Crayfish antennal neuropil. II. Periodic bursting elicited by sensory stimulation and extrinsic current in interneurons.

JOURNALOF NEUROPHYSIOLOGY 1978. Printed Vol. 41, No. 5, September

in U.S.A.

Crayfish Antenna1 Neuropil. II. Periodic Bursting Elicited by Sensory Stimulation and Extrinsic Current in Interneurons RAYMON Department

M. GLANTZ of

Biology,

Rice University,

Houston

Texas 77001

neurons and motor neurons. Rhythmic activity has been observed in neurons follow1. Intracellular recordings were obtained ing visual (1,9, 11), auditory ( 19), and mechin vivo from interneurons near the antenna1 anoreceptive (12, 23, 30) stimulation in neuropil of the crayfish brain. vertebrates (1, 19, 23) as well as in in2. Repetitive periodic bursting was elic- vertebrates (9, 11, 12, 30). The temporal ited by proprioceptive, tactile, and visual characteristics of pulse trains (other than stimuli in higher order interneurons. mean rate) have been demonstrated to have 3. The burst period was 400-700 ms. The functional significance at the crustacean burst arose from successively augmenting neuromuscular junction (29) at synapses in compound EPSPs. These EPSPs suggest the central nervous system of Aplysia (25) the recruitment of one or several presynaptic and for command interneuron activity in the neurons during the expression of repetitive crayfish (3, 8, 9). In motor neurons, patbursting. terned activity associated with organized 4. Bursting with a similar period was elic- behavior has been elicited with aperiodic ited by extrinsic current. Variations in out- sensory stimulation (12, 32, 33) and with ward current could produce variations in the regular pulse trains applied to command burst repetition rate over a narrow range. interneurons (5, 30). Sensory stimuli also produce a modest acIn quite a few instances it has been possiceleration of burst repetition rate. ble to demonstrate that the temporal char5. The bursting interneurons are recur- acteristics of a stimulus-elicited, rhythmic rently connected to other neurons. These pulse train are determined by the central connections were revealed by: a) delayed nervous system without reference to peribursts of synaptic potentials following brief odic information from primary afferents (1, current pulses to an impaled neuron, b) cross- 11, 32, 33). A well-documented example of correlation of simultaneously monitored such an analysis is the studies of Wilson cells, c) marked nonlinearities in the cur- (32) and Wilson and Wyman (33) on the orirent-frequency characteristic of these gins of the pattern generator subserving neurons. locust flight motor neurons. Further analysis 6. It is proposed that the periodic activity of the mechanisms of pattern generation has arises from weak intrinsic pacemaker prop- frequently been impeded by the large numerties in the interneurons. The bursting is ber of plausible mechanisms available in the reinforced by recurrent connections in the central nervous system. Thus it is possible brain. that the patterned discharge of an interneuron reflects the pattern of its synaptic INTRODUCTION input (11). Alternatively, the interneuron Temporal structure is a notable feature of may possess an intrinsic pattern-generating the sensory-elicited discharge of many inter- mechanism (2, 4, 13, 27). A crucial distinction is whether a direct manipulation of the rhythmically firing cell influences the phase or period of the pattern-generating process. Received for publication January 24, 1978. SUMMARY

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CONCLUSIONS

0022-3077/78/0000-0000$01.25

Copyright

0 1978 The

American

Physiological

Society

Downloaded from www.physiology.org/journal/jn by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019

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1. Repetitive bursting discharge elicited by a 5-mm oscillation of the ipsilateral antenna1 flagellum. A and lo-Hz stimulus is reduced to 3 Hz and then responses to the first and third stimulus epochs at 9 Hz. C: returned to 10 Hz. D: response of another cell to a continuous brushing stroke of the ipsilateral antenna1 flagellum. E: mild electrical shocks to the antenna1 flagellum elicit single-impulse responses of long, variable latency characteristic of some higher order interneurons. Scales: A -D, 20 mV, 500 ms; E, 20 mV, 10 ms. FIG.

