Hideho Higashi

Effects of hypoxia on rat hippocampal neurones in vitro.

1. The effects of hypoxia on the rat hippocampal CA1 neurones in tissue slices of the rat brain were studied in vitro by intracellular recording. 2. In response to superfusion of a hypoxic medium equilibrated with 95% N2‐5% CO2, a majority of the neurones showed a hyperpolarization of 5‐15 mV in amplitude and 4‐12 min in duration. The hyperpolarization was, in turn, followed by a slow depolarization which within 20 min of hypoxic exposure reached a plateau level of about 25 mV above the pre‐hypoxic resting potential. Both the initial hyperpolarization and subsequent depolarization were associated with a reduction in membrane resistance. 3. The hyperpolarization reversed in polarity at a membrane potential of ‐83 mV. There was an almost linear relationship between amplitude of the hyperpolarization and membrane potential. The hyperpolarization was markedly enhanced in potassium‐free media and was depressed in high‐potassium solutions. 4. The hyperpolarization was not significantly affected by low‐chloride or low‐sodium medium or by solution containing tetraethylammonium (10 mM), 4‐aminopyridine (1.5 mM) or caesium (3 mM). Moreover, intracellular injection of ethyleneglycol‐bis‐(beta‐aminoethylether)N,N‐tetraacetic acid (EGTA) did not alter the hyperpolarization. On the other hand, barium (0.5 mM)‐containing medium reduced the amplitude of the hyperpolarization by 20‐40%. 5. Superfusion of ouabain (5‐7 microM)‐containing medium in normoxic conditions produced hyperpolarizing and depolarizing responses similar to those elicited by hypoxic exposure. The slow depolarization was also mimicked by elevation of the extracellular potassium concentration to 10‐20 mM. 6. Evoked i.p.s.p.s were abolished within 4 min of hypoxic exposure while evoked e.p.s.p.s were maintained for about 20 min of hypoxic superfusion. Soma spikes of the neurones elicited by a depolarizing pulse were also well preserved. Their threshold was, however, raised, concomitant with a decrease in the peak amplitude. 7. When the slice was reoxygenated after 20‐40 min of hypoxic exposure, the neurones immediately began to repolarize and showed a transient hyperpolarization of 5‐10 mV in amplitude and 1‐2 min in duration. The membrane potential, input resistance and action potential returned to the pre‐hypoxic levels after 15‐20 min of reoxygenation. The amplitude of the reoxygenation‐induced hyperpolarization was not significantly changed when the membrane was hyperpolarized or depolarized. The hyperpolarization was eliminated by potassium‐free medium or solution containing ouabain (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Actions of opioids on excitatory and inhibitory transmission in substantia gelatinosa of adult rat spinal cord.

1 The actions of opioid receptor agonists on synaptic transmission in substantia gelatinosa (SG) neurones in adult (6‐ to 10‐week‐old) rat spinal cord slices were examined by use of the blind whole‐cell patch‐clamp technique. 2 Both the μ‐receptor agonist DAMGO (1 μM) and the δ‐receptor agonist DPDPE (1 μM) reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) which were monosynaptically evoked by stimulating Aδ afferent fibres. Both also decreased the frequency of miniature EPSCs without affecting their amplitude. 3 In contrast, the κ‐receptor agonist U‐69593 (1 μM) had little effect on the evoked and miniature EPSCs. 4 The effects of DAMGO and DPDPE were not seen in the presence of the μ‐receptor antagonist CTAP (1 μM) and the δ‐receptor antagonist naltrindole (1 μM), respectively. 5 Neither DAMGO nor DPDPE at 1 μM affected the responses of SG neurones to bath‐applied AMPA (10 μM). 6 Evoked and miniature inhibitory postsynaptic currents (IPSCs), mediated by either the GABAA or the glycine receptor, were unaffected by the μ‐, δ‐ and κ‐receptor agonists. Similar results were also obtained in SG neurones in young adult (3‐ to 4‐week‐old) rat spinal cord slices. 7 These results indicate that opioids suppress excitatory but not inhibitory synaptic transmission, possibly through the activation of μ‐ and δ‐ but not κ‐receptors in adult rat spinal cord SG neurones; these actions are presynaptic in origin. Such an action of opioids may be a possible mechanism for the antinociception produced by their intrathecal administration.

Regulation of DARPP-32 dephosphorylation at PKA- and Cdk5-sites by NMDA and AMPA receptors: distinct roles of calcineurin and protein phosphatase-2A.

Glutamatergic inputs from corticostriatal and thalamostriatal pathways have been shown to modulate dopaminergic signaling in neostriatal neurons. DARPP‐32 (dopamine‐ and cAMP‐regulated phosphoprotein of Mr 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Dopamine signaling is mediated in part through phosphorylation of DARPP‐32 at Thr34 by cAMP‐dependent protein kinase, and antagonized by phosphorylation of DARPP‐32 at Thr75 by cyclin‐dependent protein kinase 5. We have now investigated the effects of the ionotropic glutamate NMDA and AMPA receptors on DARPP‐32 phosphorylation in neostriatal slices. Activation of NMDA and AMPA receptors decreased the state of phosphorylation of DARPP‐32 at Thr34 and Thr75. The decrease in Thr34 phosphorylation was mediated through Ca2+‐dependent activation of the Ca2+‐/calmodulin‐dependent phosphatase, calcineurin. In contrast, the decrease in Thr75 phosphorylation was mediated through Ca2+‐dependent activation of dephosphorylation by protein phosphatase‐2A. The results provide support for a complex effect of glutamate on dopaminergic signaling through the regulation of dephosphorylation of different sites of DARPP‐32 by different protein phosphatases.

