Shingo Shoji

Inward rectifier and low-threshold calcium currents contribute to the spontaneous firing mechanism in neurons of the rat suprachiasmatic nucleus

Intracellular and voltage-clamp studies were carried out to clarify the mechanism for spontaneous firing activity in neurons of the suprachiasmatic nucleus (SCN) of rat hypothalamic brain slices in vitro. SCN neurons displayed spontaneously firing action potentials that were preceded by a depolarizing pre-potential and followed by a short spike after-hyperpolarization (AHP). Injection of inward current with a duration longer than 50 ms resulted in a depolarizing voltage “sag” on hyperpolarizing electrotonic potentials. The inward rectification was depressed by bath application of caesium (1 mM) but not by barium (500 μM). SCN neurons also showed a rebound depolarization associated with spike discharge (anodal break) in response to relaxation of hyper polarizing current injection. The rebound depolarization was reduced by nominally zero calcium. Cadmium (500 μM), cobalt (1 mM) or caesium (1 mM) but not nicardipine also depressed the rebound depolarization. Under voltage-clamp conditions, hyperpolarizing steps to membrane potentials negative to approximately −60 mV caused an inward rectifier current, probably H current (IH), which showed no inactivation with time. Bath application of caesium (1–2 mM) suppressed IH. Caesium (2 mM) depressed the slope of the depolarizing spike pre-potential, resulting in a prolongation of the interspike interval of tonic firing neurons. We conclude that both the inward rectifier current, IH, and the low-threshold calcium current contribute to the spike prepotential of spontaneous action potentials in firing neurons of the rat SCN.

cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus

Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage-clamp method, in vitro. Inward rectifier current (IH) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately −60 mV in nominally zero-Ca2+ Krebs solution containing tetrodotoxin (1 μM), tetraethylammonium (40 mM), Cd2+ (500 μM) and 4-aminopyridine (1 mM).IH developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time.IH was selectively blocked by extracellular Cs+ (1 mM). The activation of the H-channel conductance (GH) ranged between −55 and −120 mV. TheGH was 80–150 pS (n=4) at the half-activation voltage of −84±7 mV (n=4). The reversal potential ofIH obtained by instantaneous current voltage (I/V) relations was −41±6mV (n=4); it shifted to −51±8mV (n=3) in low-Na+ (20 mM) solution and to −24±4 mV (n=4) in high-K+ (20 mM) solution. Forskolin (1–10 μM) produced an inward current and increased the amplitude ofIH. Forskolin did not change the half-activation voltage ofGH. 8-Bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP, 0.1–1 mM) and dibutyryl-cAMP (0.1–1 mM) enhancedIH. 3-Isobutyl-1-methylxanthine (IBMX, 1 mM) also enhancedIH. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN.

Adenosine inhibits the synaptic potentials in rat septal nucleus neurons mediated through pre- and postsynaptic A1-adenosine receptors

Intracellular and voltage-clamp recordings were made from neurons in rat brain slices containing dorsolateral septal nucleus (DLSN), in vitro. Bath application of adenosine (100 microM) produced a hyperpolarization (2-15 mV) in 46% of DLSN neurons (AH-neurons); in the remaining 54% neurons (non-AH-neurons), no hyperpolarization to adenosine was observed. Adenosine (1-300 microM) depressed not only the excitatory postsynaptic potential (EPSP) but also the inhibitory postsynaptic potential (IPSP) and the late hyperpolarizing potential (LHP) evoked by stimulation of the hippocampal CA3 area or the fimbria/fornix pathway in both AH- and non-AH-neurons. In non-AH-neurons, adenosine did not block current responses resulting from glutamate, muscimol or baclofen applied directly to DLSN neurons. In AH-neurons, adenosine partially depressed the baclofen-induced outward current. Adenosine did not block the directly-evoked IPSP (monosynaptic IPSP) as well as the glutamate-induced (hyperpolarizing) postsynaptic potential (PSP) that is mediated by GABA released from interneurons. These results suggest that adenosine does not directly inhibit the release of GABA. The effects of adenosine was mimicked by selective A1-receptor agonists and was blocked by selective A1-receptor antagonists. Pertussis toxin (PTX) blocked the hyperpolarization induced by adenosine or baclofen applied exogenously. Adenosine consistently produced presynaptic inhibition of the EPSP even in DLSN neurons treated with PTX. We conclude that adenosine inhibits neurotransmission between the hippocampus and septum through activation of pre- and postsynaptic A1-receptors which couple with G-proteins of different PTX-sensitivity or with distinct transduction processes at pre- vs. postsynaptic sites.

Depletion of glucose causes presynaptic inhibition of neuronal transmission in the rat dorsolateral septal nucleus

The role of glucose in synaptic transmission was examined in the rat dorsolateral septal nucleus (DLSN) with single‐microelectrode voltage‐clamp and slice‐patch techniques. Removal of glucose from the oxygenated Krebs solution caused an outward current associated with an increased membrane conductance. The current‐voltage relationship (I–V curve) showed that the hypoglycemia‐induced outward current was reversed in polarity at the equilibrium potential for K+. Exposure of DLSN neurons to the glucose‐free solution for 5–20 min depressed the excitatory postsynaptic current (EPSC), the inhibitory postsynaptic current (IPSC), and the late hyperpolarizing current (LHC). Replacement of glucose with 2‐deoxy‐D‐glucose (2DG), an antimetabolic substrate, mimicked the deprivation of glucose. Mannoheptulose (10 mM) and dinitrophenol, inhibitors of glucose metabolism, also depressed the PSCs, even in the presence of 10 mM glucose. Glucose‐free perfusion did not significantly depress the glutamate‐induced inward current, indicating that the inhibition of the EPSC by the glucose‐free perfusion was presynaptic. γ‐aminobutyric acid (GABA)‐induced outward currents were depressed by the glucose‐free solution. Intracellular dialysis of DLSN neurons with a patch‐pipette solution containing 5 mM ATP attenuated the hypoglycemia‐induced outward current. Glucose‐free superfusion consistently inhibited the IPSC and the LHC without changing the GABA‐induced outward current in ATP‐treated DLSN neurons. It is suggested that glucose metabolism directly regulates the release of both excitatory amino acids and GABA from the presynaptic nerve terminals.

IL‐18 gene polymorphism confers susceptibility to the development of anti‐GAD65 antibody in Graves’ disease

Aims  This study aimed to investigate whether interleukin‐18 (IL‐18) gene polymorphisms are associated with the development of antibody against the 65‐kDa isoform of recombinant human glutamic acid decarboxylase (GAD65Ab) in patients with Graves’ disease.

Presynaptic facilitation of excitatory postsynaptic potential by glucagon in neurons of rat ventromedial hypothalamic slices

Intracellular recordings were made from neurons in rat ventromedial hypothalamus (VMH), in vitro. Application of glucagon (100 nM to 5 microM) for 2-5 min increased the amplitude of excitatory postsynaptic potential (EPSP) lasting for 10-20 min. Forskolin and 8-bromo-cyclic AMP mimicked glucagon in producing a long-lasting facilitation of the EPSP. These drugs did not affect depolarizing response produced by glutamate. 3-Isobutyl-1-methylxanthine (IBMX) potentiated the time course of glucagon-induced facilitation of the EPSP. These results suggest that glucagon facilitates the EPSP probably by increasing transmitter release through activation of adenylate cyclase.