Antidromic Modulation of a Proprioceptor Sensory Discharge in Crayfish

Michelle Bévengut, François Clarac, and Daniel Cattaert

Centre National de la Recherche Scientifique-Unité Propre de Recherche 9011, Neurobiologie et Mouvements, 13402 Marseille Cedex 20, France

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Bévengut, Michelle, François Clarac, and Daniel Cattaert. Antidromic modulation of a proprioceptor sensory discharge in crayfish. J. Neurophysiol. 78: 1180-1183, 1997. In the proprioceptive neurons of the coxo-basal chortotonal organ, orthodromic spikes convey the sensory information from the cell somata (located peripherally) to the central output terminals. During fictive locomotion, presynaptic depolarizations of these central terminals elicit bursts of antidromic spikes that travel back to the periphery. To determine whether the antidromic spikes modified the orthodromic activity of the sensory neurons, single identified primary afferents of the proprioceptor were recorded intracellularly and stimulated in in vitro preparations of crayfish nervous system. Depolarizing current pulses were delivered in trains whose frequency and duration were controlled to reproduce bursts of antidromic spikes similar to those elicited during fictive locomotion. According to their frequencies, these antidromic bursts reduce or suppress the orthodromic discharges in both position- and movement-sensitive neurons. They induce both a long-lasting silence and a gradual recovery after their occurrences. Neither the collision between the afferent and the efferent messages nor the release of serotonin by the sensory neurons can explain these results. We therefore conclude that antidromic bursts produce a peripheral modulation of the orthodromic activity of the sensory neurons, modifying their sensitivity by mechanisms yet unknown.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

In the central nervous system of both vertebrates and invertebrates, presynaptic inhibition of primary afferents is correlated with primary afferent depolarization (PAD) (Eccles et al 1962, 1963; Jiménez et al. 1988; Kennedy et al. 1974; Kirk and Wine 1984; Sillar and Skorupski 1986). Centrally, PADs reduce the amplitudes of the orthodromic sensory spikes and thereby of the transmitter release they induce, thus reducing the excitatory postsynaptic potentials in the postsynaptic neurons (Cattaert et al. 1992, 1994; Hedwig and Burrows 1996; Kirk 1985; Pearson and Goodman 1981).

In vertebrates, PADs are associated with antidromic spikes recorded from leg primary afferents in dorsal root filaments during both fictive (Dubuc et al. 1985, 1988; Gossard et al. 1989, 1991) and normal walking (Beloozerova and Rossignol 1994, 1995). In crayfish, during fictive locomotion, 90% of the primary afferents of a leg proprioceptor (the coxo-basal chordotonal organ, CBCO) receive phasic bursts of PADs (4-20 mV in amplitude) locked in phase with the locomotor rhythm (El Manira et al 1991b). Moreover, PADs of large amplitudes (15-20 mV) seen in 40% of these afferents are able to trigger antidromic spikes that travel toward the periphery in the sensory axons (El Manira et al 1991b). These spikes have no postsynaptic effect centrally (Cattaert et al. 1994; El Manira et al 1991b) but their peripheral actions are yet unknown. Therefore, we wanted to test whether the antidromic spikes were able to modify the orthodromic activities of the sensory neurons. In crayfish, the possibility of stimulating and recording intracellularly from identified CBCO afferents enables us to investigate such a mechanism.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Experiments were performed in oxygenated physiological saline on 20 in vitro preparations of the nervous system of crayfish, Procambarus clarkii (El Manira et al. 1991a; Sillar and Skorupski 1986). The preparation consisted of the last three thoracic and the first abdominal ganglia of the ventral nerve cord dissected together with all the nerves of the two proximal segments of the left fifth pereiopod (Fig. 1A). The strand of the coxo-basal chordontal organ (CBCO), containing the sensory cell bodies of this proprioceptor, also was dissected intact, and its distal end was attached to an electromagnetic puller (Ling Dynamic systems, VT101) controlled by a home-made function generator.