B:

If evidence for such a contribution can be found then, at the very least, the neuron must be a component of the pattern-generating mechanism. In tonic interneurons of the crayfish supraesophageal ganglion mechanical and visual stimuli and extrinsic outward current elicit periodic repetitive bursting. It is proposed that some of the interneurons possess an endogenous pacemaker property and that recurrent connections among the interneurons contribute to the repetitive bursting. METHODS

The dissection, stimulus, and recording procedures used in this study were virtually identical to those in the companion report (10). The only exception is that the large antenna1 muscles at the base of the anntennae were dennervated. This was done to avoid the possibility of local resistance reflexes during proprioceptive stimulation of the basal antenna1 joint. It is important to note that the experiments were carried out in vivo, in restrained but intact crayfish. Only the innervation of the green glands, mouth parts, and basal antenna1 muscles were disturbed.

The physiologic condition of the preparation was routinely checked throughout all of the experiments. A particularly useful indicator was the response of the large axons of interneurons in the circumesophageal connectives to stimulation of the antenna1 flagellum. The multiunit responses demonstrated in the companion article (10) are quite vigorous in a fresh preparation and completely absent in a pathologic animal. The pathologic state is also indicated by the loss of the eye-withdrawal reflex and the loss of neuronal responses to visual stimuli. The results presented in this report were based on studies of 61 impaled interneurons in 47 different preparations; 51 of these cells in 40 different preparations exhibited repetitive bursting with sensory stimulation. RESULTS

Repetitive bursting elicited by sensory stimulation A common feature of the interneuronal response to sensory stimulation is repetitive bursting. The bursting can be elicited with both transient and steady-state stimuli and it often exhibits strong periodicity . The periodic bursting is observed in response to vi-


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0

1.2

2.4s

FIG. 2. to a 45” deflection of Repetitive burst discharge to proprioceptive stimuli. A 1: response of interneuron the basal antenna1 joint. A,: response to a slow continuous passive antenna1 movement. B,: spontaneous bursts and small oscillations in membrane potential. B,: repetitive bursting during a mild excited state. Filled circles beneath each trace indicate 670-ms intervals. B,: autocorrelogram of low-level spontaneous activity based on a 5min sample with mean rate of 1.3 pulses/s C1, C,: faster sweep and higher gain traces of bursts elicited with stimuli as in part A. C3: autocorrelogram of bursting responses elicited as in part A,. Scales: A,, AS, B1, 20 mV, 500 ms; Bz, 40 mV, 500 ms; C1, 20 mV, 200 ms; Cz, 10 mV, 50 ms.

bration, constant pressure, joint displacement, or with constant velocity movements of a joint. Figure 1 consists of responses of a tonic vibration-sensitive higher order interneuron. The stimulus was a small (0.5 mm) horizontal oscillation of the antenna1 flagellum at about 9 Hz. Two notable features of the response are the very long latency (0.8 s) and the repetitive periodic bursting, Fig. 1A. The burst period was about 500 ms. Virtually every higher order tonic neuron, examined particularly if it had input from the antennae, exhibited delayed repetitive bursting on antennal stimulation. The periodicity was not always as pronounced as that in Fig. lA, but there was a strong tendency for the burst periods to vary between 400 and 700 ms. With repeated stimuli the bursting response may habituate or become aperiodic, Fig. 1B. In vibration-sensitive cells the repetitive bursting is usually elicited over a restricted range of stimulus frequencies, Fig. 1C. Similar results have been obtained with proprioceptive, Figs. 2,7A, tactile, Fig. lD,

and visual stimuli, Fig. 11. Repetitive bursting has also been observed following transient stimuli such as single electrical and/or mechanical shocks to the antenna1 flagellum, Figs. 6A, 7B. A variety of plausible mechanisms have been demonstrated or proposed which can generate repetitive bursting in neurons. The mechanisms include intrinsically oscillatory neurons (2, 4, 13, 17, 18, 21, 27) as well as interactions among synaptically (6, 20, 24) or electrotonically (7, 3 1) coupled cells. The data of Figs. 2-4 are derived from the same cell and they illustrate some of the similarities and differences of the periodic discharge elicited by sensory stimulation and by extrinsic current. The most effective stimulus to this neuron was a passive deflection of the basal joint of the ipsilateral antenna. Figure 2A, displays the response to a constant antenna1 displacement. Figure 2A, is the response to a slow constant-velocity movement. Similar responses but with fewer spikes were obtained by stimulation of the basal joint of


