Brain development: pervasive developmental disorders and infantile autism.
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Autistic Disorder↗
Biomedical subjects
R D Ciaranello
Publications and source records attributed to R D Ciaranello.
Isolation and characterization of a library of cDNA clones that are preferentially expressed in the embryonic telencephalon.
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.
Animals↗
A partial cDNA sequence of the Dlx-2 cDNA.
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Amino Acid Sequence↗
Molecular biology of serotonin receptors.
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.
Anxiety Disorders↗
Binding of typical and atypical antipsychotic agents to transiently expressed 5-HT1C 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.
Antipsychotic Agents↗
Chronic mianserin treatment decreases 5-HT2 receptor binding without altering 5-HT2 receptor mRNA levels.
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.
Animals↗
Developmental regulation of 5-HT2 and 5-HT1c mRNA and receptor 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.
Animals↗
Isolation and characterization of a novel cDNA clone encoding a homeodomain that is developmentally regulated in the ventral forebrain.
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.
Amino Acid Sequence↗
Genetics of major psychiatric disorders.
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.
Genetic Linkage↗
Subtractive hybridization system using single-stranded phagemids with directional inserts.
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.
Bacteriophages↗
G-protein-linked serotonin receptors in mouse kidney exhibit identical properties to 5-HT1b receptors in brain.
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.
Alkylation↗
G protein dependent alterations in [125I]iodocyanopindolol and +/- cyanopindolol binding at 5-HT1B binding sites in rat brain membranes.
Several manipulations that affect G protein/receptor coupling also alter the binding of [125I]iodocyanopindolol ([125I]ICYP) and [corrected] +/- cyanopindolol (+/- CYP) to rat brain 5-HT1B binding sites in radioligand binding assays. Inclusion of 5 mM MgSO4 in these assays results in a small but significant increase in the affinity of [125I]ICYP (from KD = 0.046 nM to KD = 0.037 nM). In contrast, 100 microM Gpp(NH)p, GTP, or GDP reduce [125I]ICYP affinity (KD = 0.056 nM with GTP) while ATP and GMP are less effective. +/- CYP affinity for 5-HT1B sites labeled by [3H]dihydroergotamine [( 3H]DE) also displays a small but significant reduction (from Ki = 1.4 nM to Ki = 3.5nM) by the inclusion of 100 microM GTP. Pre-treatment of the brain membranes with N-ethylmaleimide (NEM) in concentrations known to inactivate many G proteins reduces 5-HT1B specific binding of [125I]ICYP. The NEM induced reduction in [125I]ICYP binding can be reversed by reconstitution with purified exogenous G proteins (Go and Gi), demonstrating directly that high affinity binding of [125I]ICYP to 5-HT1B sites is dependent on G proteins. The effects of Mg2+ ion, guanine nucleotides, NEM and G protein reconstitution on [125I]ICYP and +/- CYP binding are all hallmarks of agonist binding to G protein linked receptors. The effect of GTP, however, is quantitatively much less for the binding of these pindolol derivatives than for the binding of 5-HT, a presumed full agonist at 5-HT1B sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Influence of freezer storage time on cerebral biogenic amine and metabolite concentrations and receptor ligand binding characteristics.
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.
Amygdala↗
Neurotransmitter enzyme and receptor regulation: a look back.
1. This report is a tribute to Dr. Julius Axelrod, in whose laboratory the author worked as a research associate from 1971-1974. 2. Work on the regulation of dopamine beta-hydroxylase and phenylethanolamine N-methyl transferase is reviewed, particularly studies showing how transynaptic and hormonal factors regulate enzyme synthesis and degradation respectively. 3. Work taking place in the author's laboratory is reviewed with respect to the influence Dr. Axelrod had on the author and his subsequent work.
Animals↗
Differential inactivation and G protein reconstitution of subtypes of [3H]5-hydroxytryptamine binding sites in brain.
The sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide (NEM) inactivate high affinity [3H]serotonin [( 3H]5-HT) binding to bovine and rat brain membranes in a concentration-dependent manner. In both species, 15-25% of total specific high affinity [3H]5-HT binding is relatively insensitive to NEM. This study examines the NEM sensitivity of the various high affinity [3H]5-HT binding subtypes, using selective ligands, tissues, and pharmacological masks to study each subtype. Reconstitution of NEM-inactivated binding by addition of GTP-binding proteins (G proteins, Gi and Go) is also described. Agonist binding to 5-HT1A, 5-HT1B, and 5-HT1D sites in rat brain and to 5-HT1A and 5-HT1D sites in bovine brain is sensitive to NEM. Binding of [3H]dihydroergotamine and [125I]iodocyanopindolol, both of which are weak partial agonists to 5-HT1B sites is relatively insensitive to NEM. Binding of [3H]5-HT to 5-HT1C sites in rat and bovine brain and choroid plexus is relatively insensitive to NEM. In the presence of spiperone to mask binding of 5-HT2 sites, binding of antagonist [( 3H]mesulergine) to 5-HT1C sites is also insensitive to NEM. Likewise, binding of the agonist [3H]4-bromo-2,5-dimethoxyphenylisopropylamine and of the antagonist [3H]ketanserin to 5-HT2 sites is not inhibited by NEM treatment of membranes. These findings suggest that agonist binding to 5-HT1A, 5-HT1B, and 5-HT1D sites is sensitive to NEM alkylation. Binding of neither agonist nor antagonist to 5-HT1C and 5-HT2 sites is sensitive to NEM. Inability of high concentrations of a variety of ligands to protect the sensitive binding sites against NEM inactivation indicates that the critical sulfhydryl group(s) are not located in the ligand binding domain of the NEM-sensitive binding sites. When membranes are treated with NEM, displacement of [125I]iodocyanopindolol by 5-HT is no longer sensitive to 5'-guanylyl imidodiphosphate (Gpp(NH)p). Gpp(NH)p sensitivity of agonist displacement of 5-HT1B binding to NEM-treated membranes is restored by addition of purified guanine nucleotide binding proteins (Gi plus Go). In addition, NEM-inactivated binding to 5-HT1A and 5-HT1D sites can be restored by addition of Gi plus Go. These data suggest that NEM exerts its effects on 5-HT1A, 5-HT1B, and 5-HT1D binding sites by inactivating the G protein(s) associated with the 5-HT receptor subtypes.
Animals↗
Characterization of the isozymes of bovine adrenal medullary phenylethanolamine N-methyltransferase.
Bovine adrenal medullary phenylethanolamine N-methyltransferase (EC 2.1.1.28) has been purified to apparent homogeneity. The enzymatically active monomer has a relative molecular weight of 30,000 and can be separated into at least four active charged isozymes. These isozymes, designated PNMT-1, PNMT-2, PNMT-3 and PNMT-4, have isoelectric points of 5.1, 5.2, 5.3 and 5.4, respectively. Kinetic parameters have been determined for each isozyme. The Kms for phenylethanolamine range from 11.9 to 45.9 microM; the Kms for S-adenosylmethionine range from 1.13 to 1.47 microM; and the Kis for the competitive inhibitor, S-adenosylhomocysteine, range from 0.12 to 0.22 microM. For isozymes PNMT-1 and PNMT-4, and Kms for S-adenosylhomocysteine are not significantly different. Vmax values for all of the isozymes do not change significantly in the presence of S-adenosylhomocysteine. Treatment of the purified isozymes with various endo- and exoglycosidases does not alter electrophoretic mobility. Hence, carbohydrate substitution must be minimal. No high mannan, complex sugars or terminal N-acetylglucosamine residues are present. The absence of carbohydrate is further supported by the inability of Schiff-periodic acid to stain the protein. Limited thermolysin digests of each isozyme show distinct peptide cleavage products. In conjunction with the kinetic and glycosylation data, this suggests that the isozymes of phenylethanolamine N-methyltransferase may be primary structural variants.
Adrenal Medulla↗
Brain dopamine receptor levels elevated in canine narcolepsy.
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.
3,4-Dihydroxyphenylacetic Acid↗
Multiple high-affinity [3H]serotonin binding sites in human frontal cortex.
High-affinity [3H]serotonin (5-hydroxytryptamine, 5-HT) binding sites from human frontal cortex can be divided into at least 3 pharmacological subtypes (5-HT1A, 5-HT1B and 5-HT3) based on affinity for [3H]serotonin and spiperone. All 3 sites are solubilized by 3% Triton X-100, 1% Tween-80 and can be enriched by serotonin-linked-Sepharose 4B affinity chromatography. However, 5-HT3 sites are more sensitive to heat inactivation, long-term storage, and sulfhydrylalkylation. The pharmacological profiles are distinct for the spiperone-insensitive 5-HT1B and 5-HT3 sites in both human and bovine cortex. In addition, evidence is presented for the existence of a novel, low concentration [3H]serotonin binding site in human cortex.
Aged↗