Crayfish Escape Behavior and Central Synapses. I. Neural Circuit Exciting Lateral Giant Fiber

ROBERT S. ZUCKER Department of Biological Sciences and Program in the Neurological Sciences, Staraford IJnkersity, Stanford, California 94305

CRAYFISH ARE SOUGHT as food by , birds, havior thus habituates to infrequently amphibia, and mammals, as well as man repeatecl stimuli. However, since prolonged (54, p. 460). They commonly escape these stimulation of the lateral giant at frequen- predators by darting backward and away cies up to 1 Hz results in the continued from an enemy on contact, or as the preda- appearance of tail flicks (38, 41), the re- tor approaches. This maneuver is accom- sponse lability must be located in the plished by a sudden flexion of the tail, pathways afferent to the giant- fiber, or in exerting a thrust backward and sometimes that neuron itself. upward against the aquatic medium. A sin- In 1960, Kao (25) recorded slow depolar- gle tail flexion, commonly called a tail flip izing potentials in the lateral giant axon to or flick, often suffices; a more prolonged shocks clelivered to afferent roots. The po- mode of escape, swimming, consists of a tentials could elicit spikes, and were there- periodic sequence of abdominal flexions fore excitatory postsynaptic potentials and extensions. (EPSPs). Krasne (40) showed that on repeti- In 1947, Wiersma (67) showed that stimu- tion of a these EPSPs waned with lation of any single lateral or medial giant a time course similar to behavioral habitu- fiber of the nerve cord lecl to a ra&l ab- ation. This discovery represented an impor- clominal flexion. Recently it has been shown tant neural correlate for an adaptive be- that the giant fibers excite all of the fast havior, and it seemed that to the extent flexor motoneurons of the abdomen which that the properties of the EPSP could be innervate the phasic flexor muscles (37, 38, explained, the behavioral lability coulcl be 61). It has been assumed from this evidence explained in physiological terms. that the giant fibers are the sole command This paper describes the results of an or decision fibers responsible for eliciting investigation of the pathways responsible escape behavior. Schrameck (60) has shown, for the activation of the lateral giant. The however, that many tail flicks-including seconcl paper (73) locates and analyzes the most of those in swimming-are accompa- points in the circuit responsible for the nied by giant fiber spikes in intact, un- behavioral habituation. The final paper restrainecl, chronically implanted . (74) describes the mechanism of activation Nevertheless, recent experiments by Wine of the fast flexor motoneurons by the lateral and Krasne (71) show that a phasic mechan- giant fiber. ical stimulus to the tail leads to tail flicks that are always mediatecl by lateral giant METHODS fiber activity. For this particular input, Procambarus clarkii were used for all experi- therefore, the lateral giant is a critical deci- ments. Male and female specimens about 8 cm sion fiber for the behavior. Krasne has also long were obtained from H. A. Dahl Co., found that repetition of the stimulus as Berkeley, Calif., and maintained in aerated tap- slowly as once per minute reveals a lability water tanks at room temperature until use. in the response, which fails to follow an in- Before dissection the animals were immobil- creasing proportion of the stimuli. The be- ized by cooling briefly in ice. A strip of dorsal carapace and hypodermis about 5 mm wide was Received for publication August 30, 1971. removed from the abdominal segments 1 600 R. S. ZUCKER through 6, and the underlying intestine was through the recording electrode, and this pro- excised. The fast flexor muscles were separated vision was frequently employed to stimulate cells along the midline, and the thorax and head or control their level of polarization (15). Stimu- were removed and discarded. The abdomen was lus currents were isolated from ground with a pinned ventral side up in a Lucite dish contain- W-P Instruments (Hamden, Conn.) photoelectric ing two ledges of wax, and filled with cold isolator. The only ground introduced into the oxygenated van Harreveld’s solution (66), with recording system was by a lead to the bath. Ex- the bicarbonate buffer replaced with Trisma at tracellular stimuli were obtained from Tek- PH = 7.2. Mirrors placed in the groove be- tronix pulse-generating equipment, and isolated tween the wax ledges and under the abdomen with transformers. reflected a focused light beam either directly or Procedures for locating and impaling desired obliquely through the abdomen. The ventral elements, minimizing the utilization time of nerve cord was exposed by removing the over- stimulation, and calculating the arrival time lying ribs, cuticle, and hypodermis. The remain- of spikes in presynaptic elements are described ing cross connections between the flexor muscles, in RESULTS. particularly the transverse muscles (55), were cut and trimmed, and the two sides of the RESULTS abdomen were separated slightly so that the Lateral giant response to aflewn t nerve cord was well illuminated by the light volleys: component analysis reflected from below. The ventral blood vessel was removed from the nerve cord, and the When an electrical stimulus is applied to connective tissue sheath was peeled off the Z/3, the first or second root of an abdominal 4/5, and 5/6 connectives, from which single ganglion, a depolarizing potential may %e axons were dissected with fine needles and recorded from the ipsilateral lateral giant forceps for stimulation and recording. Oblique neuron in the segment stimulated. This re- transmit ted light, directed nearly along the sponse is best seen in penetrations of the nerve cord axis, was found best for visualizing giant axon just anterior to a ganglion, ros- and separating single axons. For intracellular tral to the septal synapse with the next pos- studies, the third or fourth ganglion was de- sheathed and perineural tissue washed away terior lateral giant axon and the point with a jet of saline (51). Cell bodies and other where the major dendrite arches medially ganglionic landmarks are seen most easily in and ventrally into the ganglionic neuropil. direct transmitted light. In experiments where Such a recording is illustrated in Fig. 1. A the giant fibers might be stimulated, all third shock was delivered to the second root of the roots and sixth ganglion roots containing phasic fourth ganglion; the ipsilateral lateral giant motoneurons (45) were cut to prevent twitching of the flexor muscles. The temperature of the bath was maintained at 18 C by a Peltier cool- ing unit placed under the preparation chamber. Stimulation and recording from single axons, roots, or the nerve cord was accomplished by the use of micromanipulated suction electrodes (529, connected to a switch box which allowed either of two stimulators to be connected to any elec- trode, or any electrode could be switched to one of four Tektronix a-c preamplifiers. All signals were displayed on a conventional oscilloscope and photographed. Intracellular recordings were made with micropipettes pulled to fine tips less than 0.5 p in diameter and filled with 3 hf KCl. P--=- 1Oms The resistance was between 10 and 40 megohms. The desired elements were most easily pene- I trated by approaching the ganglion obliquely i in the vertical plane including the nerve cord. FIG. 1. Response recorded intracellularly (upper trace) from the lateral giant axon in the fourth Signals were recorded using a Bak capacitance- ganglion to stimulation of the second root of the compensated high-impedance d-c preamplifier same segment. The lower trace is an extracellular (Electronics for Life Sciences, Rockville, Md.), recording from the ipsilateral ventral nerve cortl, used differentially. This unit is provided with a 4/5 connective. The arrow marks the peak of the Wheatstone bridge circuit for passing current early component of the response. INPUT TO CRAYFISH LATERAL GIANT FIBER axon was penetrated just anterior to the ing to the antidromic invasion of the den- fourth ganglion, and t he ventral ner bve cord drite by the evoked impulse. Probing in monitored on the ipsilateral 4/5 connective. this region, the electrode was positioned so This lateral giant depolarization is the com- as to maximize this potential; whereupon a pound excitatory postsynaptic potential light tap occasionally resulted in a success- (EPSP) reported by Kao (25) and Krasne ful penetration of the dendrite of the giant (40). The inflection on the rising phase cor- fiber (cf. ref 8). Two electrodes were placed responds to the peak of the first component on the relevant afferent root (usually the of the response, designated the a-compo- second), one for stimulation and one for nent (40). The peak and shoulder of the recording. To observe the effects of con- response correspond to Krasne’s late p- and trolled activity in identified y-components. on the lateral giant, the was The maximal compound EPSPs recorded teased free from the nerve cord at least one from the lateral giant axon in over 10 ex- segment away from the ganglion of pene- periments were quite small, always under tration for stimulation, and its activity was 5 mv and frequently less than 2 mv. In order monitored by the electrode on the nerve to analyze unitary components of the re- cord. sponse, penetrations of the lateral giant This procedure yielded recordings of sub- dendrite were made close to the problable threshold potentials in giant fibers in over synaptic sites, using the reconstructions 40 specimens. The EPSPs were of much made from dye injections by Remler et al. greater magnitude than those recorded (58) and Selverston and Kennedy (61). The from the axon or proximal dendrite. Figure procedure used (see Fig. 2) involved the fol- 3 illustrates several features of typical re- lowing steps: The lateral giant axon was sponses to repetitive electrical stimulation isolated for stimulation by teasing it free of the second root. First, the a-component from the 5/6 or l/2 connective, and its reliably maintains its initial amplitude at response was morn tored by an electrode 10 Hz. The late components are quite labile placed on a connective next to the ganglion at this frequency, and decline rapidly to being studied. While repetitively stimulat- less than half their original amplitude (40). ing the giant fiber at 2 Hz, the microelec- Finally, since the compound EPSPs failed trode was lowered into ganglionic neuropil to evolve a spike, the threshold of the lat- near the ventral ipsilateral branch of the eral giant cell is extraordinarily high as seen lateral giant dendrite at its lateral bifurca- from the dendrite, exceeding 50 mv in this tion, until a region was found bearing a case. This contrasts with the 7- to 8-mv small triphasic focal potential correspond- threshold measured from axonal recordings

FIG. 2. Typical layout of electrodes and nerve cord used for these experiments. The nerve cord is left connected by its roots to the abdomen (not shown). Roman numerals refer to abdominal segments; rl is the first root, etc. See METHODS and the text for description of dissection, equipment, and procedures. GO2 R. S. ZUCKER

FIG. 3. Lateral giant dendrite responses in the fifth ganglion to repetitive stimulation of the second root of the same segment at 10 Hz. The lst, 2nd, 5th, lOth, and 30th responses are shown from left to right. The initial downward deflection is the stimulus artifact. The first fast depolarization is the a-componcn t of the response; the slower, later wave is the p-component. A separate y-component is not presen t in these responses.

