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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

Quantification of the number and distribution of GABA release sites in MLI axons. Immunolabeling of VGAT in Alexa Fluor 488–loaded MLIs was performed to identify axonal release sites. (A) Confocal image stack Z projection of an Alexa Fluor 488–filled MLI. (B) Superimposition of Alexa Fluor (magenta) and VGAT immunolabeling (green). (C) Detail of the colocalization of VGAT and Alexa Fluor 488 labeling in a varicosity. XY, YZ, and XZ projections (middle, right, and bottom panels, respectively). (D) Intensity profiles of VGAT (green line) and Alexa Fluor 488 (magenta line) along the main branch of an MLI axon. The cartoon on top represents the MLI’s soma and axonal main branch; circles indicate detected GABA varicosities. The soma produced an intense labeling while the rest of the trace showed near baseline intensity interrupted by clearly defined peaks of VGAT and Alexa Fluor intensity, corresponding to varicosities. (Inset) Criteria used for detecting release sites. Threshold = baseline + 2 SD of the VGAT intensity profile (green trace in D); width, >500 nm. GABA release sites are heterogeneously distributed along the axon. (E) Number of release sites per axonal quarter. (F) Histogram of distances between release sites (mean ± SD: 6.1 ± 5.8 µm; median: 4.4 µm; n = 390 pairs of sites). Branching points are also heterogeneously distributed along the axon. (G) Number of branches per axonal quarter. (H) Histogram of collateral’s length (mean ± SD: 24.1 ± 18.8 µm; median: 19.4 µm; n = 89 collaterals). In E and G, the gray dots represent individual cells, and the black dots show mean ± SD; n = 7 reconstructed MLIs (*, P < 0.05; **, P < 0.01).
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fig5: Quantification of the number and distribution of GABA release sites in MLI axons. Immunolabeling of VGAT in Alexa Fluor 488–loaded MLIs was performed to identify axonal release sites. (A) Confocal image stack Z projection of an Alexa Fluor 488–filled MLI. (B) Superimposition of Alexa Fluor (magenta) and VGAT immunolabeling (green). (C) Detail of the colocalization of VGAT and Alexa Fluor 488 labeling in a varicosity. XY, YZ, and XZ projections (middle, right, and bottom panels, respectively). (D) Intensity profiles of VGAT (green line) and Alexa Fluor 488 (magenta line) along the main branch of an MLI axon. The cartoon on top represents the MLI’s soma and axonal main branch; circles indicate detected GABA varicosities. The soma produced an intense labeling while the rest of the trace showed near baseline intensity interrupted by clearly defined peaks of VGAT and Alexa Fluor intensity, corresponding to varicosities. (Inset) Criteria used for detecting release sites. Threshold = baseline + 2 SD of the VGAT intensity profile (green trace in D); width, >500 nm. GABA release sites are heterogeneously distributed along the axon. (E) Number of release sites per axonal quarter. (F) Histogram of distances between release sites (mean ± SD: 6.1 ± 5.8 µm; median: 4.4 µm; n = 390 pairs of sites). Branching points are also heterogeneously distributed along the axon. (G) Number of branches per axonal quarter. (H) Histogram of collateral’s length (mean ± SD: 24.1 ± 18.8 µm; median: 19.4 µm; n = 89 collaterals). In E and G, the gray dots represent individual cells, and the black dots show mean ± SD; n = 7 reconstructed MLIs (*, P < 0.05; **, P < 0.01).

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)

Quantification of the number and distribution of GABA release sites in MLI axons. Immunolabeling of VGAT in Alexa Fluor 488–loaded MLIs was performed to identify axonal release sites. (A) Confocal image stack Z projection of an Alexa Fluor 488–filled MLI. (B) Superimposition of Alexa Fluor (magenta) and VGAT immunolabeling (green). (C) Detail of the colocalization of VGAT and Alexa Fluor 488 labeling in a varicosity. XY, YZ, and XZ projections (middle, right, and bottom panels, respectively). (D) Intensity profiles of VGAT (green line) and Alexa Fluor 488 (magenta line) along the main branch of an MLI axon. The cartoon on top represents the MLI’s soma and axonal main branch; circles indicate detected GABA varicosities. The soma produced an intense labeling while the rest of the trace showed near baseline intensity interrupted by clearly defined peaks of VGAT and Alexa Fluor intensity, corresponding to varicosities. (Inset) Criteria used for detecting release sites. Threshold = baseline + 2 SD of the VGAT intensity profile (green trace in D); width, >500 nm. GABA release sites are heterogeneously distributed along the axon. (E) Number of release sites per axonal quarter. (F) Histogram of distances between release sites (mean ± SD: 6.1 ± 5.8 µm; median: 4.4 µm; n = 390 pairs of sites). Branching points are also heterogeneously distributed along the axon. (G) Number of branches per axonal quarter. (H) Histogram of collateral’s length (mean ± SD: 24.1 ± 18.8 µm; median: 19.4 µm; n = 89 collaterals). In E and G, the gray dots represent individual cells, and the black dots show mean ± SD; n = 7 reconstructed MLIs (*, P < 0.05; **, P < 0.01).
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fig5: Quantification of the number and distribution of GABA release sites in MLI axons. Immunolabeling of VGAT in Alexa Fluor 488–loaded MLIs was performed to identify axonal release sites. (A) Confocal image stack Z projection of an Alexa Fluor 488–filled MLI. (B) Superimposition of Alexa Fluor (magenta) and VGAT immunolabeling (green). (C) Detail of the colocalization of VGAT and Alexa Fluor 488 labeling in a varicosity. XY, YZ, and XZ projections (middle, right, and bottom panels, respectively). (D) Intensity profiles of VGAT (green line) and Alexa Fluor 488 (magenta line) along the main branch of an MLI axon. The cartoon on top represents the MLI’s soma and axonal main branch; circles indicate detected GABA varicosities. The soma produced an intense labeling while the rest of the trace showed near baseline intensity interrupted by clearly defined peaks of VGAT and Alexa Fluor intensity, corresponding to varicosities. (Inset) Criteria used for detecting release sites. Threshold = baseline + 2 SD of the VGAT intensity profile (green trace in D); width, >500 nm. GABA release sites are heterogeneously distributed along the axon. (E) Number of release sites per axonal quarter. (F) Histogram of distances between release sites (mean ± SD: 6.1 ± 5.8 µm; median: 4.4 µm; n = 390 pairs of sites). Branching points are also heterogeneously distributed along the axon. (G) Number of branches per axonal quarter. (H) Histogram of collateral’s length (mean ± SD: 24.1 ± 18.8 µm; median: 19.4 µm; n = 89 collaterals). In E and G, the gray dots represent individual cells, and the black dots show mean ± SD; n = 7 reconstructed MLIs (*, P < 0.05; **, P < 0.01).
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