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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.

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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

Laser photolysis of caged Ca2+ in MLI axonal varicosities evokes ASCs. (A) MLI loaded with an intracellular solution containing Alexa Fluor 488 and the Ca2+ cage DM-nitrophen. The image corresponds to a Z projection of a stack of epifluorescence images taken at 1-µm steps. (Inset) Detail of the axon centered on the varicosity where the 405-nm laser was focused. (B) The top part of the panel corresponds to a drawing of the cell; the somatodendritic compartment is shown in magenta, and the axonal compartment is in green. The stimulated site is indicated with the arrowhead. Somatic whole-cell recordings of the laser-evoked ASCs recorded when the varicosity shown in A (inset) was stimulated. Black traces are individual sweeps; the gray trace is the average. Black dots in B and G indicate probable multi-vesicular release events. (C) The specific GABAAR antagonist, Gbz, completely blocked the laser-evoked ASCs. Representative traces of laser-evoked ASCs in control condition, during Gbz, and after 5-min wash. (D) Summary plot of the effect of Gbz (control: 27 ± 2 pA; Gbz: 1 pA ± 1 pA; after wash: 18 ± 8 pA; one-way ANOVA; **, P < 0.01; n = 4 sites). (E) Voltage dependence of ASCs is consistent with GABAAR-mediated currents. Representative traces of ASCs evoked at different holding potentials. (F) The extrapolated ASC reversal potential is not different from the EGABA predicted by the Goldman–Hodgkin–Katz equation. Continuous line, linear fit. Circles represent mean ± SEM. The extrapolated reversal potential is 2 ± 2 mV. (G) Block of Nav channels failed to affect laser-evoked ASCs. Representative traces of laser-evoked ASCs in control condition, during TTX, and after 5-min wash. (H) Summary plot of the effect of TTX on laser-evoked ASCs (control: 39 ± 9; in 1 µM TTX: 36 ± 8 pA; after wash: 44 ± 14 pA; one-way ANOVA; P > 0.05, n = 7 sites). In D and H, the black circles represent mean ± SEM for each condition, and gray dots represent the average amplitude of events evoked in individual release sites. In B, C, E, and G, arrows represent timing of the laser pulse.
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fig2: Laser photolysis of caged Ca2+ in MLI axonal varicosities evokes ASCs. (A) MLI loaded with an intracellular solution containing Alexa Fluor 488 and the Ca2+ cage DM-nitrophen. The image corresponds to a Z projection of a stack of epifluorescence images taken at 1-µm steps. (Inset) Detail of the axon centered on the varicosity where the 405-nm laser was focused. (B) The top part of the panel corresponds to a drawing of the cell; the somatodendritic compartment is shown in magenta, and the axonal compartment is in green. The stimulated site is indicated with the arrowhead. Somatic whole-cell recordings of the laser-evoked ASCs recorded when the varicosity shown in A (inset) was stimulated. Black traces are individual sweeps; the gray trace is the average. Black dots in B and G indicate probable multi-vesicular release events. (C) The specific GABAAR antagonist, Gbz, completely blocked the laser-evoked ASCs. Representative traces of laser-evoked ASCs in control condition, during Gbz, and after 5-min wash. (D) Summary plot of the effect of Gbz (control: 27 ± 2 pA; Gbz: 1 pA ± 1 pA; after wash: 18 ± 8 pA; one-way ANOVA; **, P < 0.01; n = 4 sites). (E) Voltage dependence of ASCs is consistent with GABAAR-mediated currents. Representative traces of ASCs evoked at different holding potentials. (F) The extrapolated ASC reversal potential is not different from the EGABA predicted by the Goldman–Hodgkin–Katz equation. Continuous line, linear fit. Circles represent mean ± SEM. The extrapolated reversal potential is 2 ± 2 mV. (G) Block of Nav channels failed to affect laser-evoked ASCs. Representative traces of laser-evoked ASCs in control condition, during TTX, and after 5-min wash. (H) Summary plot of the effect of TTX on laser-evoked ASCs (control: 39 ± 9; in 1 µM TTX: 36 ± 8 pA; after wash: 44 ± 14 pA; one-way ANOVA; P > 0.05, n = 7 sites). In D and H, the black circles represent mean ± SEM for each condition, and gray dots represent the average amplitude of events evoked in individual release sites. In B, C, E, and G, arrows represent timing of the laser pulse.

