Andrew A. Hicks

Synapsin I and syntaxin 1B : Key elements in the control of neurotransmitter release are regulated by neuronal activation and long-term potentiation in vivo

The messenger RNAs encoding proteins of the exocytotic machinery were measured at different times following the induction of long-term potentiation or increasing neuronal activity in the dentate gyrus of the rat in vivo. In situ hybridization revealed that from the many messenger RNAs that encode proteins involved in regulated exocytosis, only those encoding synapsin I and syntaxin 1B were specifically increased. The levels of messenger RNA encoding both synapsin I and syntaxin 1B were increased on the ipsilateral side of the dorsal dentate gyrus 2 and 5 h following the induction of long-term potentiation. Syntaxin 1B was also increased in the ventral dentate gyrus at the same time-points. On the contralateral side of the dentate gyrus there was an increase in both synapsin I and syntaxin 1B at 5 h only. All of these long-term potentiation-induced changes were prevented when the tetanus was delivered in the presence of the N-methyl-D-aspartate receptor antagonist. (D(-)-2-amino-5-phosphonopentanoic acid. Immunocytochemical staining revealed that protein levels for both synapsin I and syntaxin 1B were elevated in the mossy fibre terminal zone of CA3 5 h after the induction of long-term potentiation. In addition to these plasticity-induced changes, a transient increase in the messenger RNA encoding syntaxin 1B was observed at 2 h in conditions of high intensity stimulation of the perforant path to increase the level of cellular activation, but this change was not maintained even when high intensity stimulation was sustained for 5 h. No changes in either of the messenger RNAs were observed under low frequency stimulation and pseudotetanus at either time-points. These results show that an overall increase in neuronal excitation within a neuronal network can be differentiated from a change in synaptic strength at a specific subset of the synapses, where only synaptic plasticity leads to long-term changes in the expression of selective members of the exocytotic machinery. Altered concentrations of key vesicle proteins may thus provide the means for modulation of neurotransmitter release over long time-periods. The persistent long-term potentiation-induced postsynaptic increase in messenger RNAs encoding these presynaptic proteins has important implications for the propagation of signals downstream from the site of long-term potentiation induction in hippocampal neural networks, and highlights a candidate molecular mechanism for mediating the propagation of synaptic plasticity in such networks.

DISTINCT REGIONAL EXPRESSION OF NICOTINIC ACETYLCHOLINE-RECEPTOR GENES IN CHICK BRAIN

Four genes (alpha 2, alpha 3, alpha 4 and beta 2) have been reported as encoding subunits of the nicotinic acetylcholine receptor (nAChR) in chicken brain. The mRNAs transcribed from these genes have here been localised to particular regions using in situ hybridisation histochemistry. The beta 2 mRNA was clearly the most abundant transcript, being widely distributed throughout the chick brain. In the cerebellum, all four mRNA species were present, although they showed different cellular patterns of distribution. Only alpha 2 mRNA and beta 2 mRNA were found in significant amounts in the optic tectum. In the lateral spiriform nucleus, while alpha 2 mRNA, alpha 4 mRNA and beta 2 mRNA were all very abundant, the alpha 4 mRNA was localised to a subgroup of neurons containing alpha 2 mRNA and beta 2 mRNA. This represents the first evidence that individual cells may express two different nAChR alpha subunit genes in vivo. The distributions of the 4 mRNA species showed few common features. This suggests that other neuronal nAChR genes remain to be identified, and that these 4 genes are not generally expressed in the same cells to constitute a single macromolecular complex. The results therefore provide evidence for nAChR heterogeneity in the central nervous system.

The chicken GABAA receptor α1 subunit: cDNA sequence and localization of the corresponding mRNA

Abstract We report the sequence of a complementary DNA (cDNA) that encodes the chicken GABA A receptor α1 subunit, which is extremely homologous to mammalian α1 subunits. The distribution of α1 subunit transcripts is shown to correlate mainly, but not completely, with the previously-reported pattern of benzodiazepine type I (BZI) binding sites in the avian brain. These results suggest that the α1 subunit may not necessarily be restricted to receptors having BZI pharmacology.

