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Subcellular fractionation methods permit the isolation, purification, and/or enrichment of specific cellular compartments from complex tissue samples. Enrichment of multiple subcellular compartments from the same tissue sample permits comparisons of the spatial distribution of target proteins between specific intracellular compartments and, in some cases, can provide information about spatiotemporal processing of key cellular components. Here we describe a method to generate subcellular fractions enriched for heavy membranes and nuclei, rough and smooth endoplasmic reticulum membranes, light membranes and cytosol, synapses, and other intermediate cellular membranes from postmortem human brain tissue. These subcellular fractions can be used in a variety of downstream applications to assess the localization, relative abundance, and stoichiometry of glutamate receptor subunits along the forward trafficking pathway.

Inhibitory neurotransmission is primarily mediated by γ-aminobutyric acid (GABA) activating synaptic GABA type A receptors (GABA(A)R). In schizophrenia, presynaptic GABAergic signaling deficits are among the most replicated findings; however, postsynaptic GABAergic deficits are less well characterized. Our lab has previously demonstrated that although there is no difference in total protein expression of the α1-6, β1-3 or γ2 GABA(A)R subunits in the superior temporal gyrus (STG) in schizophrenia, the α1, β1 and β2 GABA(A)R subunits are abnormally N-glycosylated. N-glycosylation is a posttranslational modification that has important functional roles in protein folding, multimer assembly and forward trafficking. To investigate the impact that altered N-glycosylation has on the assembly and trafficking of GABA(A)Rs in schizophrenia, this study used western blot analysis to measure the expression of α1, α2, β1, β2 and γ2 GABA(A)R subunits in subcellular fractions enriched for endoplasmic reticulum (ER) and synapses (SYN) from STG of schizophrenia (N = 16) and comparison (N = 14) subjects and found evidence of abnormal localization of the β1 and β2 GABA(A)R subunits and subunit isoforms in schizophrenia. The β2 subunit is expressed as three isoforms at 52 kDa (β2(52 kDa)), 50 kDa (β2(50 kDa)) and 48 kDa (β2(48 kDa)). In the ER, we found increased total β2 GABA(A)R subunit (β2(ALL)) expression driven by increased β2(50 kDa), a decreased ratio of β(248 kDa):β2(ALL) and an increased ratio of β2(50 kDa):β2(48 kDa). Decreased ratios of β1:β2(ALL) and β1:β2(50 kDa) in both the ER and SYN fractions and an increased ratio of β2(52 kDa):β(248 kDa) at the synapse were also identified in schizophrenia. Taken together, these findings provide evidence that alterations of N-glycosylation may contribute to GABAergic signaling deficits in schizophrenia by disrupting the assembly and trafficking of GABA(A)Rs.

We propose that postmortem tissue is an underutilized substrate that may be used to translate genetic and/or preclinical studies, particularly for neuropsychiatric illnesses with complex etiologies. Postmortem brain tissues from subjects with schizophrenia have been extensively studied, and thus serve as a useful vehicle for illustrating the challenges associated with this biological substrate. Schizophrenia is likely caused by a combination of genetic risk and environmental factors that combine to create a disease phenotype that is typically not apparent until late adolescence. The complexity of this illness creates challenges for hypothesis testing aimed at understanding the pathophysiology of the illness, as postmortem brain tissues collected from individuals with schizophrenia reflect neuroplastic changes from a lifetime of severe mental illness, as well as treatment with antipsychotic medications. While there are significant challenges with studying postmortem brain, such as the postmortem interval, it confers a translational element that is difficult to recapitulate in animal models. On the other hand, data derived from animal models typically provide specific mechanistic and behavioral measures that cannot be generated using human subjects. Convergence of these two approaches has led to important insights for understanding molecular deficits and their causes in this illness. In this review, we discuss the problem of schizophrenia, review the common challenges related to postmortem studies, discuss the application of biochemical approaches to this substrate, and present examples of postmortem schizophrenia studies that illustrate the role of the postmortem approach for generating important new leads for understanding the pathophysiology of severe mental illness.

