J. comp. Physiol. 121,349-366 (1977) Journal of Comparative Physiology- A 9 by Springer-Verlag 1977

The Anatomy and Motor Nerve Distribution of the Eye Muscles in the Crayfish

DeForest Mellon, Jr. Department of Biology, University of Virginia, Charlottesville, Virginia 22901, USA

Summary. The gross anatomy of the muscles in the ci:ayfish compound eye and the distribution of brain oculomotor neurons were studied by a variety of anatomical and physiological techniques. There are 11 major mus- cles in each eye. These vary considerably in size and influence upon eye movements and in their source of motor innervation. Muscles that cause defensive eyestalk withdrawal are controlled by axons of a giant motor neuron cluster. Muscles that move the eyecup in vertical planes are innervated by cells of an anterior motor cluster, as well as by cells in the medulla terminalis. Muscles which move the eyecup horizontally are supplied by neurons of the lateral motor cluster. The separation of the oculomotor system into different neuronal groups that supply different sets of muscles thus reflects functional specializations of the component divisions.

Introduction

The eyes of decapoda and some other Eumalacostracan crustaceans are mounted on movable stalks which articulate with the head. Eye movements are effected by a variable number of muscles which can move either the entire eyestalk or its parts. The eye muscles are under control of an oculomotor system within the supraoesophageal ganglion. Neurons of this system are responsive to selected environmental stimuli, and their impulse output can initiate and maintain detailed and specific variations of eye positions in space.

Oculomotor systems of the stalk-eyed crustacea are useful objects for neu- rophysiological investigation, since they have operational features that epitomize several general problems in the nervous control of behavior. These include the generation of detailed motor patterns in the absence of proprioceptive feed- back, the integration of diverse modes of sensory input in establishing specific motor commands, and the elaboration of perceptual memory. Thus, it is not surprising that crustacean eye control has come under intensified study in recent years.

350 DeF. Mellon, Jr.

Predictable changes in position adopted by crustacean eyestalks in response to different sensory inputs have been the subject of several of these studies. Sch6ne (1951, 1954) was concerned with the relationship of eye position to both statocyst input and the background illumination perceived by the com- pound eyes. His results, obtained primarily with shrimp, but including some observations with crayfish, indicate that vertical deviation of the eyestalks from their preferred resting orientation is a function of the interaction between both visual and statocyst input. The separate reflexes act to compensate for perceived rotation of an animal around the longitudinal body axis (roll).

Recently, Sch6ne and his colleagues (Stein and Sch6ne, 1972; Sch6ne et al., 1976) and Fay (1973) have demonstrated that eye position is under additional sensory influence from proprioreceptors within the walking legs. Tilt of a platform which supports the legs is perceived by the animal as would be tilt of the body in the opposite direction. Interaction occurs between this third avenue of sensory influence and those discussed above; all three summate alge- braically (but not necessarily to equal extents) to generate the final position of the compound eyes (Fay, 1973).

A cellular analysis of crustacean eye responses to body tilt has been performed by Burrows and Horridge (1968a-c). Compensatory movements of the compound eyes of the crab result whenever the animal is tilted about either the roll axis or the transverse body axis (pitch). In either case, eye movements tend to compensate for the imposed rotation of the crab in space by counterrota- tional behavior. Intracellular recordings showed that some reflexes depend upon eight of the nine muscles present in the eyecup, and these are driven to different extents by neurons of the oculomotor system during the various experimental conditions.

Predictable eye movements in crustaceans are generated by at least three additional categories of sensory input. These include rotation about the dorso- ventral body axis (yaw), which evokes statocyst-mediated movements of both eyes in a direction opposite to the imposed rotation (Cohen and Dijkgraaf, 1961; Sandeman and Okajima, 1972, 1973a, b). Sandeman and his colleagues (Sandeman and Okajima, 1973 b; Silvey and Sandeman, 1976 a-d) have recorded electrical activity from single oculomotor neurons during stimulation of statocyst hairbeds. The functional connections between the statocyst hair sensory neurons and ipsilateral oculomotor neurons appear to be monosynaptic. Individual motor neurons receive input from sensory hairs having a preferred direction of excitation by fluid movement within the statocyst horizontal canal, and they appear to be inhibited by sensory hair neurons having an opposite stimulus preference.

