Ei-ichi Miyachi

Arginine blocks gap junctions between retinal horizontal cells

Functions of nitric oxide are of common interest among a variety of tissues, since it activates soluble guanylate cyclase to produce cGMP. Here we report that intracellular application of L-arginine, the precursor of nitric oxide, blocked gap junctions between horizontal cells of the turtle retina. The input resistances of the cells were greatly increased and the cells were thereby easily polarized by current injections through microelectrodes. This procedure enables us to plot precise I-V curves and the reversal potential of light responses was estimated at around 0 mV. These results were quite similar to those obtained by intracellular application of cGMP, suggesting that the L-arginine:nitric oxide:cGMP pathway is present in retinal horizontal cells.

Na(+) action potentials in human photoreceptors.

Mammalian photoreceptors are hyperpolarized by a light stimulus and are commonly thought to be nonspiking neurons. We used the whole-cell patch-clamp technique on surgically excised human retina to examine whether human photoreceptors can elicit action potentials. We discovered that human rod photoreceptors express voltage-gated Na(+) channels, and generate Na(+) action potentials, in response to membrane depolarization from membrane potentials of -60 or -70 mV. Na(+) spikes in human rods were elicited at the termination of a light response that hyperpolarized the potential well below -50 mV. This served to amplify the release of a neurotransmitter when a bright light is turned off, and thus selectively amplify the off response to the light signal.

Linalool suppresses voltage-gated currents in sensory neurons and cerebellar Purkinje cells

Summary.Linalool is a major component of essential oils and possesses various biological effects in sensory or central nervous systems. To investigate the pharmacological and biophysical effects of linalool on voltage-gated currents in sensory neurons, we used the whole-cell patch clamp and the Ca2+ imaging techniques. Under the voltage clamp, membrane depolarization generated time- and voltage-dependent current responses in newt olfactory receptor cells (ORCs). Linalool significantly and reversibly suppressed the voltage-gated currents in ORCs. The dose-suppression relation of linalool for the voltage-gated Na+ current could be fitted by the Hill equation with a half-blocking concentration of 0.56 mM and a Hill coefficient of 1.2. To test whether linalool suppresses voltage-gated currents in ORCs specifically or suppresses currents in other neurons generally, we next examined the effects of linalool on voltage-gated currents in newt retinal neurons and rat cerebellar Purkinje cells. Linalool suppressed the voltage-gated currents not only in retinal horizontal cells and ganglion cells but also in Purkinje cells. Furthermore, bath application of linalool inhibited the KCl-induced [Ca2+]i response of ORCs, suggesting that linalool suppresses Ca2+ currents in ORCs. These results suggest that linalool non-selectively suppresses the voltage-gated currents in newt sensory neurons and rat cerebellar Purkinje cells.

Spike encoding of olfactory receptor cells.

Olfaction begins with the transduction of the information carried by odorants into electrical signals in olfactory receptor cells (ORCs). The binding of odor molecules to specific receptor proteins on the ciliary surface of ORCs induces the receptor potentials. This initial excitation causes a slow and graded depolarizing voltage change, which is encoded into a train of action potentials. Action potentials of ORCs are generated by voltage-gated Na+ currents and T-type Ca2+ currents in the somatic membrane. Isolated ORCs, which have lost their cilia during the dissociation procedure, are known to exhibit spike frequency accommodation by injecting the steady current. This raises the possibility that somatic ionic channels in ORCs may serve for odor adaptation at the level of spike encoding, although odor adaptation is mainly accomplished by the ciliary transduction machinery. This review discusses current knowledge concerning the mechanisms of spike generation in ORCs. It also reviews how neurotransmitters and hormones modulate ionic currents and action potentials in ORCs.

