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Serotonin 2C receptor (5-HT(2C)R) exerts a major inhibitory influence on dopamine (DA) neurotransmission within the mesocorticolimbic DA pathway that is implicated in drug reward and goal-directed behaviors. 5-HT(2C)R pre-mRNA undergoes adenosine-to-inosine editing, generating numerous receptor isoforms in brain. As editing influences 5-HT(2C)R activity, individual differences in editing might influence dopaminergic function and, thereby, contribute to interindividual vulnerability to drug addiction. Liability to drug-related behaviors in rats can be predicted by their level of motor activity in response to a novel environment. Rats with a high locomotor response (high responders; HRs) exhibit enhanced acquisition and maintenance of drug self-administration compared to rats with a low response (low responders; LRs). We here examined 5-HT(2C)R mRNA editing and expression in HR and LR phenotypes to investigate the relationship between 5-HT(2C)R function and behavioral traits relevant to drug addiction vulnerability. Three regions of the mesocorticolimbic circuitry (ventral tegmental area (VTA), nucleus accumbens (NuAc) shell, and medial prefrontal cortex (PFC)) were examined. 5-HT(2C)R mRNA expression and editing were significantly higher in the NuAc shell compared with both the PFC and VTA, implying significant differences in function (including constitutive activity) among 5-HT(2C)R neuronal populations within the circuitry. The regional differences in editing could, at least in part, arise from the variations in expression levels of the editing enzyme, ADAR2, and/or from the variations in the ADAR2/ADAR1 ratio observed in the study. No differences in the 5-HT(2C)R expression were detected between the behavioral phenotypes. However, editing was higher in the PFC of HRs vs LRs, implicating this region in the pathophysiology of drug abuse liability.

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

Very limited is known about p53 expression in the normal mammalian brain and only few alternative splice variants have been reported thus far in human and rat peripheral tissues. Here, we detected eight new p53 transcripts in the human brain generated by alternative splicing, whereas two were present in the rat brain. Almost all alternative splice events occurred due to atypical splice mechanism employing direct repeats at splice sites. All discovered transcripts retain untranslated 5' area of the p53 gene and thus could be translated into peptides consisting of different functional domains.

Aims: To determine whether there are factors during apparent rapid heroin overdose death that affect agonal state and thus brain pH (index of hypoxia) that can influence neurobiological systems linked to drug abuse., Design and Methods: Brain specimens and autopsy/medical reports were investigated in subjects dying from heroin overdose (n=70) and compared to normal controls (n=45) as well as suicide victims (n=31) with a documented rapid cause of death. Detailed autopsy material was characterized as to positive and negative respiratory distress in relation to brain pH; drug toxicity and other demographic information was also evaluated. In situ hybridization histochemistry was used to study mRNA expression levels of dopamine (e.g., D2 receptor, dopamine transporter) and opioid (e.g., proenkephalin) related markers in various structures in relation to brain pH., Findings: Brain pH was generally reduced in heroin overdose cases versus normal and suicide subjects. There was, however, significant variation in heroin overdose deaths related to differences in respiratory distress that differentially altered brain pH levels. Various factors such as vomit inhalation, resuscitation, pulmonary embolism and suffocation contributed to positive respiratory distress. Elevated brain pH was observed in heroin overdose with positive alcohol toxicity suggesting potentiated alcohol-induced rapidity of heroin deaths. mRNA expression levels of the dopamine-related genes and proenkephalin were positively correlated with brain pH., Conclusions: Respiratory distress contributes to variations in the acute agonal state during heroin overdose death that differentially alters brain pH levels and significantly impacts mRNA levels. Such findings should be considered for postmortem molecular/neurochemical neurobiological studies of opiate abusers.

The regional mRNA expression pattern of 5-HT(1B) receptors has been extensively characterized in the rodent and guinea pig brain, but a detailed mapping of the 5-HT(1B) receptor mRNA expression in the human brain has not previously been performed. In the present study, the mRNA expression of 5-HT(1B) receptors was analyzed using in situ hybridization histochemistry and whole hemisphere sections of the human postmortem brain. The mRNA expression was compared with the autoradiographic distribution of 5-HT(1B) receptors. High levels of mRNA expression were found in the striatum, cortex, lateral geniculate nucleus, and raphe nuclei. The expression was higher in ventral than in dorsal striatal regions and was absent from the substantia nigra and pallidum, where high levels of 5-HT(1B) receptors were found. A layer-specific expression pattern was observed in cortical regions. The results extend previous knowledge about the localization of the 5-HT(1B) receptor in the human brain. This study provides evidence of a mismatch of the regional expression of 5-HT(1B) receptor mRNA and the 5-HT(1B) receptor distribution in human brain, similar to what has been demonstrated in other species. This is in line with the localization of this receptor subtype in nerve terminals. The results give support to species differences in the cortical mRNA expression pattern of this receptor subtype.

