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The 5-HT2C receptor2 is a prominent serotonin receptor that is uniquely expressed in the central nervous system and has been implicated in a variety of psychiatric diseases. While characterizing the 5-HT2C receptor gene, we observed that the mRNA contains a long 3' untranslated region that binds multiple brain proteins. Two proteins, molecular weights 55 and 58 kDa, were of particular interest because they were detected only in brain regions known to express the 5-HT2C receptor abundantly, namely, the hippocampus and cortex. These proteins bind with high affinity to the 5-HT2C receptor mRNA at its extreme 3' end (Kd = 1.8 nM), and binding can be specifically competed by selected regions of the 3' UTR. Furthermore, binding of the 55 and 58 kDa proteins to the mRNA is directionally specific and shows preference for an AU-rich loop containing 6 to 7 nucleotides. These results suggest the possibility that these two brain specific proteins may play a role in the post-transcriptional regulation of the 5-HT2C receptor, and that post-transcriptional control of 5-HT2C receptor expression may be an important regulatory mechanism which has not been previously reported for this serotonin receptor subtype.

Background: Genetic factors undoubtedly play a major etiologic role in autism, but how it is inherited remains unanswered. The increased incidence in males suggests possible involvement of the X chromosome., Methods: Using data from 38 multiplex families with autism (2 or more autistic siblings), we performed a multipoint sib-pair linkage analysis between autism and 35 microsatellite markers located on the X chromosome. The model included a single parameter, the risk ratio lambda xs (i.e., ratio of risk to siblings compared with the population prevalence), owing to an X-linked gene. Different lambda xs values were assumed and regions of exclusion were established., Results: The entire X chromosome could be excluded for a lambda xs value of 4. The ability to exclude an X-linked gene decreased with smaller lambda xs values, and some positive evidence was obtained with smaller values. A maximum lod score of 1.24 was obtained at locus DXS424 with a lambda xs value of 1.5., Conclusions: We were able to exclude any moderate to strong gene effect causing autism on the X chromosome. Smaller gene effects (lambda xs < 4) could not be excluded, in particular, a gene of small effect located between DXS453 and DXS1001.

Despite strong genetic influences in autism, the true mode of inheritance remains unknown. Sex differences in autism have been described in both singleton and multiplex families [Lord et al., 1982; Volkmar et al., 1993; McLennan et al., 1993; Lord, 1992]: Boys outnumber girls by 3 or 4 to 1, and so a sex-linked mode of transmission must also be considered. The key characteristic of X-linkage is that all sons of affected men are unaffected (no male-to-male transmission). In the present study, which is part of an ongoing linkage project in autism, we describe 77 multiplex autism families, 11 of who are affected cousin or half-sibling families. By using these families, it is possible to trace the path of genetic transmission and observe whether the hypothesis of X-linkage is tenable. Of 11 extended pedigrees from 77 multiplex families, six show male-to-male transmission; in these families, X-linkage can be excluded as the genetic basis for their autism. The data from the other five families are compatible with either an autosomal or an X-linked mode of transmission. The key point to emerge, then, is that autism cannot be exclusively an X-linked disorder; there must be an autosomal mode of transmission at least in some families. Thus we must consider the alternative hypotheses that autism is either entirely autosomal, or it is genetically heterogeneous, involving at least one autosomal locus with genderspecific expression, as well as a possible locus on the X-chromosome.

Previous reports have indicated that, in vivo, the serotonin-2 (5-HT2) receptor is responsive to exogenously administered glucocorticoids. The ability of the glucocorticoid receptor (GR) to influence transcription of the rat 5-HT2 receptor gene was tested in two different experimental paradigms. In both sets of experiments transcription of the 5-HT2 gene was monitored with a promoter-reporter plasmid in which the promoter for the 5-HT2 gene was driving the expression of the firefly luciferase gene. In the first, the 5-HT2 promoter-reporter plasmid was transfected directly into RS1 cells followed by dexamethasone treatment. In the second set of experiments, the cDNA encoding the GR carried on a separate expression vector was cotransfected into CCL-39 or Neuro-2a cells along with the 5-HT2 promoter-reporter plasmid. These cells were then exposed to dexamethasone. In the RS-1 and CCL-39 transfection experiments, the dexamethasone treatment caused an inhibition of transcription of the 5-HT2 promoter, whereas in the Neuro-2a cells, the dexamethasone treatment stimulated transcription from the 5-HT2 promoter. These responses were dependent on the presence of the GR. The effect of the activated GR would seem to be indirect as sequence analysis of the 4.2 kb preceding the site of transcription initiation revealed only an 11/15 nt match to a putative glucocorticoid response element (GRE), and deletion of this sequence did not alter the response to dexamethasone. Sequence analysis revealed a variety of potential response elements for other known transcription factors, including four potential AP-1 response elements.(ABSTRACT TRUNCATED AT 250 WORDS)

