Search

Transcription factors (TFs) regulate gene expression by facilitating or disrupting the formation of transcription initiation machinery at particular genomic loci. Because TF occupancy is driven in part by recognition of DNA sequence, genetic variation can influence TF–DNA associations and gene regulation. To identify variants that impact TF binding in human brain tissues, we assessed allele-specific binding (ASB) at heterozygous variants for 94 TFs in nine brain regions from two donors. Leveraging graph genomes constructed from phased genomic sequence data, we compared ChIP-seq signals between alleles at heterozygous variants within each brain region and identified thousands of variants exhibiting ASB for at least one TF. ASB reproducibility was measured by comparisons between independent experiments both within and between donors. We found that rare alleles in the general population more frequently led to reduced TF binding, whereas common alleles had an equal likelihood of increasing or decreasing binding. Further, for ASB variants in predicted binding motifs, the favored allele tended to be the one with the stronger expected motif match, but this concordance was not observed within highly occupied sites. We also found that neuron-specific cis-regulatory elements (cCREs), in contrast with oligodendrocyte-specific cCREs, showed depletion of ASB variants. We identified 2670 ASB variants associated with evidence for allele-specific gene expression in the brain from GTEx data and observed increasing eQTL effect direction concordance as ASB significance increases. These results provide a valuable and unique resource for mechanistic analysis of cis-regulatory variation in human brain tissue.

Transcription factors (TFs) orchestrate gene expression programs crucial for brain function, but we lack detailed information about TF binding in human brain tissue. We generated a multiomic resource (ChIP–seq, ATAC-seq, RNA-seq, DNA methylation) on bulk tissues and sorted nuclei from several postmortem brain regions, including binding maps for more than 100 TFs. We demonstrate improved measurements of TF activity, including motif recognition and gene expression modeling, upon identification and removal of high TF occupancy regions. Further, predictive TF binding models demonstrate a bias for these high-occupancy sites. Neuronal TFs SATB2 and TBR1 bind unique regions depleted for such sites and promote neuronal gene expression. Binding sites for TFs, including TBR1 and PKNOX1, are enriched for risk variants associated with neuropsychiatric disorders, predominantly in neurons. This work, titled BrainTF, is a powerful resource for future studies seeking to understand the roles of specific TFs in regulating gene expression in the human brain.

Introduction:Large somatic deletions of mitochondrial DNA (mtDNA) accumulate with aging in metabolically active tissues such as the brain. We have cataloged the breakpoints and frequencies of large mtDNA deletions in the human brain. Methods:We quantified 112 high-frequency mtDNA somatic deletions across four human brain regions with the Splice-Break2 pipeline. In addition, we utilized PLINK/Seq to test the association of mitochondrial genotypes with the abundance of these high-frequency mtDNA deletions. A conservative pvalue threshold of 5E−08 was used to find the significant loci. Results:One mtDNA SNP (T14798C) was significantly associated with mtDNA deletions in two brain regions, the dorsolateral prefrontal cortex (DLPFC) and the superior temporal gyrus. Since the DLPFC showed the most robust association between T14798C and two deletion breakpoints (7816–14807 and 5462–14807), this association was tested in the DLPFC of a replication sample and validated the first results. Incorporating the C allele at 14,798 bp increased the perfect/imperfect length of the repeat at the 3′ breakpoint of the two associated deletions. Conclusion:This is the first study to identify the association of mtDNA SNP with large mtDNA deletions in the human brain. The T14798C allele located in the MT-CYBgene is a common polymorphism that occurs in several mitochondrial haplogroups. We hypothesize that the T14798C association with two deletions occurs by extending the repeat length around the 3′ deletion breakpoints. This simple mechanism suggests that mtDNA SNPs can affect the mitochondrial genome structure, especially in brain where high levels of reactive oxygen species lead to deletion accumulation with aging.

Rare coding variation has historically provided the most direct connections between gene function and disease pathogenesis. By meta-analysing the whole exomes of 24,248 schizophrenia cases and 97,322 controls, we implicate ultra-rare coding variants (URVs) in 10 genes as conferring substantial risk for schizophrenia (odds ratios of 3–50, P< 2.14 × 10−6) and 32 genes at a false discovery rate of <5%. These genes have the greatest expression in central nervous system neurons and have diverse molecular functions that include the formation, structure and function of the synapse. The associations of the NMDA (N-methyl-d-aspartate) receptor subunit GRIN2Aand AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor subunit GRIA3provide support for dysfunction of the glutamatergic system as a mechanistic hypothesis in the pathogenesis of schizophrenia. We observe an overlap of rare variant risk among schizophrenia, autism spectrum disorders1, epilepsy and severe neurodevelopmental disorders2, although different mutation types are implicated in some shared genes. Most genes described here, however, are not implicated in neurodevelopment. We demonstrate that genes prioritized from common variant analyses of schizophrenia are enriched in rare variant risk3, suggesting that common and rare genetic risk factors converge at least partially on the same underlying pathogenic biological processes. Even after excluding significantly associated genes, schizophrenia cases still carry a substantial excess of URVs, which indicates that more risk genes await discovery using this approach.

Bipolar disorder is a highly heritable psychiatric disorder. We performed a genome-wide association study (GWAS) including 20,352 cases and 31,358 controls of European descent, with follow-up analysis of 822 variants with P< 1 × 10−4in an additional 9,412 cases and 137,760 controls. Eight of the 19 variants that were genome-wide significant (P< 5 × 10−8) in the discovery GWAS were not genome-wide significant in the combined analysis, consistent with small effect sizes and limited power but also with genetic heterogeneity. In the combined analysis, 30 loci were genome-wide significant, including 20 newly identified loci. The significant loci contain genes encoding ion channels, neurotransmitter transporters and synaptic components. Pathway analysis revealed nine significantly enriched gene sets, including regulation of insulin secretion and endocannabinoid signaling. Bipolar I disorder is strongly genetically correlated with schizophrenia, driven by psychosis, whereas bipolar II disorder is more strongly correlated with major depressive disorder. These findings address key clinical questions and provide potential biological mechanisms for bipolar disorder.

Subjects with schizophrenia (SZ) and bipolar disorder (BD) show decreased protein and transcript levels for mitochondrial complex I. In vitro results suggest antipsychotic and antidepressant drugs may be responsible. We measured complex I activity in BD, SZ, and controls and presence of antipsychotic and antidepressant medications, mitochondrial DNA (mtDNA) copy number, and the mtDNA “common deletion” in the brain. Complex I activity in the prefrontal cortex was decreased by 45% in SZ compared to controls (p= 0.02), while no significant difference was found in BD. Complex I activity was significantly decreased (p= 0.01) in pooled cases (SZ and BD) that had detectable psychotropic medications and drugs compared to pooled cases with no detectable levels. Subjects with age at onset in their teens and psychotropic medications showed decreased (p< 0.05) complex I activity compared to subjects with an adult age at onset. Both SZ and BD groups displayed significant increases (p< 0.05) in mtDNA copy number compared to controls; however, common deletion burden was not altered. Complex I deficiency is found in SZ brain tissue, and psychotropic medications may play a role in mitochondrial dysfunction. Studies of medication-free first-episode psychosis patients are needed to elucidate whether mitochondrial pathophysiology occurs independent of medication effects.