View larger version (25K):
[in this window]
[in a new window]
 
FIG. 1. A: In vitro preparation comprises last 3 thoracic (T3-5) and first abdominal (A1) ganglia, motor nerves innervating proximal leg muscles (promotor, PRO; remotor, REM; anterior A and posterior P levator, LEV; depressor, DEP), and coxo-basal chordotonal organ (CBCO) and nerve (CBn). Both extracellular (bullet ) and intracellular (CBT, sensory terminal of a CBCO unit) recording sites are labeled. B: during fictive locomotion, spontaneous bursts of primary afferent depolarizations (PADs) producing antidromic spikes are displayed rhythmically in a CBT (1). Simultaneous recordings from CBn and from a CBT show intracellular and extracellular antidromic spikes (bullet ) during these bursts (2). C: simultaneous recordings from CBn and from a CBT are shown during a triggered burst of antidromic spikes (stim in 1) and demonstrates that antidromic (bullet ) and orthodromic spikes are easily distinguished (2). D: superimposed recordings (n = 5) of CBn and a CBT illustrate time relationships between extracellular and intracellular spikes for orthodromic (1) and antidromic spikes (2).

Extracellular activity in the CBCO nerve was recorded using "en passant" platinum wire electrodes (200 µm in diameter) insulated with petroleum jelly (Vaseline). The left fifth ganglion was desheathed to allow intracellular recordings from CBCO sensory terminals (CBT) in the neuropile. Glass microelectrodes, filled with 3 M KCl (resistance = 10-12 MOmega ), were connected to an Axoclamp 2A amplifier (Axon Instruments). Data were recorded on a digital tape recorder (Biologic-1801) and through appropriate interface and software (Cambridge Electronic Device) directly onto a personal computer.

For each CBT, intracellular stimuli were delivered in trains whose frequency and duration were chosen. For each sequence of n trains at given parameters, a peristimulus histogram was calculated (bins of 20 ms) and normalized by dividing the bin values by n. Thus in the results, normalized histograms express the averaged number of occurrences of the orthodromic spikes per bin against time.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

During fictive locomotion, CBTs received spontaneous phasic burst of PADs (Fig. 1B1). These PADs were able to elicit antidromic spikes in the sensory afferents (Fig. 1B2). The instantaneous firing frequency of the antidromic spikes within spontaneous bursts was between 2 and 100 Hz for doublets, with a mean instantaneous firing frequency from 20 to 50 Hz (unpublished data; the mean instantaneous firing frequency in Fig. 1B was 23.34 ± SE 1.9 Hz for n = 111 spikes).

To find out whether the antidromic spikes in a given sensory neuron modified its orthodromic spiking activity, we chose to stimulate single identified primary afferents intracellularly, instead of stimulating the whole nerve because it contains nonsensory serotonergic axons (Rossi-Durand 1992) the activation of which might have altered the specific actions of the antidromic spikes.

Bursts of antidromic spikes in the CBTs (Fig. 1, C and D) were triggered by injecting trains of square pulses of depolarizing current. The amount of injected current and the duration of each pulse were set to trigger only one antidromic spike per current pulse (Fig. 1, C2 and D2). The analysis of the intracellular recording of the CBT traces enabled us to discriminate easily between the orthodromic and the antidromic spikes (respectively Fig. 1C, 1 and 2). Furthermore, the extracellular recordings of the CBCO nerve showed that the triggered antidromic spikes traveled in the nerve up to the sensory neurons of the CBCO even when orthodromic spikes were present (Fig. 1, C and D).

The effects of trains of antidromic spikes on the activity of the different cell types of the CBCO (Mill 1976) were tested for 11 position-sensitive neurons (Fig. 2) and for 10 movement-sensitive ones (Fig. 3). In both figures, under each example of recorded CBTs, a histogram of the averaged number of occurrences of the orthodromic spikes is shown for sequences of trains of stimuli at 10 and 30 Hz. In our experimental conditions, we were able to trigger antidromic spikes in all the recorded CBTs; all of them except two position-sensitive ones showed modifications of their firing activity. For the nine position-sensitive neurons that responded, three results are seen (Fig. 2). First, during stimulation, the number of occurrences of the orthodromic spikes was reduced (Fig. 2A) and then suppressed (Fig. 2B) in the CBTs. Second, the antidromic bursts produced a long-lasting silence of the orthodromic activity (300 ms duration; Fig. 2B). Third, after the cessation of the antidromic bursts, ~820 ms elapsed until the frequency of the orthodromic spikes reached a level similar to the one observed before the stimulation (Fig. 2B).