Rotation of the visual environment about a stationary animal evokes opto- kinetic nystagmus, characteristic sequelae of eye movements in which both com- pound eyes tend to track movements of the visual surround. The tracking activity is periodically interrupted as the eyes reach their extreme mechanical limit, at which time both simultaneously flick back to their starting position. Optokinetic nystagmus has been examined extensively in crabs (Horridge and Sandeman, 1964; Horridge, 1966a, b, c; Burrows and Horridge, 1968a, b, c, d; Horridge and Burrows, 1968a, b; Sandeman, Erber and Kien, 1975;

Eye Muscles in the Crayfish 351

Sandeman, Kien and Erber, 1975). Like the statocyst-evoked "vestibular" nys- tagmus, optokinetic reflexes function to stabilize retinal images. The reflex occurs in response to perceived relative motion of the animal and the visual field, regardless of whether this is caused by forced movements of the eye itself or by movements of the environmental features. The only exception to this occurs when a crab voluntarily generates eye movements during retraction or extension of the eyestalks (Horridge and Burrows, 1968c), or while the animal executes a change in direction of locomotion (Horridge and Sandeman, 1964).

It is clear from the foregoing account that the crustacean oculomotor system possesses a high degree of detailed functional complexity which is, nevertheless, amenable to experimental approach at more than one level. I have chosen to examine this system in the crayfish because of the presence of cave-dwelling forms with reduced visual structures. It will be interesting to determine the extent to which selective pressures have brought about changes in the eye motor system of such forms. The goal of the present study is to establish an anatomical and functional basis for future comparison with troglobitic species. This and the following paper provide descriptions of the major crayfish eye muscles, and their mode of innervation by specific groups of motor cells within various brain regions. A preliminary account of the spatial distribution of some oculomo- tor cells has been published (Mellon et al., 1976).

Materials and Methods

Crayfish studied were Procambarus clarkii obtained from a biological supply house and kept until used in large stainless steel holding tanks. Animals were used within two weeks of arrival and usually were not fed.

Anatomical studies of the eye musculature were performed by fine dissection in crayfish Ringer solution (Van Harreveld, 1936) to which, in some instances, 0.01% methylene blue was added. On a few occasions, the eyestalk musculature was fixed in alcoholic Bouin's solution and dissected in 70% ethyl alcohol.

The number and position of oculomotor neurons was determinated by back-filling the peripher- ally-transected motor axons with 300 mM cobaltous chloride. An animal was chilled on crushed ice for about one-half hour and then decapitated. The brain, optic tracts, and appropriate eye muscles were dissected with watchmakers' forceps and fine scissors and were isolated in Ringer in a small Sylgard-lined petri dish. The brain was pinned to the floor of the dish and a well was constructed of petroleum jelly around the appropriate muscle, so that its motor nerve protruded through the wall of the well. Ringer solution was then replaced with distilled water and the muscle nerves were cut. After a period of about 45 s, the distilled water within the well was replaced with 0.3 M cobaltous chloride. The dish was covered and placed in a refrigerator at 5 ~ for 24 to 48 h. Development was performed in fresh Ringer solution to which 2.2% ammonium sulfide was added. After development, the preparations were fixed in alcoholic Bouin's solution, dehydrated in ethyl alcohol, and cleared in methyl salicylate. In most cases, the brain was desheathed just prior to being placed in the clearing bath.

Physiology. Electrophysiological examination of motor nerve function involved en passant extracellu- lar recording of impulse traffic from restrained animals. A crayfish was chilled on ice for 30 min and clamped in a recording chamber. A small hole drilled through the dorsal exoskeleton into the pericardiai cavity served to perfuse the circulatory system with chilled Ringer. After most of the animal's blood had been replaced by Ringer, the rostrum was removed and one or both eyes were immobilized by clamps. Muscles, oculomotor and optic nerves were then exposed by fine dissection.

352 DeF. Mellon, Jr.

Intracellular electrical recordings from eye muscles were obtained by dissecting the cuticle and hypodermal tissue away from the appropriate area on the eye stalk. Glass micropipettes, filled with 2.5 M KC1 and having resistances of i540 Megohms were used. Signals were led to a high input impedance amplifier and displayed oscillographically for observation and photography.

In most instances, sufficient "spontaneous" impulse activity within the oculomotor axons was observed in the absence of specific stimulation to obtain positive correlations between electrical activity in nerves and muscles. In other cases tactile stimuli were delivered to the eyes and orbital region using a fine camels hair brush. At times, brief (0.5 ms) electrical shocks were also used to excite nerves supplying specific muscles.