A growing body of evidence suggests that abnormal elements of the cytoskeleton may be associated with the pathophysiology of schizophrenia. Isoforms of a major cytoskeleton protein, β-tubulin, were recently demonstrated to have distinct roles in neuronal differentiation and cell viability. For these reasons, we tested the hypothesis that there are differences in the expression of β-tubulin isoforms (βI-βIV) in the brain in schizophrenia, using western blot analysis in an elderly group of subjects with this illness and a control group. We found that βI-tubulin protein expression was decreased in the anterior cingulate cortex and increased in the dorsolateral prefrontal cortex, but not changed in superior temporal gyrus or hippocampus in schizophrenia. Our data supports the growing body of evidence suggesting abnormalities of the cytoskeleton in schizophrenia., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Biological psychiatry has made significant advances through the development of postmortem studies, animal models, and studies with living humans. Although these approaches each have advantages and disadvantages, the postmortem field is undergoing a significant shift toward more complex and informative methodologies. In the first part of this review, we summarize the long-standing methodologic challenges facing this field. In the second part of the article, we discuss the innovative approaches being used for postmortem studies, including laser capture microdissection and subcellular fractionization. These techniques will permit scientists working in the postmortem field to ask and answer the largest possible questions, providing new targets for drug discovery and improved treatments for severe mental illness., (Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Animal models of prenatal nicotine exposure clearly indicate that nicotine is a neuroteratogen. Some of the persisting effects of prenatal nicotine exposure include low birth weight, behavioral changes and deficits in cognitive function, although few studies have looked for neurobehavioral and neurochemical effects that might persist throughout the lifespan. Pregnant rats were given continuous infusions of nicotine (0.96mg/kg/day or 2.0mg/kg/day, freebase) continuing through the third trimester equivalent, a period of rapid brain development. Because the third trimester equivalent occurs postnatally in the rat (roughly the first week of life) nicotine administration to neonate pups continued via maternal milk until postnatal day (P) 10. Exposure to nicotine during pre- and early postnatal development had an anxiogenic effect on adult rats (P75) in the elevated plus maze (EPM), and blocked extinction learning in a fear conditioning paradigm, suggesting that pre- and postnatal nicotine exposure affect anxiety-like behavior and cognitive function well into adulthood. In contrast, nicotine exposure had no effect on anxiety-like behaviors in the EPM in adolescent animals (P30). Analysis of mRNA for the alpha4, alpha7, and beta2 subunits of nicotinic acetylcholine receptors revealed lower expression of these subunits in the adult hippocampus and medial prefrontal cortex following pre- and postnatal nicotine exposure, suggesting that nicotine altered the developmental trajectory of the brain. These long-term behavioral and neurochemical changes strengthen the case for discouraging cigarette smoking during pregnancy and clearly indicate that the use of the patch as a smoking cessation aid during pregnancy is not a safe alternative.

We compared protein expression by Western blot analysis in four areas of postmortem brain from patients with schizophrenia and control subjects for several proteins that are often used as controls for Western blot studies: beta-tubulin, actin, glyceraldehyde-3-phosphate dehydrogenase, and valosin-containing protein. We did not detect any differences in expression between subjects with schizophrenia and a comparison group. These results suggest that all four proteins are suitable loading controls for postmortem studies of schizophrenia.

Chronic abuse of cocaine is known to cause neuroadaptive changes in the nucleus accumbens (NAc) and ventral tegmental area (VTA). In addition, altered expression of the myelin-related genes MBP, MOBP, PLP1 as well as of MAL2 in NAc was recently reported by gene array analysis in brains from cocaine abusers. In the present study we used in situ hybridization to quantify transcript expression of these four genes, as well as for the myelin-related transcripts encoding quaking, EDG2, claudin-11, transferrin, CNP, and MAG in caudate, putamen, internal capsule, and NAc in postmortem brain from cocaine abusers and matched comparison subjects. Most transcripts were not different between these groups in these striatal regions, and contrary to previous reports, we did not detect any changes in the NAc. However, expression of the transcript encoding PLP1 was significantly decreased in ventral and dorsal regions of the caudate, putamen, and in the internal capsule. Additionally, expression of claudin-11 and transferrin was decreased in the caudate and internal capsule, respectively. PLP1 is expressed at very high levels in oligodendrocytes and is essential in maintaining stability of myelin sheets. Based on these findings, altered expression of PLP1 in most areas of the striatum suggests that widespread changes to the myelin structure could be associated with the adaptive changes following chronic cocaine abuse.

