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Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity.

de San Martin JZ, Jalil A, Trigo FF - J. Gen. Physiol. (2015)

Bottom Line: Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release.The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity.Finally, we show that single-site activation of presynaptic GABA(A) autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Physiologie Cérébrale, Université Paris Descartes and Centre National de la Recherche Scientifique, CNRS UMR8118, 75794 Paris, France.

No MeSH data available.


Related in: MedlinePlus

Axonal GABAARs activation in single presynaptic varicosities has a marked effect on MLI excitability. (A; left) Representative traces of laser-evoked ASPs recorded in current-clamp mode with an intracellular solution containing [Cl−]i = 15 mM. In this cell, photolysis of caged Ca2+ produced subthreshold depolarizing responses (same cell as in Fig. 6 A), with a Pspike of 0. (Right) Laser-evoked ASPs recorded in the same condition as shown in the left panel. In this cell, the ASPs induced AP firing with a probability Pspike of 0.625. (B) Summary plot of the fraction of release sites that produced active responses with 15 and 25 mM [Cl−]i. The percentage of release sites that produced active responses in each experimental condition is shown in the bars (n = 17 sites with [Cl−]i = 15 mM and 13 sites with [Cl−]i = 25 mM). (C) Example of an MLI where GABA was photolyzed from DPNI-GABA (1 ms, 2-mW pulses) in four different axonal locations. Traces are averages from four to five sweeps, with amplitudes 5.5, 4.0, 1.7, and 0.6 mV; Vm values −70.8, −70.7, −67, and −72 mV (average values during 100 ms before the laser pulse); and distances to the soma 28.9, 41, 48, and 90 µm, respectively. Gray dotted line indicates timing of the laser pulse. (D) Somatic whole-cell recordings in current-clamp mode of the responses to current injection without (left) and with (right) prepulses of caged-GABA photolysis in the axon (50-ms interval between photolysis pulse and current injection). It can be observed that Pspike increases dramatically with the GABA prepulse. (E) Pspike in control (Ctrl) and with caged-GABA prepulses (GABA) for different time intervals between the laser pulse and the current injection: 50, 100, 200, and 300 ms; **, P < 0.01; ***, P < 0.001. (F) The increase of Pspike produced by the photolysis of caged GABA was plotted as a function of the interval between the laser and the current-injection pulses. The excitability increase has a very similar time course when compared with the decay of the GABA-induced depolarizations. Gray circles are the values from individual cells; black symbols and error bars represent mean ± SEM for each time interval. The cells were held near −70 mV.
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fig7: Axonal GABAARs activation in single presynaptic varicosities has a marked effect on MLI excitability. (A; left) Representative traces of laser-evoked ASPs recorded in current-clamp mode with an intracellular solution containing [Cl−]i = 15 mM. In this cell, photolysis of caged Ca2+ produced subthreshold depolarizing responses (same cell as in Fig. 6 A), with a Pspike of 0. (Right) Laser-evoked ASPs recorded in the same condition as shown in the left panel. In this cell, the ASPs induced AP firing with a probability Pspike of 0.625. (B) Summary plot of the fraction of release sites that produced active responses with 15 and 25 mM [Cl−]i. The percentage of release sites that produced active responses in each experimental condition is shown in the bars (n = 17 sites with [Cl−]i = 15 mM and 13 sites with [Cl−]i = 25 mM). (C) Example of an MLI where GABA was photolyzed from DPNI-GABA (1 ms, 2-mW pulses) in four different axonal locations. Traces are averages from four to five sweeps, with amplitudes 5.5, 4.0, 1.7, and 0.6 mV; Vm values −70.8, −70.7, −67, and −72 mV (average values during 100 ms before the laser pulse); and distances to the soma 28.9, 41, 48, and 90 µm, respectively. Gray dotted line indicates timing of the laser pulse. (D) Somatic whole-cell recordings in current-clamp mode of the responses to current injection without (left) and with (right) prepulses of caged-GABA photolysis in the axon (50-ms interval between photolysis pulse and current injection). It can be observed that Pspike increases dramatically with the GABA prepulse. (E) Pspike in control (Ctrl) and with caged-GABA prepulses (GABA) for different time intervals between the laser pulse and the current injection: 50, 100, 200, and 300 ms; **, P < 0.01; ***, P < 0.001. (F) The increase of Pspike produced by the photolysis of caged GABA was plotted as a function of the interval between the laser and the current-injection pulses. The excitability increase has a very similar time course when compared with the decay of the GABA-induced depolarizations. Gray circles are the values from individual cells; black symbols and error bars represent mean ± SEM for each time interval. The cells were held near −70 mV.