Results

Gross Anatomy of the Eye Muscles

Figure 1 is a dorsal view of the external eye structure of P. clarkii in a posture of repose. Each stalked compound ey~ extends anterolaterally from the pro- tocephalon at an angle of about 75 ~ with the animal's midline and roughly 10 ~ above a horizontal plane passing just below the ventral wall of the eyecup. Structural support for the suspension of each eye is provided by the median ocular plate, to which are firmly attached the proximal ends of sub-cylindrical, calcified members of the eyestalks, the basophthalmites. Only the musculature of these and the distal eyecups have been examined in detail.

Descriptions of the eye muscles are available for Procambarus (Sugawara et al., 1971) and for at least two other genera of crayfish: Astacus (Schmidt, 1915) and Orconectes (Robinson and Nunnemacher, 1966). In some respects, the previous anatomical studies were not found to be sufficiently detailed for purposes of this paper. In addition, discrepancies occur between those accounts and the present discription, which identifies eleven separate muscles in the entire eyestalk of P. clarkii by an arbitrary numbering system. This is unrelated to the previous descriptions in which muscle names were assigned on the basis of assumed functions that, in at least some instances, appear to be in error. At any rate, since more than one eye muscle probably is active during most eye movements, and because the pivot point of the eye is liable to change during horizontal eye movements (Burrows and Horridge, 1968a) it appeared

] , - . . -

Fig. 1. Dorsal view of the top head region of Procambarus clarkii with the rostrum removed, illustrating the normal posture of the compound eyes

Eye Muscles in the Crayfish 353

Fig. 2. Diagram of the supraoesophageal ganglion of Procambarus. The three major groups of oculomotor neurons are indicated on both sides, and the axons of representative neurons are shown exiting the ganglion. Major regions of dendritic arborization of the different neuronal groups are included. ON, optic nerve; OCM, oculomotor nerve; ONMB, optic nerve motor bundle; DMB, distal motor bundle; AMC, anterior motor cluster; LC, lateral motor cluster; GC, giant cell cluster

safest to designate the individual muscles by number until a more thorough study could be made of their specific involvements during eye movements.

The muscles of the eyestalk are innervated by motor axons which run within the optic nerve and a separate oculomotor nerve (Fig. 2) (Hisada and Higuchi, 1973 ; Mellon et al., 1976). Axons of the giant oculomotor cells (GC) and those of the anterior motor cluster (AMC) run within the optic nerve and then, peripherally, within two small branches: the optic nerve motor bundle (ONMB) and the distal motor bundle (DMB). The oculomotor nerve (OCM) contains axons of cells in the lateral motor cluster (LC). It emerges from the dorsal surface of the supraoesophageal ganglion and passes lateral to the optic nerve as both exit the head through the epimeral foramen. Along part of its route within the eye, the OCM becomes intimately associated with branches of the ONMB.

Figure 3 shows diagrammatic dorsal views of the right eye. In Figure 3A, the two arteries and two nerve supplies of the eye are indicated in cutaway. All four structures reach the eye through a large median foramen in the anterior epimeral plate, beneath the base of the rostrum.

L Descriptions of Individual Muscles

Muscle 11. Movements of the basophthalmite relative to the head are ac- complished mainly through the action of muscle 11. This muscle has its origin on an apodeme close to the lateral margin of the epimeral foramen. It is much narrower at its origin than at its insertion, which occupies an elongate area on the dorsal part of the basophthalmite (Fig. 3B). Muscle 11 appears to be the homologue of the Attractor muscle described for Orconectes by Robinson and Nunnemacher (1966).

354 DeF. Mellon, Jr.

\ \

\ Fig. 3A-D. Dorsal view of the right eye. A the major arteries and nerves to the eye shown through the partially dissected basophthalmite. ON, optic nerve; OCM, oculomotor nerve; A.O., optic artery; A.OM., oculomotor artery; EP.F., epimeral foramen. B-D muscles of the eye, as described in the text

Muscle 12. Muscle 12 is a flat sheet and has its origin dorsally on an apodeme at the anterior edge of the basophthalmite (Fig. 3 B). This muscle runs parallel to the long axis of the eye and inserts directly on the dorsal wall of the eyecup. It appears to be the homologue of the Dorsal Retractor described by Robinson and Nunnemacher (1966). Because of its dorsal attachments, muscle 12 comprises a major suspensory element of the eyecup. Vertical movements of the eyecup relative to the basophthalmite are partly controlled by the degree of tension in this muscle. J

Muscle 13. Muscle 13 has three distinct and separate branches which originate in close proximity to one another from a sclerite near the distal termination of the basophthalmite. The three separate branches of this muscle insert in very different regions of the eyecup.

