Limits...
Functional contributions of glutamate transporters at the parallel fibre to Purkinje neuron synapse-relevance for the progression of cerebellar ataxia.

Power EM, Empson RM - Cerebellum Ataxias (2014)

Bottom Line: The enhanced PN excitability also recruited a presynaptic mGluR4 dependent mechanism that modified short term plasticity at the PF synapse.Our 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Brain Health Research Centre, University of Otago School of Medical Sciences, PO Box 56, 9054 Dunedin, New Zealand.

ABSTRACT

Background: 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. 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.

Results: PN 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.

Conclusions: Our 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.

No MeSH data available.


Related in: MedlinePlus

Location of all the major transporters and receptors at a PF-PN synapse.(A)(i) A biocytin filled Purkinje neuron post-hoc stained with streptavidin Alexa 488. (ii) A representative image of a Parallel fibre Purkinje neurons synapse showing the approximate locations of the major transporters and receptors targeted in this study. Parallel fibre synapses are located on the outer dendrites of Purkinje neurons, highlighted by the white box in (Ai). (B) An example trace showing our stimulation and recording protocol. We used a 5 mV step to calculate series and input resistance, followed by application of a pair of closely spaced stimuli to the parallel fibres and a high frequency burst stimulation (10 stimuli at 200 Hz), a protocol we repeated every 30 seconds.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4549135&req=5

Fig1: Location of all the major transporters and receptors at a PF-PN synapse.(A)(i) A biocytin filled Purkinje neuron post-hoc stained with streptavidin Alexa 488. (ii) A representative image of a Parallel fibre Purkinje neurons synapse showing the approximate locations of the major transporters and receptors targeted in this study. Parallel fibre synapses are located on the outer dendrites of Purkinje neurons, highlighted by the white box in (Ai). (B) An example trace showing our stimulation and recording protocol. We used a 5 mV step to calculate series and input resistance, followed by application of a pair of closely spaced stimuli to the parallel fibres and a high frequency burst stimulation (10 stimuli at 200 Hz), a protocol we repeated every 30 seconds.

Mentions: 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 PNs, Figure 1 and Figure 2A, hereafter called the high-frequency PF EPSC. To inhibit glutamate transport we used 50 μM TBOA. This concentration of TBOA significantly and reversibly enhanced both the peak amplitude and the duration of the high-frequency PF EPSC, Figure 2B. These changes occurred in the absence of any changes in the series resistance, input resistance or holding current of the PN (see Methods below). TBOA application also revealed a larger amplitude slow component to the high-frequency PF EPSC that was reduced by the broad spectrum mGluR1 antagonist, MCPG (0.2 mM), leaving a smaller amplitude, slow EPSC remaining, Figure 2B. In the presence of the AMPA and Kainate receptor (KA-R) antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and TBOA and MCPG (see also Table 1 for explanation),the fast peak amplitude of the high-frequency PF EPSC reduced from 980 ± 167 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 (see Figure 2A). This result indicated that the majority of the TBOA-enhanced high-frequency PF EPSC resulted from AMPA-R (α-Amino-3-hydroxy-5-methyl-4-iso xazolepropionic acid receptors) and KA-R activation. The NMDA (glutamate) receptor antagonist APV (n = 6) did not influence the amplitude or duration of the high-frequency PF EPSC in the presence of TBOA (mean values of the EPSC changed from 771 ± 66 pA for control to 1087 ± 88.3 pA in TBOA, P < 0.05, one way ANOVA, but remained unchanged at 1128 ± 124 pA in the presence of TBOA and APV, not significant in one way ANOVA multiple comparison). We also observed reversibility of the effects of 50 μM TBOA in 4 cells 15 minutes after wash back; mean values of the amplitude and duration of the 200 Hz EPSC changed from 487 ± 64 pA to 674 ± 67 pA in TBOA and back to 503 ± 65 pA (F2,11 = 14.7, P < 0.05, one way ANOVA) and 291 ± 47 ms to 615 ± 118 ms and back to 379 ± 55 ms respectively (F2,11 = 4.4, P < 0.05, one way ANOVA). The time constant of the recovery of the 2nd EPSC was also reversible, mean values changed from 19.3 ± 1.7 ms to 30 ± 3.2 and back to 22.9 ± 2.5 ms (F2,11 = 4.6, P < 0.05, one way ANOVA).Figure 1


Functional contributions of glutamate transporters at the parallel fibre to Purkinje neuron synapse-relevance for the progression of cerebellar ataxia.

