J. Exp. Biol. (1966), 44. 413-427 4 1 3With 11 text-figures

tainted in Great Britain

AN ISOLATED INSECT GANGLION-NERVE-MUSCLEPREPARATION*

BY GRAHAM HOYLE

Biology Department, University of Oregon

{Received 19 July 1965)

INTRODUCTION

Previous studies on neuromuscular transmission in insects have all been made usingpreparations in which the muscle is left in situ and a minimum amount of damage doneto the tracheal system (Hoyle, 1965 a). Success has also been achieved in the study ofan isolated muscle without its nerve supply—the dorsal longitudinal flight muscle ofLocusta migratoria (Weis-Fogh, 1956). Attempts have been made to produce fullyisolated preparations (e.g. Hoyle, 1953) but none has so far been fully successful.There are several advantages, in principle, to the use of a completely isolated prepara-tion. The muscle fibres may be much more thoroughly bathed in saline than can beachieved in situ, even with perfusion, and changes in ionic composition, addition ofdrugs, etc., can be accomplished more efficiently.

A major advantage is also offered by the fact that isolation permits examination bytransmitted light, whereas in situ work usually demands the use of reflected light. Inview of the increasing awareness of diversity in the nature of individual fibres withinthe same muscle in arthropods (Atwood & Dorai-Raj, 1964; Atwood, Hoyle & Smyth,1965) the need to visualize individual fibres clearly during intracellular penetrationhas increased and will be demanded of the most critical work. Visualization is desir-able for penetration by two electrodes at different separations for determination ofelectrical characteristics of the membrane. A further, related point, of great relevancewhere other than the thinnest muscles are concerned, is that an isolated muscle can beturned around and examined from all sides. If a muscle has a heterogeneous composi-tion this becomes of vital importance, because unusual fibres may be inaccessible toselective puncture by micro-electrodes when operations are restricted, as in in situstudies.

An insect muscle which is particularly favourable for isolation was recognized asa result of studies (Hoyle, 19656) aimed at understanding the neurological basisunderlying learning to keep a leg in the raised position, which can be accomplishedby the thoracic ganglia of certain insects Horridge (1963). In these studies it was shownthat the motor nerve output to the anterior coxal adductor (a.c.a.) muscles of grass-hoppers and locusts carries a constant tonic discharge at about 12/sec. This rate maybe raised three- or fourfold by giving electric shocks to the tibia of the leg on whichthe adductor operates, every time the spontaneous background discharge rate falls bymore than 15 % over a 10 sec. interval.

The studies described above were done with minimum dissection, but interference

• Supported by research grant N.S.F. GB-3160.27 Exp. Biol. 44, 3

414 GRAHAM HOYLE

from many sensory inputs was evidently occurring and could have been influencingthe results adversely. Progressive sectioning of nerve trunks was made in the presentwork to see how much isolation could be tolerated without loss of spontaneous activityin the motor nerve. It was found that all the nerves leaving the metathoracic ganglioncould be severed but the discharge in nerve branch 3 c (cf. Hoyle 1955) which con-tains the motor axon supplying the a.c.a. still continued in many instances. This ledto attempts to make a fully isolated ganglion-nerve-muscle preparation retaining thecapability of spontaneous activity, and of reflex and integrative functioning. Theresults were successful and are described in this paper.

The anterior coxal adductor is a flattened, fan-shaped sheet of muscle fibres attachedsolely to the sternal apophysis on its inner margin and via a short, half-disk apodemeto the coxal rim at its outer end. The muscle can be fully isolated quite easily and canbe mounted so that its force may be measured accurately (see details below). Nervebranch 3 c together with the whole mesothoracic ganglion may be freed from the restof the animal and removed along with the muscle. It was found that the preparationthus isolated would remain alive for more than 8 hr. in standard locust saline (Hoyle,1953) whilst giving good muscle-fibre membrane potentials. Stimulation of the nervebranch always leads to good contractions in the muscle.