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

JP05, also called GPR72 or GIR, is an orphan G-protein-coupled receptor, GPCR, showing significant structural similarity to the tachykinin receptors. The anatomical distribution of JP05 mRNA was first described in the central nervous system of the mouse, and recently the human JP05 orphan receptor gene has been cloned. In the present study the distribution of JP05 mRNA was examined in the human forebrain using in situ hybridization analysis. The results revealed a wide but discrete distribution of the transcript with strongly JP05 mRNA expressing cells, presumably neurons, present in the cerebral cortex (layer II), hippocampus (pyramidal CA3 neurons and granule cells), amygdala (basal and periamygdaloid cortical nuclei), in the endopiriform nucleus, diagonal band of Broca, thalamus (nucleus reuniens, parafascicular nucleus) and hypothalamus (posterior, dorsal, and around the medial mammillary). Weaker signals were detected in the deeper cortical layers and throughout the striatum. A few positive cells were evident in the raphe but not in the substantia nigra or pontine nuclei. The results indicate significant similarities between human and mouse brain with regard to JP05 mRNA expression. The distribution patterns of JP05 mRNA in the human brain suggest involvement in control of emotions and of neuroendocrine, cognitive and motor functions.

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

Understanding dopamine signaling in human behavior requires knowledge of the distribution of all molecular components involved in dopamine pathways throughout the human brain. In the present study, the relative distributions of D1 and D2 dopamine receptor mRNAs were determined by in situ hybridization histochemistry in whole hemisphere sections from normal human post mortem brains. The findings confirmed information documented from single structure examination that the highest expression of both the D1 and D2 mRNAs were localized to the striatum. The cerebral cortex expressed moderate D1 mRNA in all regions with the highest signal in the medial orbital frontal area (Brodmann areas 11, 14), the paraterminal gyrus (Brodmann area 32) and the insular cortex (Brodmann areas 13-16), whereas the D2 mRNA expression had very low cortical expression. The bed nucleus of the stria terminalis and islands of Calleja had high expression of the D1 mRNA and moderate D2 mRNA levels. Moderate to high expression of the D2 mRNA was evident in the hippocampal formation, parafascicular and paraventricular thalamic nuclei, geniculate bodies, subthalamic nucleus, and pineal gland, all of which were devoid of, or showed only faint, D1 mRNA expression. Brainstem regions, e.g. substantia nigra, red nucleus, inferior colliculus, medial lemniscus, and pontine nuclei expressed D2, but not D1, mRNA. These results emphasize the differential anatomical localization of D1 and D2 dopamine receptor mRNA neuronal populations in the human brain. The restricted expression of the D1 mRNA to the cortical mantle and to a few forebrain structures indicates a strong involvement of the D1 system in cognitive function.

Cocaine- and amphetamine-regulated transcript (CART) is a novel putative peptide neurotransmitter. We studied the expression of CART mRNA throughout the human postmortem brain by using in situ hybridization histochemistry. The cortical expression was distinct with high mRNA expression levels in the piriform cortex, dorsolateral prefrontal cortex, lateral orbital prefrontal cortex, medial orbitofrontal cortex, and middle temporal cortex, but extremely low levels in immediately adjacent cortical areas, e.g., the medial prefrontal cortex, subcallosal gyrus, and superior temporal cortex. Within the striatum, CART mRNA was only detected in the nucleus accumbens, primarily in the most medial area. No positive CART mRNA-expressing neurons were found in the dorsal caudate nucleus and putamen. High mRNA expression levels were evident within the bed nucleus of the stria terminalis as well as the amygdala (central, cortical, and medial nuclei). In the hippocampus, intense expression was found within the uncal gyrus and moderate to high levels in the CA3 and polymorphic layer of the dentate gyrus. CART mRNA expression was also detected in the locus coeruleus and dorsal raphe, but no positive labeling was apparent in the substantia nigra. Overall, the most abundant CART mRNA expression levels in the human brain were detected within in the hypothalamus (posterior, paraventricular nucleus, premammillary, tuberomamillary, dorsomedial, arcuate) and the thalamus (mediodorsal, pulvinar, anterior, zona incerta, geniculate). Rat brain specimens were also studied and many similarities to the human CART mRNA expression were evident. However, the most marked species difference was the virtual absence of the CART mRNA in the rat thalamus., (Copyright 2000 Wiley-Liss, Inc.)