Mentions: MLIs were visualized under a microscope with a 63×/0.9-NA water-dipping objective (Axio Scope; Carl Zeiss) and recorded with the patch technique under the whole-cell configuration, both in voltage and current clamp, with an amplifier (EPC 10; HEKA). The composition of the internal solution (IS) used for the high [Cl−]i experiments was as follows (mM): 90 KCl, 50 HEPES, 0.5 MgCl2, 4.25 CaCl2, 5 Na2ATP, 20 NaCl, 0.5 NaGTP, 25 KOH, 5 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N′,N′-tetrakis[(oxycarbonyl)methyl]-1,2-ethanediamine (DM-nitrophen), 0.08 Alexa Fluor 488 (or 594 for the experiments in Fig. 1, A and B), and 10 GABA (to avoid washout of intracellular GABA; Bouhours et al., 2011). KCl was replaced by 110 or 100 mM K-gluconate for the experiments with [Cl−]i = 15 and 25 mM, respectively. IS had a pH of 7.3 and an osmolality of ≈300 mOsm kg−1 H2O. Recordings were made at room temperature (22–24°C). In the experiments performed with the low [Cl−]i IS (Figs. 5–7), the membrane potential was corrected for a 12-mV liquid junction potential value (calculated with Patcher’s Power Tools for Igor Pro; F. Mendez and F. Würriehausen, Max-Planck-Institut Für Biophysikalische Chemie, 37077 Göttingen, Germany). Pipette resistance was ∼5 MΩ when filled with the high [Cl−]i IS and ∼10 MΩ when filled with the low [Cl−]i IS. Series resistance was compensated by 50%. Recordings with SR higher than 25 MΩ were discarded. Holding potentials were usually −60 mV. MLI identification was confirmed by the observation of large (0.8–1.7-nA), unclamped Na+ currents when the membrane potential was stepped from −60 to 0 mV for 2 ms (Pouzat and Marty, 1999). Recordings were filtered at 5 kHz with a Bessel filter. Data were analyzed using routines written in Igor Pro (WaveMetrics). Most data were obtained from cells located in the proximal part of the molecular layer (basket cells); however, interneurons located in the distal molecular layer (stellate cells) were also included. Reagents were purchased from Sigma-Aldrich, and gabazine (Gbz) and tetrodotoxin (TTX) were from Abcam.


Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity.

de San Martin JZ, Jalil A, Trigo FF - J. Gen. Physiol. (2015)

Axonal GABAARs activation in single presynaptic varicosities has a marked effect on MLI excitability. (A; left) Representative traces of laser-evoked ASPs recorded in current-clamp mode with an intracellular solution containing [Cl−]i = 15 mM. In this cell, photolysis of caged Ca2+ produced subthreshold depolarizing responses (same cell as in Fig. 6 A), with a Pspike of 0. (Right) Laser-evoked ASPs recorded in the same condition as shown in the left panel. In this cell, the ASPs induced AP firing with a probability Pspike of 0.625. (B) Summary plot of the fraction of release sites that produced active responses with 15 and 25 mM [Cl−]i. The percentage of release sites that produced active responses in each experimental condition is shown in the bars (n = 17 sites with [Cl−]i = 15 mM and 13 sites with [Cl−]i = 25 mM). (C) Example of an MLI where GABA was photolyzed from DPNI-GABA (1 ms, 2-mW pulses) in four different axonal locations. Traces are averages from four to five sweeps, with amplitudes 5.5, 4.0, 1.7, and 0.6 mV; Vm values −70.8, −70.7, −67, and −72 mV (average values during 100 ms before the laser pulse); and distances to the soma 28.9, 41, 48, and 90 µm, respectively. Gray dotted line indicates timing of the laser pulse. (D) Somatic whole-cell recordings in current-clamp mode of the responses to current injection without (left) and with (right) prepulses of caged-GABA photolysis in the axon (50-ms interval between photolysis pulse and current injection). It can be observed that Pspike increases dramatically with the GABA prepulse. (E) Pspike in control (Ctrl) and with caged-GABA prepulses (GABA) for different time intervals between the laser pulse and the current injection: 50, 100, 200, and 300 ms; **, P < 0.01; ***, P < 0.001. (F) The increase of Pspike produced by the photolysis of caged GABA was plotted as a function of the interval between the laser and the current-injection pulses. The excitability increase has a very similar time course when compared with the decay of the GABA-induced depolarizations. Gray circles are the values from individual cells; black symbols and error bars represent mean ± SEM for each time interval. The cells were held near −70 mV.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4664828&req=5