The contraction is effected by a single excitatory axon. In addition to this axon asecond efferent axon, also located in nerve branch 3 c, was found which when activegives rise to polarizing potentials in some of the muscle fibres and, occasionally, partialinhibition of the contraction caused by the excitor. This type of axon has been regardedby Hoyle (1965a) as a 'conditioning' axon preparing the muscle for strong contrac-tions, but by Usherwood & Grundfest (1964, 1965) as a peripheral inhibitor com-parable to those found in crustaceans (Wiersma, i960).

Three matters were considered to be worth studying with the preparation, and thereport which follows gives a preliminary account of them. First, in some preparationsthe excitatory axon still fires spontaneously at rates similar to those found in the intactanimal. The rate may reflect a previous 'learning' experience aimed at leg raising, bybeing unusually high. It can be modified by stimulation applied to various of the cutstumps. Thus the preparation may serve to permit further elucidation of the ' learning'process. Secondly, the preparation may be directly stimulated or ' driven' by electricstimulation applied to the cut ends of nerves attached to the ganglion; i.e. the motorneurons fire only during the stimulation and cease firing when it is stopped. Thiscapability permits a study of integrative processes in the ganglion. Thirdly, the secondaxon gives peripheral effects similar to those of the third axon supplying the jumpingmuscles, whose functions are currently the subject of controversy (Hoyle, 1965 a;Usherwood & Grundfest, 1964, 1965). In the isolated preparation its properties maybe studied under conditions favourable for experimentation.

MATERIALS AND METHODS

The studies have been made principally on males of the African locust Schistocercagregaria Forskal, together with some Romalea microptera, Melanoplus differentialis andS. vaga of both sexes. All the above are orthopterans, but it is considered probablethat large insects from other orders, especially Hemiptera and Coleoptera, can yield

Insect ganglion-nerve-muscle preparation 415

comparable preparations. The physiological properties of the muscles from thegrasshopper and the locust species were remarkably similar.

The dissections were made on the metathoracic legs. The insect is first laid on itsback in soft wax with all the legs spread out whilst the metathoracic legs are particularlywidely spread, rotated in the clockwise direction, and firmly fixed in wax. This extendsthe anterior coxal adductors (a.c.a.) and brings their attachments to the coxae upper-most. Next, the ventral thoracic cuticle is cut away in the central region so as to exposethe ganglion; all nerves attached to the ganglion except the third branch of the thirdnerve are severed, leaving stumps about 1 mm. long or more. The ganglion is sub-sequently washed with locust saline (Hoyle, 1953), which is frequently changed duringthe dissection.

Anterior coxaladductor muscle

Fig. 1. Location of the anterior coxal adductor muscle. Dissection of the base of the right,metathoracic leg of Sckutocerca gregaria. The animal was on its back and the leg rotatedforwards-anticlockwise. Only the thin, loose connective has been removed.

Attention is now focused on the sternal apophyseal pits. The sternal apophyses aresevered cleanly just below the invaginations and the remains of the basisternal andfirst abdominal sclerite are removed. The anterior adductor, which emerges fromunder the posterior rotator, which is also attached to the apophysis, is located (Fig. 1).The attachments of the rotator are next cut away close to their bases until the wholeof the anterior adductor is exposed. This uncovers the posterior coxal adductor, whichis also cut away. The whole of the anterior adductor can now be seen, together withthe nerve, which enters over the anterior ventral margin and immediately branchesprofusely as it enters the fan-shaped bundle.

The dorsal attachments of the sternal apophysis are now cut through, taking great27-2

416 GRAHAM HOYLE

care not to damage the muscle. After this, the apodeme is freed by cutting the distalattachment. A small piece of coxal rim may be taken along with the apodeme, pro-viding a greater area for attachment later and serving also as a handling point; or thesevered apophysis may be used. It remains only to lift the muscle gently up and cuttracheal tubes and connective tissue away. Likewise, it is necessary to see that theganglion is free from attachments. At this point the preparation may be lifted out intofresh saline.

Forcepstips force

transducer

Metathoracicganglion

Anterior coxaladductor

Fig. 2. The isolated preparation (right, metathoracic anterior coxal adductor muscle). Principalpositions of the stimulating electrodes used in eliciting responses are indicated. A singleexcitatory axon (E) and a single inhibitory-conditioning axon (I-C) innervate the muscle.