The human estrogen receptor (ER) alpha gene is transcribed from multiple promoters, generating mRNA isoforms with unique 5' ends in the untranslated region. In the present study, alternative promoters were shown to regulate the ERalpha gene expression in different neuronal populations of the human brain. By using in situ hybridization histochemistry, the A and B promoters, but not the C promoter, in the ERalpha gene were found to be active in the human forebrain. The mRNA isoform transcribed from the A promoter was expressed in low levels in most of the brain areas where ERalpha mRNA was present. In contrast, the B promoter mRNA isoform was more restricted, localized predominantly in high-expressing ERalpha mRNA regions. The gross anatomical distribution of the different mRNA isoforms analyzed with RT-PCR generally supported the results obtained by the in situ hybridization. Estrogen is known to modulate many different brain functions, such as neuroendocrine events associated with reproduction, mood, and cognition, likely to be mediated by different neuronal populations. Thus, the current findings of alternative ERalpha promoter expression in distinct neuronal populations suggest that multiple promoter usage is a possible mechanism to achieve differentiated regulation of the ERalpha expression, dependent on the cell phenotype and consequently the functions mediated by the specific neuron.

Estrogen has been shown to influence several brain functions as well as the expression of neuropsychiatric diseases. To date, two estrogen receptor (ER) subtypes have been identified, ERalpha and ERbeta. ERalpha messenger ribonucleic acid (mRNA) distribution in the human forebrain was recently characterized, and the highest expression was found in restricted areas of the amygdala and hypothalamus. However, no information exists with regard to ERbeta mRNA distribution in the human brain. To this end, the anatomical distribution pattern of ERbeta mRNA expression in the human forebrain was investigated in the present study. Overall, the ERbeta mRNA hybridization signal was relatively low, but the most abundant ERbeta mRNA areas were the hippocampal formation (primarily the subiculum), claustrum, and cerebral cortex; expression was also present in the subthalamic nucleus and thalamus (ventral lateral nucleus). In contrast to ERalpha (studied on adjacent brain sections), ERbeta mRNA expression was low in the hypothalamus and amygdala. Based on the revealed anatomical distribution of the human ERbeta gene expression, a putative role for ERbeta in the modulation of cognition, memory, and motor functions is suggested.

Acute 17beta-estradiol treatment had been shown to downregulate the 5-HT(1A) receptor mRNA expression in limbic areas of the female rat brain. The aim of the present study was to determine the effects of chronic 17beta-estradiol treatment on 5-HT(1A) receptor mRNA expression and 5-HT(1A) receptor binding in ovariectomized female rats. Using in situ hybridization histochemistry, no alterations were found on the 5-HT(1A) receptor mRNA levels after the estradiol treatment (2 weeks). Radioligand autoradiographic studies using the selective 5-HT(1A) receptor antagonist [(3)H]WAY-100635 revealed reduced receptor binding in the amygdala, hippocampus, perirhinal cortex, and motor cortex after estradiol treatment, whereas no changes were observed in the piriform or retrosplenial cortex. Thus, the previous findings together with the present results indicate that estradiol-induced alterations in 5-HT(1A) receptor mRNA expression appears within hours, but diminishes with chronic treatment when significant changes on the receptor-protein level are apparent. The effects of estradiol treatment on the 5-HT(1A) receptor binding in the limbic areas suggest that estrogen can modulate functions such as learning, memory, cognition, emotional processing, and social behavior. Consequently, estradiol modulation of 5-HT(1A) receptor circuits might be a possible pathway for the estrogen influence in the expression of psychiatric and neurological disorders such as Alzheimer's disease, affective disorders, and schizophrenia., (Copyright 2000 Wiley-Liss, Inc.)