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j 82 I

1

1 2.4

FIG. 3. a 2.0-nA inwardRepetitive burst discharge elicited with extrinsic current. A : bursting following current pulse and during a 2.0-nA outward-current pulse. Large voltage deflections during current pulses in parts A, spontaneous bursts and responses elicited with B, and D are due to imbalance of bridge circuit. B,, C1, and D: outward-current pulses of 5.0, 10.0, and 12.7 nA, respectively. B,: autocorrelogram of steady-state activity elicited with 5.0-nA current pulses, 20 ms/bin. C,: autocorrelogram of discharge elicited with lo-nA outward current, 34 ms/bin. E: membrane potential fluctuations immediately following an outward-current pulse. F: a burst of spikes elicited with 5-nA outward current. Scale: A, 20 mV, 1.0 s; B1, 20 mV, 0.5 s; C1, D, 10 mV, 500 ms; E, 5 mV, 100 ms; F, 10 mV, 20 ms.

the contralateral antenna and with tactile stimulation to the underside of the abdomen. The response began with a hyperpolarization suggestive of a compound IPSP. Several modest and successively augmenting oscillations in membrane potential preceded the appearance of repetitive bursting. Autocorrelograms, Fig. 2C3, were compiled from several data samples such as that in Fig. 2A1. The correlograms indicated a burst period of 600 ms associated with sensory stimulation. Close inspection of the bursts elicited by sensory stimuli, Fig. 2C1, CZ, reveal large compound EPSPs associated with each burst of impulses. There are no indications of IPSPs during the repolarizing phase of the burst cycle, Fig. 2C,. In the unstimulated condition the cell would occasionally exhibit weak spon-

taneous bursts, Fig. 2A1, &, with a period of 0.7% 1.7 s. Figure 2B3 is an autocorrelogram based on a LO-min sample of spontaneous activity. The autocorrelogram exhibits peaks at 720 and 1,700 ms, which estimate the most frequent periods of the spontaneous bursts. “Excited states” result in a slight membrane depolarization (2- 3 mV), enhanced spontaneous activity, Fig. 2B,, and epochs of repetitive bursting. The filled circles beneath the traces of Fig. 2B, are at 670-ms intervals. Repetitive bursting elicited by extrinsic current

The neuron shown in Fig. 2 also exhibited strong repetitive bursting following release from a pulse of inward current and on iniection of outward current, Fig. 3A. For


R. M. GLANTZ LO-

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SPIKES/CYCLE FIG.

interval grouped

4. Burst period as a function of the number of spikes per oscillatory cycle. Period was measured from the first spike of the burst to the first spike of the succeeding burst. Lines connect means data. Discharge was elicited by antenna1 displacement in A and lo-nA outward current in B.

most of the cells ex amine d the bursting observed on release from hype rpolarization was periodic, Figs. 2A, 7C,. After prolonged or intense hyperpolarization, however, long sequences of aperiodic bursts were observed, Fig. 6C. If the cell was subjected to a step of outward current (e.g., 5 nA) during an epoch of weak spontaneous bursting the bursting was enhanced and the burst period diminished by the depolarization, Fig. 3B,. . Virtually identical di scharge patterns were elicited with steps of outward current while the neuron was silent and the membrane potential flat, Fig. 3C,. With modest levels of outward current, e.g., 5- 10 nA, steadystate bursting could be maintained for lo20 s before the cell began to “adapt. ” The spikes thus elicited, exhibited an exaggerated postspike hyperpolarization and a distinct inflection on the rising phase, Fig. 3F. An autocorrelogram, Fig . 3% w.as calculated from several samples such as that in Fig. 3B,. The correlogram indicated a period of 600 ms. Thus depolarization diminishes the burst period from a minimum resting value of 720 to 600 ms. More intense outward currents, Fig. 3C1, resulted in a greater number of spikes per burst and a more tightly, organized burst structure, but the burst period actually exhibited a slight increase (to 640 ms) relative to the response to 5.0-nA outward current. Fifty-one neurons were observed to exhibit repetitive bursting with appropriate