5-HT1a receptors in the hippocampus play a critical role in modulating limbic system output. The activity and level of 5-HT1a receptors are modulated by glucocorticoid levels. The present study was undertaken to test the hypothesis that glucocorticoids attenuate the transcriptional activity of the 5-HT1a receptor gene. Using in situ hybridization and RNase protection assays, we observed a substantial increase in 5-HT1a mRNA expression after adrenalectomy in the same hippocampal regions in which 5-HT1a binding sites are increased. This increase in 5-HT1a mRNA expression occurs as early as 1 h after adrenalectomy and precedes the increase in receptor binding sites. Further in situ hybridization analysis showed that 5-HT1a mRNA is increased within individual hippocampal cells after adrenalectomy. Administration of dexamethasone completely prevents the adrenalectomy-induced elevation in hippocampal 5-HT1a receptor mRNA. Nuclear run-on assays showed that the rate of transcription of 5-HT1a mRNA after adrenalectomy increased 70% above the rate from control preparations and could be reduced to basal levels by the administration of dexamethasone. Adrenalectomy did not cause an increase in functional coupling of 5-HT1a receptors to adenylyl cyclase or phospholipase C. These results suggest that transcription of hippocampal 5-HT1a receptor mRNA is under negative regulation by corticosteroid hormones.

The serotonin2 (5-HT2) receptor has been implicated in a number of behavioral and physiological processes. It may also play a role in cellular development and differentiation, and represents a site of action of hallucinogens and certain psychotherapeutic drugs. To better understand the functions and regulation of the 5-HT2 receptor, we have undertaken a series of studies in which we attempted to identify the specific cell types that express the receptor. This was accomplished using a variety of double-labeling strategies with an antibody we raised against the rat 5-HT2 receptor protein. In this review, we recount of some of our previously published findings and present some new data in which we identify subpopulations of cholinergic neurons in the brainstem and gamma-aminobutynic acid (GABA)ergic interneurons in the cortex that express 5-HT2 receptor immunoreactivity. Developmentally, the appearance of 5-HT2 receptor immunoreactivity occurs relatively late in teh ontogeny of the cells in which it is expressed, mostly in the early postnatal period. This argues against a significant role for this receptor in early development, though it may participate in some aspect of terminal differentiation. We discuss the significance of the cell-type-specific and temporal expression of the 5-HT2 receptor in the context of current hypotheses of neuropsychiatric disorders such as schizophrenia.

We have cloned the gene encoding the rat serotonin-2 (5-HT2) receptor. The transcription unit is divided into three exons by two introns. The major 5-HT2 transcript is 5.62 kb in length and contains 1173 bases of 5'-untranslated region (5'-UTR) 1413 bases of open reading frame, and 3033 bases of 3'-UTR. Primer extension demonstrates one strong transcription initiation site 1173 nt from the start codon. Reverse transcriptase-polymerase chain reaction analysis indicates the presence of at least one additional minor site of transcription initiation 1355 nt from the start codon. There are ATA boxes 28 nt 5' to both the major and minor sites of transcription initiation. Functional promoter mapping was carried out in a transient transfection assay. This analysis reveals that there are negative attenuating elements between 2.5 and 2.3 kb from the initiation site and positive elements between 1100 and 200 nt from transcription initiation. Minimal promoter sequences are contained within 200 nt of the major site of transcription initiation. These findings suggest that the expression of the 5-HT2 receptor gene is regulated by a combination of positive and negative elements operating through the minimal promoter.

Exposure of HeLa cells stably expressing cloned human 5-hydroxytryptamine (5-HT)1A receptors (HA7 cells) to the agonist 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT) results in a loss of high-affinity binding sites and a desensitization of receptor-adenylate cyclase coupling, as measured by 5-HT1A-mediated inhibition of forskolin-stimulated adenylate cyclase activity. These responses can also be observed after exposure to forskolin, which activates cyclic AMP-dependent protein kinase A or after treatment with known activators of protein kinase C (PKC) such as phorbol-12-myristate 13-acetate (PMA). The responses elicited by exposure to 8-OH-DPAT or PMA can be blocked completely by inhibitors of PKC and also by 24-hr exposure to PMA. Preincubation of HA7 cells with 8-OH-DPAT also stimulates hydrolysis of inositol phospholipids and the production of arachidonic acid. Inhibition of phospholipase A2 with quinacrine or by removal of extracellular Ca++ blocks the agonist-mediated loss of 5-HT1A receptor binding sites. These data demonstrate that agonist-induced down regulation of the 5-HT1A receptor occurs after stimulation of both the PKC and phospholipase A2 signaling pathways, both of which may activate PKC. The subsequent response is a loss of high-affinity ligand binding sites and functional receptor coupling to adenylate cyclase.