It is held by clamping or micro-pinning the triangular apophysis in such a mannerthat the muscle overhangs a small block of hard wax (Fig. 2). The apodeme may beconveniently seized by the tips of a forceps transducer (Hoyle & Smyth, 1963) topermit registration of the force of contractions. The ganglion is gently drawn awayfrom the muscle and fixed to the wax by a small Perspex bridge. The muscle is illumi-nated by transmitted light and an intracellular glass capillary micro-electrode isintroduced into a single muscle fibre. A pair of small, tapered, chlorided-silver hookelectrodes is micro-manipulated into place under the nerve trunk 3 c. Similar electrodesare manipulated under cut nerve stumps attached to the ganglion as needed.

Recordings were made with a six-channel pen-oscillograph (Offher Dynograph)in conjunction with a Tektronix dual-beam cathode-ray oscilloscope.

RESULTS

Properties of muscle fibres at rest

When first penetrated the muscle fibres have resting potentials from 46 to 73 mV.in standard locust saline (Hoyle, 1953). Fibres showing a low resting potential at thestart of the experiment often show a gradual increase over the first half-hour. Thecause of this has not been determined, but since the muscle is under continuous

Insect ganglion-nerve-muscle preparation 417

neural excitation in these experiments at least up to the moment of severing thenerves, it may represent a slow recovery process. A high frequency of excitatory nervestimulation leads to a slow progressive depolarization (d.c. shift) in addition to thebrief junctional potentials, and this has a slow time-constant of recovery. Not all thefibres show this property, and the differences between them were sufficient to suggestthe possibility that two different kinds of muscle fibre are present. An electron-

Fig. 3 Variety of intracellularly recorded electrical responses of single muscle fibres to singleshocks applied to excitatory axon (lower traces). Mechanical responses of whole right meta-thoracic anterior coxal adductor muscles prepared fully isolated. Note markedly differentdecay rates in (6) and (c). (a)-(e) Responses of five different fibres (Romalea nacroptera) allrecorded with the same electrode from the same muscle./, g. Extremes in response duration seenin a Schutocerca gregana preparation. Note that sweep speed in g is half that in/. The responsein / is due to a junctional potential of about 25 mV. giving rise to a graded response of about22 mV. G appears to be due to a junctional potential of giant size (45 mV.) with hardly anysecondary component, k-k. Responses of R. vacroptera preparation to pair of shocks withprogressively reduced interval between stimuli. Note that the second junctional potentialgenerates a graded spike of good size, but this fails to influence the decay of the junctionalresponse. Note also that there is a markedly increased mechanical response to the second shockfor which effect there is an optimal interval. /, m, Electric responses in R. imcroptera of twoextreme types to show differences in effect of close pairing of stimuli. Note absence of facilita-tion and summation in fast-decay fibre.

microscopic investigation of the muscle fibres has been started; the fibres all havesimilar, long (6 /i) sarcomere lengths but their sarcoplasmic reticulae are of two distinctkinds, supporting this possibility.

The electric responsiveness of the membrane is of the moderately graded kind

418 GRAHAM HOYLE

(c.f. Cerf, Grundfest, Hoyle & McCann, 1959). The largest of the neurally evokedmembrane responses almost reach the zero membrane potential level, but over-shooting action potentials have not been found.

Excitatory (E) axon

Facilitation of the junctional potentials has not been found in the Schistocercapreparations, but was quite marked in some Romalea preparations. In the latter,however, it could be eliminated simply by raising the calcium ion level in the saline to5 ITIM/1. The junctional potential then had about the same height as previously afterfull facilitation. The single excitatory axon thus fails to possess, in Schistocerca, oneof the most significant criteria of' slow' axons. However, it cannot readily be assignedto the ' fast' type because many of the junctional potentials are small (less than 20 mV.).It may be termed 'intermediate', or grouped with axons of the 'slow' type.

