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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

The spatial resolution of the Ca2+ photolysis approach permits evoking ASCs from single varicosities. Light intensity distribution of the laser spot. The laser beam was reflected back to the camera with a planar mirror placed at the level of the slice chamber and recorded with the EM-CCD camera. Stack of images made by changing the focus at 0.1-µm intervals. (A and B) The XY and YZ projections of the laser spot, respectively (bars, 2 µm). The bottom graphs show the intensity profile of the spot measured on the x axis (A) and on the z axis (B) at the level of the intensity peak (1/e2 diameter for the x axis: 1.4 µm and for the z axis: 7.2 µm). (C) Stack of epifluorescence images of an Alexa Fluor–filled MLI (left) and detail of a section of the axon (right). The photolysis positions are marked with arrowheads and numbers. (D) Individual traces recorded while sequentially stimulating the positions indicated in C. Vertical dotted line indicates the timing of the laser pulse. (E) Amplitudes (normalized in 1-µm bins to the response evoked on the varicosity center) as a function of the distance between the stimulated position and the center of the varicosity (for this analysis, only the positions located on the axon were considered). Gray circles correspond to the amplitudes from individual sweeps. Continuous line shows the fit with a Gaussian function, with a half-width of 2.18 ± 0.28 µm (mean ± SEM; n = 6 sites).
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fig3: The spatial resolution of the Ca2+ photolysis approach permits evoking ASCs from single varicosities. Light intensity distribution of the laser spot. The laser beam was reflected back to the camera with a planar mirror placed at the level of the slice chamber and recorded with the EM-CCD camera. Stack of images made by changing the focus at 0.1-µm intervals. (A and B) The XY and YZ projections of the laser spot, respectively (bars, 2 µm). The bottom graphs show the intensity profile of the spot measured on the x axis (A) and on the z axis (B) at the level of the intensity peak (1/e2 diameter for the x axis: 1.4 µm and for the z axis: 7.2 µm). (C) Stack of epifluorescence images of an Alexa Fluor–filled MLI (left) and detail of a section of the axon (right). The photolysis positions are marked with arrowheads and numbers. (D) Individual traces recorded while sequentially stimulating the positions indicated in C. Vertical dotted line indicates the timing of the laser pulse. (E) Amplitudes (normalized in 1-µm bins to the response evoked on the varicosity center) as a function of the distance between the stimulated position and the center of the varicosity (for this analysis, only the positions located on the axon were considered). Gray circles correspond to the amplitudes from individual sweeps. Continuous line shows the fit with a Gaussian function, with a half-width of 2.18 ± 0.28 µm (mean ± SEM; n = 6 sites).

Mentions: The next series of experiments was designed to characterize the autoR-mediated synaptic events. To do this, we only recorded from the presynaptic cell. As in the previous set of experiments, somatic whole-cell recordings were performed with an intracellular solution containing Alexa Fluor 488, the Ca2+ cage DM-nitrophen, and high [Cl−]i to maximize the driving force for GABAA receptors (EGABA = 3 mV). Fig. 2 A shows the somatodendritic compartment and the proximal part of the axon of an Alexa Fluor–filled MLI. Scattered along the axon, it is possible to observe many elongated bright spots, or varicosities (Fig. 2 A, inset; see also Fig. 3 C), that correspond to GABA release sites (Llano et al., 1997; Forti et al., 2000; Rowan et al., 2014; Trigo et al., 2012). As in the paired experiment of Fig. 1, when the minimized laser spot (λ = 405 nm) was focused onto such an axonal varicosity, photolysis of caged Ca2+ with 100-µs pulses evoked inward currents, as recorded from the soma of the cell (Fig. 2 B; black traces are individual sweeps, and the gray trace is the average). Again, these inward currents closely resembled preminis; here, we call them ASCs (autoR-mediated synaptic currents) because, contrary to preminis, they mainly involve multi-vesicular release. In the cell shown in Fig. 2 A, photolysis was repeated seven times in the same varicosity, located at 54 µm from the center of the soma; the average amplitude of the responses was 26 ± 1 pA; its τrise was 3.1 ± 0.9 ms, its τdecay was 76.0 ± 3.4 ms, and the average latency from the laser pulse was 1.06 ± 0.23 ms. When inspecting the expanded traces, it can be observed that some of the events show multiple peaks (black circles in Fig. 2, B and G); these correspond to multiple vesicular release events, as was already described when the same approach was used to study postsynaptic currents in MLI pairs (Trigo et al., 2012).


