Original Research / Publications

Comparison of coupled and uncoupled currents during glutamate uptake by GLT-1 transporters

The transport of glutamate across the plasma membrane is coupled to the movement of cations (Na+, K+, and H+) that are necessary for glutamate uptake and transporter cycling as well as anions that are uncoupled from the flux of glutamate. Although the relationship between these coupled (stoichiometric) and uncoupled (anion) transporter currents is poorly understood, transporter-associated anion currents often are used to monitor transporter activity. To define the kinetic relationship between these two components, we have recorded transporter currents associated with stoichiometric and anion charge movements occurring in response to the rapid application of l-glutamate to outside-out patches from human embryonic kidney cells expressing GLT-1 transporters. Transporter-associated anion currents were approximately twice as slow to rise and decay as stoichiometric transport currents, but the presence of permeant anions did not slow transporter cycling. A kinetic model for GLT-1 was developed to simulate the behavior of both components of the transporter current and to estimate the capture efficiency of GLT-1. In this model the K+ counter-transport step was defined as rate-limiting, consistent with the slowing of transporter cycling after the substitution of internal K+ with Cs+ or Na+. The model predicts that in physiological conditions approximately 35% of GLT-1 transporters function as buffers, releasing glutamate back into the extracellular space after binding.

Publications / Review

Physiological characteristics of NG2-expressing glial cells.

Antibodies against the chondroitin sulfate proteoglycan NG2 label a subpopulation of glial cells within the CNS, which have a small cell body and thin radiating processes. Physiological recordings from these small cells in acute brain slices have revealed that they possess unique properties, suggesting that they may comprise a class of glial cells distinct from astrocytes, oligodendrocytes, or microglia. NG2-expressing glial cells (abbreviated as “NG2 cells” here) have a moderate input resistance and are not dye- or tracer-coupled to adjacent cells. They express voltage-gated Na+, K+ and Ca2+ conductances, though they do not exhibit regenerative Na+ or Ca2+ action potentials due to the much larger K+ conductances present. In addition to voltage-gated conductances, they express receptors for various neurotransmitters. In the hippocampus, AMPA and GABAA receptors on these cells are activated by release of transmitter from neurons at defined synaptic junctions that are formed with CA3 pyramidal neurons and GABAergic interneurons. These rapid forms of neuron-glial communication may regulate the proliferation rate of NG2 cells or their development into mature oligodendrocytes. These depolarizing inputs may also trigger the release of neuroactive substances from NG2 cells, providing feedback regulation of signaling at neuronal synapses. Although the presence of Ca2+ permeable AMPA receptors provides a pathway to link neuronal activity to Ca2+ dependent processes within the NG2 cells, these receptors also put these cells at risk for glutamate-associated excitotoxicity. This vulnerability to the sustained elevation of glutamate may underlie ischemic induced damage to white matter tracts and contribute to cerebral palsy in premature infants.

Original Research / Publications

Glutamatergic synapses on oligodendrocyte precursor cells in the hippocampus.

Fast excitatory neurotransmission in the central nervous system occurs at specialized synaptic junctions between neurons, where a high concentration of glutamate directly activates receptor channels. Low-affinity AMPA (alpha-amino-3-hydroxy-5-methyl isoxazole propionic acid) and kainate glutamate receptors are also expressed by some glial cells, including oligodendrocyte precursor cells (OPCs). However, the conditions that result in activation of glutamate receptors on these non-neuronal cells are not known. Here we report that stimulation of excitatory axons in the hippocampus elicits inward currents in OPCs that are mediated by AMPA receptors. The quantal nature of these responses and their rapid kinetics indicate that they are produced by the exocytosis of vesicles filled with glutamate directly opposite these receptors. Some of these AMPA receptors are permeable to calcium ions, providing a link between axonal activity and internal calcium levels in OPCs. Electron microscopic analysis revealed that vesicle-filled axon terminals make synaptic junctions with the processes of OPCs in both the young and adult hippocampus. These results demonstrate the existence of a rapid signalling pathway from pyramidal neurons to OPCs in the mammalian hippocampus that is mediated by excitatory, glutamatergic synapses.