as the of the

steady-state or transient sensory stimuli; 15 of these neurons were subjected to systematically varied steps of extrinsic current. All 15 exhibited bursting on release from hyperpolarization; 8 of the 15 also exhibited repetitive bursting during steps of outward current. The burst periods varied from 1.2 to 0.4 s and the periods diminished by lo30% as the intensity of the outward current increased over a narrow range. Above this narrow range of current intensit y the bursting bet ame aperiodic, Fig. 30, or the burst period increased with increasing outward current. With both sensory stimulation and extrinsic current the burst period covaries with the number of spikes per burst, Fig. 4A, B. Similar findings have been reported for pacemaker cells in the crustacean cardiac ganglion (4) the Aplysia abdominal ganglion (27), and the snail parietal ganglion (13). These results suggest that d.epolarization drives two antagonistic proce sses with respect to the determination of burst period. The depolarization, per se, resul ts in a modest acceleration of the pacemaker while the additional spikes generated by the outward current tend to decelerate the pacemaker. The consequence of this antagonism is to restrict the variations of oscillatory period to within a narrow range. Following either a sensory stimulus or a prolonged pulse of outward current, the impulse activity of the cell is transiently depressed. During this period one can detect


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A

FIG. 5. Repetitive response and subthreshold membrane potential oscillations elicited with outward-current steps of 0.5 nA in A and 1.0 nA in B and C. The seven traces in B and C are successive, 5-s samples from continuous discharge. About 3.0-5.0 s of data was deleted between samples. Scale: 10 mV, 500 ms.

small membrane oscillations with a period similar to the immediately preceding burst discharge, Fig. 3E. The depolarizing phase of these oscillations is accompanied by small subthreshold depolarizing events suggestive of remote synaptic activity. Although oscillations of this amplitude are occasionally seen in the resting cell, they are much more sporadic and with a period of about 1.5 s, Fig. 2B,. Pacemaker potentials and period of repetitive bursts About half of the cells which exhibited repetitive bursting on sensory stimulation responded to extrinsic outward current with a regular discharge, Figs. 6, 8. Inspection of the regular discharge occasionally revealed spike deletions from the train. These deletions invariably unmasked a pacemaker potential with a period in the 400- to 700-ms range, Fig. 5A, C. Similarly, spontaneously active pacemakers (4,‘14, 15,23) also exhibit spike deletions that unmask pacemaker potentials. When the discharge is accelerated by extrinsic outward current, Fig. 6B1, the pace-

a

maker potential also exhibits the expected acceleration. If driven beyond a certain rate, however, spike generation is blocked, Fig. 6Bz. The response of this neuron to a sensory stimulus (such as a single electrical shock to the antenna1 flagellum) was a series of successively augmenting compound EPSPs, Fig. 6A, which gave rise to successively stronger bursts of impulses. This pattern of EPSP augmentation is suggestive of recruitment and or facilitation of periodically firing presynaptic neurons. The bursting was followed by an accelerated pacemaker discharge which could last for up to 20 s. A similar result has been reported for pacemaker neurons in the crayfish abdominal ganglia (15). The period of the burst pattern was closely correlated to the period of the immediately preceding spontaneous discharge, Fig. 6A1, AZ, A,. Since the period of the pacemaker discharge is under the control of the cell’s membrane potential, the result would suggest that the pacemaker activity contributes to the timing of the burst pattern. Hyperpolarization of the cell however blocks the pacemaker activity with-


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FIG. 6. Responses of a spontaneous pacemaker to single electrical shocks of the antenna1 flagellum (A, D) and to steps of extrinsic current (B, C). In A, the pacemaker discharge was blocked by 2-nA inward current. Thickened trace is due to noise associated with current passage through the recording electrode. Bi, B,: responses elicited with OS- and l.O-nA outward-current steps, respectively. C: posthyperpolarization discharge following a 10.0-s lo-nA inward-current pulse. D: compound IPSPs elicited with shocks to the antenna1 flagellum. Scale: Al, 10 mV, 1.0 s; A,, Al, B1, BB, 10 mV, 500 ms; A,+ 5 mV, 500 ms; C, 10 mV, 1.0 s; D, 10 mV, 5 ms.

out significant effect on the sensory-elicited response. This result permits at least two interpretations: a) the neuron is a follower of an oscillatory process that is entirely

presynaptic to the neuron (the correlation of pacemaker rate and burst repetition rate must then be considered coincidental), b) the neuron is a member of a recurrently


