Shunting versus inactivation: analysis of presynaptic inhibitory mechanisms in primary afferents of the crayfish

Abstract

Primary afferent depolarizations (PADs) are associated with presynaptic inhibition in both vertebrates and invertebrates. In the present study, we have used both anatomical and electrophysiological techniques to analyze the relative importance of shunting mechanisms versus sodium channel inactivation in mediating the decrease of action potential amplitude, and thereby presynaptic inhibition. Experiments were performed in sensory afferents of a stretch receptor in an in vitro preparation of the crayfish. Lucifer yellow intracellular labeling of sensory axons combined with GABA immunohistochemistry revealed close appositions between GABA-immunoreactive (ir) fibers and sensory axons. Most contacts were located on the main axon at the entry zone of the ganglion, close to the first branching point within the ganglion. By comparison, the output synapses of sensory afferents to target neurons were located on distal branches. The location of synaptic inputs mediating spontaneous PADs was also determined electrophysiologically by making dual intracellular recordings from single sensory axons. Inputs generating PADs appear to occur around the first axonal branching point, in agreement with the anatomical data. In this region, small PADs (3-15 mV) produced a marked reduction of action potential amplitude, whereas depolarization of the membrane potential by current injection up to 15 mV had no effect. These results suggest that the decrease of the amplitude of action potentials by single PADs results from a shunting mechanism but does not seem to involve inactivation of sodium channels. Our results also suggest that GABAergic presynaptic inhibition may act as a global control mechanism to block transmission through certain reflex pathways.

Fig. 1.

Experimental setup. A, Thein vitro preparation of the crayfish thoracic locomotor system consisting of ganglia 3–5 (G3, G4, G5) dissected together with motor nerves of the proximal muscles and the coxo-basal chordotonal organ (CBCO). B, Enlargement of the region indicated by the dashed-line box showing a sensory axon terminal and postsynaptic motoneurons in the ganglion.ME1, Proximal electrode; ME2, distal electrode. The terms proximal and distal will always refer to this relative disposition. Distal processes of a sensory terminal are closer to the motoneurons (MN). C, Staining of all CBTs in the left 5th ganglion of crayfish. The localization of the CBTs within the ganglion is represented inC1. Most of the CBTs have the same anatomy, and all of them are more or less superimposed (2). A single CBT on which GABA-ir boutons were found was reconstructed from slices (3). The box represents the zone in which close appositions between sensory axons and GABA-ir fibers were found (Fig. 2A).

Fig. 2.

Localization of GABA-immunoreactive boutons on Lucifer yellow-filled CBCO sensory axons, using confocal microscope analysis of a 30-μm-thick slice. Only projection views are given. A, Global view of the slice showing CBCO sensory axons backfilled with Lucifer yellow (green) and GABA-immunoreactive structures (red). Scale bar, 5 μm.B, Details of GABA-immunoreactive boutons particularly abundant in the vicinity of a CBCO axon (asterisk) stained by Lucifer yellow. Note, however, that GABA-immunoreactive boutons are aligned and follow the CBCO axon. Scale bar, 2 μm.C, Detailed view of five GABA-immunoreactive boutons (arrows) that are in close apposition with the best stained CBCO axon. Scale bar, 2 μm.

Fig. 3.

Localization of zones of synaptic contacts between sensory axons and target motoneurons. A, A monosynaptically connected CBT and an MN were recorded intracellularly and injected with Lucifer yellow. The two neurons were reconstructed in 3D using a laser-scanning confocal microscope. Zone of close appositions were found after the branching point, both on the main neurite (small arrows) and on small branches of the MN (open arrow). Scale bar, 20 μm. B, High magnification of a possible zone of close apposition between a sensory process and the main neurite of the MN. Scale bar, 4 μm.C, High magnification of a zone of close apposition between a branch of the sensory axon and a neurite of the MN. Scale bar, 10 μm.

Fig. 4.

Active propagation of spikes in CBCO sensory axons in the region of the first branching point. A, Schema of the experimental procedure using dual intracellular recordings (ME1, 100 μm before the branching point; ME2, 100 μm after the branching point) from a single sensory axon in the region of the first branching point (arrow). B, Superimposed intracellular recordings from ME1 and ME2 (CBT, bottom trace) and extracellular recordings from the CBCO sensory nerve (CBn, top trace).

Fig. 5.