Publications / Review

Clearance of glutamate inside the synapse and beyond

The heated debate over the level of postsynaptic receptor occupancy by transmitter has not been extinguished – indeed, new evidence is fanning the flames. Recent experiments using two-photon microscopy suggest that the concentration of glutamate in the synaptic cleft does not attain levels previously suggested. In contrast, recordings from glial cells and studies of extrasynaptic receptor activation indicate that significant quantities of glutamate escape from the cleft following exocytosis. Determining the amount of glutamate efflux from the synaptic cleft and the distance it diffuses is critical to issues of synaptic specificity and the induction of synaptic plasticity.

Original Research / Publications

Glial contribution to glutamate uptake at Schaffer collateral-commissural synapses in the hippocampus

Astrocytes in the hippocampus express high-affinity glutamate transporters that are important for lowering the concentration of extracellular glutamate after release at excitatory synapses. These transporters exhibit a permeability to chaotropic anions that is associated with transport, allowing their activity to be monitored in cell-fee patches when highly permeant anions are present. Astrocyte glutamate transporters are highly temperature sensitive, because L-glutamate-activated, anion-potentiated transporter currents in outside-out patches from these cells exhibited larger amplitudes and faster kinetics at 36 degreesC than at 24 degreesC. The cycling rate of these transporters was estimated by using paired applications of either L-glutamate or D-aspartate to measure the time necessary for the peak of the transporter current to recover from the steady-state level. Transporter currents in patches recovered with a time constant of 11.6 msec at 36 degreesC, suggesting that either the turnover rate of native transporters is much faster than previously reported for expressed EAAT2 transporters or the efficiency of these transporters is very low. Synaptically activated transporter currents persisted in astrocytes at physiological temperatures, although no evidence of these currents was found in CA1 pyramidal neurons in response to afferent stimulation. L-glutamate-gated transporter currents were also not detected in outside-out patches from pyramidal neurons. These results are consistent with the hypothesis that astrocyte transporters are responsible for taking up the majority of glutamate released at Schaffer collateral-commissural synapses in the hippocampus.Glial contribution to glutamate uptake at Schaffer collateral-commissural synapses in the hippocampus

Original Research / Publications

Glutamate Release Monitored with Astrocyte Transporter Currents during LTP

Long-term potentiation (LTP) of synaptic transmission in the CA1 region of the hippocampus is thought to result from either increased transmitter release, heightened postsynaptic sensitivity, or a combination of the two. We have measured evoked glutamate release from Schaffer collateral/commissural fiber terminals in CA1 by recording synaptically activated glutamate transporter currents in hippocampal astrocytes located in stratum radiatum. Although several manipulations of release probability caused parallel changes in extracellular field potentials and synaptically activated transporter current amplitudes, induction of LTP failed to alter transporter-mediated responses, suggesting that LTP does not alter the amount of glutamate released upon synaptic stimulation.

Original Research / Publications

Glutamate transporter currents in Bergmann glial cells follow the time course of extrasynaptic glutamate.

Glutamate transporters in the central nervous system are expressed in both neurons and glia, they mediate high affinity, electrogenic uptake of glutamate, and they are associated with an anion conductance that is stoichiometrically uncoupled from glutamate flux. Although a complete cycle of transport may require 50-100 ms, previous studies suggest that transporters can alter synaptic currents on a much faster time scale. We find that application of L-glutamate to outside-out patches from cerebellar Bergmann glia activates anion-potentiated glutamate transporter currents that activate in <1 ms, suggesting an efficient mechanism for the capture of extrasynaptic glutamate. Stimulation in the granule cell layer in cerebellar slices elicits all or none alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor and glutamate transporter currents in Bergmann glia that have a rapid onset, suggesting that glutamate released from climbing fiber terminals escapes synaptic clefts and reaches glial membranes shortly after release. Comparison of the concentration dependence of both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor and glutamate transporter kinetics in patches with the time course of climbing fiber-evoked responses indicates that the glutamate transient at Bergmann glial membranes reaches a lower concentration than attained in the synaptic cleft and remains elevated in the extrasynaptic space for many milliseconds.

Original Research / Publications

Synaptic activation of glutamate transporters in hippocampal astrocytes

Glutamate transporters in the CNS are expressed in neurons and glia and mediate high affinity, electrogenic uptake of extracellular glutamate. Although glia have the highest capacity for glutamate uptake, the amount of glutamate that reaches glial membranes following release and the rate that glial transporters bind and sequester transmitter is not known. We find that stimulation of Schaffer collateral/commissural fibers in hippocampal slices evokes glutamate transporter currents in CA1 astrocytes that activate rapidly, indicating that a significant amount of transmitter escapes the synaptic cleft shortly after release. Transporter currents in outside-out patches from astrocytes have faster kinetics than synaptically elicited currents, suggesting that the glutamate concentration attained at astrocytic membranes is lower but remains elevated for longer than in the synaptic cleft.

