The essence of excitation.

The amino acid glutamate is the major excitatory neurotransmitter in a range of organisms from Caenorhabditis elegans to mammals, and it mediates the information processing that underlies essentially all behavior. Recent advances in our understanding of glutamate storage and release now illuminate how this ubiquitous amino acid can function as a signalling molecule.

Uptake of glutamate into synaptic vesicles by an inorganic phosphate transporter.

Previous work has identified two families of proteins that transport classical neurotransmitters into synaptic vesicles, but the protein responsible for vesicular transport of the principal excitatory transmitter glutamate has remained unknown. We demonstrate that a protein that is unrelated to any known neurotransmitter transporters and that was previously suggested to mediate the Na(+)-dependent uptake of inorganic phosphate across the plasma membrane transports glutamate into synaptic vesicles. In addition, we show that this vesicular glutamate transporter, VGLUT1, exhibits a conductance for chloride that is blocked by glutamate.

Regulation of pro-opiomelanocortin gene transcription in individual cell nuclei.

A nonrepetitive complementary RNA probe specific for an intervening sequence of the rat pro-opiomelanocortin (POMC) gene primary transcript was used to analyze the hormonal regulation of POMC gene transcription in individual cell nuclei in the rat pituitary by in situ hybridization. This probe recognized only full-length POMC heterogeneous nuclear RNA, as verified by Northern blots of pituitary RNA. When pituitary sections were hybridized with this 3H-labeled POMC intron A probe, silver grains were predominantly localized over the nuclei of cells that expressed POMC in the anterior and intermediate lobes. Adrenalectomy increased both the average grain density over corticotroph nuclei and the number of cells in the anterior pituitary with significant numbers of silver grains over their nucleus. Dexamethasone administration to intact or adrenalectomized rats results in the rapid (within 30 minutes) disappearance of silver grains over the nuclei of corticotrophs in the anterior lobe, suggesting that POMC gene transcription had been inhibited. However, adrenalectomy or dexamethasone administration did not alter the silver grain density over nuclei of intermediate lobe melanotrophs. Thus, this in situ hybridization assay utilizing an intervening sequence-specific POMC probe can measure rapid physiological changes in POMC heterogeneous nuclear RNA in individual cell nuclei.

Molecular cloning and expression of a high affinity L-proline transporter expressed in putative glutamatergic pathways of rat brain.

We have used the polymerase chain reaction (PCR) with degenerate oligonucleotides derived from two conserved regions of the norepinephrine and gamma-aminobutyric acid transporters to identify novel Na(+)-dependent transporters in rat brain. One PCR product hybridized to a 4.0 kb RNA concentrated in subpopulations of putative glutamatergic neurons including mitral cells of the olfactory bulb, pyramidal cells of layer V of the cerebral cortex, pyramidal cells of the piriform cortex, and pyramidal cells of field CA3 of the hippocampus. Transient expression of the cognate cDNA conferred Na(+)-dependent L-proline uptake in HeLa cells that was saturable (Km = 9.7 microM) and exhibited a pharmacological profile similar to that for high affinity L-proline transport in rat brain slices. The cloned transporter cDNA predicts a 637 aa protein with 12 putative transmembrane domains and exhibits 44%-45% amino acid sequence identity with other members of the emerging family of neurotransmitter transporters. These findings support a synaptic role for L-proline in specific excitatory pathways in the CNS.

Distinct spatial localization of specific mRNAs in cultured sympathetic neurons.

We examined the subcellular distribution of specific mRNAs in cultured sympathetic neurons. Under appropriate conditions, sympathetic neurons extend both axons and dendrites that are distinguishable by light microscopic and immunocytochemical criteria. In situ hybridization revealed a differential localization of mRNA within dendrites. mRNA encoding MAP2 was abundant in cell bodies and distributed nonhomogeneously throughout the dendritic compartment, but was not detected in axons. In contrast, mRNAs encoding GAP-43 and alpha-tubulin were restricted to the cell body and largely excluded from dendrites as well as axons. Detergent extraction revealed that most dendrite-associated mRNA encoding MAP2 was associated with the Triton X-100 insoluble fraction of the cell. The subset of mRNAs present in the dendritic compartment may encode proteins involved in the morphogenesis and remodeling of dendrites.

The expression of vesicular glutamate transporters defines two classes of excitatory synapse.

The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.

Identification, characterization, and molecular cloning of a novel transporter-like protein localized to the central nervous system.