The NMDA subtype of glutamate receptors are heteromeric complexes comprised of multiple subunits encoded by at least seven different genes (NR1, NR2A-2D and NR3A-3B), and differential expression of these subunits alters the pharmacological and electrophysiological properties of NMDA receptors. NR3A is a recently identified unique modulatory subunit that decreases NMDA receptor current and calcium influx. In rodents, NR3A is developmentally expressed, displaying robust expression early in development that declines with age, reaching low levels in the adult brain. A distinct and highly selective pattern of expression is observed in the developing and mature rodent brain, suggesting that NR3A may play a very specific role in NMDA receptor-mediated processes. NR3A expression in other species, however, is unknown. Therefore, we examined the expression of NR3A mRNA and protein in the adult macaque brain. Our results indicate that NR3A mRNA is expressed throughout much of the adult primate brain, and at high levels in specific brain regions including the neocortex, substantia nigra par compacta and cerebellum, as well as select areas of the hippocampus, amygdala, thalamus and hypothalamus. Western blot analysis reflects that this protein is translated and expressed in multiple brain regions. In contrast to the rat mRNA, our results suggest that NR3A transcript is widely expressed in the adult primate brain. Particular enrichment in some brain areas may reflect brain-region or circuit-specific functions for this NMDA receptor subunit.

The nicotinic cholinergic system exerts potent modulatory effects on glutamatergic neurotransmission, an effect mediated in part by increased glutamate release following activation of presynaptic nicotinic cholinergic receptors. Ionotropic glutamate receptor agonists also stimulate release of acetylcholine, suggesting that these neurotransmitter systems reciprocally regulate one another. We investigated an interface between the nicotinic cholinergic and glutamatergic systems by measuring nicotinic receptor subunit transcript expression following administration of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an antagonist of the AMPA and kainate subtypes of glutamate receptors. Using [(35)S] in situ hybridization, we measured expression of alpha 2, alpha 3, alpha 4, alpha 5, alpha 7, beta 2, beta 3, and beta 4 nicotinic receptor subunit transcripts in the rat forebrain. Following 7 days of treatment with vehicle or CNQX (1 mg/kg/day or 10 mg/kg/day), changes in nicotinic receptor subunit transcript expression were restricted to subunits that form heteromeric receptors. We found increased levels of transcripts for alpha 2 and beta 2 nicotinic receptor subunits in the hippocampus, decreased alpha 4 subunit transcripts in the medial habenula and amygdala, and increased beta 2 subunit transcripts in the septum and piriform cortex. We did not detect changes in expression of transcripts for the alpha 7 subunit, which forms homomeric nicotinic receptors. Our findings indicate that expression of nicotinic cholinergic receptor subunit transcripts are regulated in a subunit- and region-specific fashion by CNQX, an antagonist of non-NMDA ionotropic glutamate receptors., (Copyright 2004 Wiley-Liss, Inc.)

Glutamate is the primary excitatory neurotransmitter in the central nervous system, regulating numerous cellular signaling pathways and controlling the excitability of central synapses both pre- and postsynaptically. Localization, cell surface expression, and activity-dependent regulation of glutamate receptors in both neurons and glia are performed and maintained by a complex network of protein-protein interactions associated with targeting, anchoring, and spatially organizing synaptic proteins at the cell membrane. Using in situ hybridization, we examined the expression of transcripts encoding the AMPA receptor subunits (GluR1-GluR4) and a family of AMPA-related intracellular proteins. We focused on PDZ-proteins that are involved in the regulated pool and anchoring AMPA subunits to the cell membrane (PICK1, syntenin), and those maintaining the constitutive pool of AMPA receptors at the glutamatergic synapse (NSF, stargazin). In addition, we studied a fifth protein, KIAA1719, with high homology to the rat PDZ protein ABP, associated with the clustering of AMPA receptors at the glutamate synapse. The AMPA subunits showed significant differences in regional expression, especially in the neocortex, thalamus, striatum, and cerebellum. The expression of other proteins, even those related to a specific AMPA subunit (such as ABP and PICK1 to GluR2 and GluR3), often had different distributions, whereas others (like NSF) are ubiquitously distributed in the brain. These results suggest that AMPA subunits and related intracellular proteins are differentially distributed in the macaque brain, and in numerous structures there are significant mismatches, suggesting additional functional properties of the associated intracellular proteins.., (Copyright 2003 Wiley-Liss, Inc.)

