Ken Kadota

Rapid structural remodeling of shaft synapses associated with long-term potentiation in the cat superior cervical ganglion in situ.

Synaptic plasticity associated with long-term potentiation was studied electrophysiologically and ultrastructurally in the cat superior cervical ganglion in situ. The preganglionic nerve fiber was stimulated at 10 Hz for 50 s for conditioning and then at 1 Hz for 1-3 h to monitor changes in the postganglionic compound action potential (PGP). The present material has shown the long-term potentiation (LTP), around 120% of the control, which lasted for up to 3 h. Fifteen of 18 ganglia (83%) have shown LTP. Ultrastructural studies demonstrated the synaptic structural remodeling: (1) The preganglionic nerve terminals ordinarily made mainly asymmetrical type of shaft synapses directly with dendrites of the ganglion cells that lacked dendritic spines; (2) conditioning tetanus rapidly remodeled simple shaft synapses into perforated ones characterized by perforations in the postsynaptic density (PSD), some of which had synaptic spinules associated with the perforated PSDs, i.e. spinule-synapses; (3) a rapid increase in the number of both structures was detected immediately after the tetanus. Perforated synapses and the spinule-synapses increased from 5% and 0% in the control to 27 and 9% at 0 min, respectively. Spinule-synapses occurred about one-third of the perforated shaft synapses; (4) Increased numbers of restructured shaft synapses was maintained for 15 min in ganglia expressing LTP; (5) Remodeled synapses did not increase in ganglia that did not express LTP or ganglia that were activated at 0.5 or 1 Hz. It was suggested a rapid increase in the number of remodeled synapses associated with the onset of LTP and its durability at its earlier phases in the cat SCG.

Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D1 and D2 receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D1 receptor antagonist, [3H]SCH 23390, and a dopamine D2 receptor antagonist, [3H]‐spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant (KD) of 0.65 and 0.5 nM, respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [3H]SCH 23390 and [3H]spiroperidol in a stereoselective and concentration‐dependent manner with an appropriate rank order potency for dopamine D1 or D2 receptors. The regulations of the agonist binding by guanyl‐5‐ylimidodiphosphate were observed. ADP ribosylation of the CVs with [32P]NAD demonstrated predominant labeling of bands of Mr 47,000–52,000, 42,000–45,000, and 40,000‐39,000, which corresponded to the known molecular weights of the α subunits of Gs and Gi proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D2 receptor agonists, was present in the CVs. These findings suggest that the dopamine D1 and D2 receptors in the CVs couple with adenylate cyclase via Gs/Gi protein.

Characterization of [3H]5-hydroxytryptamine and [3H]spiperone binding sites in clathrin-coated vesicles from bovine brain

Coated vesicles prepared from bovine brain cerebral cortex exhibited [3H]5-hydroxytryptamine (5-HT, serotonin) and [3H]spiperone binding activities. The binding activities were localized in the inner core vesicles. Binding reached an equilibrium level by 30-45 min at 30 degreesC, and was reversed by the addition of 100 microM 5-HT for [3H]5-HT binding or 10 microM ketanserin for [3H]spiperone binding. The saturation binding experiments indicated a single class of binding sites for [3H]5-HT and [3H]spiperone with apparent Kd values of 2.4 and 1.75 nM, respectively. The binding of [3H]5-HT was displaced by 5-HT and 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), but not by ketanserin. The binding of [3H]spiperone was displaced by spiperone and ketanserin but not by 5-HT or 8-OH-DPAT even at 1 mM. The coated vesicles were shown by immunoblotting assay to contain alpha-subunits of GTP-binding proteins, Galphas, Galphai2, Galphai3, Galphao and Galphaq/11. Forskolin-stimulated adenylate cyclase activity in the coated vesicles was inhibited to 80% of the control level by 5-HT or 8-OH-DPAT. These results suggested that 5-HT1A and 5-HT2A receptors are present in bovine brain coated vesicles and that the 5-HT1A receptors are coupled to adenylate cyclase activity via GTP binding proteins.

Localization of a 82 kDa protein in postsynaptic density and its association with cytoskeletons

A fraction of synaptic junctional complex (SJC) was prepared from rat synaptosomes and served as antigen material to produce monoclonal antibodies (Mab) for examining the component proteins of the SJC. An antibody, Mab SJ-8, was obtained, which recognized a protein with a molecular weight of 82,000 Da in the SJC preparation by immunoblot analysis. The immunohistochemical localization of the 82 kDa protein was studied with the rat cerebellum. Mab SJ-8 labeled the peripheral areas of the Purkinje and granule cells. Small punctate areas were also stained in the molecular layer with SJ-8. Intracellular localization of the protein was examined with rat brain synaptosomes. Immunoelectron microscopy demonstrated that Mab SJ-8 strongly labeled the postsynaptic density (PSD) and also a fibrous network spreading out of it. However, the antibody did not label the pre- or post-synaptic membrane or the cleft material.

