PubMed Health⌕ Search

Biomedical subjects

S Mennerick

Publications and source records attributed to S Mennerick.

34 records · Page 2Linked to original sources
PubMed1998

Neuronal expression of the glutamate transporter GLT-1 in hippocampal microcultures.

To address the question of the relative contributions of glial and neuronal glutamate transport in the vertebrate CNS, we studied the distribution of forebrain glutamate transporters in rat hippocampal microcultures, a preparation in which physiological functions of glutamate transporters have been well characterized. Two of the three transporters, GLAST (EAAT1) and EAAC1 (EAAT3), are localized to microculture glia and neurons, respectively, as expected. However, we find strong immunoreactivity for the third glutamate transporter GLT-1 (EAAT2), a putatively glial transporter, in microculture neurons and in a small subset of microculture glia. Indistinguishable immunohistochemical staining patterns for GLT-1 were obtained with antibodies directed against both the N terminal and C terminal of the GLT-1 protein. Double-labeling experiments suggest that neuronal GLT-1 protein is primarily localized to the dendrites of excitatory neurons. Neuronal electrogenic transport currents in response to D-aspartate applications were occluded by the selective GLT-1 inhibitor dihydrokainate. In contrast, glia exhibited a larger transporter current density than did neurons, and the glial transport current was less sensitive to dihydrokainate. Neuronal transport currents were potentiated less than were glial currents when the chaotropic anion thiocyanate was substituted for gluconate in the whole-cell recording pipette, consistent with the previously reported lower anion permeability of EAAC1 and GLT-1 compared with that of GLAST. After microculture glia were rendered nonviable, excitatory autaptic currents (EACs) were prolonged in the presence of dihydrokainate, suggesting that neuronal GLT-1 is capable of participating in the clearance of synaptically released glutamate. Our results suggest that the initially proposed characterization of GLT-1 as a purely glial transporter is too simplistic and that under certain conditions functional GLT-1 protein can be expressed in brain neurons. The study suggests that changes in GLT-1 levels that occur with pathology or experimental manipulations cannot be assumed to be glial.

ATP-Binding Cassette Transporters↗
PubMed1998

Temporal ordering of pathogenic events following transient global ischemia.

Rats were subjected to transient global ischemia (four vessel occlusion) and time-related changes in the selectively vulnerable hippocampal field CA1 were characterized. The assessment included ex vivo field responses to afferent stimulation, silver staining, calpain-induced spectrin breakdown, chromatolysis, and cell death, beginning at 6 h post-ischemia and continuing until total disintegration of the pyramidal cells occurred several days later. The earliest change observed was a modest increase in the slope and amplitude of field CA1 potentials (at 6 h). The hyperresponsiveness was most apparent at higher stimulation currents and persisted unchanged at 16 h post-ischemia. Three effects became detectable within 24 h, post-ischemia: (a) an increase in concentrations of calpain-mediated, spectrin breakdown products; (b) enhanced silver staining in the deep pyramidal neurons of the field CA1 with lesser, though still apparent, staining of stratum radiatum, and (c) a decrease in amplitude and slope of field CA1 responses to afferent stimulation. Both the concentration of spectrin breakdown products and the intensity of silver staining progressively increased to a maximum at four days post ischemia, while the amplitude and slope of the field responses dropped to a very low level between 24 and 48 h. Disturbances of Nissl staining were finally evident at 48 h, with nearly complete disappearance of staining at five days post-ischemia. This study provides the first demonstration of a close and early temporal relationship between calpain proteolysis, subcellular damage to the pyramidal cells and their loss of function following global ischemia, prior to their eventual death.

Animals↗
PubMed1998

Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant and neurotoxin.

Extensive research has failed to clarify the mechanism of action of nitrous oxide (N2O, laughing gas), a widely used inhalational anesthetic and drug of abuse. Other general anesthetics are thought to act by one of two mechanisms-blockade of NMDA glutamate receptors or enhancement of GABAergic inhibition. Here we show that N2O, at anesthetically-relevant concentrations, inhibits both ionic currents and excitotoxic neurodegeneration mediated through NMDA receptors and, like other NMDA antagonists, produces neurotoxic side effects which can be prevented by drugs that enhance GABAergic inhibition. The favorable safety record of N2O may be explained by the low concentrations typically used and by the fact that it is usually used in combination with GABAergic anesthetics that counteract its neurotoxic potential.