The anatomical distribution of the transcript encoding calcyon, a 24 kDa membrane protein associated with coupling D1-like dopamine receptor activation to potentiated intracellular calcium release, was examined using in situ hybridization in the macaque brain. Calcyon mRNA is found to be abundantly distributed throughout the primate brain. In neocortex, moderately dense, diffuse signal is found in all areas, with increased intensity present in a superficial isodense band corresponding generally to cortical layers II and III. Increased intensity of signal is also seen in the pyramidal cell layers of medial prefrontal and anterior cingulate cortex. Calcyon mRNA is present abundantly in subcortical limbic areas such as the nucleus accumbens, septum, hypothalamus, amygdala, and hippocampus. Moderate calcyon transcript expression is seen in caudate and putamen, with lower levels in globus pallidus. Thalamic nuclei, including the reticular nucleus, express low to moderate levels. Very dense expression is noted in the substantia nigra pars compacta. Numerous brainstem regions express this transcript, notably monoaminergic nuclei including the locus coeruleus and dorsal raphe. The cerebellum has detectable levels of expression in both cortex and deep nuclei. Although calcyon is hypothesized as a means for D1-like receptors to modulate "cross-talk" with other neurotransmitter receptor systems, it is notable that abundant calcyon transcript is detected in brain regions not associated with D1-like neurotransmission, particularly the substantia nigra pars compacta and other dopamine-synthesizing cell groups. A substantial proportion of this may relate to the reported association of calcyon with the D5 receptor, or in addition, may suggest that calcyon has a wider role as a regulator of intracellular signal transduction., (Copyright 2003 Wiley-Liss, Inc.)

Transcriptional profiles within discrete human brain regions are likely to reflect structural and functional specialization. Using DNA microarray technology, this study investigates differences in transcriptional profiles of highly divergent brain regions (the cerebellar cortex and the cerebral cortex) as well as differences between two closely related brain structures (the anterior cingulate cortex and the dorsolateral prefrontal cortex). Replication of this study across three independent laboratories, to address false-positive and false-negative results using microarray technology, is also discussed. We find greater than a thousand transcripts to be differentially expressed between cerebellum and cerebral cortex and very few transcripts to be differentially expressed between the two neocortical regions. We further characterized transcripts that were found to be specifically expressed within brain regions being compared and found that ontological classes representing signal transduction machinery, neurogenesis, synaptic transmission, and transcription factors were most highly represented.

The behavioral and neurochemical effects of switching from typical to atypical medications have not been evaluated in the rodent models of tardive dyskinesia. Thus, we treated rats with haloperidol-decanoate for 12 weeks, and assessed the effects of additional treatment with olanzapine, haloperidol, clozapine, or vehicle on vacuous chewing movements and expression of transcripts for dopamine receptors, tyrosine hydroxylase, delta-opioid receptor, prodynorphin, preproenkephalin, glutamic acid decarboxylase-65 (glutamic acid decarboxylase (GAD)-65) and GAD-67 and N-methyl-D-aspartate (NMDA) receptor subunits in the striatum and its efferent pathways. Haloperidol-decanoate induced vacuous chewing movements extinguished following an additional 4 weeks of treatment with vehicle, olanzapine or haloperidol, but not clozapine. Post-treatment, vacuous chewing movements in the clozapine group were significantly higher than the vehicle, olanzapine and haloperidol groups. GAD-67 mRNA expression in the globus pallidus was decreased following additional treatment with olanzapine or haloperidol, but not clozapine. Changes in expression of other transcripts were not detected. These findings demonstrate important differences in the effects of typical and atypical antipsychotics on chronic vacuous chewing movements.

Glutamate receptors have multiple roles in the central nervous system. Recent evidence suggests that the iontropic glutamate receptors are critical during brain development, particularly for corticogenesis, neuronal migration, and synaptogenesis. In this study, we examined subunit mRNA expression and binding sites of the NMDA, AMPA, and kainate receptors from gestational weeks 8-20 in human fetal brain. Expression of glutamate receptors was high during several periods in these brains. Different levels of expression of each NMDA, AMPA, and kainate receptor subunit transcripts were present during development, with a greater abundance of NR1, NR2B, NR2D, GluR7, and KA1 mRNA at most gestational ages. Binding sites for NMDA, AMPA, and kainate receptors were all detected, but each had a unique pattern of expression. These results demonstrate that glutamate receptors are expressed early in human brain development, and undergo complex changes over time consistent with their role in normal development.