(40). Part of this difference may be due which the giant was penetrated; however, to damage inflected by the dissection, but responses in one ganglion could be seen to most of it is attributable to the difference stimulation of any abdominal segment. De- expected in recording from one or the other polarization in lateral giant fibers could be of the two spatially distinct regions: the elicited by brushing the hairs or by air bub- synaptic zone and the spike-initiating site bles blown from a pipette and allowed to (see DISCUSSION). strike the pleural plates of the same and Increasing the intensity of single shocks next posterior segment to that recorded (the delivered to afferent roots results in graded second root field). The responses to such augmentation of both early and late compo- stimuli (Fig. 5A) resembled in their shape nents (Fig. 4). This effect of recruiting ad- and amplitude the potentials evoked by ditional sensory axons indicates that there second root shocks. Occasionally, in freshly are a variety of presynaptic elements that dissected preparations, such stimuli elicited excite the lateral giant and contribute to escape responses. Finally, in several animals both phases of the response. Very weak it was possible to stimulate single hairs stimuli can elicit either an a- or B-compo- naturally and observe their unitary EPSPs nent in isolation, testifying to their inde- in the lateral giant. One such experiment is pendence. Before exploring the detailed shown in Fig. 5B. composition of these components, it is neces- Stimulation of the cutaneous pressure re- sary to answer the more general question: ceptors of the ventral soft cuticle (52), the which afferent svstems contribute to the sensory setae of the swimmerets (7), or the pathways exciting the lateral giant neuron? proprioceptors in the appendages (1, 7, 69) sometimes evoked a few depolarizing de- Sensory modalities exciting flections in the lateral giant. Stimulation of laic9-al giant these receptors invariably involved local To identify the sensory pathways activat- water currents or sl ight vibra tions, howe ver, ing the giant cell, an electrode was placed and it is likelv tha t these ex cited some tac- in the dendrite and receptors from each tile hairs, which in turn caused the re- sensory modality were stimulated in turn sponses observed. The ventral nerve cord by their adequate stimuli while monitoring stretch receptors (24, 34) can be activated the sensory response in the appropriate af- by extending the telson; this operation had ferent roots. The carapace tactile receptors no effect on the giant fiber. In five prepara- (47) were found to provide very strong exci- tions, the pleural plates were trimmed off, tation to the lateral giant in over 20 experi- the ribs cracked at their lateral junctions ments. The largest responses were elicited with the carapace, and the half dorsal cara- by natural stimuli to the ipsilateral first or pace of all segments rotated around a longi- second root fields of the ganglion (69) in tudinal axis through the swimmeret coxa so INPUT TO CRAYFISH LATERAL GIANT FIBER 603

A ’ 12mv IOms 4-4

- -6 10ms

FIG. 5. Lateral giant dcndri te responses to tactile stimulation of hairs on the ipsilateral pleural plate of the same and next posterior segments. As stimuli consisted of air bubbles striking the carapace. Upper trace is intracellular recording from the fourth ganglion; lower trace is monitor of afferent activity in the second root to the same ganglion. A, and A., are the first and third of a series of stimuli delis: wed at 0.5 Hz. The late part of the EPSP declines on rcpctition of the stimulus. B: from a different . Unitary EPSP in the lateral giant dendrite (upper trace) caused by discharges in a single tactile FIG. 4. Lateral giant dendrite responses to in- receptor on the ipsilateral fourth pleural plate. The creasing numbers (A to C) of affercnt f&r-s re- afferent spike is recorded in the second root (middle cruited by increasing the intensity of a shock trace) and in the nerve cord, z/3 connective. The delivered to the second root. Stimulation and re- receptor was excited by gentle water currents. cording from the fourth segment. Upper trace is intracellular record; lower trace is a monitor of the affercnt volley recorded by a proximal root electrode. (11, 13) of the same and next posterior segments were exposed (see ref 10 for dissec- that both the ventral surface of the Nero tion) and stretched with hooks; no response cord and the dorsum of the animal were was observed in the lateral giant, even facing upward. The muscle receptor organs when other controlled by these re- 604 R. S. ZUCKER ceptors were obtained (14). In the same tactile hair population, at least in the third experiments, the dorsal nerve branch con- and fourth abdominal segments. Similar taining the stretch receptor afferents was conclusions arise from the recent behavioral stimulated electrically; or else the branch experiments of Wine and Krasne (71) and to the phasic extensor muscles was stimu- Larimer et al. (46), who also show that lated. In both cases a second root monitor there is no input to this system from the provided evidence that all axons present in head or most of the thorax. Thus, stimula- the stimulated branch were responding; in tion of an afferent root is approximately neither case was a response seen in the lat- functionally equivalent to delivering a natu- eral giant. This experiment eliminates the ral phasic tactile stimulus to the crayfish possibility of recurrent activation of the carapace or the medium surrounding it. giant fiber on stimulation of extensor moto- neurons. Finally, the caudal photoreceptor Properties of excitatory synapses onto apparently does not excite the lateral giant, lateral giant since it would be firing regularly under the EARLY COMPONENTS. The first part of the conditions of these experiments (3 1) and lateral giant response to afferent stimulation would have led to the appearance of peri- consists of monosynaptic electrically in- odic EPSPs in the giant, which were never duced PSPs from tactile afferent fibers. Fig- observed. It may be concluded, then, that ure 6 illustrates the effects of these direct the only significant sensory pathway acti- connections. In A, the first hump in the vating the lateral giant escape system is the lateral giant response to weak electrical