The serotonin-2 (5-HT2) receptor subtype is implicated in several behavioral and physiological processes, and may be the site of action of hallucinogens and certain psychotherapeutic drugs. To better understand the function and regulation of 5-HT2 receptors, it is necessary to determine the specific brain regions and cell types expressing them. By double immunofluorescence using a polyclonal antibody raised against the rat 5-HT2 receptor in conjunction with an antibody against choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine, we have shown that cholinergic neurons in the rat laterodorsal and pedunculopontine tegmental nuclei express 5-HT2 receptors. In contrast, there was little co-localization of 5-HT2 and ChAT immunoreactivity in neurons of the basal forebrain or striatum, even though the 5-HT2- and ChAT-positive cells in these regions overlapped extensively. These findings are discussed in relation to the potential interaction between cholinergic and serotonergic systems in sleep regulation, hallucinogenesis and the pathophysiology of neuropsychiatric disorders.

In this study, we investigated the regional and temporal emergence of 5-hydroxytryptamine2 receptor immunoreactivity in the developing rat brain. In a qualitative immunocytochemical analysis using an antibody against the rat 5-hydroxytryptamine2 receptor protein, we visualized cells expressing the receptor in the pontine tegmentum, caudate nucleus, basal forebrain, hippocampus and neocortex of developing rats. Three potentially important periods in the developmental regulation of 5-hydroxytryptamine2 receptors were identified: the time of onset, a period of accelerated expression and hyper-elaboration, and a period of regression. In general, the onset of 5-hydroxytryptamine2 receptor immunoreactivity occurred relatively late in the ontogeny of cells in these regions, in the late prenatal and early postnatal periods. Following the perinatal onset of receptor expression, there was a rapid increase in the number of immunoreactive neurons during the first week after birth. In neocortex, there appeared to be a relative over-expression of the receptor, with an elevated density and hyper-elaboration of immunopositive neurons relative to the adult, reaching a peak at the end of the second week. There was then a gradual decrease in both the density and morphological complexity of cortical 5-hydroxytryptamine2-labelled neurons, until the adult pattern of expression was achieved at about four weeks of age. In all areas studied, cells positive for the 5-hydroxytryptamine2 receptor were first detected within the regions in which they would ultimately reside, and after the known periods of cell proliferation for these regions. These observations would argue against a role for the 5-hydroxytryptamine2 receptor as a transducer of the early developmental influences of serotonin in the central nervous system, but leave open the possibility that the receptor may participate in regulating some aspect of terminal differentiation or late maturation of the neurons on which it is found. The identification of important developmental periods in the ontogeny of 5-hydroxytryptamine2 receptors suggests time-points at which events that disrupt the normal ontogenetic pattern of expression could produce long-lasting effects on central serotonergic neurotransmission.