Hyperpolarizing and depolarizing actions of dopamine via D-1 and D-2 receptors on nucleus accumbens neurons

The effect of dopamine (DA) on the nucleus accumbens neurons in guinea-pig brain slices was studied by intracellular recordings. DA caused a hyperpolarization in 28% of the neurons tested, a depolarization in 11%, and a hyperpolarization followed by a depolarization in 53%. The remaining neurons were unaffected. Analyses of the responses revealed that the DA hyperpolarization was produced by activation of the D-1 receptor and associated with an increase in potassium conductance, whereas the DA depolarization was generated by activation of the D-2 receptor and accompanied by a decrease in potassium conductance. DA uptake inhibitors augmented both the hyperpolarizing and depolarizing responses, while cyclic adenosine monophosphate selectively enhanced the former.

Metabotropic mGlu5 receptors regulate adenosine A2A receptor signaling.

Dopamine, by activating dopamine D1-type receptors, and adenosine, by activating adenosine A2A receptors, stimulate phosphorylation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32,000) at Thr-34. In this study, we investigated the effect of metabotropic glutamate (mGlu) receptors on DARPP-32 phosphorylation at Thr-34 in neostriatal slices. A broad-spectrum mGlu receptor agonist, trans-ACPD, and a group I mGlu receptor agonist, DHPG, stimulated DARPP-32 phosphorylation at Thr-34. Studies with mGlu receptor antagonists revealed that the effects of trans-ACPD and DHPG were mediated through activation of mGlu5 receptors. The action of mGlu5 receptors required activation of adenosine A2A receptors by endogenous adenosine. Conversely, the action of adenosine A2A receptors required activation of mGlu5 receptors by endogenous glutamate. Coactivation of mGlu5 and adenosine A2A receptors by exogenous agonists synergistically increased DARPP-32 phosphorylation. mGlu5 receptors did not require activation of dopamine D1-type receptors by endogenous dopamine, nor did dopamine D1-type receptors require activation of mGlu5 receptors by endogenous glutamate. DHPG potentiated the effect of forskolin, but not that of 8-bromo-cAMP, and stimulated DARPP-32 phosphorylation in the presence of the phosphodiesterase inhibitor IBMX, suggesting that mGlu5 receptors stimulate the rate of cAMP formation coupled to adenosine A2A receptors. The action of mGlu5 receptors was attenuated by inhibitors of extracellular signal-regulated kinase, but not by inhibitors of phospholipase C, p38, casein kinase 1, or Cdk5. The results demonstrate that mGlu5 receptors potentiate adenosine A2A/DARPP-32 signaling by stimulating the adenosine A2A receptor-mediated formation of cAMP in an extracellular signal-regulated kinase-dependent manner.

5-Hydroxytryptamine receptors of visceral primary afferent neurones on rabbit nodose ganglia

1. The electrophysiological characteristics of 5‐hydroxytryptamine (5‐HT) receptors distributed on visceral primary afferent neurones (the nodose ganglion cells of the vagus) in rabbits were investigated with intracellular recording and voltage‐clamp techniques.

Functional reorganization of sensory pathways in the rat spinal dorsal horn following peripheral nerve injury

1 Functional reorganization of sensory pathways in the rat spinal dorsal horn following sciatic nerve transection was examined using spinal cord slices with an attached dorsal root. Slices were obtained from animals whose sciatic nerve had been transected 2‐4 weeks previously and compared to sham‐operated controls. 2 Whole‐cell recordings from substantia gelatinosa neurones in sham‐operated rats, to which nociceptive information was preferentially transmitted, revealed that dorsal root stimulation sufficient to activate Aδ afferent fibres evoked a mono‐ and/or polysynaptic EPSC in 111 of 131 (≈85 %) neurones. This is in contrast to the response following Aβ fibre stimulation, where monosynaptic EPSCs were observed in 2 of 131 (≈2 %) neurones and polysynaptic EPSCs were observed in 18 of 131 (≈14 %) neurones. 3 In sciatic nerve‐transected rats, however, a polysynaptic EPSC following stimulation of Aβ afferents was elicited in 30 of 37 (81 %) neurones and a monosynaptic EPSC evoked by Aβ afferent stimulation was detected in a subset of neurones (4 of 37, ≈11 %). 4 These observations suggest that, following sciatic nerve transection, large myelinated Aβ afferent fibres establish synaptic contact with interneurones and transmit innocuous information to substantia gelatinosa. This functional reorganization of the sensory circuitry may constitute an underlying mechanism, at least in part, for sensory abnormalities following peripheral nerve injuries.