FIG. 6. Properties of the early component of the lateral giant response. Upper trace is from the lateral giant dendrite in the third (A) or fourth (B and C) ganglion; lower trace is a monitor on the ipsilatcral second root to the same ganglion (A and B) or a record of nerve cord activity in the 2/3 connective (C). A: stimulation of the second root excites two affcrcnts (only one is recorded by the root monitor) which elicit two EPSPs in the lateral giant. B: in another animal, the first afferent stimulated in the second root elicits a unitary electrical EPSP in the lateral giant. B, is a single response; B,, shows superimposed re- sponses to stimuli delivered at 10 Hz. Triangles on the upper traces point to tie calculated arrival time of the presynaptic afferent spike (see text). In C, from a different preparation, the late response to maximal second root stimulation (CJ is blocked at a repetition rate of 200 Hz, leaving the early com- ponent undiminished (C,). INPUT TO CRAYFISH LATERAL GIANT FIBER 605 second root stimulation always corres- were calculated and found to be identical in ponded to the presence of the first aff erent both experiments. impulse recorded in the root monitor. (The The brief postsynaptic potential associ- second hump of the response is due to a ated with the afferent impulse in Fig. 6A fiber in the root whose spike height was too begins at the moment the presynaptic spike small to be recorded through the root moni- arrives at the region of the microelectrode tor at the gain used.) The triangle points to penetration. Thus, the synaptic delay is es- the calculated arrival time of the afferent sentially zero. Figure GB, shows another spike seen below. This instant was calcu- afferent EPSP, with no apparent synaptic lated by measuring the distances between delay; it consists of a rapid potential fol- the two root electrodes and the microelec- lowing each spike in an afferent fiber, ex- trode (see Fig. Z), measuring the conduction cited by stimulating the second root. In velocity between the stimulating and re- Fig. 6B,, the responses to successive stimuli cording root electrodes and, assuming this to at 10 Hz are photographically superimposed be constant (see below), extrapolating the to show that the response is uniform in arrival time in the ganglion. latency, amplitude, and duration. These This technique is subject to two sources of responses represent unitary elements of the error. I) The interval between stimulation and a-component to a maximal root shock shown passage of the afferent spike past the root moni- in Fig. 6C. When shocks are delivered at tor includes an unmeasured utilization time for 200 Hz, the late components drop out en- stimulation. Normally, this utilization time is tirely, leaving the a-component unchanged. minimized by using very brief, intense stimuli. These properties of the members of the a- However, this procedure cannot be used here component were observed in over 20 prep- because such a stimulus would recruit many arations: one-to-one correspondence with other afferent spikes in the root. Instead, a just suprathreshold, long stimulus was employed, single aff erent impulses, all-or-none and and the latency was measured from the end of constant shape, brief duration, very short the stimulus pulse. If different pulses were latency, and high-frequency following, lead chosen for stimulation, with longer and weaker together to the conclusion that the a-com- shocks, the interval increased by increments cor- ponent is the sum of unitary, monosynaptic, responding to the increments in stimulus dura- electrically induced EPSPs between tactile ation. Furthermore, if a long shock adjusted as afferents and the lateral giant. described above were greatly increased in It is known (69) that at least some of the strength, the latency was shortened by the dura- tactile afferents entering a ganglion send tion of the stimulus. Thus with the procedure branches that ascend or descend in the employed, the utilization time corresponds to the stimulus duration, and it is eliminated from nerve cord to the next ganglion. It is there- the calculations by measuring latencies from fore frequently possible to record an af- the end of the shock. ferent volley in the root before it enters the 2) The assumption of constant afferent veloc- ganglion and in the cord just caudal to the ity might be incorrect. In particular, it seems ganglion, and to obtain a more accurate possible that the central afferent processes are estimate of arrival time of the vollev in the thinner than the peripheral axon. This would ganglion. Such an experiment is illustrated cause the afferent impulse to arrive later than in Fig. 7, and shows that the a-component shown in the figures. In consequence, this error of the response to a maximal root shock would cause measurements of synaptic delay, the begins at the moment of arrival of the pre- latency between the calculated arrival time of synaptic spikes in the ganglion. the presynaptic impulse and the foot of the post- synaptic potential, to be overestimated. How- LATE COMPONENTS. The late components of ever, there is evidence that this error is negligi- the lateral giant response to afferent activ- ble. On two occasions, a tactile afferent terminal ity are very different in nature from the was penetrated in a ganglion near to an inter- neuron onto which it synapsed. Stimulating the early component. Krasne (40) showed that sensory axon through a distal root electrode, the (3- and y-components are very variable while recording an impulse from a proximal in shape, wane on repetition of the stimu- root electrode as well as the central process, the lus, are of longer latency than the a-compo- central and peripheral conduction velocities nent, and seem to consist largely of shifting 606 R. S. ZUCKER

2ms

FIG. 8. Cross section of the ventral nerve cord in the 3/4 connective; 10-u section, embedded in FIG. 7. Absence of synaptic delay of early compo- paraffin, stained with Masson’s trichrome. Dorsal nent of lateral giant EPSP, shown by interpolating surface is up. MG: medial giant; LG: lateral giant; the arrival time of the afferent volley in the gan- A, B, and C: identified tactile interneurons (see glion (triangle in the figure) between its arrival text). (Photograph courtesy of D. Kennedy.) times in the root monitor (middle trace) and the nerve cord in the 4/5 connective (bottom trace). Upper trace is from the lateral giant dendrite in ron with a receptive field of tactile hairs on the fourth ganglion, whose second root was stimu- the ipsilateral second through fourth seg- lated. ments. Interneuron C (A64) is a multiseg- mental tactile interneuron responsive to unitary transient components which drop tactile stimulation of the entire abdomen. out with repetitive stimulation as the p- In thirteen experiments, one of the above and y-components decline (see Figs. 1, 3-5, interneurons was dissected free from the 6C, and 7). Since these properties suggested nerve cord with fine needles and its influ- that the late components might be poly- ence on the lateral giant was tested by stim- synaptic, it was decided to investigate the ulating it while recording from the giant possibility that interneurons excite the lat- neuron. Interneurons A, B, and C, as well eral giant. as others, did produce all-or-none, brief de- Since the only pathways exciting the lat- polarizing potentials in the giant. Such a eral giant cell are tactile ones, interneurons potential is seen as the second deflection in exciting the giant, if they exist, must be the first trace of Fig. 9. abdominal tactile interneurons. A variety Before proceeding with a detailed analy- of such interneurons have been described sis of these connections, it seemed impor- in crayfish (68, 69). Single neurons can be tant to establish that such potentials were identified and characterized by their recep- involved in the late response of the giant tive fields, locations, sizes, and response cell to peripheral stimulation. In Fig. 9, properties, and the same fibers can be lo- shocks were delivered to the second root cated again and again in different animals, and to interneuron C, and the interval be- always having the same distinct and unique tween the shocks was varied to observe the set of properties. Three such neurons which interaction between the two lateral giant were studied extensively in these experi- responses. When a peak in the S-component ments are indicated in the cross section of of the root response preceded the stimulus the ventral nerve cord shown in Fig. 8. In- to the interneuron by 3 msec or less, the re- terneuron A (A6 in ref 69) is a unisegmental sponse to the interneuron spike was blocked tactile interneuron which has recently been (third and fourth traces). Furthermore, studied extensively by Kennedy (33). Its re- when the interneuron spike preceded the ceptive field is the dorsal surface of the ip- usual time of the peak of the P-component silateral telson and uropods. Interneuron B by 3 msec or less, the peak failed to appear. (A63) is a multisegmental tactile interneu- This result, obtained in several animals, in- INPUT TO CRAYFISH LATERAL GIANT FIBER 607

portant pathway of activation of the lateral giant in response to peripheral stimulation. In Fig. lOA, interneuron A has been iso- lated on the middle trace, and its activity monitored there and in the cord monitor. Natural stimulation in its receptive field (brushing the telson) elicits spikes in the in- terneuron which are always followed by large depolarizing potentials in the lateral giant. These synaptic potentials can be seen in the third, fourth, and fifth, but not the sixth ganglion. So connections between in- terneuron A and the giant fiber are repeated in most of the abdominal segments. In Fig. lOB, the same experiment has been re- -P-- peated with interneuron C. Connections be- tween interneuron C and the lateral giant also occur in several ganglia. In Fig. IO& it is evident that a large part of the late re- sponse of the giant cell to second root stim- ulation consists of EPSPs generated by repetitive activity in interneuron C. In an- other preparation (Fig. lOC), it is shown that interneuron C contributes to both the p- and y-components of the lateral giant re- sponse. Indeed, the very existence of sepa- rable p- and y-components depends on the double-burst nature of firing in many tac- tile interneurons. Frequently, a phasic stim- ulus elicits spikes in these interneurons which cluster into two bursts and then the late component of the lateral giant response has two separate phases. When such cluster- ing of interneuron firing does not occur, a separate y-component is not discernible. These results have been obtained in 15 animals for cells A, 13, and C, as well as ++ other identified tactile interneurons. They indicate that there exists a fairly large pop- FIG. 9. Interaction betavecn lateral giant re- sponses to second root ~ollcys and spikes in tactile ulation of tactile interneurons with differ- interncuron C. The response to second root stimu- ent and often overlapping receptive fields lation is prcccdcd by a downward-going artifact; the which excite the lateral giant in each ab- interneuron response is preceded by an upward-go- dominal segment 2 through 5. ing artifact. Recordings are from the lateral giant dendrite in the fourth ganglion. Stimuli were ap- Since these identified interneurons con- plied to the ipsilateral second root to that ganglion, tribute so strongly to the normal activation and to the intcrncuron axon in the 4/5 connective. of the lateral giant by natural stimulation, it is relevant to explore the properties of (Kcates that occlusion has OccurI-et1 and that their synapses. Figure 11 illustrates the re- interneuron C is therefore part of a path- sults of a study of the synapse from inter- way shared by\ both direct stimuli and root neuron A. Electrical stimulation of the stimuli, where the lateral giant is the final isolated interneuron led to a large rapid de- common element of the pathway (62). polarization (8 mv) in the giant cell. After Figure 10 presents more direct evidence withdrawing the microelectrode and in- l-hat tactile intf3-neuroIls constitute an im- creasing the gain IO-fold, a focal potential 608 R. S. ZUCKER