View larger version (52K):
[in this window]
[in a new window]
 
FIG. 2. Spiking activity of a position-sensitive unit of CBCO (CBT) is shown during a triggered burst of antidromic spikes at 10 Hz (A) and at 30 Hz (B). Histograms represent averaged number of occurrences of orthodromic spikes per bin of 20 ms against elapsed time (n gives number of bursts analyzed).


View larger version (35K):
[in this window]
[in a new window]
 
FIG. 3. Spiking activity of a stretch-sensitive unit of CBCO (CBT) is shown when no electrical stimulation is applied (A) and when a train of antidromic spikes is triggered at 30 Hz (B) before movement of strand (MVT). Histograms as in Fig. 2. Stretch (S) and release (R) of CBCO strand are labeled.

Out of the 10 movement-sensitive neurons, 7 displayed a tonic background activity of orthodromic spikes (Fig. 3) whereas the other 3 did not (not shown). But they all produced phasic bursts of orthodromic spikes in response to either stretch or release movements of the CBCO strand. During trains of antidromic spikes, the number of occurrences of the orthodromic spikes were reduced (not shown) or suppressed (Fig. 3B). In addition, when triggered just before the movements, antidromic bursts drastically reduced not only the phasic burst responses but also the background activity (compare in Fig. 3, A and B).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

In this paper, we demonstrate that bursts of antidromic spikes in the axons of identified primary afferents of the CBCO are able to modify the orthodromic firing of these sensory neurons. According to their frequencies, antidromic bursts reduce or suppress the orthodromic discharges and induce both a long-lasting silence and a gradual recovery after their occurrences.

The reduction or the suppression of the orthodromic activity might be explained by collision between the antidromic and the orthodromic spikes within the axons, as hypothesized in vertebrates by Dubuc et al. (1988). However, two arguments are against this hypothesis. First, we demonstrated that the antidromic spikes reached the CBCO cell bodies located peripherally in the CBCO strand. Second, within the nerve, the conduction times of both types of spikes between the two recording sites range from 2.5 to 17 ms (El Manira et al. 1991a); therefore to fulfill the conditions for collision, the antidromic burst frequency would have to be ~400 Hz for the faster and ~60 Hz for the slower conducting sensory axons. In our experiments, the mean instantaneous firing frequencies of the spontaneous antidromic bursts were between 20 and 50 Hz, and we have shown that triggered antidromic bursts within this range (30Hz) were able to block totally the orthodromic spikes. In consequence, the collision hypothesis cannot explain these modifications of the sensory discharges.

Long-lasting modifications of the firing activity of the CBCO neurons can be produced by serotonin (5HT) (Rossi-Durand 1992). Because this author has suggested that the somata of 75% of the CBCO neurons contain 5HT, one could think that antidromic spikes would release 5HT at the soma sites. So far, in crustacean sensory neurons, colocalized transmitters (such as acetylcholine and 5HT) have been shown to be released only at the axon terminals (Katz et al. 1989). For the CBCO neurons, these terminals are located centrally, thus suggesting that 5HT is unlikely released at the soma locations. Even if this was the case, it should have produced either an increase of the spiking activity of the CBCO neurons or a total inhibition of the phasic bursts in response to imposed movements of the CBCO strand in >= 20% of the afferents (see Rossi-Durand 1992). Such effects were not seen in our experiments. Therefore we conclude that these long-lasting modifications of the orthodromic firing activity are not induced by 5HT.

In conclusion, bursts of antidromic spikes, similar to those produced by phasic bursts of PADs during fictive locomotion, are responsible for the peripheral modulations of the spiking activity of the CBCO neurons. The actions of the antidromic spikes are yet unknown but we may postulate that, when they reach the cell bodies of the sensory neurons, they affect either the mechano-transducing mechanisms or the characteristics of the spike initiating zone.

    FOOTNOTES

  Address reprint requests to: M. Bévengut.

  Received 28 February 1997; accepted in final form 6 May 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society