Original Research / Publications

Excitatory actions of norepinephrine on multiple classes of hippocampal CA1 interneurons

Norepinephrine (NE) causes an increase in the frequency of inhibitory postsynaptic potentials in CA1 pyramidal neurons in vitro. The possibility that this increase in tonic inhibition is caused by an excitatory effect on inhibitory interneurons was investigated through whole-cell recordings from pyramidal cells and both whole-cell and cell-attached patch recordings from visualized interneurons in acute slices of rat hippocampus. Adrenergic agonists caused a large increase in the frequency and amplitude of spontaneous IPSCs recorded from pyramidal cells in the presence of ionotropic glutamate receptor blockers, but they had no effect on either the frequency or the amplitude of action potential-independent miniature IPSCs recorded in tetrodotoxin. This effect was mediated primarily by an alpha adrenoceptor, although a slight beta adrenoceptor-dependent increase in IPSCs was also observed. NE caused interneurons located in all strata to depolarize and begin firing action potentials. Many of these cells had axons that ramified throughout the stratum pyramidale, suggesting that they are responsible for the IPSCs observed in pyramidal neurons. This depolarization was also mediated by an alpha adrenoceptor and was blocked by a selective alpha 1- but not a selective alpha 2-adrenoceptor antagonist. However, a slight beta adrenoceptor-dependent depolarization was detected in those interneurons that displayed time-dependent inward rectification. In the presence of a beta antagonist, NE induced an inward current that reversed near the predicted K+ equilibrium potential and was not affected by changes in intracellular Cl- concentration. In the presence of an alpha 1 antagonist, NE induced an inwardly rectifying current at potentials negative to approximately -70 mV that did not reverse (between -130 and -60 mV), characteristics similar to the hyperpolarization-activated current (lh). However, the depolarizing action of NE is attributable primarily to the alpha 1 adrenoceptor-mediated decrease in K+ conductance and not the beta adrenoceptor-dependent increase in lh. These results provide evidence that NE increases action potential-dependent IPSCs in pyramidal neurons by depolarizing surrounding inhibitory interneurons. This potent excitatory action of NE on multiple classes of hippocampal interneurons may contribute to the NE-induced decrease in the spontaneous activity of pyramidal neurons and the antiepileptic effects of NE observed in vivo.

Original Research / Publications

Mossy fiber growth and synaptogenesis in rat hippocampal slices in vitro

Hippocampal slices from early postnatal rat were used to study mossy fiber (MF) growth and synaptogenesis. The ability of MFs to form new giant synapses within isolated tissue slices was established by a series of experiments involving synapsin I immunohistochemistry, electron microscopy, and whole-cell recordings. When hippocampal slices from immature rats were cultured for up to 2 weeks, the distribution of giant MF terminals was similar to that found in vivo. Using a lesioning procedure, we determined that MFs in slices extend and form appropriate synaptic connections with normal target CA3 pyramidal cells. MF terminals were dispersed more widely than normal within the CA3 pyramidal layer after a lesion, but electron microscopy indicated that synaptic junctions were still primarily associated with pyramidal cell dendrites and not the somata. Establishment of functional synaptic input in vitro was confirmed by whole-cell recordings of MF-driven excitatory postsynaptic currents (50 pA to 1 nA) in pyramidal cells. The results establish for the first time that an MF projection with appropriate and functional synaptic connections can be formed de novo and not just maintained in excised hippocampal slices. The cellular dynamics underlying MF growth and synaptogenesis were examined directly by time-lapse confocal imaging of fibers selectively stained with a fluorescent membrane dye (Dil or DiO). MFs growing deep within isolated tissue slices were tipped by small (5-10 microns), active growth cones that advanced at variable rates (5-25 microns/hr). Furthermore, dynamic filopodial structures were seen at small varicosities along the length of developing MFs, which may identify nascent en passant synaptic contacts. The hippocampal slice preparations are shown to support normal development of MF connections and allow for direct visualization of the cellular dynamics of synapse formation in a mammalian CNS tissue environment.