Individuals respond differently to traumatic experiences, including their propensity to develop posttraumatic stress disorder (PTSD). Understanding individual differences in PTSD vulnerability will allow the development of improved prevention and treatment options. Here we characterized fear conditioning and extinction in rats selectively bred for differences in their locomotor response to a novel environment. Selectively bred high-responder (bHR) and low-responder (bLR) male rats are known to differ in their emotional reactivity on a range of measures of spontaneous anxiety- and depressive-like behaviors. We demonstrate that bHRs have facilitated extinction learning and retention compared with outbred Sprague Dawley rats, whereas bLRs show reduced extinction learning and retention. This indicates that bLRs are more vulnerable to PTSD-like behavior. Fibroblast growth factor 2 (FGF2) has previously been implicated in the development of these behavioral phenotypes and facilitates extinction learning in outbred animals, therefore we examined the effects of early-life FGF2 on bHR and bLR behavior. FGF2 administered on the day after birth facilitated extinction learning and retention in bHRs, but not in bLRs or control rats, during adulthood. This indicates that, under the current fear conditioning paradigm, early-life FGF2 has protective effects only in resilient animals. This stands in contrast to FGF2’s ability to protect vulnerable animals in milder tests of anxiety. These results provide a unique animal model of individual differences in PTSD-like behavior, allowing the study of genetic, developmental, and environmental factors in its expression.

•Temperament in humans can be studied as individual differences in animal models.•The hippocampus integrates the brain’s response to stress, fear and drugs of abuse.•FGF2 plays a role in anxiety, depression, PTSD and addiction-related behaviors.

The impact of a previously successful or unsuccessful experience on the subsequent acquisition of a related task is not well understood. The nature of past experience may have even greater impact in individuals with learning deficits, as their cognitive processes can be easily disrupted. Mice with a targeted disruption of the alpha and delta isoforms of the cAMP-response element-binding protein (CREB) gene (CREB(alphadelta-)-deficient mice) have a genetic vulnerability to impaired learning and memory that is highly influenced by experimental conditions. Thus, we studied the impact of prior successful and unsuccessful experiences on the degree to which CREB(alphadelta-)-deficient mice exhibit impaired spatial learning and memory in the Morris water maze (MWM). In Experiment 1, we replicated the cognitive deficit of CREB(alphadelta-)-deficient mice when given two trials per day with a 1-min intertrial interval (MWM2), and labeled this experience as a "failure." We rescued the deficit using four trials per day with a 3- to 5-min intertrial interval (MWM4) and labeled this experience a "success." In Experiment 2, a new, naive set of wild-type (WT) and CREB(alphadelta-)-deficient mice were randomly assigned to one of two sequence protocols to assess the influence of a success or a failure on subsequent performance. In Group 1, mice were first exposed to the MWM4 condition, followed by the more difficult MWM2 task. As expected, CREB(alphadelta-)-deficient mice performed well in the MWM4; they also performed well during reversal testing (MWM4R) where the goal location is changed. With this initial successful learning experience, the CREB(alphadelta-)-deficient mice then performed as well as WT mice in the MWM2, the condition in which they are known to be impaired. In contrast, CREB(alphadelta-)-deficient mice in Group 2 had an unsuccessful experience when first exposed to the MWM2 condition, and then also showed impairment in the MWM4, the condition in which they would normally perform well. This deficit was amplified when CREB(alphadelta-)-deficient mice were then tested in the reversal test. Sex differences in learning among CREB(alphadelta-)-deficient mice were amplified upon exposure to an unsuccessful learning experience. These data indicate that, under conditions of cognitive impairment, past experience can-depending on its nature-significantly facilitate or hinder future performance.

Although nociceptin/orphanin FQ (N/OFQ) influences dopamine (DA) neuronal activity, it is not known whether N/OFQ acts directly on DA neurons, indirectly by means of local circuitry, or both. We used two parallel approaches, dual in situ hybridization (ISH) and neurotoxic lesions of DA neurons by using 6‐hydroxydopamine (6‐OHDA), to ascertain whether N/OFQ and the N/OFQ receptor (NOP) mRNA are expressed in DA neurons in the ventral tegmental area (VTA) and substantia nigra compacta (SNc). In the VTA and SNc, small populations (∼6–10%) of N/OFQ‐containing neurons coexpressed mRNA for tyrosine hydroxylase (TH), the rate‐limiting enzyme for DA synthesis. Similarly, very few (1–2%) TH‐positive neurons contained N/OFQ mRNA signal. A majority of NOP‐positive neurons (∼75%) expressed TH mRNA and roughly half of the TH‐containing neurons expressed NOP mRNA. Many N/OFQ neurons (∼50–60%) expressed glutamic acid decarboxylase 65 and 67 mRNAs, markers for γ‐aminobutyric acid (GABA) neurons. In the 6‐OHDA lesion studies, NOP mRNA levels were nearly 80 and 85% lower in the VTA and SNc, respectively, on the lesioned side. These lesions appear to lead to compensatory changes, with N/OFQ mRNA levels approximately 60% and 300% higher in the VTA and SNc, respectively, after 6‐OHDA lesions. Finally, N/OFQ‐stimulated [35S]guanylyl‐5′‐O‐(γ‐thio)‐triphosphate levels were decreased in the VTA and SNc but not the prefrontal cortex after 6‐OHDA lesions. Accordingly, it appears that N/OFQ mRNA was found largely on nondopaminergic (i.e., GABA) neurons, whereas NOP mRNA was located on DA neurons. N/OFQ is in a position to influence DA neuronal activity by means of the NOP located on DA neurons. J. Comp. Neurol. 444:358–368, 2002. © 2002 Wiley‐Liss, Inc.

Agouti-related protein (AGRP) is a newly identified orexigenic peptide that acts as an endogenous antagonist of melanocortin receptors MC3 and MC4. The present study examined the time course of the orexigenic effects of synthetic AGRP (86-132). Intracerebroventricular infusion of 0.1 nmol AGRP (86-132) increased food intake by 450 ± 81 at 2 h post-injection. A second increase in non-cumulative food intake (512 ± 135) was observed at 6 h post-injection. Following a single dose of AGRP (86-132) (0.1 nmol) the increased food intake was sustained for 6 days, occurring in the light cycle of the first 2 days and subsequently switching to the dark cycle of the last 4 days. These time course profiles indicate the complexity of the mechanisms involved in AGRP-induced feeding.

In patients with major depression, abnormalities in baseline cortisol secretion and resistance to negative feedback are well established. However, it is unclear if patients with major depression have alterations in the hypothalamic-pituitary-adrenal (HPA) response to stressors. While other challenges to the HPA axis have used endocrine stimuli such as insulin-induced hypoglycemia, we now report of the response to a social stressor in patients with major depression and matched control subjects. We used the Trier Social Stress Test (TSST), a public speaking task followed by mental arithmetic challenge in front of a panel of judges. The results suggest that depressed patients manifest normal cortisol response to a social stressor, despite increased pre-stressor plasma cortisol. However, the β-endorphin response to the TSST was significantly smaller in the depressed patients compared to matched controls. These data are similar to data found with exogenous corticotropin-releasing-hormone challenge studies and suggest that elevated baseline cortisol can modulate the pituitary corticotroph response to a stressor, but that changes in adrenal sensitivity to ACTH result in a robust cortisol response to this stressor.

