus accumbens. A 50% and 20% reduction in expression within the shell region was observed at 1.2 and 1.7 mm rostral to Bregma respectively. While the core regions at these levels were unaffected, the rostral accumbens showed a trend toward an increase in expression in the cocaine-treated rats. These findings, in combination with other behavioral and neurochemical data collected on similarly treated rats, suggest that perinatal cocaine produces a long-term dampening of function in a specific population of neurons within the mesolimbic system, (Copyright 1998 Elsevier Science B.V.)

It is well established that the opioid neuropeptide and dopamine systems are altered following the use of cocaine. However very little information is available about their possible involvement during cocaine abstinence. In the present study, the mRNA expression of the dopamine receptors, D1 and D2, and the opioid peptides, prodynorphin and proenkephalin, were analyzed in the rat striatum using in situ hybridization histochemistry. Saline or cocaine (30 mg/kg, i.p.) were administered to rats once daily for 1 or 10 days. To examine cocaine abstinence, animals were treated for 10 days as described followed by a 10-day drug free period. Acute and intermittent cocaine administration elevated the prodynorphin mRNA expression in the dorsal striatum, consistent with previous reports, while the abstinent phase resulted in a significant reduction of prodynorphin mRNA levels in the ventrorostral striatum. The D1-receptor mRNA was decreased in the caudorostral striatum during cocaine withdrawal, a finding opposite to the increase observed following a single injection of the drug. Proenkephalin and the D2-receptor mRNAs were not altered during cocaine abstinence, though proenkephalin was elevated following acute but not repeated cocaine administration. These results show long-term suppression on prodynorphin and D1-receptor systems in specific striatal populations localized mainly in rostral areas during withdrawal from cocaine., (Copyright 1998 Elsevier Science B.V.)

Experimental and clinical studies suggest an involvement of the opioid neuropeptide system in psychiatric disorders. Notably, opioid peptide immunoreactivity is altered in the cerebrospinal fluid of chronic schizophrenics and manic-depressive subjects. Despite these clinical findings, few postmortem investigations have examined the association of endogenous opioid neuropeptides with schizophrenia and suicide. Anatomically, a tight interaction exists within the neostriatum between the opioid peptide (dynorphin and enkephalin) system and classical neurotransmitters such as dopamine which has been implicated in both the psychotic symptoms and the cognitive deficits that characterize schizophrenia (see review). The neostriatum is differentially organized into patch and matrix neurochemical mosaic compartments anatomically connected to limbic- and sensorimotor-related brain regions, respectively. Moreover, the human neostriatum is characterized by a heterogenous expression of the prodynorphin opioid gene: high in the patch, but low in the matrix compartment. The present results show for the first time a differential alteration of prodynorphin within distinct striatal compartments in postmortem tissue from nonschizophrenic suicide subjects. The prodynorphin patch/matrix mRNA expression was elevated in the caudate nucleus of suicide subjects as compared to normal controls and schizophrenics in which no alterations in opioid peptides or D1 and D2 mRNA expression were apparent. Altogether the findings suggest that discrete dysfunction of the endogenous opioid dynorphin system might contribute to depression and the risk of suicide in nonschizophrenic subjects.

The opiate system is involved in a wide variety of neural functions including pain perception, neuroendocrine regulation, memory, drug reward, and tolerance. Such functions imply that endogenous opioid peptides should have anatomical interactions with limbic brain structures believed to be involved in the experience and expression of emotion. Using in situ hybridization histochemistry, the messenger RNA expression of the opioid precursors, prodynorphin and proenkephalin, was studied in whole hemisphere human brain tissue. Different components of the limbic system were found to be characterized by a high gene expression of either prodynorphin or proenkephalin messenger RNA. Brain regions traditionally included within the limbic system (e.g. amygdala, hippocampus, entorhinal cortex and cingulate cortex) as well as limbic-associated regions including the ventromedial prefrontal cortex and patch compartment of the neostriatum showed high prodynorphin messenger RNA expression. In contrast, high levels of proenkephalin messenger RNA were more widely expressed in the hypothalamus, periaqueductal gray, various mesencephalic nuclei, bed nucleus of the stria terminalis, and ventral pallidum; brain regions associated with endocrine-reticular-motor continuum of the limbic system. The marked anatomical dissociation between the expression of these two opioid peptide genes, seen clearly in whole hemisphere sections, indicates that distinct functions must be subserved by the prodynorphin and proenkephalin systems in the human brain.

Expression of neuropeptide messenger RNAs in striatal neurons was studied in post mortem human brain tissue by the use of in situ hybridization histochemistry. Clusters of cells expressing high levels of prodynorphin messenger RNA, and less strikingly, preprotachykinin messenger RNA, were prominent in the caudate nucleus and were present but less pronounced in the putamen. Proenkephalin and prosomatostatin messenger RNA-containing cells were more homogeneously distributed throughout the striatum, though the latter were much sparser. The four neuropeptide messenger RNA patterns in the nucleus accumbens were rather homogeneous compared with the dorsal striatum. Of these, prodynorphin messenger RNA showed a higher level of expression per cell in the nucleus accumbens relative to the dorsal striatum. The relationship of neuropeptide-containing cell clusters to the striosomal organization was characterized by looking at the register of these markers with patterns of low acetylcholinesterase activity and dense mu opiate receptor binding. In the caudate and putamen, clusters of cells expressing high levels of dynorphin and preprotachykinin messenger RNAs were clearly in register with the striosomes. The accumbens was defined by high prodynorphin messenger RNA levels, both low and high levels of acetylcholinesterase staining, and very low to absent mu opiate receptor binding. The distribution of high-expressing prodynorphin messenger RNA-containing cells--to the patch compartment and throughout the entire ventral striatum/nucleus accumbens region--defines the limbic domain of the neostriatum and suggests particular relevance to human striatal organization and function, because the distribution of this opioid neuropeptide is considerably more compartmentalized in human than in non-human species.