Animals↗
PubMed1997

Static and dynamic membrane properties of large-terminal bipolar cells from goldfish retina: experimental test of a compartment model.

Capacitance measurements allow direct studies of exocytosis and endocytosis in single synaptic terminals isolated from bipolar neurons of goldfish retina. Extending the technique to intact bipolar cells, with their more complex morphology, requires information about the cells' electrotonic architecture. To this end, we developed a compartment model of bipolar neurons isolated from goldfish retina and tested the model experimentally. The isolated cells retained morphology similar to that of bipolar neurons in intact goldfish retina. In whole cell recordings, current relaxations in response to 10-mV hyperpolarizing voltage pulses decayed with a biexponential time course. This suggests that the cells may be described by a two-compartment equivalent circuit with compartments corresponding to the soma/dendrites (6-10 pF) and synaptic terminal (2-4 pF), linked by the axial resistance (30-60 M omega) of the axon. Four lines of evidence validate the equivalent circuit. 1) Similar estimates of somatic/dendritic and terminal capacitance were obtained whether the patch pipette was attached to the soma or to the synaptic terminal. 2) Estimates of the capacitance of the two compartments in intact cells were similar to estimates from somata and terminals that were isolated by cleavage of the connecting axon. 3) When current transients were generated from a more complete computer simulation of a bipolar neuron, analysis of the simulated transients with the use of the simple two-compartment model yielded capacitance estimates similar to those used to set up the simulation. 4) In isolated cells, the model gave estimates of depolarization-evoked increases in capacitance of the synaptic terminal that were quantitatively similar to those measured in terminals that were detached from the rest of the cell. Although in previous studies researchers have attempted to apply a similar equivalent circuit to more geometrically complex cells, morphological correlates of the equivalent-circuit compartments have been elusive. Our results demonstrate that in dissociated bipolar cells, precise morphological correlates can be assigned to the equivalent-circuit compartments. Additionally, the work shows that time-resolved capacitance measurements of synaptic transmitter release are possible in intact, isolated bipolar neurons and may also be feasible in intact tissue.

Animals↗
PubMed1996

Modulation of excitatory synaptic transmission by low concentrations of glutamate in cultured rat hippocampal neurons.

1. The effects of low micromolar concentrations of glutamate on fast excitatory synaptic responses were studied in microcultures of postnatal rat hippocampal neurons using whole-cell patch clamp recordings. 2. Glutamate depressed the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor component of excitatory autaptic currents (EACs) with an EC50 of 3.8 microM. 3. Both pre- and postsynaptic effects contributed to the depression of AMPA receptor-mediated EACs. Cyclothiazide and wheatgerm agglutinin, agents which inhibit AMPA receptor desensitization, partially reversed the depression produced by glutamate, as did pertussis toxin, an agent that blocks presynaptic inhibition mediated by metabotropic glutamate receptors. 4. In neurons in which both the AMPA and N-methyl-D-aspartate (NMDA) receptor components of EACs were examined, low concentrations of glutamate depressed the NMDA component of EACs to a greater extent. The EC50 for inhibiting the NMDA component was 1.3 microM. 5. Calcium-dependent desensitization of postsynaptic NMDA receptors contributed to the depression of NMDA receptor-mediated synaptic responses. Both depolarization of postsynaptic neurons to +70 mV to decrease Ca2+ influx via NMDA channels and inclusion of high concentrations of a calcium chelator in recording pipettes decreased the depression of NMDA receptor-mediated EACs. 6. Threo-3-hydroxy-aspartate (THA), an inhibitor of glutamate transport, depressed EACs by about 10% and increased the degree of depression produced by 2.5 microM glutamate, suggesting that glutamate transport in microcultures helps to control ambient glutamate levels. 7. Because the normal extracellular concentration of glutamate is about 1 microM, these results suggest that the ambient glutamate level is an important determinant of synaptic efficacy. Relatively small changes in extracellular glutamate can alter fast excitatory synaptic transmission by both presynaptic and postsynaptic mechanisms.