To further understand the functions of the orexin/hypocretin system, we examined the expression and regulation of the orexin/hypocretin receptor (OX1R and OX2R) mRNA in the brain by using quantitative in situhybridization. Expression of OX1R and OX2R mRNA exhibited distinct distribution patterns. Within the hypothalamus, expression for the OX1R mRNA was largely restricted in the ventromedial (VMH) and dorsomedial hypothalamic nuclei, while high levels of OX2R mRNA were contained in the paraventricular nucleus, VMH, and arcuate nucleus as well as in mammilary nuclei. In the amygdala, OX1R mRNA was expressed throughout the amygdaloid complex with robust labeling in the medial nucleus, while OX2R mRNA was only present in the posterior cortical nucleus of amygdala. High levels of OX2R mRNA were also observed in the ventral tegmental area. Moreover, both OX1R and OX2R mRNA were observed in the hippocampus, some thalamic nuclei, and subthalamic nuclei. Furthermore, we analyzed the effect of fasting on levels of OX1R and OX2R mRNA in the hypothalamic and amygdaloid subregions. After 20 h of fasting, levels of OX1R mRNA were significantly increased in the VMH and the medial division of amygdala. An initial decrease (14 h) and a subsequent increase (20 h) in OX1R mRNA levels after fasting were observed in the dorsomedial hypothalamic nucleus and lateral division of amygdala. Levels of OX2R mRNA were augmented in the arcuate nucleus, but remained unchanged in the dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, and amygdala following fasting. The time-dependent and region-specific regulatory patterns of OX1R and OX2R suggest that they may participate in distinct neural circuits under the condition of food deprivation.

Nitric oxide (NO) has been implicated in the modulation of mitochondrial respiration, membrane potential, and subsequently in apoptosis. Although the presence of a mitochondrial NO synthase (mtNOS) has been described, there is no direct evidence in vivo of the presence of NO within mitochondria. It was the aim of this study to demonstrate the in vivo production of NO within mitochondria. Using the novel fluorometric NO detection system, 4,5-diaminofluorescein diacetate (DAF-2/DA), we observed the presence of NO production in PC12 and COS-1 cells by conventional and confocal fluorescence microscopy. Part of the overall NO signal was colocalized within a subpopulation of mitochondria, labeled with the potential-dependent probe MitoTracker red. These findings demonstrate for the first time that the subcellular distribution of NO production is consistent with the presence of a mitochondrial NOS. Our results provide a new tool to directly study the modulatory role of NO in mitochondrial respiration and membrane potential, in vivo.

Orphanin FQ (OFQ) is the endogenous agonist of the opioid receptor‐like receptor (ORL‐1). It and its precursor, prepro‐OFQ, exhibit structural features suggestive of the opioid peptides. A cDNA encoding the OFQ precursor sequence in the rat recently has been cloned, and the authors recently generated a polyclonal antibody directed against the OFQ peptide. In the present study, the authors used in situ hybridization and immunohistochemistry to examine the distribution of OFQ peptide and mRNA in the central nervous system of the adult rat. OFQ immunoreactivity and prepro‐OFQ mRNA expression correlated virtually in all brain areas studied. In the forebrain, OFQ peptide and mRNA were prominent in the neocortex endopiriform nucleus, claustrum, lateral septum, ventral forebrain, hypothalamus, mammillary bodies, central and medial nuclei of the amygdala, hippocampal formation, paratenial and reticular nuclei of the thalamus, medial habenula, and zona incerta. No OFQ was observed in the pineal or pituitary glands. In the brainstem, OFQ was prominent in the ventral tegmental area, substantia nigra, nucleus of the posterior commissure, central gray, nucleus of Darkschewitsch, peripeduncular nucleus, interpeduncular nucleus, tegmental nuclei, locus coeruleus, raphe complex, lateral parabrachial nucleus, inferior olivary complex, vestibular nuclear complex, prepositus hypoglossus, solitary nucleus, nucleus ambiguous, caudal spinal trigeminal nucleus, and reticular formation. In the spinal cord, OFQ was observed throughout the dorsal and ventral horns. The wide distribution of this peptide provides support for its role in a multitude of functions, including not only nociception but also motor and balance control, special sensory processing, and various autonomic and physiologic processes. J. Comp. Neurol. 406:503–547, 1999. © 1999 Wiley‐Liss, Inc.

The recently discovered neuropeptide orphanin FQ (OFQ), and its opioid receptor‐like (ORL1) receptor, exhibit structural features suggestive of the μ, κ, and δ opioid systems. The anatomic distribution of OFQ immunoreactivity and mRNA expression has been reported recently. In the present analysis, we compare the distribution of orphanin receptor mRNA expression with that of orphanin FQ binding at the ORL1 receptor in the adult rat central nervous system (CNS). By using in vitro receptor autoradiography with 125I‐[14Tyr]‐OFQ as the radioligand, orphanin receptor binding was analyzed throughout the rat CNS. Orphanin binding sites were densest in several cortical regions, the anterior olfactory nucleus, lateral septum, ventral forebrain, several hypothalamic nuclei, hippocampal formation, basolateral and medial amygdala, central gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, vestibular nuclear complex, and the spinal cord. By using in situ hybridization, cells expressing ORL1 mRNA were most numerous throughout multiple cortical regions, the anterior olfactory nucleus, lateral septum, endopiriform nucleus, ventral forebrain, multiple hypothalamic nuclei, nucleus of the lateral olfactory tract, medial amygdala, hippocampal formation, substantia nigra, ventral tegmental area, central gray, raphe complex, locus coeruleus, multiple brainstem motor nuclei, inferior olive, deep cerebellar nuclei, vestibular nuclear complex, nucleus of the solitary tract, reticular formation, dorsal root ganglia, and spinal cord. The diffuse distribution of ORL1 mRNA and binding supports an extensive role for orphanin FQ in a multitude of CNS functions, including motor and balance control, reinforcement and reward, nociception, the stress response, sexual behavior, aggression, and autonomic control of physiologic processes. J. Comp. Neurol. 412:563–605, 1999. © 1999 Wiley‐Liss, Inc.

The Mahogany/Attractin gene (Atrn) has been proposed as a downstream mediator of Agouti signaling because yellow hair color and obesity in lethal yellow(Ay) mice are suppressed by the mahogany(Atrnmg) mutation. The present study examined the distribution of AtrnmRNA in the brain and spinal cord by in situ hybridization. AtrnmRNA was found widely distributed throughout the central nervous system, with high levels in regions of the olfactory system, some limbic structures, regions of the brainstem, cerebellum and spinal cord. In the hypothalamus, AtrnmRNA was found in specific nuclei including the suprachiasmatic nucleus, the supraoptic nucleus, the medial preoptic nucleus, the paraventricular hypothalamic nucleus, the ventromedial hypothalamic nucleus, and the arcuate nucleus. These results suggest a broad spectrum of physiological functions for the Atrn gene product.

Since the discovery of opioid peptides, several immunohistochemical and radioimmunological studies have demonstrated their localization in the gastrointestinal tract without demonstrating the localization of their common precursor. The present study describes the distribution and the colocalization of proenkephalin and prodynorphin messenger RNAs (mRNAs) in the colon of rat by in situ hybridization. Proenkephalin and prodynorphin mRNAs were found in myenteric plexus, but not in the submucous plexus or in the mucosa. In myenteric plexus, the number of neurons expressing proenkephalin is 2.5 times greater than that of the neurons expressing only prodynorphin. Furthermore, double in situ hybridization histochemistry indicates that at least three groups of opioid neurons can be distinguished, those containing proenkephalin and prodynorphin mRNAs together, and those containing only proenkephalin mRNA or only prodynorphin mRNA.