The possible link between estrogen and serotonin (5-HT) in depression was investigated using a genetic animal model of depression, the Flinders Sensitive Line (FSL) rats, in comparison to control Flinders Resistant Line rats. The mRNA levels of the estrogen receptor (ER) alpha and beta subtypes and the 5-HT(1A) and 5-HT(2A) receptors were analyzed in several limbic-related areas of ovariectomized FSL and FRL rats treated with 17beta-estradiol (0.15 microg/g) or vehicle. The FSL animals were shown to express significantly lower levels of the 5-HT(2A) receptor transcripts in the perirhinal cortex, piriform cortex, and medial anterodorsal amygdala and higher levels in the CA 2-3 region of the hippocampus. The only significant difference between the rat lines in ER mRNA expression was found in the medial posterodorsal amygdala, where the FSL rats showed lower ERalpha expression levels. Overall, estradiol treatment increased 5-HT(2A) and decreased 5-HT(1A) receptor mRNA levels in several of the examined regions of both lines. Thus, in many areas, estradiol was found to regulate the 5-HT receptor mRNA expression in the opposite direction to the alterations found in the FSL rats. These findings further support the implication of 5-HT receptors, in particular the 5-HT(2A) subtype, in the etiology of affective disorders. Moreover, the ability of estradiol to regulate the expression of the 5-HT(1A) and 5-HT(2A) receptor genes might account for the reported influence of gonadal hormones in mood and depression.

The putative neurotransmitter peptide CART has been suggested to be involved in the actions of psychostimulants. We analyzed the CART mRNA expression in mesolimbic brain areas of male and ovariectomized 17beta-estradiol- (30 microg) and vehicle-treated female rats. A gender difference was noted in the accumbens shell during basal conditions; male rats expressed higher levels of CART mRNA than both female groups. Following binge cocaine injections (3 x 15 mg/kg), elevated levels were found in the central amygdala of male but not female rats. In the medial accumbens shell CART mRNA was elevated after cocaine, but only in the non-treated females. The present results support a role of mesolimbic CART in psychostimulant drug action.

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

In the present study, we compared neuropeptide Y mRNA expression levels in the prefrontal cortex (Brodmann area 9 and 46) of subjects diagnosed with major depression, bipolar disorder and schizophrenia with those in normal controls without a psychiatric history. No correlation was found regarding neuropeptide Y mRNA expression and postmortem interval, age, gender, hemisphere side, suicide as cause of death, or the history of use of substances such as alcohol, marihuana and cocaine/amphetamine. The only significant alteration found was related to the clinical diagnosis; neuropeptide Y mRNA expression was reduced in the group of bipolar subjects as compared to the controls. Overall, the present results confirm an involvement of neuropeptide Y in affective disorders, and show for the first time a specific association between NPY and bipolar disorder.

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

To investigate the possible link between neuropeptide Y (NPY) and depression, we analyzed NPY and its receptors in different limbic-related regions in the Flinder sensitive line (FSL), a genetic animal model of depression. In situ hybridization histochemistry was used to measure mRNA expression levels of NPY and NPY receptors, Y1 and Y2, in the FSL as compared to the control Flinder resistant Line rats (FRL). In the FSL rats, NPY mRNA expression levels were significantly decreased in the nucleus accumbens and CA regions, but increased in the arcuate nucleus and anterior cingulate cortex. Y1 receptor mRNA expression was decreased in different cortical regions (retrosplenial, anterior cingulate, and occipital) and in the hippocampal dentate gyrus. Y2 mRNA expression levels did not differ between FSL and FRL animals. The effect of the antidepressant drug fluoxetine (a serotonin reuptake inhibitor) in the two rat strains was also studied. There was an increase of the NPY mRNA hybridization signal in the arcuate nucleus of both strains following the antidepressant treatment (10 micromol/kg; daily for 14 days). However, in other brain regions, fluoxetine administration caused a differential effect on the induction of NPY-related genes in the two rat strains: in the CA region and dentate gyrus NPY mRNA expression was increased in the FSL, but decreased in the FRL. In contrast, Y1 mRNA levels tended to be decreased by fluoxetine in the nucleus accumbens of the FSL rats, but increased in the FRL. These findings suggest an involvement of the Y1, but not the Y2, receptor subtype in depressive disorder. Overall, the results appear to sustain the importance of the FSL rats as an animal model of depression in view of the impairment of NPY genes and the ability of fluoxetine treatment to normalize NPY-related gene expression selectively in this strain., (Copyright 1998 Elsevier Science B.V.)