Animals↗
PubMed1996

Postsynaptic modulation of NMDA synaptic currents in rat hippocampal microcultures by paired-pulse stimulation.

1. Paired-pulse synaptic stimulation of hippocampal neurons in microcultures resulted in depression of synaptic currents mediated by both NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. However, NMDA EPSCs were more severely depressed than AMPA EPSCs. 2. Partial NMDA receptor blockade reduced paired-pulse depression of NMDA but not of AMPA synaptic currents while partial AMPA receptor blockade had no effect on paired-pulse depression of AMPA EPSCs. These results suggest that ion flux through NMDA receptors is important in paired-pulse depression of NMDA responses but has no effect on AMPA responses. 3. Low extracellular Ca2+ concentrations or positive postsynaptic holding potentials reduced paired-pulse depression of NMDA EPSCs to near that of AMPA responses. 4. Brief paired applications of exogenous glutamate to neurons produced Ca(2+)-dependent depression similar to the depression of NMDA synaptic responses and synaptic stimulation depressed responses to exogenously applied NMDA. 5. Physiological concentrations of Mg2+ prevented expression of the postsynaptic modulation of NMDA EPSCs at -70 mV, but partial relief of Mg2+ block of the NMDA channel with depolarization increased paired-pulse depression of NMDA EPSCs.

Animals↗
PubMed1996

Swelling of Müller cells induced by AP3 and glutamate transport substrates in rat retina.

Previous studies have shown that a single systemic injection of 2-amino-3-phosphonopropionate (AP3), an agonist/antagonist at metabotropic glutamate receptors, produces marked swelling of rodent Müller cells. To investigate the effects of AP3 on Müller cells, we used in vitro retinal segments prepared from 30 day old rats. Incubation with AP3 for 1 h or more caused severe swelling of Müller cells with the appearance of mitotic cellular profiles in the outer nuclear layer. The Müller cell swelling was mimicked by substrates for glutamate transporters, suggesting that AP3 may produce its effects via transport into glial cells. To determine whether AP3 is a substrate for glutamate transporters, we studied cultured rat hippocampal astrocytes using whole-cell patch clamp recordings. In hippocampal astrocytes, AP3 activated currents via an Na(+)-dependent glutamate transporter. Consistent with this, substitution of extracellular sodium with choline blocked Müller cell swelling in the rat retina. These results indicate that the acute glial swelling produced by AP3 results primarily from a fluid shift that accompanies the transport of AP3 and sodium into Müller cells.

Alanine↗
PubMed1996

Ultrafast exocytosis elicited by calcium current in synaptic terminals of retinal bipolar neurons.

Using high resolution capacitance measurements, we have characterized an ultrafast component of transmitter release in ribbon-type synaptic terminals of retinal bipolar neurons. During depolarization, capacitance increases to a plateau of approximately 30 fF with a time constant of approximately 1.5 ms. When not limited by activation kinetics of calcium current, the small pool is depleted even faster, with a time constant of 0.5 ms. After the ultrafast pool is depleted, capacitance rises with a slower time constant of approximately 300 ms. EGTA (5 mM) depresses the slower capacitance rise but leaves the ultrafast phase intact. BAPTA (5 mM) depresses both components of exocytosis. With paired-pulse stimulation, the ultrafast pool recovers from depletion with a time constant of approximately 4 s. The ultrafast component may represent fusion of docked vesicles at the base of the synaptic ribbon, while the slower component represents more distal vesicles on the ribbon.

Animals↗
PubMed1996

Components of glial responses to exogenous and synaptic glutamate in rat hippocampal microcultures.