FIG. 10. Identification of tactile intcrneurons exciting the lateral giant. Records are from four dif- ferent animals. Upper trace is microelectrode recording from the lateral giant dendrite in the fourth (A and B) or third (C) ganglion. Middle trace (in A and B) records activity in an isolated interncuron axon from the 5/6 (A and B1) or 4/5 (I$) connective. Bottom trace is a nerve cord monitor on the 3/4 (A and B) or 2/3 (C) connective. A: spikes in tactile interneuron A, elicited by natural stimulation in its receptive field, generate EPSPs in the giant neuron. B, shows the same result when interneuron C is activated tactilely. In 13, and C, second root shocks in the segment of penetration elicit activity in interneuron C which contributes to the b- and y-components of the lateral giant response. (Extracellular spikes re- touched.) representing the passage of the presynaptic tion reduced the peaks of the response by spike in the interneuron was recorded on blocking some of the unitary components. stimulating the interneuron. This gives a This result is the opposite from that found very precise measure of the arrival time of in chemically mediated synapses (9), but the presynaptic spike in the ganglion (cf. may occur in electrical synapses if the hy- ref 27), and it may be seen that there is no perpolarization spreads to the presynaptic measurable synaptic delay to the foot of the elements and blocks spikes in some of them EPSP. This synaptic transmission is also (2). Finally, it is significant that it has been stable for many minutes at stimulation rates impossible to demonstrate any change in up to 400 Hz, a property seen in no chem- the input resistance of the lateral giant ically transmitting synapse. dendrite during any portion of the response Figure 12 illustrates properties of the to peripheral stimulation (see ref 73). svnax>se between interneuron C and the lat- To summarize, convergent monosynaptic ekal &ant. In Fig. 1%4, the all-or-none, brief and polysynaptic electrical excitation onto EPSP following stimulation of the interneu- the lateral giant summates to form a large ron appears in the giant dendrite with no compound response to peripheral stimula- delay following the calculated arrival time tion. When this potential exceeds threshold of the presynaptic impulse. It also follows (Fig. 14), a spike is evoked and the animal stimulation at 400 Hz; many superimposed produces an . The escape be- responses in Fig. 12B show that the shape havior is as all-or-none as the impulse in is constant and that there is no latency jit- the giant fiber (46, 67, 71), which thus con- ter. By the same criteria applied to- the stitutes a “decision fiber” for this behav- members of the a-component, it is con- ioral response to phasic abdominal tactile cluded from over 10 similar experiments stimulation. The orthodromic spike riding that these tactile interneurons electrically on the compound EPSP in the lateral giant excite the lateral giant in each of several ab- (Fig. 14A) attains about the same peak volt- dominal ganglia. age as an antidromic impulse recorded in Figure 13 presents another result consis- the lateral giant dendrite (Fig. 14B). tent with the notion that the early and late Activation of tactile intemwurons responses of the lateral giant consist of elec- trical EPSPs. It was often observed that de- DIRECT AFFERENT EXCITATION. Since IllOSt Of polarizing the lateral giant membrane by the excitation of the lateral giant is poly- passing current outward through the re- synaptic and mediated by tactile interneu- cording microelectrode had little effect on rons, the activation of these cells was the response, while strong hyperpolariza- studied in 25 experiments in order to com- INPUT TO CRAYFISH LATERAL GIANT FIBER 609 - - I2mv

I 20mv

ISmv

FIG. 11. Properties of the EPSP in the lateral gi- Y ant from interneuron A. A: response recorded from the giant cell dendrite in the fourth ganglion (mid- FIG. 12. Properties of the EPSP in the lateral dle trace) to stimulation of the interneuron axon giant from interneuron C. Responses recorded from in the 5/6 connective. The microelectrode was with- the giant dendrite (upper trace) in the fourth (A) drawn and the gain increased in the top trace, or third (B) ganglion to stimulation of the inter- which records the field potential generated by a neuron axon in the 4/5 connective. The intcr- spike in interneuron A passing the site of the neuron activity in the 3/4 (A) or 2/3 (B) connec- microelcctrode. This gives a measure of the pre- tive is shown on the bottom trace. The response to synaptic spike arrival time, and also the membrane high frequency (400 Hz) stimulation (AJ is the potential of the lateral giant dendrite (97 mv). I?: in same as the response to single presynaptic spikes the same preparation, the EPSP follows with no (A,). B: superimposed responses to stimuli repeated decrement long trains of presynaptic stimulation at at 10 Hz. The triangle marks the calculated arrival 400 Hz. In A and B, the interneuron activity is time of the presynaptic spike at the junction. monitored on the bottom tract from the 3/4 con- nective. A B C plete the circuit of the lateral giant-medi- I lOM ated escape response. The integrative properties, activity patterns, input organi- zation, and discharge mechanisms of tactile interneurons as a class have been studied extensively (35, 36, 56, 63), but these prop- - erties have not been examined in individ- Smr ually identified units, except for the recent experiments on interneuron A (33). In the PIG. 13. Effects of polarizing currents on the lateral giant dendrite response. The upper trace following experiments, tactile interneurons records the current which was passed continuously were studied as single units, to determine through the recording electrode; 0 marks the cur- which of the many properties of the class of rent base line. The bottom trace is the intracellular all interneurons they share. recording from the fourth ganglion, whose second root was stimulated. Depolarization (B) has little Tactile inter-neurons were studied in much effect on the control response (A); hyperpolarization the same manner ;ks the lateral @lt neuron. (C) blocks some of the unitary electrical EPSPs. 610 R. S. ZUCKER

neurons in general in crayfish. They have broad receptive fields and widely graded EPSPs re- flecting a high convergence of primary afferent input. Spikes do not reset the synaptic poten- tials, so multiple firings from single EPSPs are common. Variable spike shapes are only rarely seen, indicating the presence of branch spikes and multiple spike-initiating zones within a single ganglion. Many interneurons receive in- puts from afferents entering in each of several segments and, furthermore, a volley delivered to one segment may lead to a few spikes which arise from an adjacent ganglion as well as the main burst arising from the segment stimulated. Interneurons B and C are not spontaneously active, nor are the other tactile interneurons which excite the lateral giant.

5ms The typical mode of activation of multi- segmental tactile interneurons is by mono- synaptic, chemical, antifacilitating excita- tion from primary tactile afferents. Figure 15 illustrates a very common finding. In Fig. 154 a weak second root shock excited tactile afferents which had only a small ef- fect on interneuron C. Increasing the stim- ulus intensity slightly recruited a tactile FIG. 14. Suprathrcshold orthodromic activation afferent recorded in the root monitor which of the lateral giant. In A, the upper trace records elicited one-for-one an EPSP in the inter- the intracellular potential in the dendrite in the neuron. At very low stimulus repetition whose second root was stimulated. third ganglion, rates (less than I/min), this response was The lateral giant axon was monitored in the 2/3 connective on the middle trace, and the cord ac- all-or-none. The figure shows the first, tivity was recorded from the 516 connective in the fourth, and seventh responses to stimuli ap- bottom trace. B: antidromic spike recorded from plied once per 2 sec. The EPSP waned to the same dendritic locus as in A. one-third its initial value with repetition of the stimulus, but the shape of the response Some of them have been in-jetted with fluores- remained basically unchanged (within cent dye (33, 75) and the general location of noise-level fluctuations and contamination their dendritic trees is known. Usually, an by other small PSPs). The arrival time of interneuron was isolated from the 4/5 connec- the afferent spike was calculated as dis- tive of the nerve cord, identified by its size, cussed above. The synaptic delay was con- position, receptive field, and response proper- ties, and penetrated in the third and fourth stant at about 1.5 msec for repetitive ganglion, using its focal potential during anti- stimulation at low frequency. The potential dromic impulse generation as a guide. The in- thus appears to be a unitary, chemically me- terneuron was also monitored in the 2/3 diated, and strongly antifacilitating EPSP connective. Judging from the large spike height from a tactile afferent onto interneuron C. and large synaptic potentials, penetrations of Most of the input to multisegmental tac- the smaller multisegmental interneurons were tile interneurons consists of such EPSPs. in the main axon fairly near the synaptic zone. They can be recruited sequentially by in- Dendri tic penetrations are extremely unlikely, creasing the stimulus intensity (Fig. 16), and because the dendritic trees of these cells consist of a tuft of fine processes (<5 CL) branching form a large compound EPSP which reaches ventromedially off the main axon at one point threshold a few millivolts below the appar- in the caudal third of the ganglion. ent resting potential. Further evidence for These multisegmental tactile interneurons the chemical nature of this excitation (12) share many of the properties reported for inter- is provided in Figs. 17 and 19, which show INPUT TO CRAYFISH LATERA4L GIANT FIBER 611