(i) Synoptic transmission

The single excitatory axon has a lower threshold than the 'inhibitory' one and henceits responses may be studied alone by stimulating nerve 3 c with just threshold shocks.The responses are of various magnitudes in different fibres, ranging from 5 to 60 mV.The smaller ones are pure junctional potentials (Fig. 3 a). The largest ones are com-pound, having a large junctional component together with a large graded response(Fig. 3e) (cf. Cerf et al. 1959).

The durations of the junctional potentials vary over a ten-fold range between dif-ferent fibres of the same muscle, ranging from about 5 msec, to more than 150 msec.These differences are partly the result of differences in the electrical time-constantsof the membranes of the fibres, for the short duration responses have faster rise times(Fig. 3 6). They also appear to reflect markedly different junctional events, for thereare large differences in the extent to which graded responses initiated in the membraneby the depolarizing action associated with the junctional potentials in turn causefaster rates of decay of their falling phases. In some fibres the decay is greatly increasedby even a small graded response. In others it is partially accelerated at first but thelate phase is slow (Fig. 3 A). In extreme cases, the slow decay rate of the junctionalpotential remains totally unaffected by the occurrence in the membrane of even a largegraded response (Fig. 3 m). These differences are not easy to account for in musclefibres of short length (2-4 mm.) having multiterminal innervation. Where the decayphase of the junctional potential is obliterated by a graded response it means that thetransmitter action is brief and the falling phase a purely passive, electrotonic effect.The failure to reduce it in other fibres could be due to their having a much longertransmitter action, perhaps due to a slower enzymic destruction. The effect canotherwise be explained only on the basis of a very large area of chemically, but notelectrically excitable post-synaptic membrane. The view that the transmitter substanceis destroyed at different rates is favoured. It is significant that the fibres whosejunctional potential decay is not reduced are also the ones with the longer time-constant; they may represent fibres specialized for slow following of tension upondepolarization and hence contribute only to slow and prolonged contractions of thewhole muscle.

Insect ganglion-nerve-muscle preparation 419

(ii) Mechanical responses

The mechanical response to a single excitatory impulse is a small twitch. This risesto a peak force of about 0-4 g./cm.2 in 40 msec, and decays to zero in 600-1400 msec,at 230 C. The slow decay, compared with the rapid rise suggests that the muscle, likecrustacean muscles (Dorai-Raj, 1964; Atwood & Dorai-Raj, 1964; Atwood et al. 1965),

20 40 60

15 g-

100

1 ISeconds

Fig. 4. Mechanical response of whole muscle (middle trace) and average type of electricalresponse (top trace) recorded from a single fibre with an intracellular electrode at differentfrequencies of stimulation (bottom trace). Romalea preparation.

is not homogeneous but comprises a mixture of slow, fast and possibly intermediatemuscle fibres. Fusion of twitches begins at about 8 sec. and is complete at 30/sec. butthis may well represent only the action of the fastest fibres. Subsequently tension stillrises progressively with increasing stimulus frequency, up to a maximum at 120/sec.,where it is at least 4 K g./cm.s. Considering the junctional potential nature of theexcitatory action this is a remarkably high value. Full tetanic force is not developeduntil after at least 4 sec. of stimulation at a high frequency. The late accumulation oftension may be developed by specialized slow muscle fibres. Even larger values maybe obtained by raising the calcium ion concentration in the bathing medium or adding

4-2O GRAHAM HOYLE

Ba++ ions, which render the membrane capable of producing spikes. Full relaxationafter a tetanus takes about 4 or 5 sec. The muscle can maintain its full tetanic forcefor as long as an hour without fatigue.

imtmr

Fig. 5. Example of intracellularly recorded electrical responses (upper traces) to stimulationof inhibitory-conditioning (I-C) axon alone at various frequencies. Traces second from top showtension in whole muscle: increasing tension upwards. Third trace monitors stimuli; fourthtrace is time in seconds. Schiitocerca preparation. Calibration: 10 mV, 01 g.