A synthetic peptide was used to generate antibodies against the rat serotonin-2 (5-HT2) receptor. The peptide corresponds to a unique sequence from the N-terminal extracellular portion of the receptor protein (antibody = Ab 5HT2-N). This peptide was chosen based on its theoretical antigenic index and for specificity to the 5-HT2 receptor. In dot blot analysis, antisera detected 2 ng-2 micrograms of synthetic peptide at dilutions of 1/200-1/20,000. COS-7 cells transiently transfected with a eukaryotic expression vector containing the 5-HT2 cDNA displayed intense immunoreactivity with crude and affinity-purified Ab 5HT2-N. In contrast, no immunoreactivity was seen in control experiments when: (1) non-transfected or vector transfected COS-7 cells were used; (2) pre-immune sera was substituted for primary antisera; (3) primary antisera was omitted; or (4) antiserum was pre-adsorbed to 10 microM synthetic peptide. Immunohistochemical analysis of sections of perfused rat brain revealed intense immunolabelling of a subset of neurons in regions of the ventral forebrain, dorsal hippocampus, striatum, cerebral cortex, and laterodorsal tegmental nucleus (LDT). An especially dense band of small cells was seen in layer 2 of pyriform cortex. There was a very high concentration of labelled cells in the laterodorsal tegmental nucleus. In situ hybridization histochemistry with a 5-HT2 antisense cRNA riboprobe showed a pattern of hybridization in forebrain similar to the pattern of immunolabelling with Ab 5HT2-N. Western blot analysis of proteins extracted from the LDT revealed a single protein species reacting with the antibody. This reactivity is not present in the pre-immune sera and is blocked by the synthetic antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin2 receptors have been implicated in a variety of behavioral and physiological processes, as well as a number of neuropsychiatric disorders. To specify the brain regions and specific cell types possessing serotonin2 receptors, we conducted an immunocytochemical study of the rat brain using a polyclonal serotonin2 receptor antibody. Perfusion-fixed rat brain sections were processed for immunocytochemistry and reactivity was visualized using an immunoperoxidase reaction. Numerous small, round neurons were heavily labeled in the granular and periglomerular regions of the olfactory bulb. Heavy labeling of medium-sized multipolar and bipolar neurons was also seen in olfactory regions of the ventral forebrain, including the anterior olfactory nucleus and olfactory tubercle. Other regions of the basal forebrain exhibiting high levels of immunoreactivity were the nucleus accumbens, ventral pallidum, Islands of Calleja, fundus striatum and endopyriform nucleus. Immunoreactive neurons were also seen in the lateral amygdala. A dense band of small, round cells was stained in layer 2 of pyriform cortex. In neocortex, a very sparse and even distribution of bipolar and multipolar neurons was seen throughout layers II-VI. A much more faintly labeled population of oval cells was observed in the deep layer of retrosplenial and posterior cingulate cortex, and in the granular layer of somatosensory frontoparietal cortex. A moderate number of medium bipolar and multipolar cells were scattered throughout the neostriatum, and a moderate number of pyramidal and pyramidal-like cells were seen in the CA fields of the hippocampus. Diencephalic areas showing immunolabeling included the medial habenula and anterior pretectal nucleus, with less labeling in the ventral lateral geniculate. In the hindbrain, two dense populations of large multipolar cells were heavily labeled in the pedunculopontine and laterodorsal tegmental nuclei, with lesser labeling in the periaqueductal gray, superior colliculus, spinal trigeminal nucleus and nucleus of the solitary tract. Based on the distribution, localization and morphology of immunoreactive neurons in these regions, we hypothesize that subpopulations of serotonin2 containing cells may be GABAergic interneurons or cholinergic neurons. Further, the observed distribution suggests that the physiological effects of serotonin acting through serotonin2 receptors are mediated by a relatively small number of cells in the brain. These observations may have strong functional implications for the pharmacological treatment of certain neuropsychiatric disorders.

Autism is a syndrome with multiple etiologies, as is made clear both by the evidence of neurobiological research and by the catalog of disorders that present with autistic behaviors. What remains unclear are the specific neuropathological mechanisms that produce autistic behaviors; for example, is there a common neuroanatomic pathology for all cases of autism, or can autistic behaviors emerge from different pathological sequences within the brain? Although it is premature to generalize, neuropathological studies appear to have identified common abnormalities in the cerebellum and limbic system of at least five autistic subjects. These subjects, with variable levels of mental retardation, demonstrated marked Purkinje cell loss in the cerebellar hemispheres, together with retained fetal neuronal circuitry in cerebellar nuclei and increased neuronal packing in specific regions of the limbic system, amygdala, and hippocampus. The architecture of the cerebral cortex was not affected. Although our knowledge of brain functioning is incomplete, alterations of the kind noted in the cerebellum and limbic system could reasonably produce autistic behaviors. For more detail, readers are directed to a review of cerebellar contributions to higher functions by Schmahmann (1991). Neuroimaging studies allow less resolution of brain structure than do neuroanatomic studies, and the reported findings from neuroimaging are somewhat contradictory. However, a number of investigators have reported structural abnormalities in ventricle size and cerebral hemispheric asymmetry using CT. MRI, which offers greater resolution, has uncovered some consistent findings, along with a variety of nonspecific abnormalities. Common abnormalities include reduced volume of cerebellar hemispheres and vermal lobules--findings not inconsistent with the above-mentioned neuropathological defects. It is also interesting to note that individuals with fragile X syndrome have similar cerebellar findings. PET and NMR studies of autism are at a preliminary stage, but these methodologies allow insight into the functioning of the brain, rather than simply brain anatomy. Recent PET studies indicating decreased association between paired regions of the brains of autistic subjects are of interest, particularly if they can be confirmed and refined by additional studies. Neurophysiological studies also offer insight into brain function, but are subject to numerous methodological criticisms. Nevertheless, recent reports of diminished P300 waves and absent NC components in autistic subjects seem to indicate fundamental defects in attention and secondary processing, which could help explain the self-stimulatory behaviors often seen in autism. The disturbances in brain development associated with autism can be produced in a number of ways, and at different times during development of the nervous system.(ABSTRACT TRUNCATED AT 400 WORDS)

The past decade has seen an explosive growth in our understanding of neurotransmitter receptors and the roles they play in neurotransmission. This is particularly true of the serotonin receptors where a synergy of basic science and clinical research has not only produced a deeper understanding of serotonin receptors and their actions but also resulted in the availability of new therapeutic agents useful for treating a number of psychiatric illnesses. This chapter details our current knowledge of the major subtypes of the serotonin receptor, including recent advances in the molecular biology, pharmacology, biochemistry, and clinical correlates of these receptors.