Power EM, Empson RM - Cerebellum Ataxias (2014)

Location of all the major transporters and receptors at a PF-PN synapse.(A)(i) A biocytin filled Purkinje neuron post-hoc stained with streptavidin Alexa 488. (ii) A representative image of a Parallel fibre Purkinje neurons synapse showing the approximate locations of the major transporters and receptors targeted in this study. Parallel fibre synapses are located on the outer dendrites of Purkinje neurons, highlighted by the white box in (Ai). (B) An example trace showing our stimulation and recording protocol. We used a 5 mV step to calculate series and input resistance, followed by application of a pair of closely spaced stimuli to the parallel fibres and a high frequency burst stimulation (10 stimuli at 200 Hz), a protocol we repeated every 30 seconds.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4549135&req=5

Fig1: Location of all the major transporters and receptors at a PF-PN synapse.(A)(i) A biocytin filled Purkinje neuron post-hoc stained with streptavidin Alexa 488. (ii) A representative image of a Parallel fibre Purkinje neurons synapse showing the approximate locations of the major transporters and receptors targeted in this study. Parallel fibre synapses are located on the outer dendrites of Purkinje neurons, highlighted by the white box in (Ai). (B) An example trace showing our stimulation and recording protocol. We used a 5 mV step to calculate series and input resistance, followed by application of a pair of closely spaced stimuli to the parallel fibres and a high frequency burst stimulation (10 stimuli at 200 Hz), a protocol we repeated every 30 seconds.
Mentions: 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 PNs, Figure 1 and Figure 2A, hereafter called the high-frequency PF EPSC. To inhibit glutamate transport we used 50 μM TBOA. This concentration of TBOA significantly and reversibly enhanced both the peak amplitude and the duration of the high-frequency PF EPSC, Figure 2B. These changes occurred in the absence of any changes in the series resistance, input resistance or holding current of the PN (see Methods below). TBOA application also revealed a larger amplitude slow component to the high-frequency PF EPSC that was reduced by the broad spectrum mGluR1 antagonist, MCPG (0.2 mM), leaving a smaller amplitude, slow EPSC remaining, Figure 2B. In the presence of the AMPA and Kainate receptor (KA-R) antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and TBOA and MCPG (see also Table 1 for explanation),the fast peak amplitude of the high-frequency PF EPSC reduced from 980 ± 167 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 (see Figure 2A). This result indicated that the majority of the TBOA-enhanced high-frequency PF EPSC resulted from AMPA-R (α-Amino-3-hydroxy-5-methyl-4-iso xazolepropionic acid receptors) and KA-R activation. The NMDA (glutamate) receptor antagonist APV (n = 6) did not influence the amplitude or duration of the high-frequency PF EPSC in the presence of TBOA (mean values of the EPSC changed from 771 ± 66 pA for control to 1087 ± 88.3 pA in TBOA, P < 0.05, one way ANOVA, but remained unchanged at 1128 ± 124 pA in the presence of TBOA and APV, not significant in one way ANOVA multiple comparison). We also observed reversibility of the effects of 50 μM TBOA in 4 cells 15 minutes after wash back; mean values of the amplitude and duration of the 200 Hz EPSC changed from 487 ± 64 pA to 674 ± 67 pA in TBOA and back to 503 ± 65 pA (F2,11 = 14.7, P < 0.05, one way ANOVA) and 291 ± 47 ms to 615 ± 118 ms and back to 379 ± 55 ms respectively (F2,11 = 4.4, P < 0.05, one way ANOVA). The time constant of the recovery of the 2nd EPSC was also reversible, mean values changed from 19.3 ± 1.7 ms to 30 ± 3.2 and back to 22.9 ± 2.5 ms (F2,11 = 4.6, P < 0.05, one way ANOVA).Figure 1

Bottom Line: The enhanced PN excitability also recruited a presynaptic mGluR4 dependent mechanism that modified short term plasticity at the PF synapse.Our 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Brain Health Research Centre, University of Otago School of Medical Sciences, PO Box 56, 9054 Dunedin, New Zealand.

ABSTRACT

Background: 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. 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.

Results: PN 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.

Conclusions: Our 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.

No MeSH data available.


Related in: MedlinePlus