Muscle 13a is a slender structure which inserts at a point on the dorsal eyecup proximal to the insertion of muscle 12 (Fig. 3 B). This muscle is appar- ently the homologue of the dorsal head of the Lateral Retractor muscle in

Eye Muscles in the Crayfish 355

Orconectes. Its position and attachment suggest that, like muscle 12, it provides for the suspension of the eyecup.

Muscle 13 b inserts on the lateral wall of the eyecup (Fig. 3 B). It is a short stout muscle and undoubtedly is involved in laterally-directed movements of the eyecup relative to the basophthalmite. Muscle 13 b is probably the homologue of the ventral head of the Lateral Retractor muscle in Orconectes.

Muscle 13c is a slender muscle, the probable homologue of the Lateral Rotator described for Orconectes. This muscle inserts on the floor of the eyecup, posteriorly and near the ventral midline (Fig. 3 C).

Muscle 14. Muscle 14 is composed of two slips, 14a and 14b, which originate from separate adjacent sclerotized regions of the eyestalk membrane medial and distal to the origin of muscle 12 (Fig. 3 C). They are the homologues of the Medial Retractors of Orconectes. Both slips insert close by one another on the medial wall of the eyecup. From the placement and attachment points of muscles i4a and 14b, it is tentatively concluded that both are involved in medially-directed movements of the eyecup.

Muscle 15. This is the largest muscle in the eye. It is provided with two points of origin (Fig. 3 C). The most proximal of these is an apodeme connected to the lateral boundary of the epimeral foramen. The more distal origin is a sclerotized region of the ventromedial eyestalk membrane. The fibers of muscle 15 are grouped in fascicles which are helically arranged along its length in a manner reminiscent of the deep flexor muscles of the crayfish abdomen. All fibers of this muscle insert on the dorsolateral wall of the eyecup, just proximal to the corneal region. This muscle is undoubtedly the homologue of the Abductor muscle of Orconectes. It should be noted, however, that muscle 15 of Procambarus is surely not responsible for abduction of the eyes, but, instead, mediates the rapid reflex withdrawal of the eyestalk.

Muscle 16. Muscle 16 is a long, rather slender muscle having its origin near the ventral margin of the epimeral foramen (Fig. 3D). It inserts on the floor of the eyecup at a point near the insertion of muscle 18. Muscle 16 retracts the eye and, like muscle 15, swings the eyestalk medially toward the midline position beneath the rostrum. This muscle may be the homologue of the lateral Ventral Depressor described by Robinson and Nunnemacher (1966).

Muscle 17. This muscle originates on the ventral eyestalk membrane near to one of the lateral terminations of the basophthalmite (Fig. 3 D), and it inserts on the ventromedial eyecup just proximal to the corneal margin. It appears to be the homologue of the Orconectes Ventral Retractor. Both muscles 17 and 18, described below, are instrumental ill eyecup depression during protective withdrawal.

Muscle 18. Muscle 18 is a short broad sheet which originates on the ventral eyestalk membrane distal to the origin of muscle 17. It inserts on the ventral floor of the eyecup (Fig. 3 C). This muscle is probably homologous with the medial Ventral Depressor of Orconectes.

356 DeF. Melion, Jr.

II. Innervation of the Eye Muscles

A. Anatomical Results

Methylene blue staining provided a consistent, if incomplete, picture of the distribution of OCM, ONMB, and DMB axons to the eye musculature. All muscles of the eye except 13b, 13c, and 14b are at least partially innervated by axons which run within the ONMB and/or the DMB. In addition, OCM axons supply muscles 13b, 13c, 14a and 14b. In only two cases was the number of axons supplying any one muscle easily apparent in stained preparation: muscle 13 a receives terminations of only three axons, from the ONMB. These are sufficiently small to be identified as belonging to neurons of the AMC rather than those of the GC. Two large axons supply muscle 15, and their relative diameters indicate that they do belong to GC neurons.

More precise information about the distribution of the individual AMC and GC axons was obtained by the technique of axonal backfilling with cobal- tous chloride. Figure 4 illustrates typical results obtained when this procedure was used on the motor supplies of muscles 11, 12, 13a, and 17/18. Muscle 11 is innvervated by five identified AMC cells (Fig. 4A). These are clearly cells A through E as previously catalogued by Mellon et al. (1976). They include the medial group of three neurons (A-C) and the two large dorsally located somata, D and E.