I P I I F I I I I » P F F f ) T F }

Fig. 6. Example of intracellularly recorded depolarizing responses to the I-C axon (uppertrace). Five excitatory junctional potentials are shown at beginning of record. The I-C responsewas elicited by preganglionic stimulation of the right connective with single shocks (monitoredon middle trace). Responses occurred in close pairs leading to marked summation and thespurious appearance of large junctional potentials. Some additional, later responses occurred,outlasting the period of stimulation. The output may be considered to be markedly' patterned'.Second trace, tension; third trace, I-C stimulus; fourth trace, E stimulus; fifth trace, timein sec. SMstocerca preparation. Calibration: iomV.

'Inhibitory-conditioning' (I-C) axon(i) Synoptic transmission

The second axon could occasionally be stimulated selectively by adjustment of theposition of the stimulating leads applied to N 3 c, in combination with the stimulusstrength and duration. More commonly it was excited by preganglionic stimulationof a nerve stump. It gives rise to small junctional potentials in most of the fibres; theseare principally polarizing (Fig. 5), but some are depolarizing (Fig. 6). Their maximum

Insect ganglion-nerve-muscle preparation 421•magnitudes ranged from 16 mV. polarizing to 14 mV. depolarizing, with many fibresgiving ones so small as to be difficult to detect; all possible intermediates wereencountered. The smallest I-C junctional potentials were seldom increased greatly bypolarizing the membrane with a second electrode and so cannot be simply due to themembrane potential being close to the equilibrium potential for the 'inhibitory'synaptic conductance change (c.f. Grundfest, 1962).

Electron-microscopic examination of the neuromuscular junctions of these fibreshas shown that in addition to large neuromuscular synapses, presumed excitatory,there are terminals which are extremely thin and therefore contain but a few vesicles.These could represent inhibitory terminals and explain the small size of inhibitorypotentials in some fibres.

Fig. 7. Burst firing in I-C axon (upper trace) evoked by preganglionic stimulation. Ventral nervecord stump was stimulated electrically with single shocks. Each shock gave a burst startingat a high frequency and summating markedly, then declining in frequency; stimulus interval1 /sec. Tension (middle trace) decreasing downwards. Note that retting tension relaxes slightlywith each burst. Schittocerca preparation. Calibration: 10 mV.; 0-05 g.

The inhibitory potentials are always longer in duration than excitatory ones, rangingfrom 60 to 260 msec. This is the case whether they are polarizing or depolarizing andindicates that the duration of I-C synaptic conductance change is 3-4 times as long asthe excitatory conductance change.

(ii) Mechanical responses

Stimulation of the I-C axon alone causes a slight contraction of not more than200 mg. force in some preparations (Fig. 5). In others it causes a slight relaxation ofresting tension, even when the excitatory axon has been completely silent for some timeand therefore cannot be causing a background tonic contraction (Fig. 7). In mostmuscles there is no direct mechanical effect. Combinations of 'inhibitory' withexcitatory effects lead to complex interactions (Hoyle, 1966). The contractions arepresumably caused by depolarizing I-C axon junctional responses sufficiently largeto exceed the threshold for contraction coupling. The relaxations must be the resultof a preponderance of polarizing I-C axon junctional potentials, but another require-ment is that in some muscle fibres the threshold for contraction is exceeded in theresting, slightly stretched muscle.

422 GRAHAM HOYLE

Spontaneous activity

When first isolated many preparations show no activity in the motor axons supplyingthe a.c.a. In some of these excitatory axon activity starts up spontaneously after severalminutes. This may represent the cessation of an inhibitory effect brought on by theviolent stimulation associated with extirpation. The basic frequency of the pacemaker

(o)

-j—i—rr—J—I—i—I—i—J—f—I—i—'—i—r~i—r—j—i—i—i—i—i—;—j—i—i—i—j—i—i—i—J—j—i—r—I i—t—f r~

(*)

i i i i f t i i -

(d)

I I I I I I ) I I I I I I I I

I I I I I I I

(e)

