Ken-Ichi Ito

Reversal of long-term potentiation (depotentiation) induced by tetanus stimulation of the input to CA1 neurons of guinea pig hippocampal slices

The reduction of the long-term potentiated response induced by tetanus (depotentiation (DP) of LTP) was investigated by the delivery of a train of low-frequency afferent stimuli (depotentiating stimulation: DPS) after the tetanus (100 Hz, 100 pulses) in CA1 neurons of the guinea pigs hippocampal slice. The parameters of DPS (frequencies of 1, 2, 5 and 10 Hz; number of pulses of 200 and 1000; and the time-lag after tetanus of 20 and 100 min) were altered systematically and their effects on LTP were evaluated through the analysis of the slope of field EPSP (S-EPSP) and amplitude and peak latency of population spike (A- and L-PS). DPS of 1 Hz, 1000 pulses, given 20 min after tetanus, reduced the potentiated component of S-EPSP, A-PS and L-PS by 68.5%, 80.1% and 56.1%, respectively (mean, n = 6), whereas it reduced the control response by 4.3%, 7.1%, and 1.9%, respectively (n = 6). Significantly less effectiveness was observed for DPS at higher frequencies (2-10 Hz), with smaller numbers of pulses, featuring a longer time-lag after tetanus and under APV administration. When DPS was applied before tetanus, significantly less robust LTP was observed. However, these effects were blocked by the administration of APV during DPS.

In vivo imaging of spontaneous ultraweak photon emission from a rat’s brain correlated with cerebral energy metabolism and oxidative stress

Living cells spontaneously emit ultraweak light during the process of metabolic reactions associated with the physiological state. The first demonstration of two-dimensional in vivo imaging of ultraweak photon emission from a rats brain, using a highly sensitive photon counting apparatus, is reported in this paper. It was found that the emission intensity correlates with the electroencephalographic activity that was measured on the cortical surface and this intensity is associated with the cerebral blood flow and hyperoxia. To clarify the mechanism of photon emission, intensity changes from whole brain slices were examined under various conditions. The removal of glucose from the incubation medium suppressed the photon emission, and adding 50 mM potassium ions led to temporal enhancement of emission and subsequent depression. Rotenone (20 microM), an inhibitor of the mitochondrial electron transport chain, increased photon emission, indicating electron leakage from the respiratory chain. These results suggest that the photon emission from the brain slices originates from the energy metabolism of the inner mitochondrial respiratory chain through the production of reactive oxygen. Imaging of ultraweak photon emission from a brain constitutes a novel method, with the potential to extract pathophysiological information associated with neural metabolism and oxidative dysfunction of the neural cells.

Cytoplasmic calcium elevation in hippocampal granule cell induced by perforant path stimulation andl-glutamate application

Calcium-dependent fluorescence of a Ca2+ indicator (fura-2) loaded in the slice of guinea pig hippocampus was measured by a microscope/video-camera/photometry system. Tetanic stimulation of the perforant path (PP) or application of L-glutamate caused increment of the fluorescence from the dendritic and somatic layers of the granule cells in the dentate gyrus. Magnitude of the increment depended on the frequency and intensity of the PP-stimulation or on the dose of L-glutamate. 2-Aminophosphonovaleric acid, a glutamate-receptor antagonist, suppressed both PP-stimulus-induced and L-glutamate-evoked responses, while tetrodotoxin blocked the former only. Thus the fluorescence increment should represent an elevation of Ca2+ concentration in the postsynaptic cytoplasm of the granule cells.

Age-dependent modulation of hippocampal excitability by KCNQ-channels.

Recently, mutations of KCNQ2 or KCNQ3, members of the KCNQ-related K(+)-channel (KCNQ-channel) family, were identified as cause of benign familial neonatal convulsions (BFNC). However, the exact pathogenic mechanisms of age-dependent development and spontaneous remission of BFNC remain to be elucidated. To clarify the age-dependent etiology of BFNC, we determined age-dependent functional switching of KCNQ-channels, GABAergic- and glutamatergic-transmission in rat hippocampus. The effects of inhibitors of KCNQ-channel, GABA- and glutamate-receptors on propagation of neuronal-excitability and neurotransmitter release were determined by 64-channel multielectrode-dish (MED64), whole-cell recording, in vitro release technique and in vivo microdialysis biosensor, using rat hippocampus from day of birth (P0) to postnatal-day 56 (P56). Inhibition of KCNQ-channels enhanced depolarization-induced glutamate and GABA releases during P0-P7, but not during P14-P28. Inhibition of KCNQ-channels magnified neuronal-excitability propagation from P0 to P14: maximal at P3, but this effect disappeared by P28. GABA(A)-receptor inhibition surprisingly reduced neuronal-excitability propagation during P0-P3, but not at P7. AMPA/glutamate-receptors inhibition reduced propagation of neuronal-excitability throughout the study period. KCNQ-channels inhibition shortened spike-frequency adaptation, but this stimulation was more predominant during P<7 than P>14. During the first week of life, KCNQ-channels performed as a predominant inhibitory system, whereas after this period GABAergic-transmission switched from excitatory to inhibitory function. Contrary, glutamatergic-transmission has acquired as excitatory function from P0. These findings suggest that the pathogenic mechanisms of age-dependent development and spontaneous remission of BFNC are, at least partially, associated with the interaction between age-dependent reduction of inhibitory KCNQ-channel activity and age-dependent functional switching of the GABAergic-system from excitatory to inhibitory action in neonatal CNS.