-*- 2ms A 2ms B 1 Ml 2mv 4 B -~ A b

C ,-- -A

FIG. 16. Unitary EPSPs generated by tactile af- B 3 I ferents in interneuron C. The upper trace of each pair records in tracellularly from an interneuron -- process in the third ganglion; the lower trace moni- tors the afferent volley on the ipsilateral second root to the ganglion. An electrical shock delivered dis- tally to the second root excites a single sensory -L neuron in A, whose spike in the root monitor and FIG. lli. Properties of monosynaptic afferent arrival time in the ganglion are indicated by tri- EPSPs in tactile intcrneuron C. A: weak stimulation angles. The afferent impulse elicits a delayed EPSP of the second root excites fibers (monitored in the in the interneuron. In I3 and C, the stimulus in- second root on the lower trace) which elicit small tensity is increased by small increments, recruiting depolarizing potcn t ials in the in terneuron (upper additional afferents indicated on the monitor, each trace, recorded in the fourth ganglion). B: stronger generating an EPSP in interneuron C after a delay shocks recruit a tactile afferent, whose spike is of about 1.5 msec. marked by a triangle, which elicits a large EPSP in the interneuron. The upper triangle indicates the calculated arrival time of the afferent spike. B,, B,, nature of the central synapses discussed here, and I?, are the first, fourth, and seventh responses the effects of altering the ionic media were ex- to stimulation at 0.5 Hz. The small late depolariza- plored in over 10 experiments. tions are due to occasional activation of tactile af- It has not been possible to maintain a den- ferents whose spikes are not discernible in the root dritic penetration in an interneuron while monitor. changing the bath, so it was necessary to observe either the late response of the lateral giant to that the compound EPSP can be enhanced root stimulation or the firing rate in interneu- by hyperpolarizing currents and reduced by rons as indirect measures of the effects of ions on the interneuron EPSP. Raising the Mg++ depolarization. concentration to 11 times normal and/or re- The susceptibility of synapses to alterations ducing the Ca++ concentration to one-third, and in the concentration of divalent cations has been maintaining osmolarity at normal, had no effect used as a criterion for distinguishing chemical on the strength of the suprathreshold response from electrical synaptic transmission (50). In of multisegmental tactile interneurons to root an attempt to obtain further evidence of the stimuli, or to their rate of decline to repetitive 612 R. S. ZUCKER

C;i++ deprivation can uncouple electrical syn- A apses (53), which diminishes the usefulness of this procedure for distinguishing chemical from electrical synapses. Solutions of high Ca++ (3 times normal) were ASO tested for an effect on interueuron responses to peripheral stimulation. If high Cat+ raises the thresholds of the interneurons (16, 72), it might be expected to reduce the late components of the lateral giant response selectively (see ref 42, 43). However, the effects of Ca++ were too fre- quently irreversible and, furthermore, Ca++ in- creased the thresholds of the afferent fibers being m- stimulated as well as affecting the interneurons, making the results meaningless. 20ms Not all of the afferent chemical input to tactile interneurons antifacilitates. For ex- ample, some of the input to interneuron A facilitates at moderate frequencies (33). But most of the EPSPs seen in the multisegmen- C- tal tactile interneurons do antifacilitate to 1Ona I a small fraction of their initial amplitude at low frequencies (73).

LESS COMMON FORMS OF INPUT TO TACTILE INTERNEURONS. Although most of the input 10mv I to the multisegmental tactile interneurons 1 I consists of monosynaptic chemical exci ta- tion from afferents synapsing in the same ganglion that they enter, there are other less common forms of activation. For exam- ple, maximal afferent stimuli often elicit a small early deflection in the interneuron FIG. 17. Effects of postsynaptic polarization on EPSP showing no synaptic delay, which ap- affercnt EPSPs in interneuron C. Upper trace: cur- rent passed through recording electrode. Lower parently corresponds to a small electrical trace: response of interneuron in third ganglion to component of excitation from some of the second root stimulation of the same segment; bridge afferents. In addition, stimuli applied to an balanced to compensate for electrode resistance only. afferent root of an adjacent ganglion often lead to small broad EPSPs, which can sum- stimulation. The high Mg++/low Ca++ solution mate to produce a spike arising in the mon- did slightly reduce the lateral giant EPSP i tored segment. It is very difficult to reversibly. These results were obtained even distinguish unitary components of this com- though the ganglia stimulated were desheathed and 2 1~ were allowed for diffusion to occur. I pound EPSP, so the synaptic delay cannot do not take this as evidence that the afferent be estimated. On repetition of the stimulus, to interneuron synapse is not chemical, but these EPSPs decline gradualIy, and it seems rather that some diffusion barrier exists between likely that these EPSPs are due to mono- the surface of the ganglion and the synaptic synaptic chemical synapses from afferents sites buried in dense neuropil. It is unlikely entering in the adjacent ganglion, as sug- that these cllemic;ll synapses are not influenced gested earlier by others (35). It is also some- and n lg”, in view of the hrge number by cat+ times observed that the compound EPSP of neuromuscular junctions and neuronal syn- in an interneuron contains some small fast apses which are so ‘affected (5, 22, 26, 28, 29, 44, 48, 50, 64). This is, however, not the first report transients whose latencies vary on repeti- of the failure of Me;+* and Ca++ ions to influence tion of the stimulation (see Fig. 20). It will what otherwise appears to be chemical trans- be shown below that these are generated by mission (3, 23, 65). It has also been shown that polysynaptic pathways. Finally, in three INPUT TO CRAYFISIi LATERAL GIANT FIBER 613 preparations, inhibition was observed in In Fig. 19, the amplitudes of the EPSP these interneurons. Figure 18 shows a hy- of Fig. 17 and the IPSP of Fig. 18 are plot- perpolarizing potential recorded from inter- ted as functions of the change in membrane neuron C on stimulation of the second root potential from its resting value produced by of the next posterior (fourth) ganglion. passing current through the bridge circuit. This inhibitory postsynaptic potential The relations can be well approximated by (IPSP) remained after the EPSP disap- straight lines, and reversal potentials are peared after low-frequency repetitive s tim- measured as +40 mv for the EPSP and ulation. The potential was reversed by -6 mv for the IPSP, relative to the resting passing hyperpolarizing current through the membrane potential. electrode. Also, it interacted with an EPSP Small, fast components with variable la- generated by stimulating the second root of tency were observed in the responses of the third ganglion to reduce the amplitude about one-third of the tactile interneurons of the latter. This IPSP, difficult to observe studied. Some of these may be branch spikes against the usual excitatory background, is (63), but others are apparently due to syn- probably the basis of the inhibitory inter- aptic connections between tactile interneu- actions reported in tactile interneurons ear- rons. Figure 20 illustrates a recording from lier (35, 63). The inhibition is probably interneuron B, in which a particular all-or- polysynaptic, since the latency to the IPSP none component of the response to periph- is rather long and variable. eral stimulation corresponds one-to-one to spikes occurring in interneuron C. The field potential of the spike in interneuron C, 4 which was observed after the microelectrode

a

-o,-,a ’ I +10 0 0 -10 -20 -30 0-bAI FIG. 18. IPSPs recorded from tactile interneuron P 1 C in the third ganglion, in response to stimulation tclh&tion o& mumbr#na potqtial of the second root of the fourth ganglion. A shows #mm normal (mv) the effect of postsynaptic polarization on the IPSP; traces as in Fig. 17. B: IPSP(B,) reduced the ampli- FIG.. 19. Relation between the amplitudes of the tude of an EPSP elicited 1)~ stimulating the second postsynaptic potentials in inter-neuron C and the root of the third ganglion (B.) Y when the two oc- level of polarization. Data from experiments of curred together (B,&. Figs. 17 and 18. R. S. ZUCKER I2mv A

Sms

B 1

FIG. 20. EPSP in tactile interneuron B associated with impulses in tactile interneuron C. The upper -7 I trace is an intracellular recording from cell B in the fourth ganglion. The lower trace monitors the activity in interneuron C in the 3/4 connective. Whenever cell C fires in response to stimulation of the second root of segment four (A), the spike is associated with a fast EPSP in cell B. (Spike re- touched.) Sms FIG. 21. Proper ties of excitatory connections be- was withdrawn from interneuron H, was tween tactile intcrncurons. Upper trace is an intra- smaller and differently shaped than this po- cellular recording from a tactile interneuron; lower tential (cf. Fig. 11). The latter was thus trace monitors cord activity in the 5/6 (A) or 314 identified as a synaptic potential. Several (12) connective. A: stimulation of interneuron A in the 415 connective elicits an electrical EPSP in an such connections have been observed, and unidentified tactile interneuron in the sixth gan- their properties are illustrated in Fig. 21. glion. ZQ: in another preparation, stimulation of In each experiment, one interneuron is iso- A in the 5/G connective elicits an EPSP in inter- lated from the nerve cord for stimulation neuron C in the fourth ganglion. In B,, the EPSP while recording from another. Figure 21A follows stimulation frequencies up to 200 Hz. The record on the right of B, shows the artifacts gen- shows responses from an excitatory connec- era ted lq 3 sul~thrcsl~olcl stimulus. (Spikes rc- tion between interneuron A and interneu- touched in A and B, .) ron C in the fourth segment. These EPSPs have no synaptic delay and follow high-fre- quency stimulation of the presynaptic ele- and do not exist. 0 ther possibilities exist ment, and are therefore electrically medi- which have not been explored. For instance, ated. The significance of these electrical there may be recurrent connections from interneuronal couplings will be treated in fast flexor motoneurons onto the tactile in- a later publication. terneurons. Such possibilities seem remote, Table 1 summarizes the excitatory con- however. Connections to the nongiant fast nections described among the elements in- flexor motoneurons from the giant fibers volved in generating an escape response to have been reported by Wiersma (67) and phasic abdominal mechanical stimuli. In by Kennedy and co-workers (37, 61), and are addition, the table shows several possible studied in -more detail in the third paper connections which have been looked for in this series (74). INPUT TO CRAYFISH LATERAL GIANT FIl3En Glti

TABLE 1. Excitatory connections in third abdominal gunglion among elements in escape response circuitry -- -- ~---- - __.- In t erneuron Postsynaptic Tactile -P-P Lateral Fast Flexor Neurons Afferen ts A n c Giant Motoneurons --- ~~~~~ ~______-__ Illtcrneuron c:, A, (F) 0 0 0 A In terneuron C, A (E, B) E - E 0 B In tcrneuron C, A, (E, B) E 0 C La tcral giant E E E E B 0 Fast flexor motoneurons E, B m Fast flexor 0 0 0 0 0 C, F muscles C = chemical; E = electrical; A = antifacilitating; F = facilitating; 0 = no effect; B = amplified by branch spikes. Blanks indicate interactions not tested. Parentheses mark less prominent aspects of junc- tions.