Confirmation of the localization of the human GABAA receptor α1-subunit gene (GABRA1) to distal 5q by linkage analysis

The GABAA receptor is the major inhibitory neurotransmitter receptor in the mammalian brain. To date, 14 genes that encode subunits of this receptor have been identified; these appear to be scattered throughout the human genome and are under investigation as candidate loci for a number of neurological and psychiatric disorders. We report here a highly polymorphic (dC-dA)n repeat within the human alpha 1-subunit gene (GABRA1). Typing of this marker in the Centre dEtude du Polymorphisme Humain (CEPH) panel of families confirms the previous assignment of the GABRA1 locus to the distal portion of chromosome 5q by demonstrating linkage to the markers CRI-L45 (D5S61) (Zmax = 11.00, theta max = 0.15), CRI-V1022 (D5S54) (Zmax = 7.25, theta max = 0.20), and CRI-P148 (D5S72) (Zmax = 5.71, theta max = 0.24).

Genomic mapping and evolution of human GABAA receptor subunit gene clusters

Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow G11 6NU, UK Department of Biochemistry and Molecular Genetics, Imperial College School of Medicine at St Mary’s Hospital, London W2 1PG, UK Institut für Zellbiochemie und klinische Neurobiologie, Universita ̈ts-Krankenhaus Eppendorf, Universita ̈t Hamburg, 20246 Hamburg, Germany Institut für Humangenetik, Justus-Liebig-Universita ̈t Giessen, 35392 Giessen, Germany Laboratoire de Genetique Moleculaire de la Neurotransmission et des Processus Neurodegeneratifs, Centre National de la Recherche Scientifique, 91198 Gif-sur-yvette, France Department of Cardiothoracic Surgery, Hammersmith Hospital, London W12 0HS, UK

Induction of long-term potentiation in vivo regulates alternate splicing to alter syntaxin 3 isoform expression in rat dentate gyrus

Abstract: The regulation and specificity of the interactions between the proteins involved in neurotransmitter release are obvious targets for the cellular control of synaptic plasticity. Previous research has identified one of these proteins, syntaxin 1B, as a potential target for mediating the propagation of synaptic plasticity through neural networks. The expression of syntaxin 1B is modified in the hippocampus after the induction of long‐term potentiation (LTP) and during learning. Here, we describe the identification of two other members of the syntaxin family from rat brain, syntaxins 3A and 3B, and show that they are generated from the same gene by alternate splicing. In situ hybridization and immunohistochemical staining confirm the expression of syntaxins 3A and 3B in the adult rat brain. The transcripts and proteins show a lower abundance but a similar pattern of expression as syntaxins 1A and 1B. By using quantitative competitive PCR, we show that the mRNAs that encode syntaxins 1B and 3A are increased in dentate granule cells 6 h after the induction of LTP in vivo, whereas syntaxin 3B mRNA is decreased as rapidly as 30 min, and lasts for at least 6 h, after the induction of LTP. These findings identify coordinated changes in the expression of several syntaxin isoforms with different substrate specificities and suggest that regulation of the splicing machinery by LTP induction is one of the diverse strategies used during the long‐term modification of the synapse in the vertebrate nervous system.

Brain Structure and Task-specific Increase in Expression of the Gene Encoding Syntaxin 1B During Learning in the Rat: A Potential Molecular Marker for Learning-induced Synaptic Plasticity in Neural Networks

The mRNAs encoding the synaptic vesicle proteins syntaxin 1 B and synapsin I were measured using in situ hybridization in several brain regions‐the dentate gyrus, CA3 and CA1 of the hippocampus, the parietal, the motor and prefrontal cortices and the core and shell of the accumbens‐of rats that were learning a spatial reference or working memory task on a radial arm maze. The mRNA encoding syntaxin 1B was significantly increased in all hippocampal regions in rats learning the working memory task, whereas it was increased in the prelimbic area of the prefrontal cortex and the shell of the accumbens in rats learning the spatial reference memory task. No change in mRNA encoding syntaxin 1B was observed in the motor and parietal cortices or the core of the accumbens, and the mRNA encoding synapsin I was not significantly different from that of naive caged controls or rats running the maze for continuous reinforcement in any of the brain structures examined. These results demonstrate that the gene encoding a key member of synaptic vesicle function is up‐regulated in a task‐ and brain‐specific manner during learning. They are discussed in terms of the potential role this protein may play in trans‐synaptic propagation of plasticity within specific neural networks as a function of the information required in the laying down of different types of memory.