Age‐dependent, steroid‐specific effects of oestrogen on long‐term potentiation in rat hippocampal slices

1 Long‐term potentiation (LTP) of hippocampal population spike responses and excitatory postsynaptic potentials (EPSPs) from area CA1 stratum pyramidale was induced in slices of rat hippocampus maintained in vitro following brief high‐frequency stimulation (HFS) of the Schaffer collateral‐commissural pathway. When administered to slices prior to HFS, 17β‐oestradiol (OE2), at a concentration as low as 0.1 nm, suppressed the magnitude of the resultant HFS‐induced potentiation in slices from prepubertal animals (3 and 4 weeks old) of both sexes. 2 OE2 did not suppress the induction of LTP in slices taken from the hippocampus of adult animals of either sex. 3 There was no similar suppressant effect of 17α‐oestradiol (OE1), progesterone (PRG) or testosterone (TST) on LTP in the young animals, even at a concentration 100 times greater than was effective for OE2. 4 The anti‐oestrogen compound tamoxifen (TMX; 1.0 and 10.0 μm), which acts principally at intracellular binding sites within the nucleus, was without effect in diminishing the suppressant effect of OE2 on LTP in slices from young animals. 5 The LTP observed in slices from both 3‐week‐old and adult rats was AP5 sensitive and thus was shown to be dependent on activation of NMDA receptors. Results from whole‐cell recording experiments suggested that OE2 caused the LTP‐suppressant effect through an action on NMDA‐mediated currents. 6 These data suggest an age‐dependent and possibly a surface membrane receptor‐mediated role for oestrogens in modulating the efficacy of input‐output properties of CA1 neurones produced by HFS during a critical period in development.

The mechanism of ATP-induced long-term potentiation involves extracellular phosphorylation of membrane proteins in guinea-pig hippocampal CA1 neurons ☆

The mechanism of ATP-induced long-term potentiation was studied pharmacologically using guinea-pig hippocampal slices. Application of 1-10 microM ATP for 10 min transiently depressed and then slowly augmented the synaptic transmission in CA1 neurons leading to long-term potentiation (LTP). This ATP-induced LTP was blocked by the addition of K-252b, an ecto-protein kinase inhibitor, but was enhanced by the addition of RK682, an ecto-phosphatase inhibitor, both of which do not permeate the cell membrane. These results suggest that ATP applied to the perfusate provides enough substrate for ecto-protein kinase to induce LTP through phosphorylation of extracellular domains of membrane proteins in CA1 neurons.

Two-dimensional photon counting imaging and spatiotemporal characterization of ultraweak photon emission from a rat’s brain in vivo

The process of metabolic reactions within living cells leads to spontaneous ultraweak light emission. The development of a system for highly sensitive imaging and spatiotemporal analysis of ultraweak photon emission from a rats brain is reported in this paper. The equipment used in this experiment consists of a two-dimensional photon-counting tube with a photocathode measuring 40 mm in diameter, a highly efficient lens system, and an electronic device to record time series of a photoelectron train with spatial information. The sensitivity and ability to extract spatiotemporal information from sequential data of a single photoelectron train were examined. The minimum detectable radiant flux density of the system was experimentally estimated to be 9.9 x 10(-17) W/cm2 with a 1-s observation time. Spontaneous photon emission was demonstrated from an exposed rats cortex in vivo without adding any chemical agent or employing external excitation. An image of ultraweak photon emission was compared with one obtained after cardiac arrest. The intensity after cardiac arrest was depressed to approximately 60% of before that. The regional properties of time courses of emission intensity were also demonstrated, indicating the potential usefulness for spatiotemporal characterization of photon emission with mapping of physiological information such as oxidative stress. This technology constitutes a novel method, with the potential to extract pathophysiological information from the central nervous system.