EXPRESSION OF GAP JUNCTION CONNEXIN36 IN ADULT RAT RETINAL GANGLION CELLS

Electrophysiological and ultrastructural studies have demonstrated that gap junctions connect diverse types of neurons in the central nervous system, permitting direct electrical and metabolic coupling. A member of gap junction channel subunit connexin36 (Cx36), is probed for the location of cell-to-cell communication in the mammalian retina, where gap junction networks of major classes of neurons are present. We present an analysis of the expression and localization of Cx36 protein in adult Wistar rat retina, using a newly generated polyclonal antibody against a sequence in the predicted cytoplasmic loop of the Cx36 amino acid alignment, deduced from the cDNA sequence. The affinity-purified antibody, recognizing a single 36-kDa protein, consistently labeled discrete puncta of subcellular structures likely to be associated with gap junctions in the inner plexiform layer, and also cytoplasm within somata and dendrites of retinal amacrine and ganglion cells, following examination with various fixation protocols and double labeling immuno-fluorescence. These results provide that prominent cell-to-cell communication appears in mature excitatory neurons such as retinal ganglion cells, in addition to inhibitory amacrine cells, mediated by gap junctions in the adult retina.

Protective Effect Against 17β-Estradiol on Neuronal Apoptosis in Hippocampus Tissue Following Transient Ischemia/Recirculation in Mongolian Gerbils via Down-Regulation of Tissue Transglutaminase Activity

We analyzed the protective effect of 17β-estradiol (17β-ED) injection against delayed neuronal death in the hippocampus tissue of the brain in Mongolian gerbils after transient ischemia/recirculation treatment, especially in relation with bcl-2 gene expression and enzymatic activity changes of caspase-3 and tissue transglutaminase (tTGase). Daily intraperitoneal injection of 17β-ED to the animal after the ischemia stimulated the expression of an apoptosis suppressor gene, bcl-2, in the hippocampal tissue for a week. The gradually increasing apoptotic enzyme activity of caspase-3 and increased number of TUNEL positive fragmented neuronal nuclei caused by ischemic attack in the gerbil brain were clearly suppressed by 17β-ED administration. The reduced activity and enzyme protein of tTGase, a neurodegenerative marker of apoptosis in the hippocampus after ischemia, were also restored to nearly normal levels by 17β-ED injection. These results suggest that daily 17β-ED administration to the gerbil after transient ischemic insult with progressing neuronal deteriorative changes in hippocampus tissue can effectively prevent apoptotic changes through a molecular cascade involving gene expression regulation.

Modulation by hyperpolarization-activated cationic currents of voltage responses in human rods

We used the whole-cell patch-clamp recording technique on surgically excised human retina to examine whether human rod photoreceptors express hyperpolarization-activated cationic currents (I(h)) and to analyze the effects of I(h) on rods voltage responses. Hyperpolarizing voltage steps from a holding potential of -60 mV evoked a slow inward-rectifying current in both rods in retinal slices and isolated rods. The slow inward-rectifying currents induced by hyperpolarization were markedly reduced by 3 mM Cs(+) (a blocker of I(h)) in the bath, but not by 3 mM Ba(2+) (an anomalous rectifier K(+) current blocker) or 1 mM SITS (a Cl(-) current blocker). A concentration-response curve for block by Cs(+) of the inward currents could be fitted by the Hill equation with a half-blocking concentration (IC(50)) of 41 microM and a Hill coefficient of 0.91. The time course of the inward current activation was well described at all recorded voltages by the sum of two exponentials. Under current-clamp conditions, injection of steps of current, either hyperpolarizing or depolarizing, elicited an initial rapid voltage change that was followed by a gradual decay in the voltage response. The decay in the voltage responses was eliminated by bath application of 3 mM Cs(+). The voltage dependence, pharmacology, and kinetics of the slow inward-rectifying currents described above suggest that human rods express I(h). We suggest that I(h) becomes activated in the course of large hyperpolarizations generated by bright-light illumination and may modify the waveform of the photovoltage in human rods.