During the course of large scale purification of the D1 dopamine receptor from rat brain, a protein of approximately 87,000 daltons (p87) was observed to copurify with the D1 receptor through four chromatographic steps. To characterize the nature of this protein, bovine and rat cDNA clones were isolated and sequenced. The bovine and rat clones were highly conserved (98.5% identity). Each clone possessed an open reading frame of 2226 base pairs encoding a protein of 742 amino acids (calculated MW of 82,500), containing three stretches of peptide sequence obtained from p87 sequence analysis. Comparison of the deduced peptide sequence of this protein with those found in available databanks revealed that it was a novel protein related to the family of nutrient transport proteins from eukaryotes and bacteria, including, the mammalian facilitated glucose transporters, the yeast transporters for maltose, lactose, and glucose, and the proton-driven bacterial transporters for arabinose, xylose, and citrate. In addition p87 also shares with these transporters a similar hydropathicity profile that suggests the presence of 12 transmembrane segments. The mRNA for p87 appears to be localized primarily, if not exclusively, to the central nervous system. Northern blot analysis reveals a message of approximately 4.8 kb in cortex, hippocampus, brain stem, and cerebellum, but no detectable signal in peripheral tissues such as spleen, liver, kidney, lung, heart, or skeletal muscle. Evidence form Western blot analysis and immunohistochemistry suggests that this protein may be expressed in intracellular organelles or the membrane of synaptosomes rather than plasma membrane. Based on its structure and properties, p87 appears to define a new class of transporter-like proteins.

Transthyretin: a choroid plexus-specific transport protein in human brain. The 1986 S. Weir Mitchell award.

Plasma transthyretin (TTR, formerly called prealbumin) is a 55-kd protein that participates in the plasma transport of both thyroxine and retinol (vitamin A). TTR concentrations are disproportionately high in human ventricular CSF, suggesting that TTR is either selectively transported across or synthesized de novo within the blood-CSF barrier. To address this question, we adopted a molecular genetic approach; after isolating a cDNA clone encoding human TTR, we previously demonstrated specific TTR messenger RNA (mRNA) synthesis in rat choroid plexus. We have now extended these investigations to the human brain. Northern analysis of postmortem brain homogenates revealed abundant TTR mRNA in choroid plexus, but not in cerebellum or cerebral cortex. Choroid plexus mRNA was readily translated into TTR preprotein in an in vitro translation system. An immunocytochemical survey of human postmortem brain sections revealed the presence of TTR protein specifically and uniquely in the cytoplasm of choroid plexus epithelial cells; these results were corroborated at the mRNA level by an extensive survey of whole rat-brain sections by in situ hybridization. Therefore, within the mammalian CNS, TTR is the first known protein synthesized solely by the choroid plexus, suggesting a special role for TTR in the brain or CSF. Whether this function differs from its established plasma transport functions is presently unknown.

Expression of alpha 2-adrenergic receptor subtypes in the mouse brain: evaluation of spatial and temporal information imparted by 3 kb of 5' regulatory sequence for the alpha 2A AR-receptor gene in transgenic animals.

The present studies characterize the expression of the alpha 2A, alpha 2B and alpha 2C adrenergic receptor subtypes via in situ hybridization analysis of messenger RNA expression in the adult mouse brain, as well as the pattern of expression of alpha 2A adrenergic receptor messenger RNA at embryonic day E9.5, the earliest time for detection of the messenger RNA encoding this receptor subtype. alpha 2A adrenergic receptor messenger RNA is highly expressed in the sixth layer of the cortex and the locus coeruleus, alpha 2B adrenergic receptor messenger RNA predominantly in the thalamus and in the Purkinje layer of the cerebellum, and alpha 2C adrenergic receptor messenger RNA in the putamen caudate region of the mouse brain. Both alpha 2A and alpha 2C adrenergic receptor messenger RNA demonstrate strong expression in the amygdaloid complex, hypothalamus, olfactory system and the hippocampal formation. To develop a molecular understanding of the unique cellular expression of messenger RNA encoding the alpha 2A adrenergic receptor subtype, 2.83 kb of the upstream regulatory sequence for the alpha 2A adrenergic receptor gene was fused to the LacZ gene as a reporter gene and expression of beta-galactosidase activity was assessed in transgenic offspring. Although the spatial expression of the transgene in the adult brain often overlaps that for the endogenous alpha 2A adrenergic receptor, both ectopic expression and the absence of appropriate expression were noted; in contrast five of the six lines show temporal expression characteristic of the endogenous alpha 2A adrenergic receptor gene. The present studies provide the first characterization of messenger RNA localization for the three alpha 2 adrenergic receptor subtypes in the mouse CNS. Because the functional roles of the prazosin-sensitive alpha 2B adrenergic receptor and alpha 2C adrenergic receptor subtypes have been masked in most earlier physiological and pharmacological analyses of alpha 2 adrenergic receptor function, identifying the multiple loci alpha 2 adrenergic receptor subtype expression is an important prelude to understanding the functional roles of these three independent receptor populations in the mouse CNS. The findings in the transgenic animals indicating that approximately 3 kb of regulatory sequence has imparted faithful temporal but not spatial expression for the alpha 2A adrenergic receptor gene suggest that additional regulatory information is necessary for appropriate cell specific expression of messenger RNA for the alpha 2A adrenergic receptor subtype.