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

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

The spatial resolution of the Ca2+ photolysis approach permits evoking ASCs from single varicosities. Light intensity distribution of the laser spot. The laser beam was reflected back to the camera with a planar mirror placed at the level of the slice chamber and recorded with the EM-CCD camera. Stack of images made by changing the focus at 0.1-µm intervals. (A and B) The XY and YZ projections of the laser spot, respectively (bars, 2 µm). The bottom graphs show the intensity profile of the spot measured on the x axis (A) and on the z axis (B) at the level of the intensity peak (1/e2 diameter for the x axis: 1.4 µm and for the z axis: 7.2 µm). (C) Stack of epifluorescence images of an Alexa Fluor–filled MLI (left) and detail of a section of the axon (right). The photolysis positions are marked with arrowheads and numbers. (D) Individual traces recorded while sequentially stimulating the positions indicated in C. Vertical dotted line indicates the timing of the laser pulse. (E) Amplitudes (normalized in 1-µm bins to the response evoked on the varicosity center) as a function of the distance between the stimulated position and the center of the varicosity (for this analysis, only the positions located on the axon were considered). Gray circles correspond to the amplitudes from individual sweeps. Continuous line shows the fit with a Gaussian function, with a half-width of 2.18 ± 0.28 µm (mean ± SEM; n = 6 sites).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: The spatial resolution of the Ca2+ photolysis approach permits evoking ASCs from single varicosities. Light intensity distribution of the laser spot. The laser beam was reflected back to the camera with a planar mirror placed at the level of the slice chamber and recorded with the EM-CCD camera. Stack of images made by changing the focus at 0.1-µm intervals. (A and B) The XY and YZ projections of the laser spot, respectively (bars, 2 µm). The bottom graphs show the intensity profile of the spot measured on the x axis (A) and on the z axis (B) at the level of the intensity peak (1/e2 diameter for the x axis: 1.4 µm and for the z axis: 7.2 µm). (C) Stack of epifluorescence images of an Alexa Fluor–filled MLI (left) and detail of a section of the axon (right). The photolysis positions are marked with arrowheads and numbers. (D) Individual traces recorded while sequentially stimulating the positions indicated in C. Vertical dotted line indicates the timing of the laser pulse. (E) Amplitudes (normalized in 1-µm bins to the response evoked on the varicosity center) as a function of the distance between the stimulated position and the center of the varicosity (for this analysis, only the positions located on the axon were considered). Gray circles correspond to the amplitudes from individual sweeps. Continuous line shows the fit with a Gaussian function, with a half-width of 2.18 ± 0.28 µm (mean ± SEM; n = 6 sites).
Mentions: The next series of experiments was designed to characterize the autoR-mediated synaptic events. To do this, we only recorded from the presynaptic cell. As in the previous set of experiments, somatic whole-cell recordings were performed with an intracellular solution containing Alexa Fluor 488, the Ca2+ cage DM-nitrophen, and high [Cl−]i to maximize the driving force for GABAA receptors (EGABA = 3 mV). Fig. 2 A shows the somatodendritic compartment and the proximal part of the axon of an Alexa Fluor–filled MLI. Scattered along the axon, it is possible to observe many elongated bright spots, or varicosities (Fig. 2 A, inset; see also Fig. 3 C), that correspond to GABA release sites (Llano et al., 1997; Forti et al., 2000; Rowan et al., 2014; Trigo et al., 2012). As in the paired experiment of Fig. 1, when the minimized laser spot (λ = 405 nm) was focused onto such an axonal varicosity, photolysis of caged Ca2+ with 100-µs pulses evoked inward currents, as recorded from the soma of the cell (Fig. 2 B; black traces are individual sweeps, and the gray trace is the average). Again, these inward currents closely resembled preminis; here, we call them ASCs (autoR-mediated synaptic currents) because, contrary to preminis, they mainly involve multi-vesicular release. In the cell shown in Fig. 2 A, photolysis was repeated seven times in the same varicosity, located at 54 µm from the center of the soma; the average amplitude of the responses was 26 ± 1 pA; its τrise was 3.1 ± 0.9 ms, its τdecay was 76.0 ± 3.4 ms, and the average latency from the laser pulse was 1.06 ± 0.23 ms. When inspecting the expanded traces, it can be observed that some of the events show multiple peaks (black circles in Fig. 2, B and G); these correspond to multiple vesicular release events, as was already described when the same approach was used to study postsynaptic currents in MLI pairs (Trigo et al., 2012).

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