Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons

A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 ± 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 µM and a maximum increase of 51.2 ± 4.4%. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons.

Cerebellar granule neurons; Excitatory amino-acid transporter type 2; Patch-clamp; Glutamate; Whole-cell voltage-clamp

Authorship

R.A. Mafra

Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas , Departamento de Bioquímica e Imunologia, Belo Horizonte, Minas Gerais, BrazilUniversidade Federal de Minas GeraisBrazilBelo Horizonte, Minas Gerais, BrazilUniversidade Federal de Minas Gerais, Instituto de Ciências Biológicas , Departamento de Bioquímica e Imunologia, Belo Horizonte, Minas Gerais, Brazil

Universidade Federal da Paraíba, Centro de Ciências Exatas e da Natureza , Departamento de Biologia Molecular, João Pessoa, Paraíba, BrazilUniversidade Federal da ParaíbaBrazilJoão Pessoa, Paraíba, BrazilUniversidade Federal da Paraíba, Centro de Ciências Exatas e da Natureza , Departamento de Biologia Molecular, João Pessoa, Paraíba, Brazil

R.M. Leão

Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto , Departamento de Fisiologia, Ribeirão Preto, São Paulo, BrazilUniversidade de São PauloBrazilRibeirão Preto, São Paulo, BrazilUniversidade de São Paulo, Faculdade de Medicina de Ribeirão Preto , Departamento de Fisiologia, Ribeirão Preto, São Paulo, Brazil

P.S.L. Beirão

J.S. Cruz

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (4)

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Figure 1.
L-Glutamate-elicited current in granule neurons. A, Whole-cell current evoked by 1 mM L-glutamate in neurons held at -60 mV. The record represents the mean current from eight cells. The continuous line was drawn by eye. B, Bar graph showing the mean steady-state amplitude of whole-cell currents evoked in eight cells by 1 mM L-glutamate (dark gray column) and after washing out (light gray column). Error bars indicate SEM. *P < 0.01 compared to washout cells (two-tailed Student t-test).

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Figure 2.
Dose- and voltage-dependent currents due to the perfusion of granule neurons with L-glutamate. A, I-V relationships for L-glutamate-sensitive currents measured during 100-ms voltage ramps from -100 to +25 mV. L-Glutamate concentration was 1) 10 µM, 2) 100 µM, 3) 300 µM, and 4) 1000 µM. B, Average dose-response curve for the L-glutamate-sensitive current in five cells. The smooth curve is the best-fit: maximum percent increase = 51.2 ± 4.4% and K_m = 18.3 ± 0.9 µM (N = 5).

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Figure 3.
Dependence of L-glutamate-evoked currents on external Na⁺ in granule neurons. A, Representative I-V relationships obtained when N-methyl-D-glucamine (NMDG) was substituted for Na⁺ in a granule neuron. The result is the difference in current obtained by subtracting the current during perfusion of L-glutamate (1 mM) from the current in the absence of L-glutamate. Five different cells were used and all presented the same result. B, Bar graph showing the composite data for five cells. There was no statistically significant difference among the potentials examined. Data are reported as means ± SEM.

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Figure 4.
Pharmacology of L-glutamate-evoked current in granule neurons. A, I-V relationships obtained under the following conditions: 1) 1 mM L-glutamate, 2) 1 mM L-glutamate in the presence of 500 µM dihydrokainate (DHK), and 3) DHK washout and maintenance of 1 mM L-glutamate. All three waveforms were digitally subtracted from the control. B, The same experiment as A but with the addition of 1 mM DHK in the presence of 500 µM L-glutamate. 1) Waveform obtained in response to L-glutamate; 2) 500 µM L-glutamate + 1 mM DHK, and 3) after DHK washout. C, Composite data where a is the control, b after 1 mM L-glutamate, c 1 mM L-glutamate + 500 µM DHK, and d DHK washout. Data are the average of five cells. Error bars indicate the SEM. D, Same as in C but using 1 mM DHK and 500 µM L-glutamate. Data are the average for five different cells. Error bars indicate the SEM. The asterisks denote P < 0.05 compared to control (paired t-test).

Figure 1. L-Glutamate-elicited current in granule neurons. A, Whole-cell current evoked by 1 mM L-glutamate in neurons held at -60 mV. The record represents the mean current from eight cells. The continuous line was drawn by eye. B, Bar graph showing the mean steady-state amplitude of whole-cell currents evoked in eight cells by 1 mM L-glutamate (dark gray column) and after washing out (light gray column). Error bars indicate SEM. *P < 0.01 compared to washout cells (two-tailed Student t-test).

Figure 2. Dose- and voltage-dependent currents due to the perfusion of granule neurons with L-glutamate. A, I-V relationships for L-glutamate-sensitive currents measured during 100-ms voltage ramps from -100 to +25 mV. L-Glutamate concentration was 1) 10 µM, 2) 100 µM, 3) 300 µM, and 4) 1000 µM. B, Average dose-response curve for the L-glutamate-sensitive current in five cells. The smooth curve is the best-fit: maximum percent increase = 51.2 ± 4.4% and K_m = 18.3 ± 0.9 µM (N = 5).

Figure 3. Dependence of L-glutamate-evoked currents on external Na⁺ in granule neurons. A, Representative I-V relationships obtained when N-methyl-D-glucamine (NMDG) was substituted for Na⁺ in a granule neuron. The result is the difference in current obtained by subtracting the current during perfusion of L-glutamate (1 mM) from the current in the absence of L-glutamate. Five different cells were used and all presented the same result. B, Bar graph showing the composite data for five cells. There was no statistically significant difference among the potentials examined. Data are reported as means ± SEM.

Figure 4. Pharmacology of L-glutamate-evoked current in granule neurons. A, I-V relationships obtained under the following conditions: 1) 1 mM L-glutamate, 2) 1 mM L-glutamate in the presence of 500 µM dihydrokainate (DHK), and 3) DHK washout and maintenance of 1 mM L-glutamate. All three waveforms were digitally subtracted from the control. B, The same experiment as A but with the addition of 1 mM DHK in the presence of 500 µM L-glutamate. 1) Waveform obtained in response to L-glutamate; 2) 500 µM L-glutamate + 1 mM DHK, and 3) after DHK washout. C, Composite data where a is the control, b after 1 mM L-glutamate, c 1 mM L-glutamate + 500 µM DHK, and d DHK washout. Data are the average of five cells. Error bars indicate the SEM. D, Same as in C but using 1 mM DHK and 500 µM L-glutamate. Data are the average for five different cells. Error bars indicate the SEM. The asterisks denote P < 0.05 compared to control (paired t-test).

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Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons

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