Ontogeny of D1A and D2 dopamine receptor subtypes in rat brain using in situ hybridization and receptor binding.

The prenatal and postnatal ontogeny of D1A and D2 dopamine receptors was assessed by in situ hybridization of messenger RNAs encoding the receptors and by radioligand binding autoradiography. On gestational day 14, signals for D1A and D2 dopamine receptor messages were observed in selected regions in ventricular and subventricular zones which contain dividing neuroblasts, and in intermediate zones that contain maturing and migrating neurons. Specifically, D1A and D2 dopamine receptor message was observed in the developing caudate-putamen, olfactory tubercle, and frontal, cingulate, parietal and insular cortices. Additionally, D1A dopamine receptor messenger RNA was found in the developing epithalamus, thalamus, hypothalamus, pons, spinal cord and neural retina; D2 dopamine receptor messenger RNA was also observed in the mesencephalic dopaminergic nuclear complex. Gene expression of D1A and D2 dopamine receptor subtypes in specific cells as they differentiate precedes dopamine innervation and implies that receptor expression is an intrinsic property of these neurons. The early expression of dopamine receptor messenger RNA suggests a regulatory role for these receptors in brain development. While the signal for both messages increased in the intermediate zones on gestational day 16, it decreased in the ventricular and subventricular zones, and was no longer apparent in these zones by gestational day 18. By gestational day 18, abundant D1A or D2 dopamine receptor messenger RNA was observed in cell groups similar in location to those observed in the adult brain. On gestational day 18, D1A dopamine receptor message was noted in the neural retina, anterior olfactory nucleus, the insular, prefrontal, frontal, cingulate, parietal and retrosplenial cortices, the olfactory tubercle, caudate-putamen, lateral habenula, dorsolateral geniculate nucleus, ventrolateral and mediolateral thalamic nuclei, and the suprachiasmatic and ventromedial nuclei of the hypothalamus. D2 dopamine receptor message was observed on gestational day 18 in the insular, prefrontal, frontal and cingulate cortices, the olfactory tubercle, caudate-putamen, ventral tegmental area, substantia nigra, and the intermediate lobe of the pituitary. At birth, expression of messenger RNA for both dopamine receptor subtypes in the striatum approximated that seen in mature rats. In contrast, D1A and D2 receptor binding, measured with [3H]SCH-23390 and [3H]raclopride, respectively, was low at birth and progressively increased to reach adult levels between days 14 and 21. The in situ hybridization data showing early prenatal expression of messenger RNA for the D1A and D2 dopamine receptors are consistent with the hypothesis that these receptors have a regulatory role in neuronal development.(ABSTRACT TRUNCATED AT 400 WORDS)

Localization of vesicular glutamate transporters and neuronal nitric oxide synthase in rat nucleus tractus solitarii.