We determined the affinities of clozapine and 21 other typical and atypical antipsychotic agents for the cloned 5-hydroxytryptamine-1C (5-HT1C) receptor. For these studies, 5-HT1C receptors were transiently expressed in COS-7 cells using the vector pSVK3-5HT1C. We discovered that clozapine and several other putative typical and atypical antipsychotic agents (loxapine greater than tiosperone greater than SCH23390 greater than fluperlapine greater than rilapine greater than chlorpromazine) had relatively high affinities (7-30 nM) for the cloned 5-HT1C receptor. Other antipsychotic agents (risperidone greater than tenilapine greater than mesoridazine greater than thioridazine greater than cis-fluphenthixol) had intermediate affinities (30-100 nM), whereas many other antipsychotics (fluphenazine greater than spiperone greater than amperozide greater than melperone greater than thiothixene greater than haloperidol, metoclopramide, pimozide, domperidone, sulpiride) had low affinities (greater than 500 nM) for the cloned 5-HT1C receptor. The results indicate that although several putative atypical antipsychotic agents have high affinities for the cloned rat 5-HT1C receptor, the spectrum of drug binding does not correlate with the atypical nature of these compounds.

In order to isolate genes involved in development of the mammalian telencephalon we employed an efficient cDNA library procedure. By subtracting an adult mouse telencephalic cDNA library from an embryonic day 15 (E15) mouse telencephalic cDNA library we generated two subtracted libraries (ES1 and ES2). We estimate that ES1 contains between 200 and 600 different cDNA clones, which approximates the number of genes that are preferentially expressed in the E15 telencephalon, compared to the adult telencephalon. Northern analysis of 20 different cDNA clones shows that 14 of these are expressed at least 5-fold more in the E15 telencephalon than the adult telencephalon. Limited sequencing of the 14 differentially expressed clones reveals that 10 have no significant identity to sequences in GenBank and EMBL databases, whereas the other 4 have significant sequence identity to vimentin, histone 3.3, topoisomerase I and the B2 repeat element. In situ hybridization using one of the differentially expressed cDNAs, TES-1, demonstrates that it is transiently expressed in the anlage of the basal ganglia. In situ hybridization with another differentially expressed cDNA clone, TES-4, shows that it is specifically expressed in differentiating cells of the neural axis with a distinctive rostral-caudal temporal pattern. These findings, and the methods that we have developed, provide a framework for future investigations of the genetic control of telencephalon development.

A complementary DNA, Tes-1, of a novel homeodomain protein has been cloned, and its pattern of expression has been characterized. It is a structural homolog of Distal-less, a homeodomain-encoding gene in D. melanogaster. Its expression is developmentally regulated and is limited to structures in the head. Within the central nervous system of the midgestation mouse embryo, it is expressed exclusively in the ventral forebrain. It is likely that Tes-1 plays a regulatory role in the development of this complex neural structure.

Rats were injected with 15 mg/kg (i.p.) mianserin or vehicle (saline) for 4, 10 or 21 days and 5-HT2 receptor binding and mRNA levels measured. Treatment with mianserin induced a substantial decrease in 5-HT2 radioligand binding (44-59% decrease; P less than 0.05 vs. control). No changes in the amount of 5-HT2 or, as a control, beta-actin mRNAs were found (P greater than 0.05 vs. control). These results indicate that mianserin decreases 5-HT2 radioligand binding without altering steady-state 5-HT2 mRNA levels.

We investigated the regulation of 5-HT2 and 5-HT1c receptors and their corresponding mRNAs during rat brain development. This study showed that 5-HT2 and 5-HT1c receptors increased markedly during ontogeny. 5-HT2 receptors, measured with [3H]ketanserin or [125I]lysergic acid diethylamide binding, increased 8-fold between embryonic day 17 (E17) and postnatal day 13 (P13). 5-HT2 receptor mRNA levels, quantified by probing Northern blots of total RNA with a synthetic oligonucleotide cDNA probe, multiplied 13-fold between E17 and P5. The developmental pattern of 5-HT2 receptor and mRNA expression appeared to correlate with the serotonergic hyperinnervation of the cortex which occurs between P2 and P17. 5-HT1c receptors, measured with [125I]lysergic acid diethylamide under site-specific conditions, increased 2-fold between E17 and P27, 5-HT1c mRNA increased 5-fold between E17 and P27. Interestingly, the developmentally induced variations in 5-HT1c receptors did not precisely correlate with mRNA alterations. Further study of the factors responsible for these alterations could help to explain the molecular and biochemical mechanisms responsible for modulating receptor levels in vivo.