Mentions: In young MLIs, axonal varicosities are both the source and the target of GABAergic signaling. This makes the disentangling of the causal sequences of the events following the activation of axonal GABAA receptors particularly difficult. To separate pre- from postsynaptic mechanisms, we combined electrophysiological recordings with laser calcium photolysis in individual varicosities. Uncaging with a minimized laser spot allowed us to induce GABA release with a high spatial resolution and therefore to characterize the signals evoked from a single varicosity. We first designed experiments to assess the feasibility of using laser photolysis of Ca2+ to record autoR-mediated single-site responses in MLIs. To do this, we recorded two putative connected MLIs with a high chloride IS. The IS of the presynaptic cell also contained the calcium cage DM-nitrophen and Alexa Fluor 594; the IS of the postsynaptic cell contained Alexa Fluor 488. The fluorescent dyes were included for neurite identification. Because of the advantageous morphology of juvenile MLIs, which display a planar orientation along the parasagittal plane, direct online inspection under the microscope a few minutes after break-in readily distinguishes dendrites, which are thick and short, from the axon, which is thinner and longer and can be followed up to a few hundred micrometers (Trigo et al., 2012). When a putative synaptic contact (black arrowhead in Fig. 1 A, right) between the presynaptic axon (in green) and the postsynaptic dendrite (in magenta) was localized under LED excitation, we focused the 405-nm laser and photolyzed DM-nitrophen (see Trigo et al., 2012). Photolysis of DM-nitrophen induces a fast and homogeneous calcium transient in the presynaptic varicosity and, subsequently, a fast postsynaptic current (Fig. 1 B, bottom) that can be blocked by GABAARs antagonists (not depicted). As predicted from previous work (Pouzat and Marty, 1999; Trigo et al., 2010), concomitantly to the postsynaptic current we recorded a presynaptic current that has a smaller amplitude and a longer rise time (Fig. 1, B, top, and C), very similar to the spontaneous axonal miniature synaptic currents (preminis) described previously (Trigo et al., 2010). It is unlikely that the evoked currents represent spillover of GABA because previous EM experiments have shown that presynaptic GABAARs are located in the synapse (Trigo et al., 2010). In the pair shown in Fig. 1 (A and B), the latency to the beginning of the presynaptic current is virtually the same as the latency to the postsynaptic current (1.6 ms). The pre- and postsynaptic average latencies in the four recorded pairs are shown in Fig. 2 C (left), the pre- and postsynaptic average τrise are shown in Fig. 1 C (middle), and the pre- and postsynaptic average amplitudes are shown in Fig. 1 C (right).


