Original Research / Publications

The density of EAAC1 (EAAT3) glutamate transporters expressed by neurons in the mammalian CNS.

The extracellular levels of excitatory amino acids are kept low by the action of the glutamate transporters. Glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are the most abundant subtypes and are essential for the functioning of the mammalian CNS, but the contribution of the EAAC1 subtype in the clearance of synaptic glutamate has remained controversial, because the density of this transporter in different tissues has not been determined. We used purified EAAC1 protein as a standard during immunoblotting to measure the concentration of EAAC1 in different CNS regions. The highest EAAC1 levels were found in the young adult rat hippocampus. Here, the concentration of EAAC1 was ∼0.013 mg/g tissue (∼130 molecules μm⁻³), 100 times lower than that of GLT-1. Unlike GLT-1 expression, which increases in parallel with circuit formation, only minor changes in the concentration of EAAC1 were observed from E18 to adulthood. In hippocampal slices, photolysis of MNI-D-aspartate (4-methoxy-7-nitroindolinyl-D-aspartate) failed to elicit EAAC1-mediated transporter currents in CA1 pyramidal neurons, and D-aspartate uptake was not detected electron microscopically in spines. Using EAAC1 knock-out mice as negative controls to establish antibody specificity, we show that these relatively small amounts of EAAC1 protein are widely distributed in somata and dendrites of all hippocampal neurons. These findings raise new questions about how so few transporters can influence the activation of NMDA receptors at excitatory synapses.

Original Research / Publications

Astrocyte glutamate transporters regulate metabotropic glutamate receptor-mediated excitation of hippocampal interneurons.

Clearance of extracellular glutamate is essential for limiting the activity of metabotropic glutamate receptors (mGluRs) at excitatory synapses; however, the relative contribution of transporters found in neuronal and glial membranes to this uptake is poorly understood. Hippocampalinterneurons located at the oriens-alveus border express mGluR1alpha, a metabotropic glutamate receptor that regulates excitability and synaptic plasticity. To determine which glutamate transporters are essential for removing glutamate at these excitatory synapses, we recorded mGluR1-mediated EPSCs from oriens-lacunosum moleculare (O-LM) interneurons in acute hippocampal slices. Stimulation in stratum oriens reliably elicited a slow mGluR1-mediated current in O-LM interneurons if they were briefly depolarized to allow Ca2+ entry before stimulation. Selective inhibition of GLT-1 [for glutamate transporter; EAAT2 (for excitatory amino acid transporter)] with dihydrokainate increased the amplitude of these responses approximately threefold, indicating that these transporters compete with mGluRs for synaptically released glutamate. However, inhibition of all glutamate transporters with TBOA (DL-threo-b-benzyloxyaspartic acid) increased mGluR1 EPSCs >15-fold, indicating that additional transporters also shape activation of these receptors. To identify these transporters, we examined mGluR1 EPSCs in mice lacking GLAST (for glutamate-aspartate transporter; EAAT1) or EAAC1 (for excitatory amino acid carrier; EAAT3). A comparison of responses recorded from wild-type and transporter knock-out mice revealed that the astroglial glutamate transporters GLT-1 and GLAST, but not the neuronal transporter EAAC1, restrict activation of mGluRs in O-LM interneurons. Transporter-dependent potentiation of mGluR1 EPSCs led to a dramatic increase in interneuron firing and enhanced inhibition of CA1 pyramidal neurons, suggesting that acute or prolonged disruption of transporter activity could lead to changes in network activity as a result of enhanced interneuron excitability.

Original Research / Publications

Synaptic signaling between GABAergic interneurons and oligodendrocyte precursor cells in the hippocampus

Oligodendrocyte precursor cells (OPCs) express receptors for many neurotransmitters, but the mechanisms responsible for their activation are poorly understood. We have found that quantal release of GABA from interneurons elicits GABA(A) receptor currents with rapid rise times in hippocampal OPCs. These currents did not exhibit properties of spillover transmission or release by transporters, and immunofluorescence and electron microscopy suggest that interneuronal terminals are in direct contact with OPCs, indicating that these GABA currents are generated at direct interneuron-OPC synapses. The reversal potential of OPC GABA(A) currents was -43 mV, and interneuronal firing was correlated with transient depolarizations induced by GABA(A) receptors; however, GABA application induced a transient inhibition of currents mediated by AMPA receptors in OPCs. These results indicate that OPCs are a direct target of interneuronal collaterals and that the GABA-induced Cl(-) flux generated by these events may influence oligodendrocyte development by regulating the efficacy of glutamatergic signaling in OPCs.

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.

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

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.