Fig. 8. Simple and complex discharges in isolated preparation. Schistocerca vaga. (a) Example ofsmooth, regular discharge. (6) Spontaneous disruption and complex pattern. Note that I-Caxon fires at same time as E axon firing rate increases, (c) I-C burst accompanied by centralinhibition of E. This record suggests an association aimed at peripheral inhibition, though it isnot very effective, (d) A burst of I-C activity closely accompanying increased frequency of firingof E. («) Prolonged I-C axon firing accompanying a decreased rate of firing in E Upper traces,intracellular recordings; middle traces, tension; lower traces, time in seconds. Calibrations:io mV.; c i g.

is 8-12 per sec. in the intact animal before extirpation, and similar frequencies areencountered in the isolated preparation (Fig. 8). Since the activity in the excitatoryaxon appears to continue relatively unchanged in frequency and pattern followingextirpation, it must be located in the same ganglion as the motomeuron, possibly in

Insect ganglion-nerve-muscle preparation 423

the neuron itself. However, the frequency may be much higher than this, or it mayfluctuate between wide limits, as is also the case in the intact animal. In some prepara-tions spontaneous firing of the excitatory axon never occurs, but bursts of impulsesmay be elicited by preganglionic stimulation (see below). The effects could be causedby prolonged central inhibition, or they may be a result of deterioration.

The I-C axon is usually silent in these isolated preparations. However, it may fireimpulses spontaneously in bursts, sometimes with a slow rhythm, at about one burstevery 30 sec, with a frequency of impulses within the bursts of 5-8/sec.; but suchactivity is sporadic. It also may fire for prolonged periods, especially when the excita-tory axon is firing uninterruptedly at a relatively high (7-15/sec.) frequency. Thefunctional role of the I-C axon will be considered in the second paper.

1—i—i—»—r T—r~\—1—r 1—(—r~i—1—r 1—(—r—(—rFig. 9. Central inhibition of spontaneous discharge of excitatory axon by single shock appliedto ventral nerve cord stump. Upper trace shows output of sealer at every tenth junctionalpotential. Larger, upward deflexions are artifacts of print-out unit occurring at 10 sec.intervals. Trace second from top is intracellular record. Third trace, tension; fourth tracemonitors stimuli; fifth trace is time in seconds. Note abrupt nature of onset and recovery frominhibition. Schutocerca preparation. Calibration: 10 mV.; 02 g.

Sometimes the smooth, regular spontaneous discharge of the E axon breaks up intoa series of slowly changing, patterned bursts. The change may be triggered by a shockapplied to a cut nerve trunk, or it may occur without stimulation. The I-C axon mayalso enter the picture and fire in bursts. Some of the patterns are reminiscent of thoseseen in the intact animal (Hoyle, 1966). Examples of such discharges are given inFig. 8.

The potential value of such activity lies in the fact that it may afford clues as to thestorage capacity of the ganglion for complex information related to behaviour. Theactivity may represent the spontaneous expression of neural' centres' involved in some

424 GRAHAM HOYLE

kinds of complex motor acts. However, so far this activity has been bewildering in itscomplexity, as the few examples given in Fig. 8 will show, and it has not so far beenpossible to assign any well-defined meaning to any of them.

p . PregangUonically-evoked activity

In a silent preparation the excitatory axon may usually be induced to fire by stimula-ting some of the cut nerve stumps. It is most readily driven by exciting the stump ofN5 on the same side, but all of the major nerves have at some time been foundeffective. It is possible that owing to the small size of the ganglion, stimulus escapecould be causing the excitation by affecting the motorneuron directly. However,several points argue against this being the case. For example, electric stimulationapplied to the ventral nerve cord, no matter how strong, rarely evokes a response andusually suppresses a spontaneously occurring one (Fig. 8). Since this stimulation is aslikely to lead to stimulus escape as any other, and since it causes a negative responsecompatible with natural behaviour, one is justified in considering the stimulation tobe indirect. The response to stimulation of the whole nerve cord, mediated by thegiant axons in the cord, is activation of leg extensors accompanied by central sup-pression of flexors and of the coxal adductors. This is comparable to the suppressionfound in the isolated preparation.