Previously we reported that glutamate and neuronal nitric oxide synthase (nNOS) colocalize in neurons of the nucleus tractus solitarii (NTS). That finding provided anatomical support for the suggestion that nitric oxide and glutamate interact in cardiovascular regulation by the NTS. Here we test the hypothesis that nNOS colocalizes with vesicular glutamate transporters (VGluT1 and VGluT2) in the NTS. Immunoreactivity (IR) for VGluT better identifies glutamatergic terminals than does glutamate-IR, which may label metabolic as well as transmitter stores of the amino acid. We used fluorescent immunohistochemistry combined with confocal laser scanning microscopy to study IR for VGluT1, VGluT2 and nNOS in rat NTS. A high density of VGluT1-IR positive fibers was present in the gracilis and cuneatus nuclei while in the NTS we found a moderate density in the lateral and interstitial subnuclei and a low density in the dorsolateral, ventral and intermediate subnuclei. The medial, central, commissural and gelatinosus subnuclei contained few VGluT1-IR containing fibers. Thus, VGluT1 containing fibers are not prominent in portions of the NTS where cardiovascular afferent fibers terminate. In contrast, we found a high density of VGluT2-IR containing fibers in the gelatinosus subnucleus and subpostremal area and a moderate density in cardiovascular regions such as the dorsolateral and medial subnuclei as well as in the central and lateral subnuclei. We found a low density in the ventral, intermediate, interstitial and commissural subnuclei. VGluT1-IR and VGluT2-IR rarely colocalized in fibers within the NTS. VGluT1-IR did not colocalize with nNOS, but VGluT2-IR and nNOS-IR colocalized in fibers in all NTS subnuclei. When compared with the other NTS subnuclei, the dorsolateral, gelatinosus and subpostremal subnuclei had higher frequencies of colocalization of VGluT2-IR and nNOS-IR. VGluT2-IR positive fibers were also apposed to nNOS-IR positive fibers throughout the NTS. These data support our hypothesis and confirm that glutamatergic fibers in the NTS contain nNOS.

Intervening sequence-specific in situ hybridization: detection of the pro-opiomelanocortin gene primary transcript in individual neurons.

We describe here the in situ hybridization procedure which we have used to detect the pro-opiomelanocortin (POMC) gene primary transcript in nuclei of individual neurons in the periarcuate region of the hypothalamus. An exon-intron RNA probe was used to detect POMC primary transcript and mature mRNA in nuclear extracts of nucleic acids using a sensitive S1 nuclease protection assay. The levels per cell of nuclear primary transcript were similar to those seen in the anterior pituitary, suggesting that intervening sequence in situ hybridization should be feasible. A nonrepetitive complementary RNA probe specific for the first intervening sequence of the rat POMC gene (POMC IVS-A) was used to detect the POMC primary transcript in hypothalamic tissue sections by in situ hybridization. The distribution of nuclear localized autoradiographic grains was similar to that previously reported for immunocytochemically defined POMC neurons, suggesting that the procedure is also effective in brain cells.

Distribution of alpha 2-adrenergic receptor subtype gene expression in rat brain.

alpha 2-Adrenergic receptors in brain are important presynaptic modulators of central noradrenergic function (autoreceptors) and postsynaptic mediators of many of the widespread effects of catecholamines and related drugs. alpha 2-Adrenergic agonists are currently used as antihypertensives and preanesthetic agents, but new subtype-selective alpha 2-adrenoceptor agonists and antagonists have additional therapeutic application potential. Three genes encoding specific alpha 2-adrenoceptor subtypes (alpha 2A, alpha 2B, and alpha 2C) have been isolated and characterized. RNA blotting indicates that all three are expressed in rat brain. This study used in situ hybridization with 35S-labeled RNA probes to map the distribution of alpha 2-adrenoceptor subtype gene expression in rat brain. alpha 2A mRNA was most abundant in the locus coeruleus, but was also widely distributed in the brain stem, cerebral cortex, septum, hypothalamus, hippocampus and amygdala. alpha 2B mRNA was observed only in the thalamus. alpha 2C mRNA was mainly localized to the basal ganglia, olfactory tubercle, hippocampus, and cerebral cortex. These mRNA distributions largely agree with previous findings on the alpha 2-adrenoceptor distributions in the rat brain, but suggest that the localization patterns for each receptor subtype are unique. The expression of alpha 2A mRNA in noradrenergic neurons indicates that this subtype mediates presynaptic autoreceptor functions. Furthermore, the localization of alpha 2A mRNA in noradrenergic projection areas suggests that this receptor may also have an important role in mediating postsynaptic effects. The precise physiological and pharmacological roles of the alpha 2-adrenoceptor subtypes are still largely unknown, but it is expected that in situ hybridization coupled to various methods to identify the transmitter phenotypes of the subtype-expressing neurons will help to clarify these important issues in the near future.

Location and molecular cloning of D1 dopamine receptor.