Changes in spontaneous firing patterns of rat hippocampal neurones induced by volatile anaesthetics.

1. The effects of the volatile anaesthetics, halothane, isoflurane and enflurane, on rat hippocampal CA1 and CA3 neurones in in vitro preparations were studied by intracellular recording methods. 2. The three anaesthetics, at concentrations similar to those used clinically (0.2‐1.2 mM), initially increased and then subsequently decreased the spontaneous firing of CA1 neurones without affecting the resting membrane properties or the EPSPs evoked by focal stimuli. 3. The anaesthetics at these concentrations depressed both the fast after‐hyperpolarization of the soma spike and the post‐tetanic hyperpolarization induced by repetitive stimulation. They also decreased the IPSPs evoked by focal stimuli. 4. The threshold for spike generation was gradually elevated by as much as 4‐6 mV during application of the anaesthetics at these concentrations. The subthreshold potential oscillations (which are likely to be associated with periodic alterations in non‐inactivating Ca2+ and Na+ currents) were enhanced in the low concentrations (0.2‐0.5 mM), but were depressed in the high concentrations (0.8‐1.2 mM). 5. The results suggest that the transient increase in the firing frequency was caused by a depression of both the spike after‐hyperpolarization and the post‐tetanic hyperpolarization, and that the reduction of spontaneous firing was mainly due to an elevated threshold for spike generation. 6. The three anaesthetics altered the pattern of spontaneous spike‐firing in CA3 neurones from solitary spiking to burst firing without affecting the resting membrane properties. 7. The effects of the anaesthetics on the active membrane properties and the postsynaptic potentials in CA3 neurones were similar to the effects in CA1 neurones. 8. In the majority of CA3 neurones, soma spikes elicited by depolarizing current pulses were followed by a Ca2+‐dependent after‐depolarization, which was in turn followed by a prolonged after‐hyperpolarization (post‐burst hyperpolarization). The anaesthetics facilitated the after‐depolarizing potential, while they depressed the post‐burst hyperpolarization. Combination of the two effects would give rise to the highly stereotyped burst (about 1 Hz in frequency) in the presence of the volatile anaesthetics.

Differential regulation of dopamine D1 and D2 signaling by nicotine in neostriatal neurons

Nicotine, acting on nicotinic acetylcholine receptors (nAChRs) expressed at pre‐synaptic dopaminergic terminals, has been shown to stimulate the release of dopamine in the neostriatum. However, the molecular consequences of pre‐synaptic nAChR activation in post‐synaptic neostriatal neurons are not clearly understood. Here, we investigated the effect of nAChR activation on dopaminergic signaling in medium spiny neurons by measuring phosphorylated DARPP‐32 (dopamine‐ and cAMP‐regulated phosphoprotein of Mr 32 kDa) at Thr34 (the PKA‐site) in mouse neostriatal slices. Nicotine produced dose‐dependent responses, with a low concentration (1 µm) causing a sustained decrease in DARPP‐32 Thr34 phosphorylation and a high concentration (100 µm) causing a transient increase in DARPP‐32 Thr34 phosphorylation. Depending on the concentration of nicotine, either dopamine D2 or D1 receptor signaling was predominantly activated. Nicotine at a low concentration (1 µm) activated dopamine D2 receptor signaling in striatopallidal/indirect pathway neurons, likely by activating α4β2* nAChRs at dopaminergic terminals. Nicotine at a high concentration (100 µm) activated dopamine D1 receptor signaling in striatonigral/direct pathway neurons, likely by activating (i) α4β2* nAChRs at dopaminergic terminals and (ii) α7 nAChRs at glutamatergic terminals, which, by stimulating the release of glutamate, activated NMDA/AMPA receptors at dopaminergic terminals. The differential effects of low and high nicotine concentrations on D2‐ and D1‐dependent signaling pathways in striatal neurons may contribute to dose‐dependent actions of this drug of abuse.

Reorganization of the primary afferent termination in the rat spinal dorsal horn during post-natal development

To study the reorganization of the primary afferent input in the spinal dorsal horn during post-natal development, synaptic responses evoked by large Abeta and fine Adelta afferents were recorded from substantia gelatinosa (SG) neurons in slices obtained from immature (post-natal days 21-23) and mature rats (post-natal days 56-60). Threshold stimulus intensities and conduction velocities (CVs) of Abeta and Adelta afferents were determined by intracellular recordings of the antidromic action potentials from dorsal root ganglion (DRG) neurons isolated from immature and mature rats. In immature rats, excitatory postsynaptic currents (EPSCs) were elicited by stimulation sufficient to activate Abeta afferents in the majority of SG neurons (64.9%, 24 of 37 neurons), while most EPSCs observed in mature rats were elicited by stimulation of Adelta afferents (62.5%, 25 of 40 neurons). These observations suggest that the primary afferents innervating SG neurons were reorganized following maturation; Abeta afferents were the predominant inputs to the SG neurons in the immature state, thereafter Adelta afferents were substituted for the Abeta afferents to convey sensory information to the SG neurons. This relatively slow reorganization of the sensory circuitry may correlate with slow maturation of the SG neurons and with a delay in the functional connections of C afferents to the SG neurons.