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.

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.

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.

L-proline and L-pipecolate induce enkephalin-sensitive currents in human embryonic kidney 293 cells transfected with the high-affinity mammalian brain L-proline transporter.

The high-affinity mammalian brain L-proline transporter (PROT) belongs to the GAT1 gene family, which includes Na- and Cl-dependent plasma membrane carriers for neurotransmitters, osmolites, and metabolites. These transporters couple substrate flux to transmembrane electrochemical gradients, particularly the Na gradient. In the nervous system, transporters clear synapses and help to replenish transmitters in nerve terminals. The localization of PROT to specific excitatory terminals in rat forebrain suggests a role for this carrier in excitatory transmission (). We investigated the voltage regulation and electrogenicity of this novel transporter, using human embryonic kidney (HEK) 293 cells stably transfected with rat PROT cDNA. In physiological solutions between -140 and -40 mV, L-proline (PRO) and its six-member ring congener L-pipecolate (PIP) induced inward current. The current-voltage relationship and the variance of current fluctuations were similar for PRO- and PIP-induced current, and the ratio of induced variance to the mean current ranged from 20 to 60 fA. Des-Tyr-Leu-enkephalin (GGFL), a competitive peptide inhibitor of PROT, reduced the rat PROT-associated current to control levels. GGFL alone did not elicit currents, and the GGFL-sensitive substrate-induced current was absent in nontransfected cells. Finally, GGFL inhibited PROT-mediated transport only when applied to the extracellular face of PROT. These data suggest that (1) PROT uptake is electrogenic, (2) individual transporter currents are voltage-independent, and (3) GGFL is a nonsubstrate inhibitor that interacts either with an extracellular domain of PROT or in an externally accessible pore.

Localization of the brain-specific high-affinity l-proline transporter in cultured hippocampal neurons: molecular heterogeneity of synaptic terminals.

The expression of a brain-specific, high-affinity Na+-(and Cl--)dependent l-proline transporter in subpopulations of putative glutamatergic pathways in mammalian brain suggests a physiological role for this carrier in excitatory neurotransmission (Fremeau et al. , Neuron 8: 915-926, 1992). To assess further the cell-type and subcellular localization of PROT, we examined its distribution in low-density cultures of embryonic rat hippocampus. PROT immunoreactivity was detected beginning at 8 days in culture in a highly punctate pattern localizing to a subset of synaptic terminals. PROT was not detected at GABAergic terminals but was specifically localized to a subset of excitatory nerve terminals. PROT-labeled terminals showed partial apposition to AMPA-type and NMDA-type glutamate receptor clusters. Immunolabeling of isolated neurons grown in microisland cultures revealed that PROT was expressed by 60% of cultured hippocampal neurons. Individual microisland cultures were immunopositive for either PROT or glutamic acid decarboxylase, but never both. In the expressing pyramidal neurons, PROT was targeted to all presynaptic terminals. These findings indicate that PROT contributes to the molecular heterogeneity of glutamatergic terminals and suggest a novel presynaptic regulatory role for PROT in excitatory transmission at specific glutamatergic synapses.

The mammalian brain high-affinity L-proline transporter is enriched preferentially in synaptic vesicles in a subpopulation of excitatory nerve terminals in rat forebrain.

The expression of a brain-specific high-affinity Na+-dependent (and Cl--dependent) L-proline transporter (PROT) in subpopulations of putative glutamatergic neurons in mammalian brain suggests a physiological role for this carrier in excitatory neurotransmission (). To gain insights into potential sites where PROT may function, we used a C-terminal domain antipeptide antibody to determine the regional distribution and subcellular localization of PROT in rat forebrain. PROT immunoreactivity was seen in processes having a regional light microscopic distribution comparable to that of known glutamatergic projections within the cortex, caudate putamen nucleus (CPN), hippocampal formation, and other forebrain regions. In all regions examined by electron microscopy (cortex, CPN, and the stratum oriens of CA1), PROT labeling was observed primarily within subpopulations of axon terminals forming asymmetric excitatory-type synapses. Immunogold labeling for PROT was detected in close contact with membranes of small synaptic vesicles (SSVs) and more rarely with the plasma membrane in these axon terminals. Subcellular fractionation studies confirmed the preferential distribution of PROT to synaptic vesicles. The topology of PROT in synaptic vesicles was found to be inverted with respect to the plasma membrane, suggesting that PROT-containing vesicles are generated by a process involving endocytosis from the plasma membrane. Because PROT lacks any of the known characteristics of other vesicular transporters, these results suggest that certain excitatory terminals have a reserve pool of PROT associated with SSVs. The delivery of PROT to the plasma membrane by exocytosis could play a critical role in the plasticity of certain glutamatergic pathways.