Muscle 12 is supplied by four laterally situated AMC cells, F- I (Fig. 4B). The precipitated cobalt is less intense in both this photograph and that of Figure 4 C, possibly due to the longer distances required for migration of cobalt from these more distally situated muscles.

The motor supply of muscle 13 a is shown in Figure 4C. The three AMC neurons filled appear to be cell D and two of the medial cluster. Although precise identification of individual cells within the medial group is difficcult, these two neurons are probably A and C. Muscle 13a thus shares its entire motor supply with muscle 11.

Axons of four ventrally placed AMC neurons, cells J -M, exit the optic nerve within the DMB near the medulla terminalis, and they are probably distributed only to muscle 18. Figure 4D shows the results of backfilling the DMB with cobalt. The difficulties encountered in attempting to selectively fill the axons to individual muscles of this group precluded a more precise anatomi- cal determination of the motor neuron distribution.

The three cells of the giant cluster were briefly noted in a previous paper (Mellon et al., 1976). The dendritic arborization of each of these three neurons is sufficiently unique to allow individual identification on morphological grounds alone. Figure 5A shows a cell-designated G l -on the right hand side of a preparation in which it was the only giant neuron visualized by backfilling the ONMB. The axon, neuritic segment, and two stout club-like dendrites which give this cell a characteristic appearance diverge from a common junction. The dendrites course ventrally across the medial margin of large fiber tracts comprising rostral extensions of the circumoesophageal connectives. The den- drites of G1 are further characterized by profuse tufts to which they give rise along their course.

Eye Muscles in the Crayfish 357

Fig. 4A-D. Visualization of selected AMC neurons produced by axonal backfilling of the motor supplies to (A) muscIe 11, (B) muscle 12, (C) muscle 13a, (D) muscle 17/18

Figure 5 B is a photograph illustrating cells G2 and G3. The dendritic trees of these two neurons from the same side have very similar branching configura- tions. These spread laterally and posteriorly from the junction of the axon and neuritic segment and appear to wrap around the rostral extensions of the circumoesophageal connectives. There are no apparent dendritic tufts such as occur on cell G~. The primary difference between Gz and G3 is in dendritic branch diameter those of G2 being roughly twice as large as the corresponding processes of G3. In addition, the plane of the branch distribution of G2 is positioned dorsal and ouside to that of cell G3. Selective axonal backfilling indicates that cell G t supplies muscles 15 through 18. Cell G2 innervates only muscle 15, while cell G3 supplies all of the other three (Fig. 5C-D).

B. Electrophysiology

Confirmation of the anatomical observations was obtained by recording electri- cal activity simultaneously from different efferent pathways and specific eye

358 DeF. Mellon, Jr.

Fig. 5A-D. Dendritic morphology of cobalt-filled GC neurons. A Dorsal view of Ga on the right side of brain. Soma at right; neuritic segment runs ventrally and out of focal plane. B G2 and G3 backfills from a fortuitous preparation in which G 1 did not fill. C visualization of G1 and G3 obtained by backfilling the motor supply to muscles 16/17. D G1 and G2 backfills of the motor supply to muscle 15

musles. During the course of these studies, it was found that, as in the crab (Burrows and Horridge, 1968c), individual fibers of the different muscles are not always physiologically homogeneous. While some muscle fibers clearly re- ceive terminals from all axons constituting the motor supply to a particular muscle, others do not.

Figure 6 illustrates electrical records obtained simultaneously from intracellu- lar electrodes within fibers of muscles 11, 12, and 13a, and from a suction

Eye Muscles in the Crayfish 359

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Fig. 6A-C. Correlation between electrical activity in selected fibers of muscle 12 (A, top trace), muscle 11 (B, bottom trace), muscle 13 a (C, bottom trace), and the ONMB. In each case, identified pairs of nerve and muscle events are numbered for clarity. Calibrations: 200 ms: 20 mV

electrode placed on the ONMB. Five distinct amplitude classes of excitatory junction potentials (EJP'S) obtained from a fiber of muscle 11 (Fig. 6) are temporally correlated with five classes of nerve impulses, supporting the predic- tions based upon the anatomical data for this muscle. These recordings were obtained in a fiber from the medial portion of muscle 11. As in the eye muscles of the crab (Burrows and Horridge, 1968c) phasic and tonic motor systems exist in parallel and the individual muscle fibers clearly fall into either one category or the other. In muscle 11, laterally placed muscle fibers are supplied by fewer axons, and they exhibit little, if any, tonic activity.