We review recent studies on the inheritance of affective disorders and schizophrenia. Both groups of diseases have substantial genetic and non-genetic components, and their inheritance does not fit simple Mendelian models. Linkage studies implicate the Xp28 and 11p15 regions as potential disease loci in affective illness, and the 5q11-q13 region in schizophrenia. These conclusions are highly controversial, however, and must be viewed as extremely tentative.

Both the 5-hydroxytryptamine2 (5-HT2) and 5-HT1c receptors may be regulated by a large number of endogenous and exogenous factors. The 5-HT2 receptors, for example, may be decreased by acute and chronic treatment with many antipsychotic agents, some antidepressants, and receptor-specific agonists. Similar to the 5-HT2 receptor, the 5-HT1c receptors may be decreased by acute and chronic treatment with the antidepressant mianserin. The 5-HT2 receptors appear to increase during perinatal development and are reported to be elevated in the frontal cortex and hippocampus of victims of suicide; the 5-HT1c receptors display supersensitivity following ablation of serotonergic terminals with 5,7-dihydroxytryptamine. The molecular details responsible for these changes remain unknown, though with the recent cloning of the cDNAs for the 5-HT2 and 5-HT1c receptors the occasion is particularly favorable for mechanistic studies aimed at determining how these alterations occur. Preliminary information suggests that developmentally induced alterations in 5-HT2 and 5-HT1c receptors may be due to transcriptional regulation while changes caused by mianserin treatment might be due to posttranslational processes (e.g., proteolysis, internalization, phosphorylation, covalent alterations). Insights into the molecular means by which 5-HT receptors are regulated could have profound influences on our understanding of pharmacologic, developmental, and psychopathologic processes.

We describe a subtractive hybridization protocol which is designed to permit subtractions between cDNA libraries. The method uses single-stranded phagemids with directional inserts as both the driver and the target. We modified the M13 phagemid vector pBluescript for the directional cDNA cloning and subtractive hybridization. Two simplified methods for efficient construction of directional cDNA libraries are also described. Using a model system, we found that one round of subtractive hybridization results in a 5,000-fold specific subtraction of abundant molecules. We used two methods to quantify the efficiency and verify the specificity of the subtraction. In order to obtain these subtraction efficiencies, it was necessary to develop a method to purify the single-stranded DNA to homogeneity. The single-stranded purification involved using potassium iodide (KI) density centrifugation, restriction endonuclease digestion and phenol extraction in the presence of magnesium. We describe the several advantages of using directional inserts for the subtraction procedure.

The serotonin 1b (5-HT1b) receptor is thought to mediate both pre- and postsynaptic actions of serotonin. Until recently 5-HT1b sites were thought to be present only in rodent brain. We now report the presence of high-affinity [125I]iodocyanopindolol [( 125I] ICYP) binding sites in the mouse renal medulla with properties identical to those of brain 5-HT1b receptors. In vitro receptor autoradiography demonstrates that [125I]ICYP binding is highly localized to the outer stripe of the renal medulla. Association and dissociation kinetics, saturation analysis and competition displacement analyses indicate that renal medullary [125I]ICYP binding sites exhibit identical properties with brain 5-HT1b receptors. Incubation of renal medullary or brain membranes with guanylimidodiphosphate results in a decreased affinity of 5-HT1b sites for 5-HT and [125I]ICYP; this can be reversed by the addition of a purified mixture of G proteins (Gi/Go). Treatment of brain or kidney membranes with N-ethylmaleimide results in a decrease in 5-HT1b binding which can also be restored by reconstitution with purified G proteins. Adenylyl cyclase from renal medullary homogenates or minces can be stimulated more than 3-fold by forskolin and attenuated by 5-HT. These results indicate that mouse kidney contains high-affinity 5-HT1b receptors with identical properties to those found in brain. These are localized in the outer stripe of the renal medulla and are functionally coupled to adenylyl cyclase inhibitor (Gi) G-proteins.

The authors found that platelet serotonin concentrations were significantly elevated in patients with chronic schizophrenia and in patients with bipolar major depressive disorder. High-affinity serotonin uptake was significantly reduced only in patients with bipolar major depressive disorder. Thrombin-induced release of serotonin from platelets in any patient group was not different from that of normal control subjects. Platelet serotonin storage in chronic schizophrenic patients was also not different from that in normal control subjects. These platelet findings could not be explained by age, sex, or medication variables. The authors suggest that the pharmacodynamics of platelet serotonin may be different in chronic schizophrenia than in bipolar major depressive disorder.