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

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

Laser photolysis of caged Ca2+ in MLI axonal varicosities evokes ASCs. (A) MLI loaded with an intracellular solution containing Alexa Fluor 488 and the Ca2+ cage DM-nitrophen. The image corresponds to a Z projection of a stack of epifluorescence images taken at 1-µm steps. (Inset) Detail of the axon centered on the varicosity where the 405-nm laser was focused. (B) The top part of the panel corresponds to a drawing of the cell; the somatodendritic compartment is shown in magenta, and the axonal compartment is in green. The stimulated site is indicated with the arrowhead. Somatic whole-cell recordings of the laser-evoked ASCs recorded when the varicosity shown in A (inset) was stimulated. Black traces are individual sweeps; the gray trace is the average. Black dots in B and G indicate probable multi-vesicular release events. (C) The specific GABAAR antagonist, Gbz, completely blocked the laser-evoked ASCs. Representative traces of laser-evoked ASCs in control condition, during Gbz, and after 5-min wash. (D) Summary plot of the effect of Gbz (control: 27 ± 2 pA; Gbz: 1 pA ± 1 pA; after wash: 18 ± 8 pA; one-way ANOVA; **, P < 0.01; n = 4 sites). (E) Voltage dependence of ASCs is consistent with GABAAR-mediated currents. Representative traces of ASCs evoked at different holding potentials. (F) The extrapolated ASC reversal potential is not different from the EGABA predicted by the Goldman–Hodgkin–Katz equation. Continuous line, linear fit. Circles represent mean ± SEM. The extrapolated reversal potential is 2 ± 2 mV. (G) Block of Nav channels failed to affect laser-evoked ASCs. Representative traces of laser-evoked ASCs in control condition, during TTX, and after 5-min wash. (H) Summary plot of the effect of TTX on laser-evoked ASCs (control: 39 ± 9; in 1 µM TTX: 36 ± 8 pA; after wash: 44 ± 14 pA; one-way ANOVA; P > 0.05, n = 7 sites). In D and H, the black circles represent mean ± SEM for each condition, and gray dots represent the average amplitude of events evoked in individual release sites. In B, C, E, and G, arrows represent timing of the laser pulse.
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fig2: Laser photolysis of caged Ca2+ in MLI axonal varicosities evokes ASCs. (A) MLI loaded with an intracellular solution containing Alexa Fluor 488 and the Ca2+ cage DM-nitrophen. The image corresponds to a Z projection of a stack of epifluorescence images taken at 1-µm steps. (Inset) Detail of the axon centered on the varicosity where the 405-nm laser was focused. (B) The top part of the panel corresponds to a drawing of the cell; the somatodendritic compartment is shown in magenta, and the axonal compartment is in green. The stimulated site is indicated with the arrowhead. Somatic whole-cell recordings of the laser-evoked ASCs recorded when the varicosity shown in A (inset) was stimulated. Black traces are individual sweeps; the gray trace is the average. Black dots in B and G indicate probable multi-vesicular release events. (C) The specific GABAAR antagonist, Gbz, completely blocked the laser-evoked ASCs. Representative traces of laser-evoked ASCs in control condition, during Gbz, and after 5-min wash. (D) Summary plot of the effect of Gbz (control: 27 ± 2 pA; Gbz: 1 pA ± 1 pA; after wash: 18 ± 8 pA; one-way ANOVA; **, P < 0.01; n = 4 sites). (E) Voltage dependence of ASCs is consistent with GABAAR-mediated currents. Representative traces of ASCs evoked at different holding potentials. (F) The extrapolated ASC reversal potential is not different from the EGABA predicted by the Goldman–Hodgkin–Katz equation. Continuous line, linear fit. Circles represent mean ± SEM. The extrapolated reversal potential is 2 ± 2 mV. (G) Block of Nav channels failed to affect laser-evoked ASCs. Representative traces of laser-evoked ASCs in control condition, during TTX, and after 5-min wash. (H) Summary plot of the effect of TTX on laser-evoked ASCs (control: 39 ± 9; in 1 µM TTX: 36 ± 8 pA; after wash: 44 ± 14 pA; one-way ANOVA; P > 0.05, n = 7 sites). In D and H, the black circles represent mean ± SEM for each condition, and gray dots represent the average amplitude of events evoked in individual release sites. In B, C, E, and G, arrows represent timing of the laser pulse.
Mentions: In young MLIs, axonal varicosities are both the source and the target of GABAergic signaling. This makes the disentangling of the causal sequences of the events following the activation of axonal GABAA receptors particularly difficult. To separate pre- from postsynaptic mechanisms, we combined electrophysiological recordings with laser calcium photolysis in individual varicosities. Uncaging with a minimized laser spot allowed us to induce GABA release with a high spatial resolution and therefore to characterize the signals evoked from a single varicosity. We first designed experiments to assess the feasibility of using laser photolysis of Ca2+ to record autoR-mediated single-site responses in MLIs. To do this, we recorded two putative connected MLIs with a high chloride IS. The IS of the presynaptic cell also contained the calcium cage DM-nitrophen and Alexa Fluor 594; the IS of the postsynaptic cell contained Alexa Fluor 488. The fluorescent dyes were included for neurite identification. Because of the advantageous morphology of juvenile MLIs, which display a planar orientation along the parasagittal plane, direct online inspection under the microscope a few minutes after break-in readily distinguishes dendrites, which are thick and short, from the axon, which is thinner and longer and can be followed up to a few hundred micrometers (Trigo et al., 2012). When a putative synaptic contact (black arrowhead in Fig. 1 A, right) between the presynaptic axon (in green) and the postsynaptic dendrite (in magenta) was localized under LED excitation, we focused the 405-nm laser and photolyzed DM-nitrophen (see Trigo et al., 2012). Photolysis of DM-nitrophen induces a fast and homogeneous calcium transient in the presynaptic varicosity and, subsequently, a fast postsynaptic current (Fig. 1 B, bottom) that can be blocked by GABAARs antagonists (not depicted). As predicted from previous work (Pouzat and Marty, 1999; Trigo et al., 2010), concomitantly to the postsynaptic current we recorded a presynaptic current that has a smaller amplitude and a longer rise time (Fig. 1, B, top, and C), very similar to the spontaneous axonal miniature synaptic currents (preminis) described previously (Trigo et al., 2010). It is unlikely that the evoked currents represent spillover of GABA because previous EM experiments have shown that presynaptic GABAARs are located in the synapse (Trigo et al., 2010). In the pair shown in Fig. 1 (A and B), the latency to the beginning of the presynaptic current is virtually the same as the latency to the postsynaptic current (1.6 ms). The pre- and postsynaptic average latencies in the four recorded pairs are shown in Fig. 2 C (left), the pre- and postsynaptic average τrise are shown in Fig. 1 C (middle), and the pre- and postsynaptic average amplitudes are shown in Fig. 1 C (right).

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