Electrical records from muscle 12 are more complex. In the general, the lateral portion of this muscle is tonically active, some individual fibers exhibiting EJP's which are correlated with four different impulse heights from the ONMB (Fig. 6B). Other fibers, however, are appreciably different in the nature of their activity. Tonic EJP's are present, but no more than two classes of these can be positively correlated with distinct ONMB impulses. At least one EJP type persists even following section of the entire optic tract and the OCM (Fig. 7D). The motor neuron responsible for this category of junctional activity

9 has its soma in the medulla terminalis, as visualized by backfilling the motor supply of muscle 12 with cobalt, and its activity is uniquely affected by illumina- tion of the compound eye (Mellon and Lorton, 1977).

The branch of the ONMB which innervates muscle 13a is sufficiently long that its activity can be directly recorded by suction electrode. Three sizes of motor impulses are recorded from this nerve, and all are temporally correlated with EJP's in individual fibers of the muscle (Fig. 6 C). These data agree numeri- cally with findings from axonal backfilling experiments on muscle 13 a. However; in order to verify that some of the motor axons which supply muscles 11 and 13a are shared, simultaneous extracellular records were obtained in pairs from both of these structures and from muscle 12. These data are shown in Figure 7A-C, and confirm that some portions of muscle 11 and muscle 13a

360 DeF. Melon, Jr.

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Fig. 7. A-C paired extracellular records from muscles 11, 12, and 13a as described in the text. D intracellular records from a fiber of muscle 12 (bottom trace) and extraceliular records from its motor supply, in an isolated eye. Nerve record retouched for photographic clarity. E intracellular records from a fiber of muscle 18 (bottom trace) and extracellular records from the DMB. Correla- tions between at least one impulse type and EJP's are indicated by asterisks. Calibrations: 200 ms; 20 mV

are act ivated in concert, while the motor supply to muscle 12 is distinctly separate f rom that of the other two.

Electrophysiological recordings showed that only muscle 18 is suppl ied by axons of the DMB. F igure 7E shows s imultaneous records obtained from a muscle fiber and f rom the DMB. One AMC axon clearly supplies this fiber, a l though records f rom others may show either no activity or correlated EJP's f rom up to three different axons.

Conf i rmat ion of giant cell axon distr ibut ion was obtained by s imultaneous electrical recordings from the ONMB and muscles 15 through 18. Evidence that cell G3 supplies muscles 16, 17, and 18- to the exclusion of muscle 15- i s shown in F igure 8. A large ONMB axon, generating impulses with at least twice the ampl i tude of those in any AMC axon is phasical ly driven by moderate tacti le st imulat ion of the head, especially in the orbital region. Impulses in this cell are correlated one-for-one with discrete, facil itating EJP's in fibers f rom muscles 16, 17, and 18 (Fig. 8A, E -F ) but not with EJP's f rom muscle 15 (Fig. 8 B, D).

Intense tacti le st imulat ion of the head, including the eye itself, evokes activity in another very large ONMB neuron. Impulses in this cell are correlated with

Eye Muscles in the Crayfish 361

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Fig. 8. A-D simultaneous records from fibers in muscles 15 and 17 and from the ONMB during strong tactile stimulation of the eyes and anterior head. In A, the large spike in the nerve (N) record is from the axon of cell G3. In B, eye stimulation evokes responses not only from G3 but from a larger axon as well, cell Gz. Records in C and D from another preparation. Arrows mark pair of spikes in G2 which generate EJP's only in muscle 15. E-F intracellular record pairs from muscles 16, 17, and 18 to demonstrate common innervation by cell Ga. Calibrations: 200 ms; 50 mV

junction potentials in muscle 15 alone (Fig. 8B, D). The identity of this giant motor axon, therefore, must be G2, since the only alternative, cell Gt, supplies muscles 16-18 in addition to muscle 15 (cf. Fig. 9). Cell G1 could not be activated by tactile stimulation of the head or eyes. However, the presence of a functioning axon in addition to G3 supplying the fibers of muscle 15 was demonstrated by electrically stimulating the ONMB (Fig. 9A-D). Paired intracellular electrical records were obtained from individual fibers in muscle 15 and each of the other three withdrawal muscles while the optic nerve was stimulated with 0.5 ms electrical shocks. At minimal stimulus intensities an EJP was evoked from the fiber in muscle 15 alone. When a slight increase in stimulus intensity was effected, an EJP was generated in each of the fibers from muscles 16, 17 and 18. Simultaneously, a step increase occurred in the amplitude of the response recorded from muscle 15. These results show that a single low threshold ONMB axon innervates muscle 15. A second motor neuron, exhibiting a slightly higher stimulus threshold, is distributed to all four of the muscles under examination. It is concluded that the axon with the lowest threshold is G2, and the cell common to all four of the muscles is G1.