Modulation by cGMP of the voltage-gated currents in newt olfactory receptor cells

Effects of cGMP on voltage-gated currents in the somatic membrane of isolated newt olfactory receptor cells were investigated using the whole-cell mode of the patch-clamp technique. Under voltage clamp, membrane depolarization generated time- and voltage-dependent current responses, a transient inward current and a sustained outward current. When cGMP or a membrane permeant analog of cGMP, 8-p-chlorophenylthio-cGMP (CPT-cGMP), was applied to the recorded cell, the amplitude of the transient inward current increased markedly, but that of the sustained outward current did not change significantly. When each current was isolated by pharmacological agents, 0.1 mM CPT-cGMP increased the peak amplitude of a Na(+) current (I(Na)) by approximately 40%, a T-type Ca(2+) current (I(Ca,T)) by approximately 40%, and an L-type Ca(2+)current (I(Ca,L)) by approximately 10%; however it did not change significantly the amplitude of a delayed rectifier K(+) current (I(K)). A selective cGMP-dependent protein kinase inhibitor, KT5823, blocked the enhancement by cGMP of I(Na) and I(Ca,T), suggesting that cGMP increases these currents via cGMP-dependent phosphorylation. Under current-clamp conditions, application of CPT-cGMP lowered the current threshold of action potentials induced by current injection, and increased the maximum spike frequency in response to strong stimuli. We suggest that cGMP may lower the threshold in olfactory perception by decreasing the current threshold to generate spikes, and also prevent the saturation of odor signals by increasing the maximum spike frequency.

Patch-clamp recording of human retinal photoreceptors and bipolar cells.

Photoreceptors and retinal bipolar cells are considered as nonspiking neurons; however, we recently showed that human rod photoreceptors can generate sodium action potentials in response to membrane depolarization from membrane potentials of −60 or −70 mV ( Kawai et al., Neuron30 [2001] 451). We performed patch‐clamp recording of human cone photoreceptors and retinal bipolar cells to examine whether functional voltage‐gated sodium channels are expressed in these cells as well as rod photoreceptors. Under current‐clamp conditions, the injection of depolarizing current steps into a cone photoreceptor‐induced marked action potentials. These action potentials were blocked by 1 µM tetrodotoxin, a voltage‐gated sodium channel blocker. Under voltage‐clamp conditions, depolarizing voltage steps‐induced a fast transient inward current in several bipolar cells (n = 4/78). This current was activated from −70 to +20 mV (maximal at −10 mV) and inactivated within 5 ms. The 10–90% rise time of this current was shorter than another inward current (less than one‐hundredth). These results indicate that human cones and bipolar cells express voltage‐gated sodium channels as rod photoreceptors. Sodium channels may serve to amplify the release of a neurotransmitter and to accelerate the light–dark change in photosignals.

Requirement of the tumour suppressor APC for the clustering of PSD‐95 and AMPA receptors in hippocampal neurons

Mutations in the adenomatous polyposis coli (APC) gene are associated with familial adenomatous polyposis and sporadic colorectal tumours. The APC gene is expressed ubiquitously in various tissues, especially throughout the large intestine and central nervous system (CNS). In the CNS, the expression of the APC protein is highest during embryonic and early postnatal development. APC associates through its C‐terminal region with postsynaptic density (PSD)‐95, a neuronal protein that participates in synapse development. Here, we examined the involvement of APC in synaptogenesis. In cultured hippocampal neurons, both overexpression of a dominant‐negative construct that disrupts the APC–PSD‐95 interaction and knockdown of APC expression using small interfering RNA (siRNA) inhibited the clustering of PSD‐95 and a glutamate receptor subunit, and reduced alpha‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate (AMPA)‐induced activity of AMPA receptors; however, the clustering of an N‐methyl‐d‐aspartate (NMDA) receptor subunit was unaffected. These results are suggestive of APC involvement in the development of glutamatergic synapses.