fig7: Axonal GABAARs activation in single presynaptic varicosities has a marked effect on MLI excitability. (A; left) Representative traces of laser-evoked ASPs recorded in current-clamp mode with an intracellular solution containing [Cl−]i = 15 mM. In this cell, photolysis of caged Ca2+ produced subthreshold depolarizing responses (same cell as in Fig. 6 A), with a Pspike of 0. (Right) Laser-evoked ASPs recorded in the same condition as shown in the left panel. In this cell, the ASPs induced AP firing with a probability Pspike of 0.625. (B) Summary plot of the fraction of release sites that produced active responses with 15 and 25 mM [Cl−]i. The percentage of release sites that produced active responses in each experimental condition is shown in the bars (n = 17 sites with [Cl−]i = 15 mM and 13 sites with [Cl−]i = 25 mM). (C) Example of an MLI where GABA was photolyzed from DPNI-GABA (1 ms, 2-mW pulses) in four different axonal locations. Traces are averages from four to five sweeps, with amplitudes 5.5, 4.0, 1.7, and 0.6 mV; Vm values −70.8, −70.7, −67, and −72 mV (average values during 100 ms before the laser pulse); and distances to the soma 28.9, 41, 48, and 90 µm, respectively. Gray dotted line indicates timing of the laser pulse. (D) Somatic whole-cell recordings in current-clamp mode of the responses to current injection without (left) and with (right) prepulses of caged-GABA photolysis in the axon (50-ms interval between photolysis pulse and current injection). It can be observed that Pspike increases dramatically with the GABA prepulse. (E) Pspike in control (Ctrl) and with caged-GABA prepulses (GABA) for different time intervals between the laser pulse and the current injection: 50, 100, 200, and 300 ms; **, P < 0.01; ***, P < 0.001. (F) The increase of Pspike produced by the photolysis of caged GABA was plotted as a function of the interval between the laser and the current-injection pulses. The excitability increase has a very similar time course when compared with the decay of the GABA-induced depolarizations. Gray circles are the values from individual cells; black symbols and error bars represent mean ± SEM for each time interval. The cells were held near −70 mV.
Mentions: MLIs were visualized under a microscope with a 63×/0.9-NA water-dipping objective (Axio Scope; Carl Zeiss) and recorded with the patch technique under the whole-cell configuration, both in voltage and current clamp, with an amplifier (EPC 10; HEKA). The composition of the internal solution (IS) used for the high [Cl−]i experiments was as follows (mM): 90 KCl, 50 HEPES, 0.5 MgCl2, 4.25 CaCl2, 5 Na2ATP, 20 NaCl, 0.5 NaGTP, 25 KOH, 5 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N′,N′-tetrakis[(oxycarbonyl)methyl]-1,2-ethanediamine (DM-nitrophen), 0.08 Alexa Fluor 488 (or 594 for the experiments in Fig. 1, A and B), and 10 GABA (to avoid washout of intracellular GABA; Bouhours et al., 2011). KCl was replaced by 110 or 100 mM K-gluconate for the experiments with [Cl−]i = 15 and 25 mM, respectively. IS had a pH of 7.3 and an osmolality of ≈300 mOsm kg−1 H2O. Recordings were made at room temperature (22–24°C). In the experiments performed with the low [Cl−]i IS (Figs. 5–7), the membrane potential was corrected for a 12-mV liquid junction potential value (calculated with Patcher’s Power Tools for Igor Pro; F. Mendez and F. Würriehausen, Max-Planck-Institut Für Biophysikalische Chemie, 37077 Göttingen, Germany). Pipette resistance was ∼5 MΩ when filled with the high [Cl−]i IS and ∼10 MΩ when filled with the low [Cl−]i IS. Series resistance was compensated by 50%. Recordings with SR higher than 25 MΩ were discarded. Holding potentials were usually −60 mV. MLI identification was confirmed by the observation of large (0.8–1.7-nA), unclamped Na+ currents when the membrane potential was stepped from −60 to 0 mV for 2 ms (Pouzat and Marty, 1999). Recordings were filtered at 5 kHz with a Bessel filter. Data were analyzed using routines written in Igor Pro (WaveMetrics). Most data were obtained from cells located in the proximal part of the molecular layer (basket cells); however, interneurons located in the distal molecular layer (stellate cells) were also included. Reagents were purchased from Sigma-Aldrich, and gabazine (Gbz) and tetrodotoxin (TTX) were from Abcam.

Bottom Line: Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release.The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity.Finally, we show that single-site activation of presynaptic GABA(A) autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Physiologie Cérébrale, Université Paris Descartes and Centre National de la Recherche Scientifique, CNRS UMR8118, 75794 Paris, France.

No MeSH data available.


Related in: MedlinePlus