362 DeF. Mellon, Jr.

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Fig. 9. A-C simultaneous intracellular record pairs from muscle 15 and muscles 16, 17 and 18 in response to electrical stimulation of the ON. D simultaneous records from muscle 15 and 16 in response to maximal brief, electrical stimulation of the ipsilateral circumoesophageal connec- tive. Similar records in response to (E) subthreshold and (F-H) suprathreshold electrical stimuli in another preparation. Synaptic failure occurred shortly before record H was photographed, Calibra- tions: A-D, 50 ms and 50 mV; E-H, i0 ms and 25 mV

What is the normal pathway for excitation of G1 ? The intimate anatomical association of dendritic branches from this neuron with fiber tracts in the circum- oesophageal connectives suggested an obvious possibility, namely, ascending giant fiber activity. This expectation was born out by results of experiments in which the ipsilateral connective was electrically stimulated while recording simultaneously from muscles 15 and 16 (Fig. 9E-H). Junction potentials in fibers from both muscles appeared simultaneously at threshold voltage for an exceedingly large action potential in the electrical records from the connective, possibly originating in the medial giant axon. Whatever the identity of the presynaptic neuron, the synapse with the motor neuron (G~) is very labile in the radically dissected preparations required by these experiments. This prop- erty proved useful in determining which of the following alternatives offered the most reasonable explanation of the results. Simultaneous generation of junction potentials in muscles 15 and 16 might be caused either by excitation of a single shared motor neuron (G1) or by two separate motor neurons (G2 and GB) excited in common by a single presynaptic neuron. To decide which of these two alternatives best accounted for the experimental observations, a continuous train of single shocks, suprathreshold for the large connective im- pulse, was delivered to the ipsilateral connective at a frequency of one Hz. When failure of synaptic transfer occurred after several minutes, responses in both muscles disappeared together. This finding strongly implicates a common final efferent path and supports the contention that cell G1 is normally activated by a neuron or neurons within the ipsilateral connective.

Identification of LC motor neurons on an individual cellular basis has not yet been attempted. Nonetheless, we wished to confirm electrophysiologically the methylene blue staining data that suggested LC axons supply muscles 13 b, 13c, 14a, and 14b. Accordingly, various fibers in these four muscles were sampled by intracellular electrode while simultaneously recording from the ipsi- lateral OCM. The results are shown in Figure 10. In each case, some EJP's

Eye MuscIes in the Crayfish 363

A

r-'al' ~ , . L .t Ja_ . . . . IL. Ii.T , t . , L . L i [ I+ I . . . . I L , , , ~ , JU , , . i l , , l , t ih ,L t l , t . . . . l i ,* i i , . i i l - ' . . i L :2 i+~r .;.i. I : " r l ' . , - - r . r ' , - " r+ F +r+~r- - i ~ - .n -+~ +~m . . . . ,~ ' ' ~' ' ~ t , "T ' r - , -+" I r

~ I * j 9 i J .a

l" - I ' [ . . . . . F[ ?i r r l -- " t " - - - rq~-+?p ~ r r ' [ " -TVW+I[ ~" / / 9 +"

C /

~=' -~:"1 rl . . . . , r "U ' "n -~ . . . . " -+ j (~ ' ' l r l . . . . . ,r- , , ,r '?-] , . . . . l r - r -1 . . . . ,1 -+, l , - c ,~ , - :

E i ' i ; ,~ ,T~ Irql , 'T ' I1~ Ir' l ; ; ; "*; , . : , 1 [ .+ lu .+ l ] * I t . J . . .UL , ,*~ . . L~ ' ' ' / '"'""'[" r [ . . . . . . . r r ' ,, . re, ; '- .*, , "" rlTl'~' iT ' j , I~l~[P."~, [" ' '~' 'J

Fig. 10. A -D s imultaneous electrical records from OCM (top trace) and single fibers in muscle 13b (A), 13c (B), 14a (C) and 14b (D). E records from ONMB and a fiber of muscle 14a. At least two ONMB axons evoke EJP's in 14a (asterisks). Cal ibrat ions: 200 ms; 50 mV

evoked in individual muscle fibers are correlated with a particular class of nerve impulse in the OCM record. The included records also demonstrate that muscle 14a is supplied by at least two axons of the ONMB (Fig. 10E). These data support the anatomical results and implicate LC neurons in the control of horizontally-directed eye movements.