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*ccc

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*3

.

c c2

FIG. 7. Synaptic potentials, and repetitive bursting elicited by a steady deflection ipsilateral basal antenna1 joint, AZ. A3 is an expanded view of a burst in part A,. mechanical shock to the cephalic carapace. Stimulus time indicated by arrow. BB, B,: response elicited with a lOO-ms, 5-nA lOO-ms, lo-nA outward-current pulse. B,: C,: posthyperpolarization responses following 5.0-nA inward-current pulses. Scales: 10 mV, 100 ms; B1, 10 mV, 200 ms; BB, 10 mV, 500 ms; B3, B4, 10 mV, 100 ms; C,

coupled population of cells with similar characteristics. Removing the pacemaker contribution of one cell in a coupled population would not necessarily modify the population output. Evidence consistent with the presence of recurrent connections among first-order interneurons of the antenna1 neuropil has been presented in the companion report (10). Furthermore, it has been shown that first-order cells can burst in near synchrony in response to sensory stimulation and steps of extrinsic current. Figures 7-9 present additional data consistent with the presence of recurrent connections among repetitively bursting interneurons. Repetitive recurrent

bursting excitation

and

As noted previously multimodal interneurons exhibit periodic bursting in response to a variety of sensory stimuli, but may respond to steps of extrinsic outward current with a more regular discharge, Fig. 8. Figure 7A, displays the response to an im-

of the telson, Al, and the response to a strong B,: responses elicited with a outward-current pulse. C1, Al, AZ, 20 mV, 500 ms; As, 10 mV, 500 ms.

posed dorsal flexion of the telson and 7A2, the response to a passive flexion of the ipsilateral basal antenna1 joint. The highfrequency burst of EPSPs associated with sensory stimulation is shown at higher gain and sweep speed in Fig. 7A3. Repetitive bursting was also generated with single mechanical shocks to the cephalic carapace, Fig. 7B1. Brief (100 ms) pulses of outward current elicit a high-frequency burst of impulses in this interneuron, Fig. 7B,. Furthermore, repetitive bursts of EPSPs appear as a delayed response to the current pulse. The bursts are initiated at 350-400 ms after onset of the current pulse and the succeeding EPSP bursts exhibit a similar period, Fig. 7B3, B4. The results are suggestive of recurrent synaptic activity. A similar suggestion is obtained by examining the posthyperpolarization rebound. This response frequently consists of several bursts at a period of about 600 ms, Fig. 7C,. If the impulses fail to occur during this period, Fig. 7CZ, one observes subthreshold EPSPs with


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a9 .

0

5 CURRENT

IOnA

FIG. 8. Nonlinear current-frequency characteristic of interneuron described in Fig. 7. Outward-current intensity is stated in nanoamperes at the left of each trace. Discharge frequency as a function of outward current plotted at lower right. Filled circles indicate reciprocal of first interspike interval, open circles indicate reciprocal interspike interval observed 1.0 s after onset of current step. Scales: left column, 20 mV, 500 ms; right column, mV, 10 ms.

amplitude and time course similar to the events that follow depolarizing pulses. An additional observation that is consistent with the argument of recurrent excitation is the markedly nonlinear currentfrequency relationship, Fig. 8, lower right. Evidence for weak recurrent connections among repetitively bursting interneurons was also obtained by analysis of simultaneously recorded extracellular impulse activity. Figure 9A displays the activity of a large neuron and several smaller units observed in the circumesophageal connective.

is of 10

The response was elicited with a constant antenna1 deflection, which commenced at the onset of the trace. The large neuron exhibited strong repetitive bursting with a period of about 650 ms, Fig. 9C. One of the smaller neurons also exhibited bursting, but it was largely aperiodic, Fig. 9D. The crosscorrelation between the cells, Fig. 9B, indicates a relatively strong monosynaptic connection from the large to the small cell and a much weaker recurrent connection. The conditional probability of a small spike occurring 3 ms after a large spike was 0.19.