Glutamatergic dysfunction has been suggested as a possible substrate of the pathophysiology of schizophrenia. Of the multiple glutamate receptors, those most commonly implicated in schizophrenia are the ionotropic subtypes, the NMDA, AMPA, and kainate receptors. The expression of the glutamate receptors has been determined at multiple levels of gene expression in postmortem brain samples from schizophrenics and controls; while results have not been entirely consistent from study to study, several generalizations have emerged from this literature: (1) The AMPA receptor is abnormally decreased in expression in the schizophrenic hippocampus, involving decreased levels of subunit transcripts and protein levels, as well as binding sites, (2) similar changes are seen for kainate receptor expression in the hippocampus, and (3) the obligate NMDA receptor subunit, NMDAR1, may be abnormally expressed in some cortical regions in schizophrenia. These data support the hypothesis of abnormal glutamatergic neurotransmission involving the ionotropic glutamate receptors in schizophrenia.

The high affinity of 5-HT6 receptors for atypical antipsychotic drugs, and their localization in limbic and cortical regions of the brain, suggest that they might play a role in the pathophysiology of schizophrenia. To determine if this receptor is regulated by antipsychotics, rats were injected with clozapine (20 mg/kg/day), haloperidol (2 mg/kg/day), or vehicle daily for 2 weeks, and 5-HT6 receptor mRNA levels were measured by in situ hybridization. Clozapine but not haloperidol significantly decreased 5-HT6 expression in all subfields of the hippocampus. No drug effects were observed in cortical or forebrain structures. These results suggest that downregulation of this receptor in the hippocampus might be a characteristic of atypical antipsychotic drugs, although this hypothesis will require testing with other atypical antipsychotics.

The dopamine (DA) hypothesis of schizophrenia, which was based largely on evidence that pharmacological manipulations of DA systems influence the symptoms of schizophrenia, is undergoing a transformation due to our knowledge of the anatomy and pharmacology of additional subtypes of dopamine receptors. New research links the multiplicity of D2-like receptors to divergent neuroanatomic sites of suspected pathology in schizophrenia. We hypothesize that this research suggests that D2 receptors in the basal ganglia are the likely site of extrapyramidal symptoms and not antipsychotic effects. Rather, D3 receptors of the mesolimbic system are a likely site of antipsychotic effects, and D2 and D4 receptors in the medial temporal lobe and limbic cortical areas are the sites of additional antipsychotic effects. This work also suggests that divergent DA receptor circuits are likely associated with the pathophysiology of this disorder.

Postmortem analysis of brain chemistry and anatomy in the study of psychiatric disorders has enjoyed renewed interest recently. There are myriad difficulties in establishing a brain bank, including proper diagnosis and confounding neuropathology. These difficulties may become more pronounced in a long-term domiciliary facility. In order to begin to address neuropathologic considerations, we performed neuropathological examinations on each patient included in our bank of 20 brains. Forty-five per cent of all patients had significant and unexpected neuropathology. Selection of patients to be included in postmortem studies requires careful screening to enhance accurate neuropathological assessment.

The tone and regulation of the brain dopaminergic projections are, in part, determined by the presence or absence of dopamine (DA) autoreceptors: rate of DA synthesis and turnover, as well as both pattern and rate of neuronal firing, are modulated by the expression and activity of these autoreceptors. The expression of dopaminergic receptors in the midbrain DA cell groups, presumably reflecting DA autoreceptors, was determined in the brains of the rat, Old World monkey, and human. In the rat, both the substantia nigra (A9) and the ventral tegmental area (A10) appear to express DA autoreceptors. In the monkey and human, however, only the projections arising from the substantia nigra express these receptors; the limbic projections originating in the ventral tegmental area lack this substrate for DA autoregulation. These results indicate that in the human, the nigrostriatal and mesocorticolimbic dopamine systems may be differentially autoregulated.