Rat pineal hydroxyindole-O-methyltransferase is controlled similarly to adrenal medullary phenylethanolamine N-methyltransferase. S-adenosylmethionine (SAM), the in vivo cofactor utilized by the enzyme to convert N-acetylserotonin to melatonin, protects this methyltransferase against tryptic proteolysis in vitro. Furthermore, in vivo studies suggest that the nucleoside itself is controlled by glucocorticoids. Hypophysectomy decreases hydroxyindole-O-methyltransferase levels as compared with control animals, while dexamethasone and SAM administration restore enzyme levels toward control values. In vitro proteolytic studies further demonstrate that, although N-acetylserotonin does not stabilize the enzyme against trypsinization, this substrate acts synergistically with SAM to confer greater stabilization than observed with SAM alone.

[3H]Dihydroergotamine (DE) labels a population of binding sites in rat brain membranes with an affinity of approximately 70 pM in both hippocampus (maximal binding at saturation [Bmax] = 340 fmol/mg of protein) and cerebral cortex (Bmax = 250 fmol/mg of protein). Specific binding typically comprises about 97% of total binding at the Kd of the radioligand when nonspecific binding is determined in the presence of 100 nM unlabeled DE. Association kinetics at 37 degrees C are consistent with a uniform association rate constant for all sites labeled. Specific binding is completely reversible with addition of excess unlabeled DE, but dissociation does not proceed with simple first-order kinetics, suggesting the presence of more than one discrete binding site. Competition studies with selective drugs reveal alpha adrenergic, 5-HT1A and 5-HT1B components of [3H]DE specific binding. When phentolamine (500 nM) is included to block alpha receptors and DPAT (100 nM) or spiroxatrine (500 nM) is included to block 5-HT1A receptors, specific binding is exclusively to sites with drug affinities characteristic of 5-HT1B receptors. Under these 5-HT1B-selective conditions, [3H]DE binding is about 90% specific, with a Kd of about 50 to 60 pM and a Bmax of 96 fmol/mg of protein in hippocampus and 77 fmol/mg of protein in cortex. [3H]DE binding to 5-HT1B sites is very slowly dissociable, with a T1/2 of greater than 2 h at 37 degrees C. 5-HT1B antagonists and DE itself yield competition curves at [3H]DE-labeled 5-HT1B sites that are adequately fit assuming a single site in nonlinear regression analysis. Competition by the agonists 5-HT and RU 24969 at 5-HT1B sites are often best described by two site fits. Addition of 100 microM guanylyl 5'-imidodiphosphate appears to convert nearly all 5-HT1B sites to those having low affinity for agonists while having a much smaller effect on the binding of [3H]DE. This suggests that the 5-HT1B site exists in two interconvertable agonist affinity states and is yet another member of the G-protein-linked receptor family. In agreement with previous studies using other radioligands, no 5-HT1B sites can be detected in bovine, porcine or human hippocampus membranes.

We and others have shown that in vitro tryptoline (tetrahydro-beta-carboline) formation accounts for the apparent-N-methylating activity of a brain enzymatic preparation using 5-methyltetrahydrofolic acid (5-MTHF) as a cofactor and tryptamines or catecholamines as substrates. This paper demonstrates that N5,10-methylenetetrahydrofolate reductase (methylene reductase) is responsible for this in vitro tryptoline formation with human brain enzymatic preparations. Others have described a folate-responsive psychosis which was associated with markedly reduced methylene reductase activity. Therefore, we also have examined this enzymatic activity in autopsied brains from chronic schizophrenics and controls. There were no statistically significant differences between activities for schizophrenics and controls in the six brain regions studied. Thus, although it is possible that some subgroup of schizophrenics may be characterized by abnormal methylene reductase activity, there does not appear to be a general association between the two.

This report describes the pharmacologic characterization of [3H]serotonin binding activity solubilized from bovine frontal cortical membranes. The ability of a number of serotonin (5-HT) and lysergic acid diethylamide (LSD) analogs to compete with [3H]serotonin and D-[3H]LSD for binding to membrane and solubilized 5-HT1 sites has been investigated. The results indicate that the solubilized binding site is probably of the 5-HT1B type. Fifteen of the 21 compounds tested exhibit nearly identical affinity for membrane or solubilized 5-HT1 binding sites. However, some important differences were observed, and these may help elucidate the molecular structure of the binding site. In particular, some N-substituted tryptamine analogs show a markedly lower affinity for solubilized 5-HT1 sites compared to their binding to intact membranes. Further, the solubilized site does not distinguish stereoisomers of LSD: both D- and L-LSD bind to solubilized 5-HT1 sites with comparable high affinities, whereas D-LSD has a markedly higher affinity for the membrane 5-HT1 site. Methiothepin, which binds to the 5-HT1 site primarily through its amine groups, has virtually no affinity for the solubilized receptor, whereas it is quite potent at competing for [3H]serotonin binding to membrane sites. These observations lead to the conclusions that in bovine cortical membranes, the 5-HT1 site contains both indole and amine attachment sites. After solubilization, the indole attachment site retains its binding properties, but the amine attachment site has been significantly altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Brains from breed and age-matched canines stored at -80 degrees C for between 3 and 44 months showed a time-dependent decline in the concentration of norepinephrine in the amygdala and dopamine and norepinephrine in the caudate. No changes were seen in the density or ligand affinities of prazosin or spiperone binding sites in the same areas nor were there changes in quinuclidinyl benzylate binding sites in the frontal cortex. The changes in dopamine concentrations in the caudate were not accompanied by changes in the concentrations of dopamine metabolites. The chromatograms from which the dopamine and norepinephrine concentrations were estimated contained several unidentified, amperometrically detectable, extraneous peaks which increased in size in the older tissue samples. These results suggest that the decline in dopamine and norepinephrine concentrations was not the result of enzymatic breakdown, but probably the result of chemical decomposition. These findings have significance for the measurement of dopamine and norepinephrine concentrations in autopsied brains kept frozen in storage.

