Physiology and Morphological Correlates of Excitatory Transmission are Preserved in Glutamine Transporter SN1-Depleted Mouse Frontal Cortex.

Glutamine is an astroglia-derived precursor of the neurotransmitter glutamate, and its astroglia-to-neuron transfer is controlled by distinct glutamine transporters on the astrocytic and neuronal sites. In this study, we focused on the role of astrocytic glutamine efflux-mediating system N transporter SN1 in the maintenance of glutamatergic neurotransmission by analyzing the electrophysiological parameters ex vivo in the brain slices from control mice and mice in which vivo-morpholino technique was used to diminish SN1 protein. The glutamatergic transmission was characterized by electrophysiological recordings, ultrastructure of neuron terminals, and determination of proteins related to glutamate synaptic transmission: synaptophysin, synaptotagmin, and vit1A. The space-restricted ∼51,5% reduction of SN1 protein did not affect the expression of the neuronal glutamine transporter SAT2. SN1 depletion resulted in a reduction of field potentials (FPs), unaltered frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs/mEPSCs), and presented a tendency towards a decrease of long-term potentiation (LTP). Ultrastructurally, preserved number of synaptic vesicles, primarily localized centrally of the cell body, correlates with unchanged levels of synaptic proteins. Collectively, the study indicates that glutamatergic transmission proceeds relatively independently of the SN1 - mediated glutamine transfer to the synapse.

Repeated blockade of 5-HT7 receptors depresses glutamatergic transmission in the rat frontal cortex.

The effects of the intraperitoneal administration of the 5-HT7 receptor antagonist SB 269970 were studied in the rat frontal cortex. In ex vivo slices prepared from rats receiving 14 daily doses of the drug (1.25 mg/kg) the mean frequency and the mean amplitude of glutamate-mediated, spontaneous excitatory postsynaptic currents (sEPSCs) recorded from layer II/III pyramidal neurons, were decreased. In contrast, single administration of SB 269970 affected neither the frequency nor the amplitude of sEPSCs. Treatment with SB 269970 did not affect membrane excitability of pyramidal cells. These data indicate that repeated, but not single, treatment with SB 269970 results in an attenuation of glutamatergic transmission in the frontal cortex, most likely due to a combination of pre- and postsynaptic mechanisms.

Repeated administration of imipramine attenuates glutamatergic transmission in rat frontal cortex.

The effects of repeated administration of a tricyclic antidepressant, imipramine, lasting 14 days (10 mg/kg p.o., twice daily), were studied ex vivo in rat frontal cortex slices prepared 48 h after last dose of the drug. In slices prepared from imipramine-treated animals the mean frequency, and to a lesser degree the mean amplitude, of spontaneous excitatory postsynaptic currents recorded from layer II/III pyramidal neurons, were decreased. These effects were accompanied by a reduction of the initial slope ratio of pharmacologically isolated N-methyl-D-aspartate to AMPA/kainate receptor-mediated stimulation-evoked excitatory postsynaptic currents. Imipramine treatment also resulted in a decrease of extracellular field potentials evoked in layer II/III by stimulation of underlying sites in layer V. These results indicate that chronic treatment with imipramine results in an attenuation of the release of glutamate and an alteration in the postsynaptic reactivity of ionotropic glutamate receptors in rat cerebral cortex.

Tetraethylammonium-induced long-term potentiation in layer V horizontal connections of rat motor cortex.

The possibility for the induction of long-lasting synaptic plasticity by the potassium channel blocker tetraethylammonium (TEA) was investigated in adult rat motor cortex in vitro. Brief application of TEA (25 mM) resulted in a long-term potentiation (LTP(K)) of field potentials evoked in layer V intralaminar connections by 59+/-17%. This effect could be prevented by preincubation with nifedipine, a voltage-dependent calcium channel blocker (20 microM), but not by N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (APV, 100 microM). LTP(K) induction resulted in a smaller relative potentiation of a response to the second pulse of a pair (60 ms interpulse interval). These results indicate a potential of layer V horizontal connections for a NMDA receptor-independent form of persistent synaptic enhancement.

Past and present hepatitis G virus infections in areas where hepatitis C is highly endemic and those where it is not endemic.

We reported previously on an area in Japan where over 30% of the inhabitants were positive for hepatitis C virus (HCV) antibody. In the present study, clinical features of hepatitis G virus (HGV) infection in this area of high endemicity were compared to those in an area where HCV is not endemic. A total of 400 individuals were selected randomly from those who were medically screened for liver disease in 1993; 200 were from the high-endemicity area, and the other 200 were from the no-endemicity area. HGV RNA was measured by reverse transcription and PCR with primers in the 5' noncoding region. Antibody to HGV envelope protein E2 was measured by an enzyme-linked immunosorbent assay. Prevalence of any HGV marker in the high-endemicity area (32%) was significantly (P < 0.0001) higher than that in the no-endemicity area (6%); similar differences, 32% versus 3% (P < 0.0001), had been observed for HCV markers (HCV RNA and HCV antibody). In areas of both high and no endemicity, HCV markers were significantly more prevalent in individuals with any HGV marker than in those without HGV markers, and age-specific prevalence of HGV markers was distributed similarly to that of any HCV marker. Among possible routes of HGV transmission that were analyzed, folk medicine was significant in the high-endemicity area, but blood transfusion was the major route in the no-endemicity area. The rate of accompanying viremia in HGV infection (15%) was significantly lower than that in HCV infection (78%) (P < 0.0001). In conclusion, HGV infection was highly prevalent in the area of high HCV endemicity and was closely associated with HCV infection. HGV seemed to be transmitted via the practice of folk medicine as well as blood transfusion. HGV resulted in a chronic carrier state less frequently than did HCV.

