Localization And Regulation of DARPP-32 M. Schalling, M. Djurfeldt, M. Herrera-Marschitz, H. Hallman, Karolinska Institute and The Rockefeller University We have mapped the rat brain with regard to DARPP-32 (dopamine- and adenosine 3é:5é-monophosphate-regulated phosphoprotein) gene expression using a 35S-labeled oligonucleotide probe complementary to mRNA coding for rat DARPP-32. 1-2 · 106 cpm of probe ranging from 5-20. 108 cpm/μg were used on each section. Labeling was observed in close correlation with DARPP-32 immunohistochemistry. Thus, strong labeling could be seen, with overnight exposures to x-ray film, in the caudate nucleus, nucleus accumbens, and piriform cortex. Furthermore, strong labeling of ependymal cells around the third ventricle was observed and, after longer exposure, labeling was also evident in two bands throughout rat cerebral cortex. To evaluate possible regulation of DARPP-32 gene expression, animals were treated with the neurotoxins 6-OH-DA and ibotenic acid, agonists and antagonists with high affinity to D1 and D2 receptors, respectively, and combinations of the above. In addition, the response to the classical catecholamine depleting drug reserpine was analyzed. Adjacent sections were analyzed for changes in gene expression of DARPP-32, substance P (SP), dynorphin (DYN), and GAD using an IBAS image analysis system. Two levels of DARPP-32 mRNA were observed in the caudate nucleus. In preliminary studies, the higher level, localized in patches, showed some change in mRNA levels after drug treatment, whereas the caudate as a whole exhibited only minor variations. Marked changes in SP, DYN, and GAD were observed in several of these models. It is concluded that DARPP-32 has a more stable gene expression than several other molecules involved in signal transduction. We are therefore carrying out a more detailed analysis with a computerized image analysis system to reveal possible discrete regional effects. The GABAA Receptor: Subunit Heterogeneity and Transient Expression in Mammalian Cells Peter R. Schofield, Dolan B. Pritchett, Sanie Ymer, Harry Sondheimer*, Martin Köhler, Pia Werner, Brenda D. Shivers, Helmut Kettenman*, and Peter H. Seeburg ZMBH & Department of Neurobiology*, University of Heidelberg, Heidelberg, FRG GABA (y-aminobutyric acid), the major inhibitory neurotransmitter in the vertebrate brain, mediates neuronal inhibition by binding to the GABA/benzodiazepine (GABAA) receptor and opening an integral chloride channel. The GABAA receptor is the target for therapeutically important drugs, such as benzodiazepines and barbiturates, which allosterically modulate GABAmediated neuronal inhibition. The structure of the a and ẞ subunit cDNAs of the GABAA receptor has shown the existence of a ligand-gated receptor superfamily (Schofield et al., Nature 328:221-227, 1987). Two additional a subunits have been identified that share 70 percent identity. When these a subunits are expressed in oocytes, together with the B subunit, functionally distinct receptor subtypes are generated that are characterized by a thirtyfold difference in their GABA sensitivity (Levitan et al., submitted). Two newẞ subunit cDNAs, which share 75 percent sequence identity, have been isolated by homology screening. Interchange of ẞ subunits, as well as a subunits can generate a large family of functionally distinct receptor subtypes. Highly homologous receptor subtypes which differ functionally appear to be a common feature of brain receptors; differential expression of such receptor subtypes may be a major mechanism that contributes to synaptic plasticity. Human cDNAs encoding the a and B subunits have been expressed transiently in cultured mammalian cells. Electrophysiological recording shows that both a and ẞ subunits are responsive to GABA and are potentiated by barbiturates. Transient versus permanent expression of ion channels in mammalian cells offers significant advantages: the same cells can be used for both pharmacological and electrophysiological studies; cells are not subject to seasonal variation; microinjection is not required; the cell attached patch clamp is very sensitive; cells express high receptor densities; and permanent cell lines can be established. In Situ Hybridization Histochemistry as a Tool to J.M. Séquier, P. Malherbe, W. Hunziker, H. Möhler, Pharmaceutical and Central Research Departments, F. Hoffmann-La Roche & Co., Ltd., CH-4002 Basle, Switzerland The sensitivity and cellular resolution of in situ hybridization histochemistry make it the method of choice for locating mRNA encoding specific cell constituents such as receptor glycoproteins and for studying the regulation of their expression. 