Discussion

In the crayfish, axons of motor neurons that influence eye movements are distributed to ipsilateral muscles, which they supply via three anatomically dis- tinct tracts: two branches of the optic nerve and a separate oculomotor nerve. This anatomical separation may reflect a segregation of the eye muscles into functional subsets. The oculomotor nerve supplies, apparently to the exclusion of others, four muscles which, on anatomical grounds, must move the eyecup horizontally. The non-giant motor axons of the optic nerve supply muscles which probably are responsible for vertical movements of the eyecup. An appar- ent further subdivision of this group is the distal separation of axons which provide for, respectively, eyecup elevation (ONMB) and eyecup depression (DMB). Finally, axons of the giant motor cluster (GC) undoubtedly effect both depression and withdrawal of the entire eyestalk during protective reflexes by which the eyes are withdrawn to a secure position beneath the rostrum.

364 DeF. Mellon, Jr.

These anatomical arrangements appear to be at variance with the organiza- tional framework of the crab oculomotor system in which, according to Burrows and Horridge (1968a), motor axons from both optic and oculomotor nerves intermingle and innervate all muscle blocks in the eye. However, knowledge of the distribution of individual motor neurons within the crab eye is about as incomplete as our present understanding of the involvement of different crayfish eye muscles in the various compensatory reflexes. It is thus possible that the differences are more apparent than real. The mechanisms of crustacean eye movements are complex. As Burrows and Horridge point out, no hinge is present at the eyecup-eyestalk joint, and the eyecup, which is suspended by the tonic activity of several muscles, is consequently free to move within three planes of freedom. They note, "It is possible that a muscle with no detectable change in impulse frequency may, nevertheless, contribute to a move- ment as the balance at the flexible joint is changed by other muscles." These observations emphasize the need for caution in the interpretation of anatomical data alone when making conclusions about the contributions of individual eye muscles to different eye movements.

The anatomy of the giant motor axons which drive protective eye withdrawal reflexes is interesting. Two types of dendritic branching morphology are exhib- ited by specific cells of this group, suggesting questions which concern the electrophysiological consequences of specific structural arrangements. For exam- ple, what is the functional significance of the paired, club-like dendrites of G1 as opposed to that of the more open branching configuration which charac- terizes G2 and G37 Is this an obligatory function of the large size of GI axon, or of the specific nature of the transmission process at this synapse? The tufts which occur in profuse numbers at the termination of the G1 dendrites have a superficial resemblance to the dendritic spines of cells in the vertebrate central nervous system. Dendritic spines have been viewed as a mechanism for injecting postsynaptic current into the main axis of large dendritic trunks (such as those on cerebellar Purkinje cells) without severely lowering their input impedance. The advantages gained by this sort of morphological arrangement are a greater longitudinal spread of synaptic potentials within the dendrite and an increased safety factor for the generation of dendritic action potentials (Llinfis and Hillman, 1969).

The dendritic morphology of cell G1 also superficially resembles that of the crayfish motor giant axon at the latter's synapses with the giant fibers in the ventral nerve cord. These are rectifying electrical synapses, and were first studied physiologically by Furshpan and Potter (1959). Using cobalt ionto- phoresis, Mittenthal and Wine (1973) have recently been able to visualize the synaptic connections made by the motor giant axon with both the lateral and medial giant fibers. The morphology of the postsynaptic fiber at the region of functional contact is characterized by fine projections and tufts that appear to be involved in intimate association with the presynaptic axon. It is probable that the postsynaptic projections penetrate invaginations of the presynaptic mem- brane, as originally shown by Robertson (1953). On morphological grounds, therefore, it seems as if theGx dendrites are suitably designed for the injection of large amounts of synaptic currents and the passive or active transmission

Eye Muscles in the Crayfish 365

of resultant potential change along the paired dendritic branches with minimal amplitude decrement. It is hoped that electrophysiological studies now underway with cell G 1 will provide sufficient functional evidence to understand the ana- tomy of this interesting neuron and the basis for those differences in dendritic branch configuration exhibited by cells G2 and G3.

I am grateful to Mr. Gene Lorton for his technical assistance with some phases of this work. I also thank Sharon Greene for executing the illustration in Figure 1 and Susan Snarez for illustrating Figure 2. This work was supported by USPHS research grant NS04989.

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