Molecular Biology of Nicotinic Acetylcholine Receptors from Chicken Muscle and Brain

While it has been completely established that the AChR** as isolated from the electroplaques of electric fish is composed of four different polypeptides, such that its subunit structure is α2βγδ (as reviewed in detail elsewhere in this Volume), the evidence for this structure has so far been less complete for the related AChRs from the skeletal muscle of various vertebrates (Dolly and Barnard, 1984). Of course, sequencing of the cDNAs encoding several of these has revealed strong amino acid homology between their subunits and those of the Torpedo AChR (summarised by Kubo et al., 1985). However, what appears to be a full complement of the subunits of a muscle AChR has been identified, via their DNAs, so far only in calf (Kubo et al., 1985) and even there a difference from the α2βγδ structure has been revealed. Thus, an additional cDNA of bovine muscle was recently identified (Takai et al., 1985), which encodes a fifth polypeptide, designated the e subunit. Electrophysiological measurements on Xenopus oocytes injected with combinations of subunit-specific mRNAs, in conjunction with Northern blot analysis, have shown that the s subunit replaces the γ subunit during muscle development (Mishina et al., 1986). These findings have led to the conclusion that the fetal and adult forms of the receptor are α2βγδ and α2βδe respectively in their subunit complements. Even in this case, although there is considerable evidence on the subunits present in an AChR species isolated from bovine muscle (Conti-Tronconi et al., 1982a), a correspondence of each of the putative cDNA products with an identified subunit and a proof that they form two different oligomers as just noted, has not yet been demonstrated.

The Expression of Syntaxin1B/GR33 mRNA Is Enhanced in the Hippocampal Kindling Model of Epileptogenesis

Abstract: Syntaxin, a protein required for the docking of synaptic vesicles, may be involved in the manifestation of synaptic plasticity. The possible involvement of syntaxin in epileptogenesis was investigated by assessing the expression levels of syntaxin1B/GR33 mRNA by in situ hybridization at different stages of hippocampal kindling epileptogenesis and after the induction of generalized seizures. Densitometric analysis of the autoradiograms revealed that the expression was not changed in pyramidal and granular neurons of the hippocampal formation 24 h after the first kindling stimulation. However, the mRNA levels in CA1, CA3, and fascia dentata neurons were bilaterally enhanced after six afterdischarges and remained at this elevated level during the whole period along which afterdischarges were elicited. An immunoassay was unable to reveal a clear significant increase of syntaxin1B/GR33 protein levels in hippocampus homogenates of fully kindled animals. The use of syntaxin1B‐specific antibodies is necessary to draw definite conclusions on the changes at the protein level. At long term, 4 weeks after the last kindling‐elicited generalized seizure, no significant alterations in transcript levels could be detected. The results suggest that the induction of kindling epileptogenesis is associated with an enhanced expression of syntaxin1B/GR33, but this enhanced expression is not necessary for persistence of kindling‐induced synaptic plasticity.

Brain α-Neurotoxin-Binding Proteins and Nicotinic Acetylcholine Receptors

The study of the nicotinic acetylcholine receptor (AChR) of skeletal muscle and fish electric organ has been greatly facilitated by the application of the α-neurotoxins (postsynaptic toxins) of elapid and hydrophid snake venoms, such as α-bungarotoxin (α-BTX), introduced by C. Y. Lee (Lee, 1973). These polypeptide toxins bind to the receptor with KD values 10−9 and 10−12 M, causing blockade of function. It has been found that the peripheral (vertebrate muscle and electric organ) type of AChR invariably has two of these high-affinity α-toxin binding sites, one on each of the α subunits of its α2βγδ pentameric structure (for reviews see Dolly and Barnard, 1984; Popot and Changeux, 1984) . However, attempts to use α-bungarotoxin as a probe for the more poorly-characterised, neuronal nicotinic receptors have caused a great deal of confusion.