35S-cRNA probes have been used to localize, in rat brain, mRNAs coding for: (1) a- and ẞ-subunits of the rat GABA/benzodiazepine receptor complex; (2) the proto-oncogene c-fos- a marker of cellular activity-expressed in animals with seizures induced by DMCM, an inverse agonist of the GABA/benzodiazepine receptor; (3) calbindin D28, a vitamin D-dependent calcium-binding protein. a- and ẞ-subunits of the GABA/benzodiazepine receptor were expressed to a high degree in neuronal perikarya of numerous brain regions including the olfactory bulb, cerebral cortex, hippocampus, dentate gyrus, and cerebellum. Expression of c-fos after seizures was rapid and occurred above all in the accessory olfactory bulb, cerebral cortex (e.g., primary olfactory cortex), dentate gyrus, amygdala, choroid plexus, and cerebellum. mRNA encoding calbindin D28 occurred in cerebellar Purkinje cells, dentate gyrus granule cells, as well as other brain regions and kidney. Current investigations focus on (a) the molecular heterogeneity of GABA/benzodiazepine receptor, (b) the pharmacological regulation of DMCM-induced c-fos expression and its time course, (c) the regulation and possible function of calbindin D28 in the CNS. Signal Transduction in the Olfactory System: The Odorant-Stimulated Adenylate Cyclase P.B. Sklar, E. Krowiak, and S.H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Olfactory neurons respond to many odorants by the generation of electrical signals. The discovery of an odorant-stimulated adenylate cyclase (Pace et al., 1985) suggests that these signals are transduced through a receptorlinked adenylate cyclase. Characterization of adenylate cyclase activity in isolated chemosensory cilia reveals high levels of cyclase activity which is stimulated approximately twofold by GTP and approximately fivefold by guanosine 5'-(3-0-thio)triphosphate and forskolin. The olfactory adenylate cyclase displays Michaelis-Menten kinetics, with a Km of 185 μM and a Vmax of 1.5 nmol/mg/min, dependence on MgATP, and a pH optima of 7.5-8.0. Calcium reduces GTP-stimulated activity. Odorants augment enzyme activity 30-65 percent above the GTP-stimulated basal level in a tissue-specific and GTP-dependent manner. Blocking G-protein activity with GDPBS decreases the odorant elevation 30 percent. Odorants vary considerably in their influence upon olfactory adenylate cyclase activity. Most fruity, floral, minty, and herbaceous odorants stimulate the enzyme and display similar potencies in activating the adenylate cyclase. Putrid odorants and odorous chemical solvents do not stimulate enzyme activity. In homologous series of pyrazine, thiazole, and pyridine odorants, compounds with the longest hydrocarbon side chains are best able to enhance enzyme activity, although hydrophobicity alone does not fully determine the ability of an odorant to activate adenylate cyclase activity. Observation of an odorant-sensitive, tissue-specific adenylate cyclase, combined with the recent description of a cyclic-nucleotide activated channel in olfactory cilia (Nakamura and Gold, 1987), suggests that odorant-activation of adenylate cyclase is a fundamental mechanism of olfactory transduction. Repression of the Expression of Functional Nicotinic Acetylcholine Receptors by Antisense RNAs and an Oligonucleotide K. Sumikawa and R. Miledi University of California, Irvine It was recently discovered that gene expression in prokaryotes and in various cells, including Xenopus oocytes and mammalian cells, can be selectively inhibited by antisense RNA, that is, RNA which is complementary to a target RNA. This inhibition sometimes involves a hybridization between messenger RNA (mRNA) and its counterpart antisense RNA, which results in an inhibition of mRNA translation. Antisense RNAs can thus be used for identifying a gene product of interest, and studying its function as well as its role in early development. To examine the applicability of antisense RNAs to the study of neurotransmitter receptors, which are key molecules in synaptic communication and may also play an important role in the formation of synaptic connections, we have examined the effect of antisense RNAS on the functional expression, in Xenopus oocytes, of the multi-subunit nicotinic acetylcholine receptor of the electric organ of Torpedo. Four antisense RNAs, were synthesized from cDNA clones coding for the four subunits of the acetylcholine receptor of Torpedo electroplaques, were used to study the effect on the expression of functional Torpedo acetylcholine receptors in Xenopus ooctyes. All antisense RNAs inhibited the appearance of functional receptors in the oocyte's surface membrane for at least 1 week. This inhibition was specific, because the antisense RNAs did not block the expression of the Cl- channels, also encoded by Torpedo electroplaque mRNA. Experiments with incomplete antisense RNAs, and a synthetic oligonucleotide, indicate that covering the ribosome binding site, or the initiation condon, in the mRNA is not a necessary requirement for efficient blocking. Thus, the use of antisense RNAs combined with the Xenopus ooctye system provides a novel approach to screen cDNA libraries for the genes coding for multi-subunit neurotransmitter receptors. |