DISCUSSION out by the greater amplitude of the spike Functiontll annfon7y of lrrfcd recorded from near the proximal lateral ginn t neuron giant dendrite (85-100 mv) as compared to those spikes seen from the distal dendrite Certain properties of the potentials gen- (40-70 mv). erated in the lateral giant could be corre- lated with the position of the recording Organization of input onto microelectrode in the neuron. For example, lateral giant recordings from the most ventrolateral den- In the present study, it has not been pos- drites always contained the largest and most sible to demonstrate anything but electrical distinguishable unitary EPSPs. This part of excitation of the lateral giant in response the dendritic tree lies in the field of en- to tactile stimulation. This input is pre- trance and divergence of the second and dominantly polysynaptic, and highly con- first root afferent axons (30), and also in the vergent. The experiments eliminate an region through which many of the tactile alternative proposal suggested by Krasne interneurons course (see Fig. 8; also ref 69). (40) on the basis of preliminary results. He It seems highly probable that most of the thought that the fast transient unitary all- electrical junctions on the lateral giant den- or-none components of the late response drite are ‘located in this part of the den- represented branch spikes in the lateral dri tic tree. giant’s dendritic tree. However, the fact The position of the spike-initiating zone that each such brief depolarization can be can also be inferred from the results. The associated with an immediately preceding threshold for initiating orthodromic spikes spike in an interneuron known to connect is between 52 and 57 mv in the dendritic electrically with the giant fiber precludes recordings of suprathreshold orthodromic this hypothesis and confirms the notion that responses illustrated in Fig. 14. However, all of these potentials are electrical EPSPs. the same threshold measured in the main One wonders, then, why the input to the axon just rostra1 to the dendrite is about giant neuron is organized in this fashion. 7-S n& (40). These results indicate gener- The answer may lie in the fact that the ally that the latter recording site is much giant fiber, like so many other large cells closer to the spike-initiating zone than the (20, 39, 49), 1tas a very high threshold, par- former, which in turn is much closer ticularly when measured from the synaptic to the major synaptic zone for tactile zone. Chemical EPSPs summate very non- activation. This interpretation is also borne linearly, espe&lly when they are large (57) 616 R. S. ZUCKER and they approach their equilibrium level sic mechanical stimulus (70). Both cells only asymptotically. Moreover, chemical have large dendrites with several specialized EPSPs in tactile interneurons from tactile synaptic regions receiving input from dis- afferents have a reversal potential of only crete sources (4, 8). Both neurons are ex- 40 mv, and especially when corrected for cited by highly convergent monosynaptic the estimated distance between the record- and polysynaptic pathways, and the excita- ing electrode and synaptic site (see below), tion is at least partially electrical (17). The the equililbrium potential is below the lateral giant and Mauthner cells each re- threshold for lateral giant excitation. Such ceive recurrent collateral inhibition from chemical synapses could never excite spikes both members of the pair (19, 59), although in the giant. Highly convergent electrical the synaptic mechanisms are not identical. input from afferents and several repetitively Finally, in both neurons, the spike-initiat- firing interneurons (Fig. 22) seems to be the ing locus is distant from the excitatory syn- only possible way to activate the lateral aptic region. Consequently, both cells have giant. If such huge giant fibers with high high thresholds when measured from the thresholds are really necessary for the rapid distal dendrites receiving excitatory input and coordinated excitation of motoneurons (18) . by electrical transmission (see 74), then the circuit used by the crayfish to mediate sin- Oymixntion of connections to and gle escape responses seems the most appro- clmong tactile interneurons priate. The functional anatomy of the tactile in- Many properties of the lateral giant neu- terneurons in each ganglion is relatively ron and the escape circuit associated with simple. Kennedy (32) has shown that spikes it bear a striking resemblance to that of the that arise in any ganglion are initiated Mauthner neuron in goldfish. Both cells within one 35-p region along the axon. This are very large paired premotor neurons single spike-initiating zone is probably at which run the full length of the neuraxis. the point where the relatively sparse den- A single spike in either cell elicits a rapid, dritic tree joins the axon (33, 75). Most powerful, coordinated, and highly stereo- electrode penetrations are within about 50 typed escape behavior in response to a pha- p of this point; I assume an average dis- tance of 25 p. In the third paper in this series (74), the space constant (A) of a pro- cess 20 p in diameter was calculated to be 350 p. The tactile interneurons are about the same size, and presumably have a sim- ilar space constant. Thus the electrode is about 0.1 a from the base of the dendritic tree and the most probable spike-initiating zone. Since synapses are dispersed along the FIG. 22. Schematic circuit of elements and con- short dendrites, a reasonable estimate for nections concerned with generating a rapid tail flex- the distance to the center of the synaptic ion to phasic mechanical abdominal disturbances. locus is about 0.2 h. This accounts for the Large circles represent single neurons; squares en- success, at least in some experiments, of compass populations of similar neurons. Small open circles arc antifacilitating chemical synapses finding a reversal potential for the excita- or junctions. Filled circles arc facilitating neuromus- tory synapses from the tactile afferents, and cular junctions. Electrical junctions are shown as for the IPSPs of unknown origin. bars; the one to the motor giant is rectifying. Fine The bulk of the input to tactile interneu- lines indicate that by and large, multisegmental in- terneurons arc interconnected and are excited by rons is chemical, monosynaptic, afferent, some unisegmental interneurons. The separate path- and excitatory. Massive input is not needed ways generating the a- and fl-components of the lat- to reach the fairly low thresholds of 3-7 eral giant response arc indicated. TR: tactile reccp- mv. Since the electrode is about 0.2 h from tors; A, B, C: identified tactile intcrneurons; LG: lateral giant; MoG: motor giant; FFMN: fast flexor the synapses, the actual site of the EPSP motoneurons. The fast flexor musculature is drawn generation would show an EPSP only eO.2 on the right. or 1.22 times larger than that actually re- INPUT TO CRAYFISH LATERAL GIANT FIBER 617 corded by the microelectrode (6); that is, experiments must be performed rapidly on EPSPs up to 10 mv might be generated. a dissected and gradually deteriorating This is still a respectably small fraction of preparation, which is still recovering from the equilibrium potential, which is esti- a strongly habituated state. It is not surpris- mated to be 40 mv degraded by 0.2 A (6), or ing that command fibers are often difficult about 33 mv. Chemical transmission be- to excite with natural stimuli. These con- tween aff eren ts and interneurons also pro- siderations give rise to the advantages of vides a convenient point for generating using intracellular techniques to identify adaptive behavioral habituation. The evi- the input to command and decision fibers. dence for ascribing this aspect of escape be- In addition, the present findings shed havior to this physiological mechanism is some light on the typical difficulty of elicit- presented in detail in the next paper (73). ing behavioral responses by stimulating The input to tactile interneurons from sensory interneurons. If the escape response adjacent segments has been described ear- circuit is at all typical, it appears that it is lier (35). These multisegmental pathways necessary for a minimal fraction of a popu- may partially overcome the effect of colli- lation of interneurons of a class (an “exci- sion of interneuron spikes initiated in sepa- tation cluster,” ref 21) to be active before rate ganglia, and thus make the interneuron any behavioral effects of stimulation will more responsive to massive stimulation, appear. Stimulation of single primary sen- and more effectively excite the lateral giant. sory interneurons, even at high frequencies, The electrical connections between tactile may be a quite inefficient strategy for dis- interneurons mav function similarly. Evi- covering the types of behavior in which dence will be presented in a later publica- these interneurons normally participate. tion that these junctions actually function Rather, it may be necessary to identify a to cause clustering of impulses in tactile in- hypothetical class or cluster of interneurons, terneurons. The function of inhibition in and to stimulate a number of them selec- these interneurons is at present unknown. tively and simultaneously. The anatomical dispersion of the members of a class often Circuit for one type of escape behavior, renders this approach unfeasible. In that and implications for eficient case, as in the present one, it becomes neces- neurophysiological strategies sary to work with the next higher hierar- The circuit of Fig. 22 provides a cascade chical tier of neurons whose activity effects of electrical input to effectively excite a are describable, and to study the subthresh- high-threshold command fiber, allowing for old effects of members of the lower class input-specific modifiability of behavior (73), of interneurons on the higher ones. and guaranteeing secure and rapid excita- tion of a population of motoneurons in ex- SUMMARY ecuting a stereotyped response (74). The lateral giant sits at the crest of the circuit The afferent limb of the neural circuit for escape responses to caudal phasi .c t actile mediating escape response in crayfish is de- stimulation. Like m anv command or deci- scribed. When a phasic mechanical stimulus sion neurons, its behavioral effect is easily is applied to the tail, crayfish frequently demonstrated by direct stimulation, but its evade the disturbance by executing a single normal route of activation is difficult to dis- rapid tail flexion. This behavior is con- cover. If only extracellular techniques are trolled by the lateral giant fiber, which used, stimuli must be chosen carefully to elicits each tail flip by a single discharge. penetrate the neural filters of sensory pro- The lateral giant is activated by tactile cessors and activate the giant fiber. Given stimulation of the pit hair receptors on the the habituation of the behavior, which re- surface of the abdominal carapace. Other sults from the properties of the pathways sensory systems do not excite this neuron, which excite the lateral giant (73), the sen- even subliminally. sory activation is nearly always rendered By recording in tracellularly from the lat- subthreshold by the drastic handling in- eral giant dendrite, it is shown that-all ex- volved in dissection. Hence physiological citatory input to the lateral giant elicited 618 R. S. ZUCKER