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B

I I luti -100

II Y I I II 0

I IOOms

C

FIG. 9. Correlation of simultaneously monitored bursting cells. A: repetitive burst discharge of several neurons recorded extracellularly at the origin of the circumesophageal connective near the brain. Scale: 1 mV, 500 ms. Response elicited with steady deflection of the ipsilateral antenna1 flagellum. B: cross-correlogram of large -+ small neurons in part A, 1 ms/bin. C, D: autocorrelograms of large and small neurons, respectively, 10 mslbin.

Inhibition

in bursting

interneurons

An additional feature of interneurons that exhibit repetitive bursting is stimuluselicited inhibition. This is most clearly seen in the oscillograph traces initiated with electrical shocks to the antenna1 flagellum, Figs. 60, 10. The short (2-5 ms) constant latency indicates that the inhibition is derived from primary afferent input. In many instances the compound IPSP produces a modest depolarization of the resting cell membrane, Fig. lOA, lower trace. If the cell is subjected to subthreshold outward current, however, the PSP inverts indicating a reversal potential below the spike threshold. The time course of the compound IPSP is similar to the time course of the discharge of descending interneurons, Fig. 1OB. A similar result is obtained in neurons inhibited by pulses of illumination, Fig. 11A. Furthermore, delayed poststimulus bursts in connective axons, Fig. lOC, D are also associated

with a compound

IPSP in these same inter-

neurons* The frequent association of stimulus-elicited inhibition and repetitive bursting, Figs. 2, 6, 11, suggests that the two phenomena may be related. This is further supported by the marked tendency of virtually all tonic interneurons to exhibit repetitive bursting on rebound from extrinsic inward current, Figs. 3, 6, 7. These results raise the possibility that reciprocal inhibition may play a role in burst generation (20, 24). Although occasional antiphasic bursting among pairs of interneurons has been noted, more thorough analysis consistently yields unconvincing results. Summary Of results Sensory stimulation elicits delayed periodic bursting in higher order interneuTons. The initial poststimulus response is a series of successively augmenting bursts of compound EPSPs. The burst period was 400-


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FIG. 10. IPSPs and correlated connective discharge elicited with electrical shocks (1.0 ms duration) to the ipsilateral antenna1 flagellum. A: lower trace, IPSP at membrane resting potential; upper trace, inverted response polarity associated with a 3.0-nA outward-current bias. B-D: upper traces are intracellularly recorded IPSPs, lower traces are simultaneous responses of axons in the ipsilateral circumesophageal connective. Scales: A, 2.5 mV, 20 ms; B, 2.5 mV, 10 ms; C, D, 2.5 mV, 100 ms.

A

B

FIG. 11. IPSPs and postinhibitory bursting elicited by pulses of illumination (downward deflection in lower trace) to the ipsilateral eye. Middle trace in part A is recording from the ipsilateral circumesophageal connective. Scales: A, 10 mV, 200 ms; B, 10 mV, 500 ms.