Although glia are known to be sensitive to exogenously applied neurotransmitter substances, little is known about how glia respond to neuronal activity on the millisecond time scale of individual synaptic events. We have explored the electrophysiological effects of excitatory neuronal signaling on glial cells in rat hippocampal microcultures. Exogenous applications of glutamate analogs to islands of glia revealed a large ionotropic AMPA receptor-mediated current and a smaller current mediated by electrogenic glutamate uptake. Glia demonstrated no evidence of NMDA or metabotropic receptor-mediated currents or membrane conductance changes. Despite the dominant contribution of AMPA receptors to exogenous glutamate applications in glia, AMPA receptor currents contributed only approximately 20% to the response of glia to endogenous glutamate release from solitary excitatory neurons. Electrogenic glutamate uptake contributed strongly to glial responses to neuronal stimulation. In addition, some glia exhibited a residual current is response to neuronal stimulation that was not attributable to calcium-dependent transmitter release or to neuronal potassium efflux. These results help provide a context for understanding the role of glial transporters and receptors in nervous system signaling.

Animals↗
PubMed1995

Paired-pulse modulation of fast excitatory synaptic currents in microcultures of rat hippocampal neurons.

1. Paired-pulse modulation of excitatory non-N-methyl-D-aspartate (non-NMDA) receptor-mediated autaptic currents and conventional monosynaptic (interneuronal) excitatory postsynaptic currents (EPSCs) was investigated in microcultures of rat hippocampal neurons, where polysynaptic influences are eliminated. 2. Most autaptic currents and EPSCs exhibited paired-pulse depression in response to paired stimuli. Depression was sensitive to the level of transmitter release, which was varied by manipulating extracellular Ca2+ and Mg2+ concentrations. Paired-pulse facilitation emerged in many cells at low levels of transmitter release. 3. Paired-pulse depression and facilitation could be differentially expressed at two distinct postsynaptic targets of a single presynaptic cell, and the form of modulation was not dependent upon the transmitter phenotype of the postsynaptic cell. 4. Paired-pulse depression recovered exponentially with a time constant of approximately 5 s, although in most neurons a much faster component of recovery was detected. Recovery from paired-pulse facilitation was well described by a single exponential of 380 +/- 57 ms. 5. Under conditions of robust paired-pulse depression of evoked responses, spontaneous autaptic and postsynaptic currents (sEPSCs, presumed miniature EPSCs) occurred at an enhanced frequency immediately following evoked responses. The decay of the frequency increase mirrored the time course of recovery from paired-pulse facilitation of evoked responses examined under conditions of reduced transmitter release. 6. Several lines of evidence suggested a large presynaptic component to paired-pulse depression. In eight out of nine cells no depression in sEPSC amplitudes was detected following conditioning stimulation. Simultaneously recorded glial glutamate uptake currents showed depression similar to neuronal evoked EPSCs. Finally, NMDA receptor-mediated EPSC paired-pulse depression at positive potentials was similar to non-NMDA EPSC depression. 7. Neither adenosine nor glutamate feedback onto presynaptic receptors is likely to mediate paired-pulse depression, because neither competitive nor non-competitive inhibitors of the actions of these agents diminished paired-pulse depression.

Animals↗
PubMed1995

Passive and synaptic properties of hippocampal neurons grown in microcultures and in mass cultures.

1. We used whole cell recordings to compare passive membrane properties and synaptic properties of postnatal rat hippocampal neurons grown for 7-15 days in either conventional mass cultures or on physically restricted microisland cultures. Despite matching microisland and mass culture cell across several variables, there were significant differences between neurons in the two groups regarding passive membrane characteristics and synaptic properties. 2. Microisland neurons displayed significantly faster charging of the membrane capacitance than mass culture counterparts matched with microisland neurons for age, somal diameter, and transmitter phenotype. When we used a two-compartment equivalent circuit model to quantify this result, microisland neurons displayed approximately half the distal capacitance of mass culture neurons. These data suggest that microisland neurons elaborate less extensive neuritic arborizations than mass culture neurons. 3. Evoked synaptic responses were enhanced on microislands compared with mass cultures. Excitatory and inhibitory autaptic currents were more frequent and displayed larger amplitudes on single-neuron microislands than in matched mass culture neurons. 4. In recordings from pairs of neurons in the two environments, we observed a significantly higher probability of obtaining a monosynaptic response on two-neuron microislands than in matched mass culture pairs (85% vs. 42%). Evoked excitatory postsynaptic currents were also significantly larger in the microisland environment, with evoked excitatory synaptic currents from two-neuron microislands exhibiting a mean amplitude 20-fold larger than mass culture monosynaptic responses. 5. The differences in evoked synaptic responses were not reflected in differences in the amplitude or frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs). Analysis of mEPSC rise times, decay times, and peak amplitudes within individual cells suggests that electrotonic filtering is not an important contributor to the variability of peak amplitudes and decay times of synaptic currents in cells of either culture environment. However, composite data across neurons in both cultures reveal a significant correlation between mEPSC rise and decay times. 6. Out results suggest that the microisland preparation may be a useful tool for exploring factors that influence synapse formation and development. Additionally, the preparation is a particularly convenient model for the study of single-neuron-mediated synaptic events.

