Abstract
BackgroundRapid uptake of glutamate by neuronal and glial glutamate transporters (EAATs, a family of excitatory amino acid transporters) is critical for shaping synaptic responses and for preventing excitotoxicity. Two of these transporters, EAAT4 in Purkinje neurons (PN) and EAAT1 in Bergmann glia are both enriched within the cerebellum and altered in a variety of human ataxias.ResultsPN excitatory synaptic responses and firing behaviour following high frequency parallel fibre (PF) activity commonly encountered during sensory stimulation in vivo were adversely influenced by acute inhibition of glutamate transporters. In the presence of a non-transportable blocker of glutamate transporters we observed very large amplitude and duration excitatory postsynaptic currents accompanied by excessive firing of the PNs. A combination of AMPA and mGluR1, but not NMDA, type glutamate receptor activation powered the hyper-excitable PN state. The enhanced PN excitability also recruited a presynaptic mGluR4 dependent mechanism that modified short term plasticity at the PF synapse.ConclusionsOur findings indicate that reduced glutamate transporter activity, as occurs in the early stages of some forms of human cerebellar ataxias, excessively excites PNs and disrupts the timing of their output. Our findings raise the possibility that sustaining cerebellar glutamate uptake may provide a therapeutic approach to prevent this disruption and the glutamate excitotoxicity-induced PN death that signals the end point of the disease.
Highlights
Rapid uptake of glutamate by neuronal and glial glutamate transporters (EAATs, a family of excitatory amino acid transporters) is critical for shaping synaptic responses and for preventing excitotoxicity
Inhibition of glutamate transport enhanced high frequency stimulus-evoked parallel fibre EPSCs and revealed a slow EPSC with mGluR1 dependent and independent components A burst of ten, high frequency (200 Hz) stimulations to the PFs aimed to mimic the in vivo behaviour of PFs [32] and evoked a large amplitude long-lasting EPSC in cerebellar Purkinje neurons (PN), Figure 1 and Figure 2A, hereafter called the high-frequency PF EPSC
In the presence of the AMPA and Kainate receptor (KA-R) antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and DL-threo-beta-Benzyloxyaspartic acid (TBOA) and MCPG,the fast peak amplitude of the high-frequency PF EPSC reduced from 980 ± 167 Peak Amplitude (pA) in control to 112 ± 36 pA, n = 4, P < 0.001, t-test, whilst the slower component EPSC reduced from 65 ± 16 pA to 2 ± 1.2 pA
Summary
Rapid uptake of glutamate by neuronal and glial glutamate transporters (EAATs, a family of excitatory amino acid transporters) is critical for shaping synaptic responses and for preventing excitotoxicity. Glutamate clearance relies upon glutamate transporter carrier proteins (excitatory amino acid transporters, EAATs) located in close proximity to the synapse [1] that couple glutamate movement with the Na+ electrochemical gradient, H+ movement and a countertransported K+ ion [2] These transporters behave like ligand gated ion channels, since glutamate activates their. The main output neurons of the cerebellar cortex, the Purkinje neurons (PN), are highly enriched for EAAT4 [12] present exclusively within the PN somatodendritic compartment [12] and perisynaptic regions [13] This location means it powerfully influences both climbing fibre (CF) and parallel fibre (PF) synapse responses [14]. EAAT3 ( known as EAAC1) is negatively modulated by its interacting protein, glutamate transporter-associated protein 3-18 (GTRAP3-18) [23] and is known to transport cysteine into cells to produce neuroprotective glutathione [24,25]
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