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700 ms. The bursting discharge is labile. In excitatory connections or pacemaker aceach cell a variety of stimulus procedures tivity or both. It is proposed that the period applied to different receptive fields can elicit of repetitive bursting elicited by sensory qualitatively similar responses but of vary- stimuli is determined by the temporal charing impulse frequency. The same applies to acteristics of the weak intrinsic pacemakers the discharges elicited by the excited state. that are endogenous to these neurons. The The bursting may be preceded by a com- bursting itself is attributed to the recurrent pound IPSP with a time course similar to that connections. This model is consistent with of the stimulus elicited, descending volley all of the data presented in this and the in the ipsilateral connective. In about half companion report. A qualitative examinaof the neurons tested extrinsic outward cur- tion of the model vis-a-vis the more salient rent elicits repetitive bursting with a period observations is presented below. similar to that observed with sensory stimuAn important finding is that the overall patlation. The period of the burst discharge tern of the discharge and the period of the elicited by outward current is slightly shorter burst are relatively independent of the locus than the spontaneous period, but may not or magnitude of the sensory stimulus and/or vary systematically with variations in out- the level of extrinsic current. These results ward current. On terminating a prolonged follow from the assumption that the burstpulse of outward current subthreshold ing always arises from recurrent activation oscillations in membrane voltage are ob- of pacemaker neurons, many of which are served. The oscillation period is similar to only indirectly influenced by the stimulus. the immediately preceding burst period. These pacemakers will tend to fire at their The oscillations are associated with small natural period and thus constrain the burst subthreshold events suggestive of synaptic period to within a narrow range. The various activity. Similar oscillations and EPSPs methods of activation merely provide suffican be observed following depolarizing cient depolarization to release a discharge pulses of 100 ms duration. pattern intrinsic to the population. The Repetitive bursting with a period of about periodic bursting following a pulse of inward 500 ms is also elicited following hyper- current results from pacemaker activation polarization. If the impulses fail during the during the initial rebound. The rebound rebound period, subthreshold EPSPs may spikes activate other members of the popube observed. lation which, in turn, reactivate the stimuA few neurons exhibit a regular discharge lated cell. The augmentation of successive either spontaneously or on depolarization. compound EPSPs following sensory stimulaThe discharge is timed by a pacemaker po- tion is a reflection of recruitment and or tential that is revealed by occasional spike synaptic facilitation within the pacemaker deletions. The period of the pacemaker po- population. tential can be controlled with extrinsic curThe proposed recurrent connections can rent. When these cells burst in response to also explain the prolonged responses followsensory stimuli, the burst repetition rate ing single electrical and mechanical shocks is correlated to the rate of the immediately to the antenna1 flagellum and the observed preceding regular train. nonlinearities in the current-frequency reThe current-frequency relationship of lation. these cells is markedly nonlinear. Alternative mechanisms of periodic burstCross-correlation of two simultaneously ing that have been proposed in the past monitored bursting pulse trains indicates are not consistent with our observations. asymmetric recurrent connections simi- The bursting neurons do not exhibit the lar to those previously found among first- essential properties of intrinsically oscillaorder interneurons. Evidence for antiphasic tory bursting neurons as observed in Crusbursting and/or reciprocal inhibitory con- tacea (17;. 18, 26, 28) and mollusks (2, 13, nections was sought but not obtained. 16, 21, 27). Particularly notable in this regard are the absence of large subthreshold oscillations in membrane potential and the DISCUSSION narrow range of burst repetition rates in the Virtually all of the neurons examined in discharge elicited with sensory stimuli and/ or extrinsic current. this study exhibited evidence for recurrent


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Reciprocal inhibition (20, 24) and serial inhibition (6) coupled with postinhibitory rebound have also been proposed as a basis of sustained oscillations. The interneurons of the crayfish brain exhibit strong inhibition on sensory stimulation and the inhibition is correlated with the activity of other interneurons. During repetitive bursting, however, there are no unambiguous indications of IPSPs during the silent intervals. Furthermore, in nearly 50 studies involving recordings from bursting cells in the brain and simultaneous recordings from bursting neurons in the connectives we have not observed a single case of clear antiphasic bursting. On the contrary, the bursting of simultaneously monitored cells has always been nearly synchronous. Further analysis either by correlating impulses in one cell to PSPs in another (10) or by correlations of spike trains between neurons indicate only excitatory connections among descending elements. A model of burst formation based on electrotonic synapses (7, 31) comes somewhat closer to meeting the conditions observed in the crayfish brain. In the trigger group neurons of Tritonia ganglia (7, 31) bursting is produced by mutual excitation across nonrectifying electrotonic junctions among 30 neurons. The bursts are terminated by postspike hyperpolarizations, which are generated nearly simultaneously by the synchronously firing neurons at the end of the burst. The specifics of this model however are inappropriate to the present data on two counts: a) the successive intraburst intervals do not exhibit the expected accelerando pattern, and b) the postspike hyperpolarizations are of brief duration (50-100 ms) compared to the burst period (400-700 ms). The function of the repetitive bursting is unknown, but two possibilities come to mind. The first follows from the fact that at least half of the neurons in the sampled