Components of a calmodulin-dependent protein kinase cascade. Molecular cloning, functional characterization and cellular localization of Ca2+/calmodulin-dependent protein kinase kinase beta.

Ca2+/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV, respectively) require phosphorylation on an equivalent single Thr in the activation loop of subdomain VIII for maximal activity. Two distinct CaMKI/IV kinases, CaMKKalpha and CaMKKbeta, were purified from rat brain and partially sequenced (Edelman, A. M., Mitchelhill, K., Selbert, M. A., Anderson, K. A., Hook, S. S., Stapleton, D., Goldstein, E. G., Means, A. R., and Kemp, B. E. (1996) J. Biol. Chem. 271, 10806-10810). We report here the cloning and sequencing of cDNAs for human and rat CaMKKbeta, tissue and regional brain localization of CaMKKbeta protein, and mRNA and functional characterization of recombinant CaMKKbeta in vitro and in Jurkat T cells. The sequences of human and rat CaMKKbeta demonstrate 65% identity and 80% similarity with CaMKKalpha and 30-40% identity with CaMKI and CaMKIV themselves. CaMKKbeta is broadly distributed among rat tissues with highest levels in CaMKIV-expressing tissues such as brain, thymus, spleen, and testis. In brain, CaMKKbeta tracks more closely with CaMKIV than does CaMKKalpha. Bacterially expressed CaMKKbeta undergoes intramolecular autophosphorylation, is regulated by Ca2+/CaM, and phosphorylates CaMKI and CaMKIV on Thr177 and Thr200, respectively. CaMKKbeta activates both CaMKI and CaMKIV when coexpressed in Jurkat T cells as judged by phosphorylated cAMP response element-binding protein-dependent reporter gene expression. CaMKKbeta activity is enhanced by elevation of intracellular Ca2+, although substantial activity is observed at the resting Ca2+ concentration. The strict Ca2+ requirement of CaMKIV-dependent phosphorylation of cAMP response element-binding protein, is therefore controlled at the level of CaMKIV rather than CaMKK.

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).

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.

A novel nonopioid action of enkephalins: competitive inhibition of the mammalian brain high affinity L-proline transporter.

The high affinity L-proline transporter (PROT) is a member of the family of Na+ (and Cl-)-dependent plasma membrane transport proteins that comprises transporters for several neurotransmitters, osmolytes, and metabolites. The brain-specific expression of PROT in a subset of putative glutamatergic pathways implies a specialized function for this novel transporter and its presumed natural substrate L-proline in excitatory synaptic transmission. However, definitive studies of the physiological role(s) of high affinity L-proline uptake have been precluded by the lack of specific uptake inhibitors. Here, we report that Leu- and Met-enkephalin and their des-tyrosyl derivatives potently and selectively inhibited high affinity L-proline uptake in rat hippocampal synaptosomes and in PROT-transfected HeLa cells. High concentrations of the opiate receptor antagonist naltrexone did not block the inhibitory actions of these peptides, arguing against an involvement of opioid receptors. Des-tyrosyl-Leu-enkephalin elevated the apparent K(m) of L-proline transport in transfected HeLa cells without altering the V(max). PROT-transfected HeLa cells did not accumulate [3H]Leu-enkephalin above background levels, demonstrating that enkephalins are not substrates for PROT. These findings indicate that enkephalins competitively inhibit mammalian brain PROT through a direct interaction with the transporter protein at or near the L-proline binding site. The high potency and specificity of des-tyrosyl-Leu-enkephalin make this compound a useful tool for elucidating the structure-function properties and physiological role(s) of PROT.