The lateral division of the central nucleus of the amygdala (CEAl) and the oval nucleus of the bed nucleus of the stria terminalis (BSTov) have been linked closely anatomically and functionally. To determine whether these regions may be subdivided further on a neurochemical basis, dual in situ hybridization was used to determine the colocalization of corticotropin‐releasing hormone (CRH), enkephalin (ENK), or neurotensin (NT) with glutamic acid decarboxylase isoforms 65 and 67 [used concurrently as a marker for γ‐aminobutyric acid GABA] in these nuclei. It was found that, for both regions, each peptide invariably was localized in a GABAergic cell. Although there was a similar overlap in the distribution of NT with ENK in the BSTov and CEAl, it was observed that CRH and ENK rarely were colocalized in either nucleus. To determine whether these distinct neuronal populations could be activated differentially, male rats were given a systemic injection of interleukin‐1β (IL‐1β; 5 μg/kg, i.p.), a stimulus that results in a robust increase in c‐fos mRNA expression in the BSTov and CEAl. The neurochemical identity of these activated neurons showed striking similarities between the BSTov and the CEAl; All IL‐1β‐responsive cells were GABAergic, the majority of c‐fos‐ positive cells expressed ENK mRNA (BSTov, 81%; CEAl, 94%), and some expressed NT mRNA (BSTov, 23%; CEAl, 22%), whereas very few expressed CRH mRNA (BSTov, 4%; CEAl, 1%). These data provide evidence for the existence of discrete neural circuits within the BSTov and CEAl, and the similarities in the patterns of neurochemical colocalization in these nuclei are consistent with the concept of an extended amygdala. Furthermore, these data indicate that intraperitoneal IL‐1β recruits neurochemically distinct pathways within the BSTov and CEAl, and it is suggested that this differential activation may mediate specific aspects of immune, limbic, and/or autonomic processes. J. Comp. Neurol. 413:113–128, 1999. © 1999 Wiley‐Liss, Inc.

Although much has been learned about the mechanisms of ligand selectivity between different opioid receptor subtypes, little is known about the common opioid binding pocket shared by all opioid receptors. The recently discovered orphanin system offers a good opportunity to study the mechanisms involved in the binding of opioid versus nonopioid ligands. In the current study, we adopt a “gain of function” approach aimed at shifting the binding profile of the orphanin FQ receptor toward that of the opioid receptors. After two rounds of mutagenesis, several orphanin FQ receptor mutants can be labeled with the opiate alkaloid [3H]naltrindole and show greatly increased affinities toward the opiate antagonists naltrexone, nor-binaltrophine HCl, and (−)-bremazocine. These orphanin FQ receptor mutants also display stereospecificity similar to that of opioid receptors. Furthermore, the orphanin FQ receptor mutant that has the best affinities toward the opioid alkaloids shows, in the presence of GTP and high salt concentration, an affinity-shift profile similar to that of the δ receptor. Most strikingly, the same mutant exhibits naltrindole-sensitive etorphine-stimulated [35S]guanosine-5′-O-(3-thio)triphosphate binding, whereas the effect of etorphine on GTP binding cannot be inhibited by naltrindole in the wild-type receptor. Our results indicate that 1) several residues in the orphanin FQ receptor are critical to its selectivity against the opiate alkaloids, particularly antagonists; and 2) mutating these residues to those of the opioid receptor at the corresponding position preserves the agonist/antagonist nature of opiate alkaloids as they interact with the mutant receptor. It is reasonable to hypothesize that the corresponding residues in the opioid receptors may form a functional common binding pocket for opiate alkaloids. These findings may be helpful to medicinal chemists in designing ligands for the orphanin FQ receptor based on the structure of the opiate alkaloids.

Abstract:Structural elements of the rat μ‐opioid receptor important in ligand receptor binding and selectivity were examined using a site‐directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the μ, δ, and κ receptors (Asn150to Ala, His297to Ala, and Tyr326to Phe) and two designed to test for μ/δ selectivity (Ile198to Val and Val202to Ile). Mutation of His297in transmembrane domain 6 (TM6) resulted in no detectable binding with [3H]DAMGO (3H‐labeled d‐Ala2,N‐Me‐Phe4,Gly‐ol5‐enkephalin), [3H]bremazocine, or [3H]ethylketocyclazocine. Mutation of Asn150in TM3 produces a three‐ to 20‐fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β‐endorphin1–31, JOM‐13, deltorphin II, dynorphin1–13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor‐binaltorphamine. In contrast, the Tyr326mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val202to Ile in TM4 produced no change on ligand affinity, but Ile198to Val resulted in a four‐ to fivefold decreased affinity for the μ agonists morphine and DAMGO, with no change in the binding affinities of κ and δ ligands.

OBJECTIVE: To determine whether depressed patients demonstrate hypothalamic-pituitary-adrenal (HPA) axis activation during the late afternoon and evening, a time when the HPA axis is usually quiescent in normal subjects. METHODS: We administered metyrapone, an 11-βhydroxylase inhibitor of cortisol synthesis, to normal controls and depressed patients between 4 and 10 PM. Metyrapone blockade of cortisol secretion would amplify any HPA axis secretion. RESULTS: In 10 normal control subjects, administration of metyrapone lowered plasma cortisol levels to a mean of 36 nmol/L. No rebound corticotropin or β-endorphin secretion was seen in these normal controls between 4 and 10 PM, supporting the existence of a period of minimal endogenous corticotropin releasing factor drive. Compared with a group of placebo-treated depressed patients (n=10), metyrapone-treated depressed subjects (n=17) had significantly decreased plasma cortisol concentrations. However, in contrast to normal controls treated with metyrapone, metyrapone-treated depressed patients demonstrated rebound corticotroph secretion, particularly between 7:30 and 10 PM (P=.036 for patients vs normal controls for β-endorphin secretion from 4:30 to 10 PM). CONCLUSION: These data support the hypothesis of increased corticotropin releasing factor drive in the evening in depressed subjects and are in agreement with the longstanding observation of "early escape" from dexamethasone suppression between 4 and 11 PM in depressed patients.

• A rate-sensitive fast-feedback inhibition of stress-induced corticotropin secretion by glucocorticoids is well documented in rats. Studies in patients with Cushing's disease or adrenal insufficiency have also supported the existence of fast feedback in humans. However, few studies exist in normal healthy subjects or depressed patients. This study compared fast-feedback inhibition of β-endorphin/β-lipotropin secretion by hydrocortisone in 16 control subjects and 16 depressed patients. A fast-feedback effect of hydrocortisone on β-endorphin/β-lipotropin secretion during the hour of the hydrocortisone infusion was demonstrated in control subjects. Depressed patients demonstrated no increase in β-endorphin/β-lipotropin concentrations during the infusion. These data suggest a decreased sensitivity to glucocorticoid fast feedback in depressed patients and complement existing studies demonstrating decreased sensitivity to proportional feedback by dexamethasone in depressed patients. We believe the data presented herein are the first demonstration that abnormal feedback occurs at the level of the brain rather than pituitary in depressed patients.