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

In situ hybridization histochemistry was used to investigate acute estrogen effects on serotonin 5HT1A receptor mRNA levels in limbic-related brain areas in the female ovariectomized rat. Acute administration of 17 beta-estradiol (10 micrograms) decreased 5HT1A receptor mRNA expression levels within the medial amygdala (after 2 and 24 h), piriform cortex (after 2 and 24 h), and perirhinal cortex (after 24 h). No changes in 5HT1A mRNA levels were observed in hippocampus or retrosplenial cortex. The findings suggest specific regional effects of estrogen on 5HT functions mediated through regulation of the 5HT1A gene.

In the present study, estrogen receptor (ER)alpha and ER beta genes were found to be differentially expressed in discrete subregions of the rat amygdaloid complex. The amygdala nuclei showing predominant ER alpha mRNA expression included the posterolateral cortical nucleus, amygdala hippocampal area, and lateral dorsolateral nucleus, whereas the amygdala areas with predominant ER beta mRNA expression were the medial anterodorsal and central nuclei. Both ER alpha and ER beta mRNAs were highly expressed in the medial posterodorsal nucleus. In addition to the discrete anatomical expression patterns, there appeared to be a differential regulation by estradiol of the ER alpha and ER beta mRNAs. Two weeks of estradiol (170 microgram total) treatment decreased ER alpha mRNA expression levels in the arcuate, ventromedial hypothalamus, and posterolateral cortical amygdala nucleus, but increased ER beta mRNA in the arcuate. In the medial amygdala nuclei, only ER beta mRNA levels were altered (reduced) by estradiol treatment. These results suggest that estrogen can modulate behaviors and functions mediated by the amygdala and hypothalamus via differentially regulated ER subtypes.

Considerable attention has been given to the association of the D3 dopamine receptor subtype and limbic function based on the abundant localization of D3 receptor sites and mRNA expression in the islands of Calleja and nucleus accumbens in experimental animals. Though most human anatomical studies have focused on the role of D3 receptors in limited brain structures, detailed information about the overall anatomical organization of the D3 receptor in the human brain is still, however, not available. In the current study, we examined the anatomical distribution of D3 receptor mRNA expression at different levels of the human brain in whole hemisphere horizontal cryosections using in situ hybridization. This approach made it possible to establish for the first time the wide and heterogenous expression of the D3 receptor gene throughout the human brain. As expected, the most abundant D3 mRNA expression levels were found in the islands of Calleja and discrete cell cluster populations within the ventral striatum/nucleus accumbens region. High levels were also evident within the dentate gyrus and striate cortex. Low to moderate D3 mRNA expression levels were apparent in most brain areas including all other cortical regions (highest in the anterior cingulate/subcallosal gyrus), caudate nucleus, putamen, anterior and medial thalamic nucleus, mammillary body, amygdala, hippocampal CA region, lateral geniculate body, substantia nigra pars compacta, locus coeruleus, and raphe nuclei. While the current anatomical map of D3 receptor mRNA expression in the human brain does confirm previous reports that D3 receptors may play important roles in limbic-related functions such as emotion and cognition, the findings also suggest other non-limbic functions for D3 mRNA-expressing cell populations such as processing of motor and sensory information.

Many neurobiological functions have been ascribed to the NPY Y1 receptor subtype, but autoradiographic analysis has failed to detect Y1 binding sites in most human brain areas, in contrast to the rat. We examined the regional distribution of Y1 mRNA-containing cells in the post-mortem human brain to clarify if there is a major species difference in terms of the existence of Y1 receptors in the human telencephalon, in particular the striatum and cortex. In situ hybridization experiments revealed widespread distribution of Y1 mRNA signals in all layers of most limbic and neocortical regions, predominantly in layer IV (most cortical regions) and layer VI. The striatum showed moderate Y1 receptor mRNA expression levels with intensely expressing cells localized to the nucleus accumbens. The highest Y1 receptor mRNA expression was apparent within the dentate gyrus, and the lowest in the subiculum, parahippocampal gyrus, cerebellum, and thalamus. In vitro autoradiography using [125I]Leu31Pro34-PYY and [125I]PYY with NPY (13-36) or Leu31Pro34-NPY, confirmed the presence of low Y1-like binding in the human brain despite abundant Y1 mRNA expression. However, using a rat model of the human autopsy process, it was apparent that the inability to reveal high Y1- versus Y2-like receptors in the human brain was related in part to marked reductions of Y1-like, but not Y2-like, receptors within a 4 h post-mortem delay. Altogether, the results indicate that the Y1 receptor gene is abundant in the human brain and this receptor may have important roles in cognitive, limbic and motor function.