Characterization and distribution of the neuronal glutamate transporter EAAC1 in rat brain.

The extracellular concentration of glutamate and other related excitatory amino acids (EAA) is regulated by the action of transporter proteins located on either presynaptic terminals or adjacent astroglial processes. Recent molecular advances have led to the cloning of three separate cDNAs encoding for Na(+)-dependent glutamate transporters; two are thought to be primarily glial in origin (GLAST and GLT-1) and the third (EAAC1) is localized to neurons in the brain and other nonneural tissues. An EAAC1 cDNA was initially cloned from rabbit small intestine (13). In this study, we report isolation and characterization of the homologous clone from rat brain. Northern blot hybridization revealed high levels of EAAC1 mRNA in rat brain and kidney and low levels in heart, lung, and skeletal muscle. Transient expression of EAAC1 in HeLa cells resulted in an increase in Na(+)-dependent high-affinity L-[3H]glutamate and D-[3H]aspartate transport. The pharmacological profile of EAAC1 was very similar to that reported for the rabbit and human EAAC1 homologues. Transport activity was potently inhibited by D- and L-threo-beta-hydroxyaspartate and L-trans-pyrrolodine-2,4-dicarboxylate. Dihydrokainate and L-alpha-aminoadipate did not inhibit transport at concentrations below 1 mM. Oligonucleotide cDNA probes (45-mer) were constructed and labeled with 35S-ATP for film- and emulsion-based in situ hybridization of rat brain. EAAC1 mRNA had the highest density in the cerebellar granule cell layer, hippocampus, superior colliculus, and neocortex. Sections that were emulsion-dipped and counterstained with cresyl violet revealed EAAC1 labeling localized exclusively over neuronal cell bodies, including some nonglutamatergic neurons such as spinal cord ventral horn cells.

Human brain-specific L-proline transporter: molecular cloning, functional expression, and chromosomal localization of the gene in human and mouse genomes.

L-Proline fulfills several of the classic criteria used to identify amino acid neurotransmitters, including the presence of a high affinity, Na(+)- (and Cl-)-dependent synaptosomal transport process and the Ca(2+)-dependent release of exogenously loaded radiolabeled L-proline from brain slices and synaptosomes after K(+)-induced depolarization. However, studies to define the role of L-proline in discrete pathways in the mammalian brain have been precluded by the inability to block its biosynthesis or high affinity transport in nervous tissue. We report the molecular cloning, functional expression, and chromosomal localization of a human brain-specific high affinity L-proline transporter (hPROT). The pharmacological specificity, kinetic properties, and ionic requirements of hPROT clearly distinguish this carrier from the other Na(+)-dependent plasma membrane carriers that transport L-proline. Multiple tissue Northern blot analysis revealed a prominent approximately 4-kb mRNA transcript in human brain tissue, whereas no specific hybridizing species were detected in peripheral tissue. An antipeptide antiserum directed against the carboxy-terminus of the predicted hPROT protein identified a single, broad immunoreactive protein of 68 kDa on immunoblots of synaptosomal membranes from various human brain regions. In contrast, no specific labeling was detected on immunoblots of membranes from human liver, kidney, or heart. A differential distribution of hPROT mRNA and protein was observed in the human corpus striatum, consistent with the hypothesis that the hPROT protein is synthesized in neuronal cell bodies in an extrastriatal location and axonally transported to the corpus striatum. These findings warrant the consideration of a synaptic regulatory role for this transporter and its presumed natural substrate, L-proline, in the mammalian central nervous system.

Mammalian brain-specific L-proline transporter. Neuronal localization of mRNA and enrichment of transporter protein in synaptic plasma membranes.