This paper reviews recent developments in dopamine receptor biology, with an emphasis on our recent understanding of the anatomical locations of where various genes for the dopamine receptors are expressed. At least five genes encoding dopamine receptors have been discovered. These five receptors have distinct pharmacological profiles as well as unique neuroanatomical distributions. While D1 and D2 receptors are widely expressed in many neural systems, the novel D3 and D4 receptors appear to be concentrated within parts of the limbic system, and the D5 receptor has a limited and unusual distribution. The distinct distributions of each of the dopamine receptors in brain facilitates a new understanding of potential neurochemical and neuroanatomical substrates of psychiatric illnesses such as schizophrenia, permits an understanding of observed actions and side effects of psychotropic agents, and provides clues for the rational development of new drugs with effects in the dopaminergic system.

Ventricular enlargement is one of the most consistently documented neurobiological abnormalities in schizophrenia. The timing of the development of this abnormality in the course of schizophrenic illness and its relationship to neuropsychological dysfunction and premorbid adjustment is, however, unclear. To address these questions, we examined the relationship between ventricle-brain ratio (VBR), premorbid adjustment, and neuropsychological function, in 23 acutely exacerbated chronic schizophrenic inpatients. We observed that larger ventricles were associated with better current neuropsychological test performance, better premorbid cognitive ability, greater cognitive deterioration, better childhood premorbid social function, and greater decline in social function from premorbid levels. These data suggest that at least two developmental processes may operate in the genesis of cognitive and social dysfunction in schizophrenia: (1) childhood onset associated with poor premorbid childhood function, low educational achievement, lower intelligence quotient (IQ) and variably with VBR; and (2) adolescent onset associated with relatively normal childhood social function, higher academic achievement and IQ and increased VBR. Ventricular enlargement may reflect a late developmental or degenerative pathological process in schizophrenia.

The effects of chronic cocaine exposure on dopamine D1 and D2 receptor gene expression in the human brain were studied in postmortem samples from chronic cocaine abusing and matched control subjects. Using in situ hybridization of receptor autoradiography to examine messenger ribonucleic acid (RNA) and binding sites, respectively, neither D1 nor D2 receptor expression was found to be changed in the nucleus accumbens, caudate, putamen, or substantia nigra of the cocaine-exposed subjects. Although chronic cocaine exposure can produce alterations in dopaminergic neurotransmission, sustained compensatory changes in dopamine receptor expression do not appear to occur in the human.

D1, a subtype of the dopamine receptors, is widely distributed in the nervous system and has been shown to be positively coupled to adenylate cyclase. Using a combination of in vitro receptor autoradiographic and in situ hybridization techniques, the present study examines the co-distribution of D1 receptor binding sites and D1 receptor mRNA in adjacent rat brain sections. D1 receptor binding sites were labeled using the selective antagonist [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzaz epin- 7-ol (SCH23390) (4.6 nM), in the presence of 1 microM ketanserin, while the D1 receptor mRNA was visualized with a 35S-labeled riboprobe corresponding to a region between transmembrane domains III and VI of the rat D1 receptor (base pairs 383-843). Analysis of serial sections suggested a good agreement between D1 receptor binding and mRNA in several brain regions, including the paleocortex, caudate-putamen, nucleus accumbens, amygdala, and suprachiasmatic nucleus. Marked discrepancies between D1 receptor binding and mRNA were observed in other brain regions including the entopeduncular and subthalamic nuclei, substantia nigra (pars reticulata), hippocampus, and cerebellum. While technical considerations may contribute to these results, much of the discordance between the distributions is probably due to the differential localization of D1 receptor mRNA in cell bodies and receptor binding sites on fibers and may provide insights into receptor synthesis, transport, and membrane insertion. In the basal ganglia, for instance, D1 receptors are synthesized in the striatum and are either transported to efferent projections in areas such as the substantia nigra, or remain localized in striatal cells bodies. Ibotenic acid lesions in the striatum are consistent with these conclusions and demonstrate a coordinate loss of D1 receptor binding and mRNA in the caudate-putamen that is accompanied by a degeneration of fibers projecting to substantia nigra and a loss of D1 binding in the pars reticulata. Neurons in the dentate gyrus and in the granular layer of the cerebellum, on the other hand, synthesize D1 receptors and transport them entirely to either their dendritic or axonal fields, respectively, in the molecular layer. This analysis provides a better understanding of dopaminergic receptor systems in the CNS and their anatomical organization.