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

Dysfunction of dopamine neural systems is hypothesized to underlie neuropsychiatric disorders and psychostimulant drug abuse. At least three dopamine systems have been characterized in the brain-nigrostriatal, mesolimbic, and mesocortical. Abnormalities of nigrostriatal dopamine neurons cause motor impairment leading to Parkinson's disease, whereas dysfunction of mesolimbic and mesocortical dopamine neurons are most implicated in psychotic disorders such as schizophrenia and in drug addition. One of the primary neural sites of action of potent antipsychotic agents and psychostimulant drugs of abuse are dopamine receptors and dopamine transporters which, respectively, mediate the induction and termination of dopamine's actions. Very limited information is, however, available about which particular set of dopaminergic cells in the human brain actually express the genes for these dopamine-specific proteins. In this study, we observed that the dopamine transporter and D2 receptor messenger RNAs are differentially expressed within the human mesencephalon: highest expression in ventral subpopulations of the substantia nigra pars compacta neurons with lowest expression in the mesolimbic/mesocortical ventral tegmental area and retrorubral cell groups. These findings suggest that motor- and limbic-related mesencephalic neurons in the human brain differ in the degree of dopamine transporter and D2 receptor gene expression.

The acute and long-term effects of a single injection of psychomotor stimulants (amphetamine (1.5 mg/kg i.p.), cocaine (30 mg/kg i.p.) and GBR 12909 (10 mg/kg i.p.)) were studied with in situ hybridization histochemistry to assess alterations in the mRNA expression of enkephalin, dynorphin and substance P in the striatum. The greatest alterations on mRNA levels of enkephalin, dynorphin and substance P were observed 2 h following the first administration of each drug compared to that observed following a second challenge injection 14 days later. Of the drugs tested, the dopamine uptake inhibitory agents cocaine and GBR 12909 acutely elevated mRNA levels of all three neuropeptides, while amphetamine elevated mRNA levels of substance P only. A second challenge administration of the stimulants 14 days subsequent to the initial single injection re-elevated the mRNA level of substance P. An overall tolerance is speculated to account for diminution of the enkephalin and dynorphin responses to a challenge injection while a relative sensitization is suggested for the enkephalin response due to a reduction in the baseline level of expression produced by the first injection. The data also show that there are regional variation within the striatum following systemic administration of psychomotor stimulants, with greater elevations in the sensorimotor dorsolateral striatum than in the ventromedial 'limbic' nucleus accumbens region.

The effects of acute and repeated amphetamine administration on mesolimbic dopamine (DA) neurons was assessed by studying DA and cholecystokinin (CCK) release in the nucleus accumbens (Acc), as well as effects on mRNA genes regulating DA and CCK synthesis in ventral tegmental area (VTA) cells in rats. Amphetamine (1.5 mg/kg) markedly increased extracellular levels of DA in the medial Acc (assessed by in vivo microdialysis) in drug-naive animals, about twice the amount released in animals repeatedly administered the drug for the previous 7 days (twice daily). CCK overflow was found to mirror the DA responses in that the very transient elevation of CCK monitored in drug-naive animals was attenuated in those with prior amphetamine use. The attenuation of both DA and CCK overflow in the medial Acc was found to be associated with a decrease in the number of CCK mRNA-positive VTA neurons (assessed by in situ hybridization histochemistry). Although the number of cells expressing CCK mRNA were decreased, the gene expression in those positive CCK and tyrosine hydroxylase mRNA cells in the VTA was significantly increased. The CCK mRNA neurons in the VTA were positively identified as those projecting to the medial Acc by the local perfusion of Fluoro-gold retrograde tracer via microdialysis probes located in the Acc.

The influence of cocaine self-administration on the expression of messenger RNAs for dynorphin, enkephalin and substance P was analyzed in the rat striatum with in situ hybridization histochemistry. Cocaine, an indirect dopamine agonist, was found to differentially affect the levels of mRNA encoding these neuropeptides in different subregions of the striatum. Following a 7 day period of variable free access to cocaine, dynorphin and substance P mRNA levels were elevated throughout the striatum, but the increases were substantially greater in the dorsal striatum than in the nucleus accumbens. Enkephalin mRNA was not significantly altered in the dorsal striatum but was slightly elevated in the nucleus accumbens. These results suggest that cocaine self-administration has differential effects on striatonigral and striatopallidal projection neurons, and that these effects vary in subregions of the striatum.