N-methyl-D-aspartate receptor mediated component of field potentials evoked in horizontal pathways of rat motor cortex.

To identify potential sites of synaptic modification of intrinsic cortical circuits, the contribution of the N-methyl-D-aspartate type of glutamate receptors to field potentials evoked in horizontal and oblique intracortical pathways was examined in rat motor cortex slice preparations. Presumably monosynaptic, short latency responses with a prominent negativity (-0.4 to -2.0 mV) were recorded in both superficial (across layer III) and deep (across layer V) horizontal pathways at a distance of approximately equal to 500 microns lateral to electrical stimulation sites and in oblique V-III pathway (-0.3 to -1.6 mV). Bath application of the N-methyl-D-aspartate receptor antagonist D,L-2-amino-5-phosphonovaleric acid (100 microM) reversibly decreased field potentials. Although decreases were observed in all components of the waveform, the most pronounced effect was on the late phase of the response. D,L-2-Amino-5-phosphonovaleric acid produced on average a 22% decrease in area, 12% in initial slope and 11% in peak amplitude of responses. Combined application of 100 microM D,L-2-amino-5-phosphonovaleric acid and a non-N-methyl-D-aspartate glutamate receptor antagonist, 6-cyano-7-nitro- or 6,7-dinitro-quinoxaline-2,3- dione (10-20 microM), eliminated all but a small, early and presumably non-synaptic response. In 18 of 23 cases, the relative contribution of the D,L-2-amino-5-phosphonovaleric acid-sensitive component was unrelated to field potential magnitude, suggesting that this component is present in all fiber classes. It is concluded that glutamate is the major transmitter of horizontal connections of layers II/III and layer V, as well as in the oblique V-III pathway. While most glutamatergic transmission is relayed by other glutamate receptor subtypes, N-methyl-D-aspartate receptor activation contributes a small but consistent part of ordinary transmission in each of these pathways in vitro. The results further suggest that a potential for N-methyl-D-aspartate receptor-mediated synaptic modification exists in intrinsic horizontal pathways of both superficial and deep layers of rat motor cortex.

Acetylcholine receptor: channel-opening kinetics evaluated by rapid chemical kinetic and single-channel current measurements.

A combination of rapid chemical kinetic (quench-flow) and single-channel current measurements was used to evaluate kinetic parameters governing the opening of acetylcholine-receptor channels in the electric organ (electroplax) of Electrophorus electricus. Chemical kinetic measurements made on membrane vesicles, prepared from the E. electricus electroplax, using carbamoylcholine (200 microM-20 mM) at 12 degrees C, pH 7.0, and in the absence of a transmembrane voltage, yielded values for K1 (dissociation constant for receptor activation), phi (channel closing equilibrium constant), J (specific reaction rate for ion flux), and alpha max (maximum inactivation rate constant) of 1 mM, 3.4, 4 x 10(7) M-1 s-1, and 12 s-1, respectively. The single-channel current recordings were made with cells also from the E. electricus electroplax, at the same temperature and pH as the chemical kinetic measurements, using carbamoylcholine (50 microM-2 mM), acetylcholine (500 nM), or suberyldicholine (20 nM). Single-channel current measurements indicated the presence of a single, unique open-channel state of the E. electricus receptor, in concurrence with previous, less extensive measurements. The rate constant for channel closing (kc) obtained from the mean open time of the receptor channel is 1,100 s-1 for carbamoylcholine, 1,200 s-1 for acetylcholine, and 360 s-1 for suberyldicholine at zero membrane potential; and it decreases e-fold for an 80 mV decrease in transmembrane voltage in each case. The decrease in mean open times of the receptor channel that is associated with increasing the carbamoylcholine concentration is interpreted to be due to carbamoylcholine binding to the regulatory (inhibitory) site on the receptor. An analysis of data obtained with carbamoylcholine showed that the closed times within a burst of channel activity fit a two-exponential distribution, with a concentration-independent time constant considered to be the time constant for carbamoylcholine to dissociate from the regulatory site, and a carbamoylcholine concentration-dependent, but voltage-independent, time constant interpreted to represent the rate constant for channel opening (k0). An analysis of the mean closed time data on the basis of the minimum model gives values for K1 and k0 of 0.6 mM and 440 s-1, respectively, with carbamoylcholine as the activating ligand. The values obtained for K1, phi (= kc/k0), J, and alpha from the single-channel current measurements using electroplax are in good agreement with the values obtained from the chemical kinetic measurements using receptor-rich vesicles prepared from the same cells. These results confirm the assumed basic agreement between two entirely different methodologies and underlie the strategy of using the two techniques to obtain complementary information in time and ligand-concentration regions where only one or the other technique can be used. This agreement between results allows estimates to be made of the ko values, for both acetylcholine and suberyldicholine, from the phi values obtained from the chemical kinetic measurements and the kc values obtained in single-channel current measurements.