• Electroconvulsive therapy is accompanied by an activation of the hypothalamic-pituitary-adrenal axis, resulting in a release of β-endorphin from the anterior pituitary corticotrophs of humans. As a group, patients in our study demonstrated similar plasma β-endorphin immunoreactivity response to their initial and final treatments. However, approximately half of the patients demonstrated greater β-endorphin immunoreactivity release with their first seizure compared with their last seizure, and half of the patients demonstrated the opposite pattern. This difference was not explained by age, sex, unilateral vs bilateral treatments, sine wave vs brief pulse, or psychotropic or anticholinergic medication. Patients with constant seizure duration during the first and final treatments demonstrated a greater release of β-endorphin immunoreactivity with the final treatment compared with the first treatment. Individuals with decreasing seizure duration during the course of the electroconvulsive therapy demonstrated a decreased β-endorphin immunoreactivity response during their final treatment.

• Studies in depression using a maximal stimulatory dose of corticotropin releasing factor have concluded that elevated resting cortisol levels in depressed patients exert a negative feedback effect on the corticotroph, resulting in a decreased corticotropin response. In this preliminary report, we examine the effects of a submaximal dose of corticotropin releasing factor on the release of another corticotroph secretory product, β-lipotropin—β-endorphin. We observed a decreased β-lipotropin—β-endorphin response in depressed subjects, but a normal adrenal cortisol response. Although the total β-lirotropin—β-endorphin response was decreased, the initial secretory response did not differ between patients and normal controls. Rather, the patients appeared to turn off secretion faster. This rapid shutoff was seen in all patients regardless of resting cortisol levels, suggesting that resting cortisol levels alone do not explain the decreased response seen in depressed patients.

• This study addresses the question of whether pituitary peptides (ie,β-endorphin) show regulatory disruption in endogenous depression and, if so, does it co-occur in the same subjects who show cortisol dysregulation. Endogenously depressed patients and psychiatric controls from three centers were evaluated, when not taking medications, and studied for plasma cortisol and β-endorphin levels. Plasma samples were taken at four time points over one hour, on the basal day, and 16 hours after 1 mg of dexamethasone. From 33% to 69% of the endogenous patients were abnormal in their postdexamethasone cortisol levels, and from 50% to 69% were abnormal on postdexamethasone β-endorphin values (vs 0% and 8%, respectively, for controls). When endogenous subjects were evaluated for abnormality on both cortisol and β-endorphin, after dexamethasone, it was found that the two measures of hypothalamic-pituitary-adrenal dysfunction did not necessarily co-vary. In fact when having either abnormal β-endorphin or cortisol levels (or both) was used as a biological marker a larger number of the endogenous patients were detected than with either measure alone. Our conclusions are as follows: Plasma β-endorphin shows a circadian rhythm similar to that seen with corticotropin (ACTH) and is suppressable by dexamethasone. In many endogenous patients plasma β-endorphin levels escape from dexamethasone suppression. Many of these subjects are not cortisol escapers. When abnormality of either the β-endorphin or cortisol is considered it is clear that both levels of the hypothalamic-pituitary-adrenal axis can be dysregulated in endogenous depression.

• To study the effects of β-endorphin in chronic schizophrenia, nine male patients participated in a double-blind crossover comparison of a single intravenous 20-mg injection of β-endorphin and saline. Bolus injection of β-endorphin from an albumincoated syringe produced markedly higher plasma concentrations than did slow intravenous infusion from a non$albumincoated syringe. β-endorphin intravenously injected in nine patients produced a statistically significant increase in serum prolactin levels. In one patient, both 10 mg of morphine sulfate and 20 mg of β-endorphin produced similar increases in the alpha power of the EEG. In eight patients, β-endorphin administration was associated with a statistically significant but not clinically obvious improvement in schizophrenic symptoms.

Background & Aims:Orphanin FQ (OFQ) is a recently discovered neuropeptide that structurally resembles an opioid peptide. However, the functional role of OFQ in rat gastrointestinal tract remains unknown. Methods:We investigated the effects of OFQ on contractions of muscle strips obtained from different regions of the gastrointestinal tract. Immunohistochemical studies were performed on rat colonic tissue using OFQ antibody. Results:OFQ (10−9to 10−7mol/L) caused significant contractions in the rat colon but not in the stomach or small intestine. Tetrodotoxin, veratridine, and long-term serosal application of benzalkonium chloride completely abolished OFQ-induced colonic contractions without affecting myogenic contractions in response to carbachol. OFQ-induced contractions were not affected by naloxone, atropine, phentolamine, propranolol, methysergide, substance P antagonist, vasoactive intestinal polypeptide antagonist, apamin, and NG-nitro-L-arginine methyl ester. OFQ (10−9to 10−7mol/L) significantly reduced muscle contractions and 3H-acetylcholine release in response to electrical field stimulation in both the stomach and small intestine but not in the colon. OFQ-immunopositive neuronal fibers were found in the colonic myenteric plexus. Conclusions:These studies indicate that the mechanisms and sites of action of OFQ are region specific. OFQ inhibits cholinergic transmission in the stomach and small intestine, whereas OFQ stimulates colonic contraction possibly by inhibiting an inhibitory neural pathway within the myenteric plexus.

Disturbances in the serotonin (5-HT) system is the neurobiological abnormality most consistently associated with suicide. Hyperactivity of the hypothalmic-pituitary-adrenal (HPA) axis is also described in suicide victims. The HPA axis is the classical neuroendocrine system that responds to stress and whose final product, corticosteroids, targets components of the limbic system, particularly the hippocampus. We will review resulsts from animal studies that point to the possibility that many of the 5-HT receptor changes observed in suicide brains may be a result of, or may be worsened by, the HPA overactivity that may be present in some suicide victims. The results of these studies can be summarized as follows: (1) chronic unpredictable stress produces high corticosteroid levels in rats; (2) chronic stress also results in changes in specific 5-HT receptors (increases in cortical 5-HT2A and decreases in hipocampal 5-HT1A and 5-HT1B); (3) chronic antidepressant administration prevents many of the 5-HT receptor changes observed after stress; and (4) chronic antidepressant administration reverses the overactivity of the HPA axis. If indeed 5-HT receptors have a partial role in controlling affective states, then their modulation by corticosteroids provides a potential mechanism by which these hormones may regulate mood. These data may also provide a biological understanding of how stressful events may increase the risk for suicide in vulnerable individuals and may help us elucidate the neurobiological underpinnings of treatment resistance.

This paper offers a technique for obtaining monoamine histofluorescence in the CNS by means of formaldehyde perfusion followed by cryostat sectioning. No freeze-drying is involved. Cryostat sections are exposed to formaldehyde vapor to complete the fluorophore formation. The fluorescence thus obtained is bright, well localized, and does not require loading the animals with precursors. The anatomical distribution of the pathways is identical to that obtained with the classical technique. Furthermore, the fluorescence is reversible by sodium borohydride, and exhibits the expected changes in intensity with pharmacological manipulations. The sections can be exposed to a cold aqueous medium for as long as 15 min with minimal diffusion of fluorophore; this suggests potential for combining monoamine histofluorescence with other visualization techniques.