Comparison of the PAD and spike propagation in passive sites of CBCO axons. A, Diagram of the experimental procedure used. The more proximal microelectrode (ME1) was recorded from a site located just after the first branching point. The more distal microelectrode (ME2) was recorded 150 μm more distally in the main branch. The drawing was reconstructed after Lucifer yellow staining.B, Simultaneous intracellular recordings from ME1 and ME2. C, Enlarged superimposed ME1 (gray trace) and ME2 (black trace) traces during a spike. The recording of the more distal microelectrode displays a smaller spike with an increased time to peak, therefore indicating that the spike was passively propagated between recordings sites of ME1 and ME2. To make these differences more obvious, the mid-amplitude of both recordings have been aligned (dashed line), and the spike recorded with ME1 has been moved slightly to the right to make both spikes cross the mid-amplitude line at the same point.D, Same arrangement as in C but of a PAD instead of a spike. The amplitude of the distally recorded PAD (ME2, black trace) is slightly smaller than the more proximal recording of the same PAD (ME1, gray trace).

Fig. 6.

Time course of the decrease of the amplitude of action potential in relation to PADs. A, The amplitude of afferent action potentials decreases during the occurrence of PADs.B, The maximum decrease in the amplitude of action potentials coincides with the peak of PADs.

Fig. 7.

Comparison of PAD and spike propagation in passive sites of CBCO axons: statistical analysis. The results are analyzed from the experiment shown in Figure 5. A,B, The spike amplitude at the distal recording site is always smaller than the proximal one. A, Graph plotting the amplitude of distal against proximal spike. The lineis the linear regression curve (r = 0.93).B, Histograms of spike amplitudes recorded proximally (from ME1 in Fig. 5; open bars) and distally (from ME2 in Fig. 5; filled bars). C,D, The same analysis as in A andB but for the amplitude of PADs. C, Graph plotting distal against proximal PAD amplitude. The relationship is linear (dashed line represents the regression line;r = 0.84). The continuous linerepresents the equation y = x. The fact that most of the points are below this line indicates that PADs display a smaller amplitude at the distal than at the proximal recording site. D, The distal and proximal PAD amplitude histograms largely overlap, the distal one being slightly displaced to the left. E, Graph of the amplitude ratio of PADs and spikes recorded at two sites in the same sensory axon.

Fig. 8.

PADs reduce spike peaks at sites more proximal than ME1. A, Simultaneous intracellular recordings from ME1 and ME2 (same electrode positions as in Fig. 5). The first spike occurs in the absence of a PAD (1), whereas the second spike occurs in the presence of a PAD (2) and shows a reduced peak. B, Superimposed traces of proximal (ME1) and more distal (ME2) recordings from spike 1 (in the absence of PADs) and spike 2 (in the presence of PADs). In both the absence and presence of PADs, the amplitude of action potentials was reduced to 86% of the initial value because of the passive propagation between ME1 and ME2 recording sites, indicating that PADs did not produce any shunting of action potentials in the sensory axon between ME1 and ME2 recording sites.C, Schematic diagram representing the effect of the location of PADs on the amplitude of action potentials. If PADs occurs close to the first branching point (C1), the amplitude of action potential does not show any further decrease as they propagate from ME1 to ME2. If PADs occur more distal than the branching point, the amplitude of action potentials is decreased further (C2).

Fig. 9.

Effect of PADs on spike peaks at two locations of a CBCO sensory axon. A, Superimposed recordings from a site 100 μm before the first branching point. Positioning of the microelectrode and anatomy of the sensory terminal (reconstructed after Lucifer yellow staining) are shown in the inset. Thethin and thick traces were obtained in the absence and presence of a PAD, respectively. Note that peak values of both recordings are identical. V_m = resting membrane potential. B, Diagram of spike peak value against PAD amplitude. At this recording site, PADs have a very small but positive effect on the spike peak: the larger the PAD, the larger the spike. C, Superimposed recordings from a site 100 μm after the first branching point. Positioning of the microelectrode and anatomy of the sensory terminal (reconstructed after Lucifer yellow staining) are shown in the inset. Thethin and thick traces were again obtained in the absence and presence of a PAD, respectively. Note that the peak value in the presence of the PAD is reduced. D, Diagram of spike peak value against PAD amplitude. At this more distal recording site, PADs reduce spike peak linearly with respect to their amplitude. Recordings in A and C are from different experiments.

Fig. 10.

Shunting and inactivating mechanisms of PADs.A, Intracellular recording from a CBCO sensory axon. Two microelectrodes were placed in the same CBCO sensory axon. One was used to inject current steps (recording not shown), and the other was used to measure the voltage response. B, During the same experiment as in A, spontaneously occurring PADs reduced spike amplitudes. C, Comparison of the effects of spontaneous PADs and current injection on spike amplitude. The peak value is plotted against the membrane potential at the base of the spike. Although 1- to 17-mV-amplitude PADs (responsible for membrane potential at the base of spikes being in the range −77 to −60 mV) induce a noticeable reduction of spike peak, the injection of depolarizing current does not induce any visible reduction of the peak until the membrane potential is increased to −55 mV and above (open symbols). D, Relationship between input resistance (Rin) and the imposed membrane potential during the experiment.