by natural tactile stimulation, or by shock- typed behavior. Some general strategies for ing afferent roots, is electrically mediated. determining the functions and typical Many receptor axons excite the lateral giant routes of activation of central neurons are directly. However, most of the input is di- also suggested. synaptic, via tactile interneurons. Several tactile interneurons are strongly excited by ACKNOW'LEDGhlENTS tactile stimulation and produce large uni- tary EPSPs in the giant fiber. It is argued I am particularly grateful to Dr. Donald Kennedy for his constant encouragement and guidance pro- that the electrical mode of activation is the vided throughout all phases of this research. I am most efficient way to excite a large cell with also indebted to Mr. Donald H. Pcrkel and Dr. Kao a high voltage threshold. Liang Chow for help in preparing the manuscript, The tactile interneurons are excited to Glenda Zuckcr, Teppy Dice, and Louise Follett chemically by tactile afferents. Most of the for help with the figures, and to Eric Swan and Roy Hamilton for technical assistance. synapses antifacilitate extensively at low This work was supported by a research grant frequencies (1 /set to 1 /min). Some of the from the Public Health Service (NS-02944-11) to D. interneurons are electrically coupled. Kennedy. The full neural circuit comprises recep- The author was supported by a predoctoral fcl- lowship from the National Science Foundation. tors, uni- and multisegmental interneurons, a “decision fiber,” mo toneurons, and mus- Prcscn t address of author: Dept. of Biophysics, cles. The properties of each junction have University College London, London WClE GBT, been described and related to the stereo- England.

REFERENCES

1. BARTH, F. G. A phasic-tonic proprioceptor in Feedback inhibition of the receptors. J. CeZZzd- the t&on of the crayfish Procambarus clarkii lar Camp. Physiol. 57: 149-162, 1961. (Girard). 2. Vergleich. Physiol. 48: 181-189, 11. EYZAGUIRRE, C. AND KUFFLER, S. W. Processes 1964. of excitation in the denclri tcs and in the soma 2. BENNETT, M. V. L. Physiology of elcctrotonic of single isolatccl sensory nerve cells of the lob- junctions. Ann. N.Y. Acad. Sci. 137: 509-539, ster and crayfish. J. Gen. Physiol. 39: 87-119, 1966. 1955. 3. BIEDEBACH, M., MEUNIER, J.-M., AND TAUC, L. 12. FATT, P. AND KATZ, B. An analysis of the end- Bases ioniqucs du potcnticl postsynaptique bi- plate potential recorded with an intra-cellular phasique chez 1’Aplysie. J. Physiol., Paris 60: electrode. J. Physiol., London 115: 320-370, 220, 1968. 1951. 4. BODIAN, D. The structure of the vertebrate 13. FIELDS, H. L. Propriocepti\-e control of pos- synapse. A study of the axon endings on Mauth- ture in the crayfish abdomen. J. ExijtZ. Biol. ner*s cell and neighboring centers in the gold- 44. . 455468, 1966. fish. J. Camp. Neurol. 68: 117-160, 1937. 14. FIELDS, H. L., EVOY, W. H., AND KENNEDY, D. 5. BRACHO, H. AND OKKAND, R. K. Effects of cal- role played by cfferent control of an in- cium on excitatory neuromuscular transmission vertebrate stretch receptor. J. Neurophysiol. in the crayfish. J. Physiol., London 206: 61-71, 30: 859-874, 1967. 1970. 15. FRANK, K. AND FUORTES, M. G. F. Stimulation 6. CALVIN, W. H. Dcndritic synapses and reversal of spinal motoncurones with intracellular elcc- potentials: theoretical implications of the view trodes. J. Physiol., London 134: 451-470, 1956. from the soma. L;x@. Neural. 24: 248-264, 16. FRANKENHAWSER, B. AND HODGKIN, A. L. The 1969. action of calcium on the electrical properties 7. DAVIS, W. J. Reflex organization in the swim- of squid axons. J. Physiol., London 137: 218- meret system of the lobster. I. Intrasegmental 244, 1957. reflexes. J. E~ptl. Viol. 51: 547-563, 1969. 17. FURSHPAN, E.J. “Electrical transmission” at an 8. DIAMOND, J. The activation and distribution excitatory synapse in a vertebrate . Scief2ce of GABA and L-glutamate receptors on gold- 144: 878-880, 1964. fish Mauthller ncurones: an analysis of dcn- 18. FURSHPAN, E. J. AND FURUKAWA, T. Intraccllu- dritic remote inhibition. J. Physiol., London lar and cxtraccllular responses of the se\reral 194: 669--723, 1968. regions of the Mau thner cell of the goldfX~. 9. ECCLES, J. C:. The Physiology of Synapses. New J. Neurophysiol. 25: 732-771, 1962. York: Ai:ademic, 1964. 19. FURUKAWA, T. AND FURSHPAN, E. J. Two in- 10. ECKERT, I<. 0. Reflex relationships of the ab- hibitory mechanisms in the of dominL;r] ~trct~jr receptors of the crayfish. 1. the goldfish. J. Neurophysiol. 26: 140-176, 1963, INPUT TO CRAYFISH LATERAL GIANT FIBER 619