Three opioid receptor types have been identified in the CNS and periphery that are referred to as μ, δ, and κ. The present study examines the mRNA distribution of the κ₁ receptor in the rat brain and compares it to the distribution of κ receptor-binding sites and prodynorphin mRNA using a combination of in situ hybridization and receptor autoradiographic techniques. κ₁ receptor mRNA was localized with a cRNA probe generated with a BamHI-HindIII cDNA fragment of the rat κ₁ receptor and corresponds to the last 45 bp of the protein coding region and 728 nucleotides of the 3' untranslated region. Prodynorphin mRNA was localized with a cRNA probe corresponding to a 733-bp BamHI-HincII fragment of prodynorphin. κ receptor-binding sites were labeled in one of two ways: [³H]U69,593 or [³H]bremazocine in the presence of a 300-fold excess of DAMGO and DPDPE. A high degree of correspondence between the κ₁ receptor mRNA and κ receptor binding was observed in several brain regions, including the endopiriform nucleus, claustram, nucleus accumbens, olfactory tubercle, bed nucleus of the stria terminalis, medial preoptic area, paraventricular, supraoptic, suprachiasmatic, dorsomedial and ventromedial hypothalamic nuclei, basolateral, medial and cortical amygdaloid nuclei, midline thalamic nuclei, periaqueductal grey, parabrachial nucleus, locus coeruleus, and the nucleus of the solitary tract. Differences in the localization of κ₁ receptor mRNA and binding and the relationship between the distribution of κ₁ receptor and prodynorphin mRNAs are discussed. Copyright 1994, 1999 Academic Press

THE periaqueductal gray (PAG) and rostral ventrome-dial medulla (RVM) are important brain stem pain modulating regions. Recent evidence suggests that κ opioids antagonize the effects of μ opioids in the RVM. However, the anatomical relationship between μ and κ opioid receptors in PAG and RVM is not well characterized. This study examined relationships between μ and κ opioid receptor immunoreactivity (IR) and mRNA in PAG and RVM. Brain slices were processed for either immunocytochemistry or in situhybridization. We found considerable anatomical overlap of μ and κ opioid IR and mRNA in the RVM and PAG. These results provide an anatomical basis for recent behavioral and electrophysiological findings in RVM, and suggest modulatory interactions between μ and κ opioids in PAG.

The present studies were undertaken to determine whether hippocampal glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) biosynthesis, as inferred from mRNA expression, exhibit diurnal patterns of activation which may reflect or predict changes in plasma adrenocorticosteroid levels. Animals received either adrenalectomy (ADX) or sham adrenalectomy and were sacrificed at 4-h intervals throughout the diurnal cycle. Hippocampal and control brain areas were assayed for regional GR and MR mRNA changes via semiquantitative in situ hybridization histochemical analysis. The results indicate subfield-specific significant circadian rhythms in both GR and MR mRNAs. A significant diurnal rhythm in GR mRNA expression was seen in the dentate gyrus (DG), which took the shape of a monotonic curve with a marked trough at 1500 h after lights on (1 h after lights off). A similar pattern was evident in subfield CA1, although the effect fell short of statistical significance. No rhythm was seen in CA3. In response to ADX, GR mRNA was markedly increased in both CA1 and CA3; these increases appeared to be independent of circadian influences. In contrast, ADX effects in DG were quite limited and appeared to eliminate the circadian GR mRNA trough. Bimodal diurnal rhythms in MR mRNA expression were observed in all subfields and commonly exhibited troughs at 1500 h after lights on and 0300 h after lights on. These rhythms appeared to be related to circulating steroids, as ADX eliminated both the 1500 and 0300 h troughs, resulting in flat levels of MR mRNA expression corresponding roughly to the circadian peak. Notably, no diurnal GR or MR mRNA rhythms were observed in frontoparietal cortex, nor were any GR mRNA changes seen in the dorsomedial thalamus or hypothalamic paraventricular nucleus. Indeed, ADX was ineffective in altering adrenocorticosteroid receptor mRNA expression in any extrahippocampal region examined. These results indicate that GR and MR mRNAs exhibit hippocampus-specific diurnal rhythms in expression which are controlled to a greater (MR) or lesser (GR) extent by circulating steroids. The apparent steroid sensitivity of hippocampal adrenocorticosteroid receptor populations may be involved in the expression of rhythmic changes in hippocampal function associated with HPA regulation and information processing. Copyright 1993, 1999 Academic Press

The Mahogany/Attractin gene ( Atrn) has been proposed as a downstream mediator of Agouti signaling because yellow hair color and obesity in lethal yellow( Ay) mice are suppressed by the mahogany( Atrnmg) mutation. The present study examined the distribution of AtrnmRNA in the brain and spinal cord by in situ hybridization. AtrnmRNA was found widely distributed throughout the central nervous system, with high levels in regions of the olfactory system, some limbic structures, regions of the brainstem, cerebellum and spinal cord. In the hypothalamus, AtrnmRNA was found in specific nuclei including the suprachiasmatic nucleus, the supraoptic nucleus, the medial preoptic nucleus, the paraventricular hypothalamic nucleus, the ventromedial hypothalamic nucleus, and the arcuate nucleus. These results suggest a broad spectrum of physiological functions for the Atrn gene product.

In the central nervous system, adrenal steroids bind to two different types of corticosteroid receptors: glucocorticoid (GR) or mineralocorticoid (MR). In vitro biochemical and autoradiographic techniques have been used to infer GR and MR protein abundance in the hippocampus. Adrenalectomy (ADX) is routinely performed to measure the normal receptor number in absence of corticosterone (B), which would otherwise interfere with the binding reaction, The developing rodent has low basal B levels until the third week of life. We were interested in whether removal of circulating B may have a greater impact in the developing hippocampus than in the adult animal. In this study we examined the effect of a 14-h ADX on hippocampal GR and MR binding capacity (B_max) by standard binding techniques and on gene expression by in situ hybridization. ADX was performed on Day 6, 10, 14, 18, 22, 28, 35, and 45 and on adult animals. GR B_max increased from Day 6 to adult levels by Day 22 (d6 = 159.0 ± 27; d22 = 369.1 ± 43; a = 344.8 ± 23, fmol/mg protein ± SE). In contrast, MR B_max had adult levels on Day 6 and increased above these until Day 45, when it decreased and approached adult concentrations (d6 = 83.2 ± 22; d45 = 123 ± 23; a = 76.9 ± 13, fmol/mg protein ± SE). The greatest absolute increase for both receptors occurred between Days 22 and 45 and correlated with increases in GR and MR gene expression. Moreover, age- and region-specific changes were evident in the developing hippocampus. In addition, the adult animal also exhibited an MR mRNA upregulation after 14 h of adrenalectomy. We propose extreme caution when interpreting GR and MR B_max values obtained after short-term adrenalectomy in both adult and developing animals since upregulation of these genes is evident in this short time frame. Copyright 1993, 1999 Academic Press

OBJECTIVE: To determine if corticotroph nonsuppression, as reflected by β-endorphin nonsuppression, occurs before cortisol nonsuppression (defined as a cortisol level of >140 nmol/L) when examining multiple time points in a day. SETTING: The General Medical Clinical Research Center and Inpatient Depression Research Unit, Ann Arbor, Mich. DESIGN: Multiple blood samples were obtained through an intravenous catheter around the time points of 8 AM, noon, and 4 PM and assayed for β-endorphin and cortisol. PATIENTS: Patients meeting Research Diagnostic Criteria for the diagnosis of major depressive disorder, primary and simple. A total of 73 subjects, both inpatients and outpatients, were studied. INTERVENTION: Samples were obtained both at baseline and 1 day after administration of 1 mg of dexamethasone at 11:30 PM. MEASUREMENTS AND RESULTS: Overall 39 patients (53%) demonstrated β-endorphin nonsuppression after administration of dexamethasone at any of the three time points, while only eight patients (11%) demonstrated cortisol nonsuppression at any of these time points. Cortisol nonsuppression, but not β-endorphin nonsuppression, was associated with lower concentrations of dexamethasone in plasma. Baseline cortisol and menopausal status were significantly associated with β-endorphin nonsuppression in women.