Animals↗
PubMed1995

Presynaptic influence on the time course of fast excitatory synaptic currents in cultured hippocampal cells.

Since the lifetime of synaptically released glutamate is thought to be very brief, reflecting diffusion and glutamate uptake, the decay of synaptic currents is thought to represent the average elementary lifetime of a receptor channel bound only once by transmitter molecules. We show here that the decay of evoked non-NMDA synaptic currents can reflect presynaptic factors, in particular, the prolonged action of transmitter at postsynaptic receptors under conditions of enhanced transmitter release. We show that diffusion, high-affinity glutamate uptake, and non-NMDA receptor desensitization are insufficiently rapid to limit the decays of evoked synaptic currents to those of miniature synaptic currents in microcultures of rat hippocampal cells. Our results are consistent with recent studies suggesting that during evoked release, multiple glutamate quanta can interact with overlapping postsynaptic receptor domains.

2-Amino-5-phosphonovalerate↗
PubMed1994

Glial contributions to excitatory neurotransmission in cultured hippocampal cells.

Although many glial cells possess neurotransmitter receptors and transporters, little is known about glial participation in neurotransmission. To explore this issue, we recorded neuronal autaptic and glial responses from cultured hippocampal single-neuron micro-islands. Excitatory synaptic events activate rapid electrogenic glial glutamate transporter currents similar to those elicited by exogenous glutamate in other preparations. We show here that glial transporter responses may be used to sense changes in presynaptic efficacy and that glial uptake helps to remove synaptically released glutamate, thereby contributing to the termination of excitatory synaptic currents under certain conditions. These observations provide a framework for understanding the role of glia in both normal and pathological processes.

Action Potentials↗
PubMedDate not supplied

Rapid calcium-current kinetics in synaptic terminals of goldfish retinal bipolar neurons.

Onset and offset of calcium current in response to short, depolarizing voltage pulses were studied in giant synaptic terminals of goldfish bipolar neurons. Tail-current decays were extremely rapid (80 micros at -70 mV), and exponentially slowed at more positive repolarization potentials. The amplitude of tail current following voltage pulses varied with the pulse amplitude, according to a Bolzmann distribution with a V1/2 of -27.6 mV and a slope factor of 13.8 mV. When interpreted with a Hodgkin-Huxley model, calcium-current onset was best described by m2 kinetics and a time constant (tau(m)) of 0.62 ms at -10 mV. The kinetics of calcium-current onset and offset are at least two- to threefold faster than those described in other native cells. Combined with other recent data, the results suggest that the rapid kinetics may participate in a fast signaling mode in the goldfish ON-center pathway.

Animals↗
PubMedDate not supplied

Neural activity and survival in the developing nervous system.

Recent evidence suggests that blockade of normal excitation in the immature nervous system may have profound effects on neuronal survival during the period of natural cell death. Cell loss following depression of electrical activity in the central nervous system (CNS) may explain the neuropsychiatric deficits in humans exposed to alcohol or other CNS depressants during development. Thus, understanding the role of electrical activity in the survival of young neurons is an important goal of modern basic and clinical neuroscience. Here we review the evidence from in vivo and in vitro model systems that electrical activity participates in promoting neuronal survival. We discuss the potential role of moderate elevations of intracellular calcium in promoting survival, and we address the possible ways in which activity and conventional trophic factors may interact.

Action Potentials↗