The cloning of the dopamine (DA) D2 receptor now permits the characterization and regulation of D2 messenger RNA (mRNA) in the brain. In this article, the authors describe their studies delineating the distribution of D2 receptor mRNA in the rodent and primate brain, and compare the distribution of message to D2 receptor binding sites. The effects of chronic DA agonist and antagonist treatment on D2 receptor mRNA are also presented, and provide insights into receptor regulation. Finally, the autoreceptor role of D2 receptors located in the midbrain is examined with a combination of 6-hydroxydopamine lesions and anatomic colocalization studies with tyrosine hydroxylase. These preclinical results provide a framework for subsequent investigation into the nature of D2 receptor gene expression in postmortem brains from patients with disorders putatively associated with dopaminergic dysfunction, especially schizophrenia. They also lay the groundwork for a more profound understanding of DA neurocircuitry by combining molecular biological and traditional anatomical techniques.

Several lines of evidence suggest the existence of multiple dopamine receptor subtypes, referred to as D1 and D2. The present study examines the distribution of these dopamine binding sites in the rat brain and pituitary in relation to the distribution of D2 receptor mRNA using a combination of in vitro receptor autoradiographic and in situ hybridization techniques. 3H-Raclopride and 3H-SCH23390 (in the presence of 1 microM ketanserin) were used to label D2 and D1 receptor binding sites, respectively, while a 495 bp cRNA probe synthesized from the Sac I-Bgl II fragment of a rat D2 receptor cDNA was used to visualize the D2 receptor mRNA. Analysis of adjacent tissue sections in which receptor autoradiography and in situ hybridization had been performed revealed several brain regions where the D2 binding site and corresponding mRNA appear to be similarly distributed, including the caudate-putamen, nucleus accumbens, olfactory tubercle, globus pallidus, substantia nigra, and ventral tegmental area. In the pituitary gland, D2 binding sites and mRNA appear to be codistributed with very dense levels in the intermediate lobe and individually labeled cells in the anterior lobe. Brain regions demonstrating a lack of correspondence between the distribution of the D2 binding site and D2 receptor mRNA include the olfactory bulb, neocortex, paleocortex, hippocampus, and zona incerta. Several hypotheses are discussed to explain the lack of correspondence in certain brain regions; these include the localization of receptor binding sites on both fibers and cell bodies and receptor transport. These studies provide a better understanding of the anatomical distribution of the D2 receptor and serve as a framework for future regulatory and anatomical mapping studies. By focusing on specific brain regions, such as the nigrostriatal system, hippocampus, and olfactory bulb, they provide insights into D2 receptor synthesis, transport, and insertion into cell membranes.

The total content and extent of processing of the gamma 3MSH and beta-endorphin-containing N- and C-terminal domains of proopiomelanocortin were determined in the anterior and intermediate lobes of the pituitaries and in 11 regions of the brains of three Rhesus monkeys. Most immunoreactive gamma 3MSH and beta-endorphin was located in the pituitary lobes, although significant amounts were also found in several brain regions. Sephadex column chromatography revealed that gamma 3MSH immunoreactivity was found primarily as 4K and 9K forms; no gamma 1MSH was detected. beta-Endorphin immunoreactivity was found as beta-endorphin, beta-lipotropin, and as a 5K form which may represent beta-endorphin extended N-terminally by part or all of beta-MSH. In the anterior lobe of the pituitary, the predominant products were 9K gamma 3MSH and beta-lipotropin; in the intermediate lobe, more processed forms (4K gamma 3MSH, beta-endorphin and 5K beta-endorphin) appeared to be preferentially stored. The pattern of processing in various brain regions was similar to that of the intermediate lobe of the pituitary.

The distribution of mRNA coding for the D2 dopamine receptor was studied in the rat brain by in situ hybridization. A cDNA probe corresponding to the putative third cytosolic loop and sixth and seventh transmembrane domains of the rat D2 receptor was used to generate an 35S-labeled riboprobe to hybridize to D2 receptor mRNA. D2 mRNA was found both in dopamine projection fields and in regions associated with dopamine-containing cell bodies, suggesting both postsynaptic and presynaptic autoreceptor localization. Highest concentrations of D2 mRNA were found in neostriatum, olfactory tubercle, substantia nigra, ventral tegmental area, and the nucleus accumbens. This distribution is consistent with those reported with D2 receptor autoradiography.