• With the discovery of the opiate peptides, several major avenues of research became apparent. These peptides produced a great deal of focused attention on their anatomy, biochemistry, and physiology. In this article, we present an overview of some of the main research issues and recent findings in the field of opiate peptides. The possible relationship of the opiate peptide neuronal systems to schizophrenia is discussed in light of attempts to alter schizophrenic symptoms with opiate antagonists, β-endorphin, and dialysis. It is hypothesized that if the opiate peptides are involved in schizophrenia, then their involvement with dopamine systems and/or with stress responses may be critical.

Within the large family of G-protein-coupled receptors, a picture is emerging which contrasts the binding of small ligands and the binding of peptides to the seven-helix configuration of the proteins. Because of its unique richness in both peptide and non-peptide ligands, the opioid receptor family offers several advantages for achieving a better understanding of similarities and differences in ligand/receptor interactions across different classes of agonists and antagonists. Since multiple, naturally occurring, ligands interact with the multiple receptors with varying degrees of selectivity, this family is also an excellent model for examining the structural basis of selectivity. Thus, the molecular basis of binding affinity and selectivity of the κ and the δ opioid receptors was investigated by the construction of four κ/δ chimeric receptors. The pharmacological profiles of these chimeras as well as those of the wild type κ and δ receptors were determined by their binding with several different categories of opioid ligands. A linear model was used to deduce the relative contribution of each corresponding pairs of κ-δ receptor segments to the binding of a given ligand. The results show that the κ and δ receptors bind the same opioid core differently and achieve their selectivity through different mechanisms. In addition, the interaction of a peptide ligand with a receptor appears to be different from that of a small ligand. Furthermore, these results point to a particularly important role of the second extracellular loop and the top half of transmembrane domain 4 in the binding of prodynorphin products. Together, the results suggest that these peptide receptors can be bound and activated via multiple binding pockets as a function of their own topography and the nature of the interacting ligand.

It is unclear how receptor/ligand families that are evolutionarily closely related achieve functional separation. To address this question, we focus here on the newly discovered Orphanin FQ, a peptide homologous to the opioid peptide Dynorphin, and its receptor, the Orphanin FQ receptor, which is highly homologous to the opioid receptors. In spite of this high degree of homology in terms of both ligands and receptors, there is little direct cross-talk between the Orphanin FQ system and the endogenous opioid system. Thus, the opioid peptides show either relatively low affinity or no affinity toward the Orphanin FQ receptor; conversely, Orphanin FQ has no affinity toward any of the opioid receptors. We sought to investigate the molecular basis of such discrimination by attempting to reverse it and endowing the Orphanin FQ receptor with the ability to bind opioids. We report that by mutating as few as four amino acids, we can produce a receptor that recognizes pro-Dynorphin products with very high affinity and yet still binds Orphanin FQ as well as the wild-type receptor. This suggests that the Orphanin FQ receptor has developed features that specifically exclude the opioids and that these features are distinct from those required for the high affinity binding of its own endogenous ligand.

The present study determined simultaneously the behavioural, neuroendocrine and regional brain activity, using semi‐quantitative analysis of c‐fosmRNA induction, produced by 30 min of auditory stimulation at different white noise intensities (background 60 dB, 70, 80, 90 and 105 dBA), in rats. Only the highest noise intensities (90 and 105 dB) significantly increased corticosterone release after 30 min stimulation. Behaviourally, the 105 dB noise condition reliably reduced overall activity, and moderate noise intensities (70 and 80 dB) increased sleeping time. Three distinct patterns of c‐fosmRNA induction were observed. First, following exposure to the experimental cages, a wide pattern of brain activation was obtained in experimental animals irrespective of noise intensity presentation, compared to the naive rats. Second, a number of auditory structures (cochlear nuclei, superior olivary complex, nuclei of the lateral lemniscus, inferior colliculus and the medial division of the medial geniculate body) displayed a clear intensity‐dependent increase in c‐fosinduction. Third, compared to all other conditions, the stressed rats (90 and 105 dB conditions) displayed significantly higher c‐fosinduction in relatively few areas. Particularly intense c‐fosinduction was observed in the bed nucleus of the stria terminalis, especially its anterior medial and ventral aspects, the septohypothalamic nucleus, the ventral lateral septum, the ventral portion of the dentate gyrus, a number of hypothalamic nuclei including the lateral preoptic area, the medial preoptic nucleus and the paraventricular nucleus, the median raphe and the pedunculopontine tegmental nucleus. The involvement of a number of these structures in a specific audiogenic stress responsive circuit is discussed.

Localization and regulation of mineralocorticoid receptor (MR) heteronuclear RNA (hnRNA) was assessed in rat hippocampus using an intron-directed in situ hybridization approach. The presence of hnRNA in the cell nucleus reflects recent gene transcriptional events and can be used as an index of neuronal transcriptional activation or inhibition. In the present study, sections incubated with an MR intron probe labeled cells in all regions known to contain MR mRNA. Signal generated by the intron probe was localized specifically to the nuclear compartment, consistent with recognition of hnRNA. Analysis of distribution across hippocampus indicated that MR hnRNA was particularly abundant in dentate gyrus (DG). In contrast, MR mRNA shows similar levels of expression across all hippocampal subfields. Exposure of animals to acute restraint markedly reduced MR hnRNA levels in CA1 and DG 60 min and 120 min following stress exposure, consistent with reduced gene transcription. These data support the hypothesis that stress-induced glucocorticoid secretion can rapidly affect transcription of between transcriptional changes and changes in cytoplasmic mRNA pools or stimulus-driven alterations in post-transcriptional RNA processing.