Correlation between long-term potentiation and synaptic spinule

‘CREST, Jap. Sci. Tech. Corp., *Lab. for Cellular Neurophysiol., Brain Sci. Inst., RIKEN, Wake, 351-0198, ‘Dep. ofbtomY, Hokkaido Univ. Sch. of Med, Sapporo, 060-8638, ‘Lab. for Physiol., Kanazawa Univ. Sch. of Med.. Kanazawa. 920-8640. Japan. Massive elimination of supernumerary climbing fiber (CF) Purkinje cell (PC) synapse is known to occur during postnatal cerebellar development. In rodents, the adult-type mono innervation pattern is established on around postnatal day 20 (P20). To test whether neural activity is involved in this process, TTX was delivered continuously from an implant of Elvax (ethylene-vinyl acetate copolymer) applied onto the cerebellar lobules 6-8 under Nembutal anesthesia. On P24-36, parasagittal cerebellar slices were prepared, and the number of CFs innervating each PC was estimated electrophysiologically. About 50% of PCs in lobules 6-8 (close to the Elvax) were multiply-innervated by CFs, whereas about 80% of PCs in lobules l/2 and 10 (distant from the Elvax) and PCs in vehicle-treated control cerebella were mono-innervated. These results suggest that the effect of TTX was restricted to the lobules 6-8, and that neural activity within the cerebellar cortex is required for CF synapse elimination. Furthermore, daily injection of MK801, an NMDA receptor blocker, during P15-18 resulted in persistent m!ultiple CF innervation. NMDA receptor-mediated components of excitatory synaptic currents were not found in PCs, whereas they were rich in mossy fiber (MF) to granule cell (GC) synapses. These results suggest that neural activity involving NMDA receptors at MF-GC synapses during P1.5-18 is required for CF synapse elimination during postnatal cerebellar development.

1329 Correlation between post-tetanic potentiation and synaptic spinule

Takanori Hashimoto, Yasushi Kajii, Toru Nishikawa Psychostimulants elicit a progressive and persistent enhancement of behavioral responses to these drugs (behavioral sensitization). To get an insight into the possible involvement of neuronal plasticit,y in the behavioral sensitization, we studied the effects of single administration of MAP and cocaine on the expression of a plasticity-related molecule, tissue plasminogen activator (TPA) mRNA. An acute injection of MAP and cocaine induced TPA mRNA in a subpopulation of neurons in the mediodorsal frontal cortex, agranular insular cortex and piriform cortex. Similar distribution and time course of TPA mRNA expression was seen after systemic application of nomifensine and phencyclidine which are also capable to produce sensitization. Pretreatment with dopamine receptor antagonists, haloperidol and SCH23390, inhibited MAP-induced TPA mRNA expression. Retrograde tracer study combined with in situ hybridization revealed majority of TPA mRNA expressing cells project to the medial striatum.

1411 The synaptic spinule formation attendant on the post-tetanic potentiation in the cat superior cervical ganglion in vivo

Following peripheral nerve injury, pathological states such as allodynia and/or hyperalgesia have been reported to develop in a subpopulation of patients. Although peripheral neural mechanisms are likely to contribute to the pathological state, persistence of pain after healing of the damaged tissue suggests that plastic changes in the CNS, including the spinal cord, may also play a important role in processing the pathological pain transmission. To investigate plastic changes in synaptic transmission at the spinal level, whole cell recordings were made from substantia gelatinosa (SC) and laminae IV/V neurons in the spinal cord slice with attached dorsal root dissected from rats with or without sciatic nerve transection (SNT) No significant changes in passive and active membrane characteristics, including resting membrane potential, input resistance and configuration of action potential and spike after potentials, were detected between normal and SNT rats. In the control rats, primary afferent stimulation with intensity sufficient to activate Ad afferents elicited a monosynaptic fast EPSC in the majority of SC neurons. In the SNT rats, however, a polysynaptic EPSC with a long latency was evoked by activation of afferent fibers. The stimulus intensity for eliciting the polysynaptic EPSCs was much lower than that for activation of A6 afferents, suggesting that AB afferents are responsible for generation of this response. In addition, the conduction velocity and threshold of these afferents were not significantly altered. These observations suggest that synaptic plasticity occuned in a subset of deep dorsal horn neurons, which begin to transmit sensory information’to SG. This plastic change may.

Low Incubation Temperature Favors Detection of Depolarization-Induced Coated Vesicles in Motor Axon Endings in Frog Nerve-Muscle Preparations

Depolarization-induced coated vesicles were detected in motor axon endings in frog nerve-muscle preparations stimulated at 10 degrees C but hardly in those activated at 25 degrees C.