Effect of the mono- and tetra-sialogangliosides, GM1 and GQ1b, on long-term potentiation in the CA1 hippocampal neurons of the guinea pig

Abstract Effects of the mono- and tetra-sialogangliosides, GM1 and GQ1b, on long-term potentiation (LTP) were investigated in the CA1 neurons of guinea-pig hippocampal slices. The magnitude of LTP induced by a strong tetanus (100 Hz, 100 pulses) was not significantly affected by application of either ganglioside. In contrast, when LTP was induced by a weak tetanus (100 Hz, 4 pulses), a significantly greater LTP was induced in the presence of either ganglioside. Similarly, when slices were incubated in low-Ca2+ (1.0–1.1 mM) medium for more than 2 h, the LTP was usually small or absent, but showed a significant increase in amplitude of population spike (A-PS) when the slices were incubated with either GM1 or GQ1b (4–5 µg/ml). In addition, the application of GQ1b (4 µg/ml) reversed the blocking effect of an NMDA-receptor antagonist, APP-5 (10 µM), on the induction of LTP and resulted in forming LTP. Based on these findings, we conclude that GM1 and GQ1b exert positive modulatory effects on the induction of LTP in hippocampal CA1 neurons and suggest that GM1 and GQ1b may participate in the induction of LTP as donors of Ca2+ ions.

Increases in adrenal catecholamine secretion and adrenal sympathetic nerve unitary activities with aging in rats

Changes in sympathoadrenal medullary functions with age were investigated using urethane-chloralose anesthetized Wistar rats between about 100-900 days after birth. Although secretion rates of adrenaline and noradrenaline from the adrenal gland under resting conditions varied widely among animals, they gradually increased after 300 days and reached a level 2-4 times higher at 800-900 days compared with that of 100 days. Sympathetic nerve efferent unitary activity at resting conditions was also increased during aging in a manner similar to the catecholamine secretion rates.

The long-term suppressive effect of prior activation of synaptic inputs by low-frequency stimulation on induction of long-term potentiation in CA1 neurons of guinea pig hippocampal slices

We have investigated the effects of prior activation of afferent inputs by a train of low-frequency stimulation (LFS) on the induction of long term potentiation (LTP) induced by highfrequency stimulation (tetanus, 100 Hz, 100 pulses) in CA1 neurons of guinea pig hippocampal slices. The parameters of the LFS were altered systematically: the frequency (1 or 5 Hz); the number of pulses (80, 200 or 1000); and the time lag from the LFS to the tetanus (20, 60 or 100 min). Conditioning effects of the LFS on the induction of LTP were evaluated in terms of the slope of the field excitatory postsynaptic potential (S-EPSP) and the amplitude of the population spike (A-PS). LTP could reliably be induced by 100 Hz tetanic stimulation delivered to a naive slice. In contrast, the attempt to induce LTP 60 min after LFS of 200 or 1000 pulses at 1 Hz resulted only in short-term potentiation while the LFS itself produced no significant change in the responses. The suppressive effect on LTP was significantly reduced for 1 Hz LFS with a smaller number of pulses (80 pulses), or a shorter (20 min) or longer (100 min) time lag from the LFS to the tetanus, or with LFS at a higher frequency (5 Hz). When the LFS of 1000 pulses at 1 Hz was delivered in the presence of the n-methyl-d-aspartate (NMDA) receptor antagonist AP5 (d,l-4-amino-5-phosphonovalerate, 50 μM), which was washed out after the end of the LFS, the tetanus given 60 min after application of the LFS produced stable LTP, indicating the involvement of NMDA receptor/channels in the mechanisms of this particular form of synaptic plasticity-long-term suppression of LTP.We have investigated the effects of prior activation of afferent inputs by a train of low-frequency stimulation (LFS) on the induction of long term potentiation (LTP) induced by highfrequency stimulation (tetanus, 100 Hz, 100 pulses) in CA1 neurons of guinea pig hippocampal slices. The parameters of the LFS were altered systematically: the frequency (1 or 5 Hz); the number of pulses (80, 200 or 1000); and the time lag from the LFS to the tetanus (20, 60 or 100 min). Conditioning effects of the LFS on the induction of LTP were evaluated in terms of the slope of the field excitatory postsynaptic potential (S-EPSP) and the amplitude of the population spike (A-PS). LTP could reliably be induced by 100 Hz tetanic stimulation delivered to a naive slice. In contrast, the attempt to induce LTP 60 min after LFS of 200 or 1000 pulses at 1 Hz resulted only in short-term potentiation while the LFS itself produced no significant change in the responses. The suppressive effect on LTP was significantly reduced for 1 Hz LFS with a smaller number of pulses (80 pulses), or a shorter (20 min) or longer (100 min) time lag from the LFS to the tetanus, or with LFS at a higher frequency (5 Hz). When the LFS of 1000 pulses at 1 Hz was delivered in the presence of the n-methyl-d-aspartate (NMDA) receptor antagonist AP5 (d,l-4-amino-5-phosphonovalerate, 50 μM), which was washed out after the end of the LFS, the tetanus given 60 min after application of the LFS produced stable LTP, indicating the involvement of NMDA receptor/channels in the mechanisms of this particular form of synaptic plasticity-long-term suppression of LTP.