Concentrations of dopamine D2 receptors in discrete brain areas differed significantly between dogs with the genetically transmitted form of narcolepsy, and age- and breed-matched controls. D2 receptors were assayed and quantified with Scatchard analysis using [3H]spiperone. Receptor densities in the nucleus accumbens, rostral caudate, and amygdala were consistently higher in narcoleptic animals. In amygdala, dopamine receptor abnormalities were associated with elevated dopamine and 3,4-dihydroxyphenylacetic acid concentrations, but no change in 3-methoxytyramine or homovanillic acid concentrations. These data indicate mesolimbic system involvement in canine narcolepsy and point to impaired dopamine release as a possible etiologic factor.

This paper attempts to view the autistic syndrome in the context of a disorder of brain development. The authors review some of the known or suspected causes of the autistic syndrome: maternal rubella, metabolic diseases, and heredity. Some basic principles of cellular neuroanatomy and chemical neurotransmission are sketched. The stages of human brain development from neurulation through histogenesis, cell migration, and elaboration of dendritic trees and axonal projections are described. The authors conclude that there are a limited number of developmental loci that could be disrupted and lead to the autistic syndrome, and that these most probably occur in the end stages of neuronal development, after the migrating neurons have reached their final place in the brain and have begun to elaborate communicative processes. Finally, the authors speculate on how neurochemical disturbances might alter end stage neuronal differentiation leading to the pathology of infantile autism.

The present study examines the possible involvement of alpha-adrenergic receptors in catecholamine-stimulated cAMP production in intact slices of rat striatum. Norepinephrine (NE) produces a greater stimulation of cAMP levels than does the beta-adrenergic agonist, isoproterenol (ISO), and the NE response is inhibited by both the beta-adrenergic antagonist, propranolol, and the alpha-adrenergic antagonist, phentolamine. The alpha-adrenergic agonist, 6-fluoronorepinephrine (6-FNE), has little or no effect on basal cAMP levels; however, 6-FNE causes a marked potentiation of the cAMP response to ISO. Hence, NE stimulation of cAMP levels in striatal slices appears to involve a synergistic interaction between alpha- and beta-adrenergic receptors. alpha-Receptors also potentiate adenosine stimulation of cAMP levels in striatal slices. However, in contrast to results previously reported in cerebral cortical slices, the alpha-adrenergic component of the NE response in striatal slices is not dependent on endogenous adenosine. Finally, 6-FNE interactions with adenylate cyclase in striatal homogenates differ from those observed in the slice preparation. In homogenates, 6-FNE appears to directly stimulate adenylate cyclase through a D-1 receptor. D-1 receptor involvement in catecholamine responses in the striatal slice preparation, on the other hand, appears to be minimal.

RDS-127, in a dose-related manner, induced seminal emission ex copula after intracerebroventricular (i.c.v.) administration. In mating tests initiated 6 min after i.c.v. administration, RDS-127 induced decreases in ejaculation latency and intromission frequency, with some rats ejaculating on the initial intromission. Additionally, penile reflexes were eliminated by 150 micrograms and 600 micrograms, but not by an intermediate dose. In in vitro radioligand binding studies, RDS-127 potently displaced [3H]DPAT binding to 5-HT1A sites in rat cortex (Ki = 14 +/- 4 nM) and was only moderately effective in displacing [3H]spiperone binding to dopaminergic D2 sites in rat striatum. RDS-127 was essentially ineffective at 5-HT1B sites labeled by [3H]5-HT in rat striatum (Ki = 13 000 +/- 4 000 nM). These data demonstrate that centrally administered RDS-127 mimics the previously reported alterations in sexual behavior after systemic treatment and that RDS-127 is a high affinity 5-HT1A agent with low affinity at the 5-HT1B binding site.