The expression of a high affinity Na(+)- (and Cl-) dependent L-proline transporter (PROT) in subpopulations of putative glutamatergic pathways in rat brain raises the possibility of a specific physiological role(s) for this carrier in excitatory neurotransmission (Fremeau, R. T., Jr., Caron, M. G., and Blakely, R. D. (1992) Neuron 8, 915-926). However, the biochemical properties and regional, cellular, and subcellular distribution of the PROT protein have yet to be elucidated. Here, we document the brain-specific expression and neuronal localization of rat PROT mRNA. We also report the first identification and partial biochemical characterization of the mammalian brain PROT protein. An affinity-purified antipeptide antibody was produced that specifically recognized a single 68-kDa PROT protein on immunoblots of rat and human brain tissues. Deglycosylation of rat hippocampal membranes with peptide-N-glycosidase F reduced the apparent molecular mass of the native PROT protein from 68 to 53 kDa, the size of the primary PROT translation product determined by in vitro translation of the rat PROT cDNA in the absence of microsomes. Subcellular fractionation studies demonstrated that the PROT protein was enriched in synaptic plasma membranes but absent from postsynaptic densities. A differential distribution of PROT mRNA and protein was observed in rat striatum, suggesting that the transporter protein is synthesized in neuronal cell bodies in the cortex and exported to axon terminals in the caudate putamen. These findings warrant the consideration of a novel presynaptic regulatory role for this transporter in excitatory synaptic transmission.

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.

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)

Mammalian homologs of Drosophila ELAV localized to a neuronal subset can bind in vitro to the 3' UTR of mRNA encoding the Id transcriptional repressor.

Mammalian cDNAs encoding a rat (Rel-N1) and a human (Hel-N1) neuronal RNA-binding protein have been cloned and characterized with respect to tissue specificity, neuroanatomical localization, and RNA binding specificity. Both proteins are highly similar to the product of the Drosophila elav gene, which is expressed in all neurons of the fly and is required for development of the nervous system. However, in situ hybridization of rat tissues demonstrated more restricted expression of Rel-N1 mRNA within a subset of neurons of the hippocampus, cortex, and other regions of the gray matter, but not in glial cells or white matter. In vitro RNA binding experiments demonstrated that Hel-N1 can bind to the 3' untranslated region (3' UTR) of Id mRNA, a transcript that encodes a helix-loop-helix transcriptional repressor that is abundantly expressed in undifferentiated neural precursors. Sequences characterized for Hel-N1 binding were also abundantly present in the 3' UTR of the Drosophila extramacrochaetae mRNA, which encodes an Id homolog. Thus, we have identified a potential link between a neuronal 3' UTR RNA-binding protein and regulatory transcription factors involved in neural development. These findings are interpreted in light of recent studies in which mRNA 3' UTRs were found to be important for the regulation of cell growth and differentiation.

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.

D1 dopamine receptor binding and mRNA levels are not altered after neonatal 6-hydroxydopamine treatment: evidence against dopamine-mediated induction of D1 dopamine receptors during postnatal development.

The role of dopaminergic innervation on the postnatal developmental expression of D1 dopamine receptors was investigated. Bilateral destruction of dopamine-containing neurons was achieved by treating rats intracisternally with 6-hydroxydopamine (6-OHDA) on postnatal day 3, and rats were killed on day 21. To ensure effective reduction of D1 receptor activation by residual dopamine, a group of 6-OHDA-lesioned rats was given twice daily injections of the D1 receptor antagonist SCH-23390, from day 4 to 20. D1 dopamine receptor binding was assessed in the caudate-putamen, nucleus accumbens, and olfactory tubercle by quantitative autoradiographic analysis of [3H]SCH-23390 binding. In addition, the relative amount of D1A receptor mRNA was assessed by in situ hybridization of a 35S-labeled riboprobe. In the developing rats, neither the amount of [3H]SCH-23390 binding nor the amount of D1A receptor mRNA was altered by 6-OHDA lesioning followed by chronic treatment with SCH-23390. Thus, bilateral destruction of dopamine-containing neurons and treatment with SCH-23390 in neonatal rats did not interfere with the developmental expression of D1 receptors or alter the levels of mRNA that code for this receptor protein. Treatment of intact rats with SCH-23390 from postnatal day 4 to 20 also did not alter [3H]SCH-23390 binding or levels of D1 receptor mRNA. However, adult rats treated chronically with SCH-23390 exhibited increased [3H]SCH-23390 binding but did not show a significant change in D1 receptor mRNA levels.