The distribution of the messenger RNA (mRNA) encoding the serotonin transporter was investigated in the entire extent of the ascending raphe systems of the human brainstem, using a sensitive cRNA probe. This mRNA is abundant in the neurons of the raphe nuclei, especially in the dorsal and median raphe. The subregional distribution of this mRNA closely corresponds to that described for tryptophan hydroxylase in immunocytochemical studies. It is expressed at the highest levels in ventral and ventrolateral subregions of the dorsal raphe, with lower levels of expression in the median raphe, oral pontine reticular nuclei, and the supralemniscal cell groups. These results provide a description of the distribution of this mRNA in normal subjects and will serve as the basis for future studies of the subregional distribution of this mRNA in the brains of depressed patients and those who have committed suicide. © 1996 American College of Neuropsychopharmacology

The distributions of the transcripts encoding the five dopamine receptors have been determined in the human striatum and selected regions of the neocortex. In the striatum significant levels of dopamine receptor expression are restricted to the D1, D2, and D3receptors. D1and D2receptor messenger ribonucleic acids (mRNAs) are homogeneously distributed throughout the caudate, putamen, and nucleus accumbens. D3receptor mRNA is particularly enriched in the nucleus accumbens, with moderate levels in the ventral putamen. In the prefrontal cortex D1and D4receptor mRNAs are the most abundant, although the other three transcripts are seen at lower levels. A similar pattern is seen in the temporal neocortex. In the occipital cortex, D1receptor mRNA is the most abundant, D3the rarest, while the other three transcripts are present at modest levels of expression. These data add to a growing understanding of the neuroanatomical distribution of these transcripts in the human brain. They are essential to understand in the context of the limbic circuitry of the brain, as new hypotheses of dysfunction of dopaminergic neurotransmission are advanced in psychiatry and as these receptor subtypes are targeted for development of novel pharmacological treatments.Neuropsychopharmacology (1996) 15 17–29.

The opioid peptides are derived from three prohormone precursors referred to as proopiomelanocortin (POMC), proenkephalin (ProEnk) and prodynorphin (ProDyn). Following specific cleavage, several biologically active peptides are generated that can bind the μ, δ and κ receptors. The present study examines the receptor binding affinities of the POMC, ProEnk and ProDyn peptides to the cloned μ, δ and κ receptors expressed transiently in transfected COS-1 cells. Consistent with previous findings using brain homogenates, competition studies demonstrate that no opioid peptide family can be exclusively associated with a specific opioid receptor type. Short ProEnk peptides, such as Leu- and Met-enkephalin are selective for δ, but C-terminally extended peptides such as Met-Enk-Arg-Gly-Leu and Met-Enk-Arg-Phe have a high affinity to μ, δ and κ. Similarly, Peptide E, the BAM peptides, and metorphamide have a high affinity for all three opioid receptors types. While dynorphin A peptides and α- and β-neoendorphin have a preference for κ, they also bind the cloned δ and μ receptors. Our findings do not easily fit a simple ‘message-address’ model where the Try-Gly-Gly-Phe core is extended and this gradually alters selectivity. Rather, the pattern appears more discontinuous, and would fit better with the idea of two similar but distinct cores; a Tyr-Gly-Gly-Phe Met- or Leu core that is necessary and sufficient for μ and δ but not κ and a Tyr-Gly-Gly-Phe-Met or Leu core with an Arg-X extension that is equally necessary and sufficient for κ.

BACKGROUND: The identification of novel subtypes of the dopamine receptors has renewed interest in the involvement of dopaminergic mechanisms in schizophrenia. We determined the expression of transcripts encoding the dopamine receptors in the brains of schizophrenic patients. METHODS: The levels of the messenger RNA molecules encoding the 5 dopamine receptors were quantified in postmortem brain samples from 16 schizophrenic patients and 9 control subjects. Samples from multiple regions of the prefrontal cortex, primary visual cortex, and striatum were subjected to in situ hybridization followed by quantitative image analysis. RESULTS: Expression of dopamine receptor transcripts did not differ between schizophrenic patients and controls in striatum or visual cortex. Dramatic decreases of dopamine receptor transcripts were found in the prefrontal cortex, but these changes were restricted to the D3 and D4 receptors, and localized to Brodmann area 11 (orbitofrontal cortex). CONCLUSIONS: Cortical dopaminergic neurotransmission may be disrupted in schizophrenia at the level of receptor expression. There appears to be a focal abnormality of D3 and D4 messenger RNA expression in the prefrontal cortex, with down-regulation of both, consistent with prefrontal cortical hypodopaminergia in schizophrenia.

WHEN Hughes et al.1first described the sequences of the enkephalins, they pointed out that methionine–enkephalin (met-enkephalin) could be found in β-lipotropin (β-LPH 61–65). The entire C-terminal fragment of β-LPH was subsequently found to have opiate-like properties (β-LPH 61–91 or β-endorphin)2–7, and it seemed reasonable to hypothesise that the primary biosynthetic route for met-enkephalin was β-LPH to β-endorphin and eventually to met-enkephalin. Although this hypothesis awaits biochemical evidence, immunocytochemical studies of these peptides may shed light on the question. If β-LPH and β-endorphin are, in fact, enkephalin precursors which are stored by enkephalinergic neurones, one would expect them to be visualised in met-enkephalin-positive cells. Several laboratories produced immunocytochemical maps of the distribution of enkephalin-like-immunoreactivity (enkephalin-reactivity) in mammalian brain8–11. Watson et al.12demonstrated β-LPH-reactive cells and processes in rat brain. This β-LPH reactivity has the same distribution as β-endorphin-like-immunoreactivity (β-endorphin-reactivity) as reported by Bloom et al.13and Watson et al.14. However, the distribution of the ) β-LPH/β-endorphin-reactive system on the one hand and the met-enkephalin-reactive system on the other seemed quite different. Met-enkephalin-reactivity was seen in many local cell groups throughout the neuraxis, especially the lower brain stem8–11, whereas β-LPH- and β-endorphin-reactivities were found only in a single hypothalamic cell group with long ascending and descending processes12–14. We present here biochemical and anatomical evidence for the existence of two separate opioid systems in brain—an enkephalin system and a β-LPH/β-endorphin system. We also present immunocytochemical data for the coexistence in the brain of ACTH with β-LPH/β-endorphin.

Ardrenocorticotropic hormone (ACTH), β-endorphin and the melanotropins (MSHs) are all derived from a single large precursor molecule, proopiomelanocortin (POMC)1–5by individual processing through a series of co- and post-translational modifications. Although the primary site of synthesis is in the pituitary, POMC-derived peptides have been identified in various tissues, notably the brain (see refs 6, 7 for review). A major question concerning brain POMC is whether it is synthesized within the central nervous system (CNS) itself or whether it is taken up from plasma flowing in a retrograde fashion from the pituitary. POMC peptides have been detected immunohistochemically and biochemically in the medial basal hypothalamus, the amygdala and throughout the brain stem. POMC peptide-containing cell bodies have been identified only in two cell groups, however, principally in the periarcuate region of the hypothalamus and to a lesser extent in the nucleus of the tractus solitarius8–10. These and other11,12observations have suggested that POMC peptides are synthesized locally in the medial basal hypothalamus and reach other regions of the CNS by axonal transport. Civelli et al. identified POMC mRNAs in nucleic acid extracts of rat and bovine hypothalami by solution hybridization as well as Northern gel blot analysis12, but because of the close proximity of the hypothalamus to the pituitary and the extremely low amounts of POMC mRNA being measured in the hypothalamus, the possibility of tissue contamination during dissection could not be ruled out. We report here the anatomical co-localization of POMC-related peptides and POMC-specific mRNAs to a single major cell group in the medial basal hypothalamus. The presence of POMC-specific mRNA in a POMC peptide-containing cell in the brain is strong support for POMC biosynthesis within brain tissue.

ProbeMatchDB is a web-based database designed to facilitate the search of EST/cDNA sequences or STS markers that can be used to represent the same gene across different microarray platforms and species. It can be used for finding equivalent EST clones in the Research Genetics sequence verified clone set based on results from Affymetirx GeneChips. It will also help to identify probes representing orthologous genes across human, mouse and rat on different microarray platforms.