Recently, our laboratory has purified the D1 dopamine receptor 6600 fold to near homogeneity from digitonin solubilized rat striatal membranes using sequential affinity, ion exchange, lectin, and size exclusion chromatographies. The resulting receptor preparations still retained ligand binding activity (-11,000 pmol [3H]SCH 23390 bound per mg/protein) and appeared as a single band at 70-80 kDa on SDS-PAGE. In order to learn more about the sequence and structure of this protein, we recently cloned the gene for a human CNS D1 dopamine receptor. This gene has an open reading frame of 1388 nucleotides and encoded for a protein with a deduced amino acid sequence of 446 residues. When expressed in mammalian cells the cloned D1 receptor had all the ligand binding properties expected for a D1 receptor (SCH 23390 > cis flupenthixol > raclopride and SKF 38393 > apomorphine > dopamine > quinpirole). The cloned D1 receptor was found to stimulate adenylyl cyclase but not phospholipase C. The message for this D1 dopamine receptor was found in caudate, putamen, frontal cortex, and hippocampus, but not in substantia nigra, heart, or kidney. These accomplishments now will allow the pursuit of biochemical studies of the receptor protein as well as investigations into structure/function relationship of the receptor using a molecular biological techniques.

Distribution of beta 3-adrenoceptor mRNA in human tissues.

The beta 3-adrenoceptor is a G protein-coupled receptor which mediates metabolic functions of the endogenous catecholamines epinephrine and norepinephrine. Questions exist regarding distribution of the beta 3-adrenoceptor in human tissue. In order to examine the distribution of beta 3-adrenoceptor mRNA in human tissues, we used sensitive and specific RNase protection assays without previous PCR amplification in an extensive list of human tissues. We confirm the presence of beta 3-adrenoceptor mRNA in human white fat from several locations, gall bladder, and small intestine, as well as extend the distribution of beta 3-adrenoceptor mRNA to previously uncharacterized human tissues such as stomach and prostate. The presence of beta 3-adrenoceptor mRNA in human white adipose tissue has important implications regarding possible use of beta 3-adrenoceptor selective agonists as anti-obesity agents, and the demonstration of beta 3-adrenoceptor mRNA in a number of gastrointestinal tissues and prostate raises the question of the role of the beta 3-adrenoceptor in motility and secretory processes.

In situ hybridization: identification of rare mRNAs in human tissues.

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non-selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32P or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

In situ analysis of myelin basic protein gene expression in myelin-deficient oligodendrocytes: antisense hnRNA and readthrough transcription.

Mice homozygous for the recessive mutation myelin deficient (mld) exhibit a severe deficit in the synthesis of myelin basic protein (MBP). The primary defect in mld mice is an unusual rearrangement of the MBP gene. The mld MBP locus consists of two tandem MBP genes that span approximately 90 kb of DNA; the upstream gene contains an extensive inversion of its 3' region, while the downstream gene appears identical to the wild-type gene. In this report, the aberrant expression of the mld MBP locus was examined by in situ hybridization with MBP genomic and cDNA probes. In situ hybridization with a single-stranded genomic probe from the inverted region of the mld MBP gene revealed that primary transcripts initiating from the upstream MBP promoter elongate through the inverted region generating abundant antisense MBP heterogeneous nucleus RNA (hnRNA) transcripts in mld oligodendrocytes. Little or no antisense MBP RNA was detected in cytoplasmic regions in mld brain sections indicating that the antisense MBP hnRNA transcripts are not processed and transported out of the nucleus. Furthermore, we provide evidence that these abnormal transcripts elongate through the downstream MBP gene's promoter region, suggesting that transcription of the downstream mld MBP gene is inhibited by readthrough transcripts that originate at the upstream promoter.

Secretin stimulates cyclic AMP formation in the rat brain.

The effects of secretin on cyclic AMP levels in the rat brain were determined. Incubation of rat brain frontal cortex slices with secretin or the structurally related peptides peptide histidine leucine (PHI) or vasoactive intestinal polypeptide (VIP) in the presence of 10 mM theophylline resulted in a dose-dependent increase in the cyclic AMP levels. The half-maximal increase in cyclic AMP occurred using a 1 microM dose of secretin or a 2 microM dose of PHI or VIP. Preincubation of slices with secretin-(5-27) produced a dose-dependent inhibition of the secretin but not VIP- or PHI-stimulated increase in the cyclic AMP content. Also, in receptor binding studies, secretin-(5-27) produced a dose-dependent inhibition (Ki = 400 nM) of 125I-secretin but not of 125I-VIP binding to rat brain membranes. Guanyl-5'-yl imidodiphosphate decreased the affinity of radiolabelled secretin binding as a result of an increased rate of dissociation of bound 125I-secretin. These data suggest that secretin receptors in the rat brain may be coupled to adenylate cyclase in a stimulatory manner and that secretin-(5-27) may function as a central secretin receptor antagonist.