population are command cells for motor activity in the thoracic appendages (unpublished observations). The repetitive bursting may, therefore, contribute to the timing of motor activity in the chelipeds or walking legs. This speculation is clearly at odds with the current view that motor rhythms are generated in the segmental ganglion containing the motoneurons. In this view sensory input and command cells merely provide the trigger or steady bias to activate the local pattern generators (5, 30, 32, 33). It should be pointed out, however, that the experiments which provide the evidence for this view demonstrate that local oscillators are present and that intersegmental oscillators are not necessary (for an important exception see Ref. 6). If the oscillatory mechanisms are distributed, however, it is possible that intersegmental oscillators may play an important role. Furthermore, in systems of distributed oscillators it is not surprising to find that a system can continue to exhibit a periodic function after the removal of one source of periodic information. A second possible role for the periodic activity arises from the fact that in several systems repetitive bursting is a more efficient means of transmitting information across synapses than continuous impulse trains of the same mean rate (3,8,9,25, 29). In studies of decapod command neurons (3, 8, 9), however, it has not been determined whether the temporal properties of behaviorally effective pulse trains coincide with the temporal properties of pulse trains elicited by physiological stimuli. ACKNOWLEDGMENTS

I thank Drs. Arnold Eskin and Daniel Johnston for their helpful comments during the progress of these studies. I thank Howard Wood and Kathy Tuggle for their efforts in establishing the software for the Interdata 7/16. This work is supported by National Science Foundation Grant BNS-76-80507.

REFERENCES 1. ADRIAN, E. D. AND MATHEWS, R. The action of light on the eye. Part III. The interaction of retinal neurons. J. Physiol. London 65: 273-298, 1928. 2. ARVANITAKI, A. AND CHALAZONITIS, N. Electrical properties and temporal organization in oscillatory interneurons. In: Symposium on Neurobiology

of Invertebrates, edited by J. Salanki. New York: Plenum, 1967, p. 169-199. 3. ATWOOD, M. C. AND WIERSMA, C. A. GI Command interneurons in the crayfish central nervous system. J. Exptl. Biol. 46: 249-261, 1967. 4. BULLOCK, T. AND TERZUOLO, C. A. Activity of neurons in crustacean heart ganglion. J. Physiol. London 138: 341-364, 1957.


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O., POON, M., AND STENT, G. S. An oscillatory neuronal circuit generating a locomotor rhythm. Proc. Natl. Acad. Sci. U.S. 73:

3734-3738, 7. GETTING,

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P. A. AND WILLOWS, A. 0. D. Modification of neuron properties by electrotonic synapses. II. Burst formation by electrotonic synapses.

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Relaxation after a voltage step of inhibitory synaptic current elicited by nerve stimulation (crayfish neuromuscular junction).

In vivo imaging of calcium accumulation in fly interneurons as elicited by visual motion stimulation.

Periodic behavioral changes during hippocampal theta rhythm elicited by septal stimulation in rats.

Habituation of mechanoreceptive interneurons in the crayfish.

ATP-sensitive K+ channels mediate an IPSP in dorsal horn neurones elicited by sensory stimulation.

Physical and physiological properties of the crayfish antennal flagellum.

Sensory antennal organization in an ant and a wasp.

Integration in the crayfish antennal neuropile: topographic representation and multiple-channel coding of mechanoreceptive submodalities.

Patterned sensory nerve stimulation enhances the reactivity of spinal Ia inhibitory interneurons.

Gating current "fractionation" in crayfish giant axons.

Cardiovascular responses elicited by electrical brain stimulation in the rabbit.

Diverse thalamocortical short-term plasticity elicited by ongoing stimulation.

Enhancement of electrically elicited startle by amygdaloid stimulation.

Intercellular junctions of antennal gland epithelial cells in the crayfish, Orconectes virilis. A freeze-fracture study.

Intrusive Thoughts Elicited by Direct Electrical Stimulation during Stereo-Electroencephalography.

An antennal-derived mechanosensory pathway in the cockroach: descending interneurons as a substrate for evasive behavior.

Sensory stimulation by time-varying magnetic fields.

Mechanisms Underlying Population Response Dynamics in Inhibitory Interneurons of the Drosophila Antennal Lobe.

Modeling Inhibitory Interneurons in Efficient Sensory Coding Models.

Sensory inhibition of flexion producing interneurons in lobster abdomen.

Potential distribution in three-dimensional periodic myocardium--Part II: Application to extracellular stimulation.

Periodic current oscillation catalyzed by δ-MnO2 nanosheets.

Temporal Prediction in lieu of Periodic Stimulation.

The effect of sensory feedback on crayfish posture and locomotion: II. Neuromechanical simulation of closing the loop.