20. HI~NNVA~AN, E., SOM.JT:N, G., AND CARIV:NTER, I. The twitch system. .7.E?cfltl. Biol. 43: 21 l-227, D. 0. Functional significance of cell size in 1965. spinal motoncurons. .I. Neurof)hysiol. 28: !XO- 39. KERNEIA, 1). Input resistance, clcctrical cx- 580, 1965. citability, and size of ventral horn cells in cat 21. HORRIDCE, G. A. Interneurons. London: Frcc- . Science 152: 1637-1640, 3966. man, 1968. 40. KRASNE, F. B. Excitation and habituation of 22. HOYLE, G. The effects of some common cations the crayfish escape reflex: the depolarization re- on neuromuscular transmission in insects. J. sponse in lateral giant fibers of the isolated Physiol., London 127: 90-103, 1955. abdomen. J. Exptl. Biol. 50: 2946, 1969. 23. HUGI’IES, G. M. AND TAUC, L. A unitary bi- 41. KRASNE, F. B. AND WOODSMALL, K. S. Waning phasic postsynaptic potential (B.P.S.P.) in Aply- of the crayfish escape response as a result of sia brain. J. Physiol., London 179: 27-28I?, 1965. repeated stimulation. Animal Behavior 17: 416- 24. HUGHES, G. M. AND WIERSMA, C. A. G. Neu- 424, 1969. ronal pathways and synaptic connexions in the 42. KUPFERMANN, I., CASTELLUCCI, V., PINSKER, H., abdominal nerve cord of the crayfish. J. Exptl. AND KANDEL, E. Neuronal correlates of habitu- Sol. 37: 291-301, 1960. ation and dishabituation of the gill-withdrawal 25. KAO, C. Y. Postsynaptic electrogenesis in scp- reflex in Aplysia. Science 167: 1743-1745, 1970. tatc giant axons. II. Comparison of medial and 43. KUPFERMANN, I. AND KANDEL, E. R. Neural lateral giant axons of crayfish. J. Neurophysiol. controls of a behavioral response mediated by 23: 618-635, 1960. the abdominal ganglion of Aplysia. Science 164: 26. KATZ, B. AND MILEDI, R. A study of spontane- 847-850, 1969. ous miniature potentials in spinal motoneurons. 44. KUSANO, K. AND HAGIWARA, S. On the integra- J. Physiol., London 168: 389422, 1963. tive synaptic potentials of Onchidium nerve 27. KATZ, B. AND MILEDI, R. The measurement of cell. Japan. J. Physiol. 11: 96-101, 1961. synaptic delay, and the time course of acetyl- 45. LARI~IER, J. L. AND KENNEDY, D. Innervation choline release at the neuromuscular junction. patterns of fast and slow muscle in the uropods Proc. Roy. Sot., London, Ser. B lG1: 483-495, of crayfish. J. Exptl. Biol. 51: 119-133, 1969. 1965. 46. LARIMER, J., EGGLESTON, A., MASUKAWA, L., AND 28. KATZ, B. AND MILEDI, R. The effect of calcium KENNEDY, D. The different connections and on acetylcholinc release from motor nerve ter- motor outputs of lateral and medial giant fibers minals. Proc. Roy. Sot., London, Ser. B. 161: in the crayfish. J. Exptl. Biol. 54: 391402, 1971. 49G-503, 1965. 47. MELLON, DEF., JR. Electrical responses from 29. KEHOE, J. Single presynaptic neuronc mediates dually innervated tactile receptors on the thorax a two component postsynaptic inhibition. Na- of the crayfish. J. Exptl. Biol. 40: 137-148, 1963. ture 22 1: 866-868, 1969. 48. MILEDI, R. AND SLATER, C. R. The action of 30. KENDIG, J. J. Structure and function in the calcium on neuronal synapses in the squid. J. third abdominal ganglion of the crayfish Pro- Physiol., London 184: 473498, 1966. cambarus clarkii (Girard). J. Exptl. 2001. 164: 49. NAKAJIMA, S. AND ONODERA, K. Membrane l-20, 1967. properties of the stretch receptor neuroncs of 31. KENNEDY, I). Physiology of photoreceptor neu- the crayfish with particular reference to mech- rons in the abdominal nerve cord of the cray- anisms of sensory adaptation. J. Physiol., LOW fish. J. Gen. Physiol. 46: 551-572, 1963. don 200: 165-185, 1969. 32. KENNEDY, D. Input and output connections of 50. NICHOLLS, J. G. AND PURVES, D. Monosynaptic single arthropod neurons. In: Physiological and chemical and electrical connexions between sen- Biochemical Aspects of Nervous Integration, sory and motor cells in the central nervous sys- edited by F. D. Carlson. Englewood Cliffs, N.J.: tem of the leech. J. Physiol., London 209: 647- Prentice-Hall, 1968, p. 285-306. 667, 1970. 33. KENNEDY, D. Crayfish interneurons. Physiolo- 51. OTSUKA, M., KRAVITZ, E. A., AND POTTER, D. D. gist 14: 5-30, 1971. Physiological and chemical architecture of a 34. KENNEDY, D., EVOY, W. H., AND FIELDS, H. L. lobster ganglion with particular reference to The unit basis of some crustacean reflexes. gamma-aminobutyrate and glutarate. J. Neul-o- Symp. Sot. Exptl. Biol. 20: 75-109, 1966. physiol. 30: 725-752, 1967. 35. KENNEDY, D. AND MELLON, DEF., JR. Synaptic 5 -*9 PABST, H. AND KENNEDY, D. Cutaneous mcch- activation and receptive fields in crayfish inter- anorcceptors influencing motor output in the neurons. Con+ Biochem. Physiol. 13: 275-300, crayfish abdomen. 2. Vergleich. Physiol. 57: 1964. 190-208, 1967. 36. KENNEDY, D. AND PUSTON, J. 13. Activity pat- 53. PENN, R. D. AND LOEWENSTEIN, W. R. Uncou- terns of interneurons in the caudal ganglion of pling of a nerve cell membrane junction by the crayfish. J. Gen. PhysioZ. 43: 655-670, 1960. calcium-ion removal. Sciezzce 15 1: 88-89, 1966. 37. KENNEDY, D., SELVERSTON, A. I., AND REVILER, 54. PENNAK, R. W. Fresh-water Invertebrates of M. P. Analysis of restricted neural networks. the United States. New York: Ronald, 1953. Science 164: 1488-1496, 1969. 55c . PILGRIM, R. L. C. AND WIERSMA, C. A. G. Ob- 38. KENNEDY, D. AND TAK~DA, K. Reflex control servations on the skeleton and somatic muscu- of abdominal flexor muscles in the crayfish. lature of the abdomen and thol*ax of Procam- 620 R. S. ZUCKER

barus clarkii (Girard), with notes on the thorax for freshwater crustaceans. Proc. Sot. Exptl. of Panulirus interruptus (Randall) and Astacus. Biol. Med. 34: 428432, 1936. J. Morphol. 113: 453-487, 1963. 67. WIERSMA, C. A. G. Giant nerve fiber systems 56. PRESTON, J. B. AND KENNEDY, D. Integrative of crayfish. A contribution to comparative synaptic mechanisms in the caudal ganglion of physiology of synapse. J. Neurophysiol. 10: 23- the crayfish. J. Gen. Physiol. 43: SW6Sl, 1960. 38, 1947. 57. RALL, W. Theoretical significance of dendritic 68. WIERSMA, C. A. G. AND BUSH, B. M. H. Func- trees for neuronal input-output relations. In: tional neuronal connections between the tho- Neural Theory and Modeling, edited by R. F. racic and abdominal cords of the crayfish, Pro- Reiss. Stanford: Stanford Univ. Press, 1964, p. cambarus clarkii (Girard). J. Camp. Neural. 73-97. 121: 207-235, 1963. 58. REMLER, M., SELVERSTON, A., AND KENNEDY, D. 69. WIERSMA, C. A. G. AND HUGHES, G. M. On the Lateral giant fibers of crayfish: location of so- functional anatomy of neuronal units in the mata by dye injection. Science 162: 281-283, abdominal cord of the crayfish, Procambarus 1968. clarkii (Girard). J. Camp. Neurol. 116: 209-228, 59. ROBERTS, A. Recurrent inhibition in the giant 1961. fibre-system of the crayfish and its effect on the 70. WILSON, D. M. Function of giant Mauthner’s excitability of the escape response. J. Exptl. neurons in the lungfish. Science 129: 841-842, Biol. 48: 545-567, 1968. 1959. 60. SCHRAMECK, J. E. Crayfish swimming: alter- 71. WINE, J. J. AND KRASNE, F. B. The organization nating motor output and giant fiber activity. of escape behaviour in the crayfish. J. Exptl. Science 169: 698-700, 1970. Biol. 56: l-18, 1972. 61. SELVERSTON, A. I. AND KENNEDY, D. Structure 72. WRIGHT, E. B. AND TOMITA, T. A study of and function of identified nerve cells in the the crustacean axon repetitive response. II. The crayfish. Endeavour 28: 107-l 13, 1969. effect of cations, sodium, calcium (magnesium), 62. SHERRINGTON, C. S. Some functional problems potassium and hydrogen (pH) in the external attaching to convergence. Proc. Roy. Sot., Lon- medium. J. Cellular Camp. PhysioZ. 65: 211- don, Ser. B 105: 332-362, 1929. 228, 1965. 63. TAKEDA, K. AND KENNEDY, D. The mechanism 73. ZUCKER, R. S. Crayfish escape behavior and of discharge pattern formation in crayfish in- central synapses. II. Physiological mechanisms terneurons. J. Gen. Physiol. 48: 435453, 1965. underlying behavioral habituation. J. Neuro- 64. TAUC, L. Transmission in invertebrate and physiol. 35: 621-637, 1972. vertebrate ganglia. Physiol. Rev. 47: 521-593, 74. ZUCKER, R. S. Crayfish escape behavior and 1967. central synapses. III. Electrical junctions and 65. TAUC, L., EPSTEIN, R., AND MALLART, A. Action dendrite spikes in fast flexor motoneurons. des ions Mg++ et Cat+ sur les potentiels post- J. Neurophysiol. 35: 638-651, 1972. synaptiques uni taires chez 1’ApZysie. J. Physiol., 75. ZUCKER, R. S., KENNEDY, D., AND SELVERSTON, Paris 57: 284, 1965. A. I. Neuronal circuit mediating escape re- 66. VAN HARREVELD, A. A physiological solution sponses in crayfish. Science 173: 645-650, 1971.