Suppression of the spontaneous excitatory discharge is also the commonest resultof stimulating the anterior connectives, though another common result is a combina-tion of initial suppression followed by late excitation in a continued burst of stimuli.

Interesting results have been obtained by combining stimuli to various nerves, buta full appraisal of such effects needs a more extensive study than has yet been made.

Inhibitory-conditioning

Stimulation of the connectives with the mesothoracic ganglion, especially that onthe ipsilateral side, not only suppresses spontaneous excitatory activity, but also oftencauses the I-C axon to fire. The rate of firing may be only loosely related to thestimulus frequency; sometimes a single shock will evoke a long burst. At other timesa similar stimulus will give rise to a single impulse and the fibre can be driven 1:1 upto frequencies as high as 150/sec. Later the same fibre may be completely unresponsiveto similar treatment. It is apparent that either the intervening synapses are very labile,or else spontaneous inhibitory and excitatory effects within the ganglion, and perhapssome effects of deterioration, combine to alter the properties. When the I-C axon isresponding to stimulation of the connectives between meso- and metathoracic gangliain a i : 1 manner, if the frequency of stimulation is raised to above about 50/sec theE axon also fires, giving a burst of impulses (Fig. 9). A few seconds of stimulationmust be applied before the burst occurs, and the E axon frequency is not related tothe stimulus frequency.

Preliminary report on conditioning of the spontaneous discharge

The existence of the spontaneous firing in the E axon makes it possible to carry outconditioning experiments aimed at altering the maintained mean frequency of thedischarge. Provided such changes persist over long periods they constitute a simple

Insect ganglion-nerve—muscle preparation 425

form of learning. In view of the critical nature of this kind of experiment, and theneed for careful statistical controls, no definitive statements may yet be made. How-ever, some encouraging indications of the possibility of effecting long-term changesunder conditions which may permit the physiological analysis of underlying eventshave been obtained.

Fig. 10. Dual response to preganglionic stimulation. Single stimuli were applied to the stumpof the right anterior connective. At low frequencies (up to io/sec.) they caused the I-C axonalone to fire i : i. At a higher frequency (20/sec.) applied for 2 sec., a late burst of excitatoryaxon impulses was evoked in addition. Upper trace, intracellular recordings; middle trace,tension; lower trace, stimulation. Sckistocerca preparation. Calibration: 10 mV.; 0-5 g.

1 1 1 1—>—1—1—1 1 1 1 i 1—1 1 1 1 1 1 1 1 1 1 1 -1—1 1 1 1 1 1 1—1 1 1 1 1—1 1 1 1 1 1

~~r-1—1—1—1—r~

1 1 1 1 1 1 ~T 1 1—1—1—1—1—1—1—

Fig. 11. Spontaneous discharge in isolated Schittocerca preparation. Note that frequency isdeclining slowly. After selectively timed shocks were applied to cut stump of N5 for a total of17 min., frequency was raised to middle leveL After an additional period of similar treatment itwas raised to the level shown in the bottom record. Upper traces, intracellular recording;middle traces, tension; lower trace, time in seconds. Calibration: 10 mV.; 1 g.

Single electric shocks of increasing strength were applied to the cut stump of thecrural nerve (N 5) on the same side until they just called forth a small reflex increasein the excitatory axon output to a.c.a. Shocks of this strength were then given everytime the mean spontaneous activity fell over a 10 sec. period by 20% from the mean

426 GRAHAM HOYLE

value over the immediately preceding 10 sec. period. This experiment is comparable*to that which was often successful in raising the output frequency in the semi-intactpreparation where stimuli were applied to the whole tibia (Hoyle, 1965 A).

Unfortunately, most experiments of this kind have so far given less satisfactoryresults than those done on the semi-intact preparations. There are larger variations inmean frequency and prolonged inhibition frequently occurs. Only two successfulexamples in which persistent changes occurred have been experienced so far (examplein Fig. 10) and the possibility that in both cases the rise in frequency obtained couldhave been due to chance alone cannot be ruled out.

DISCUSSION

Little can usefully be said in discussion about the above results at the present timesince most of them are of a preliminary nature. However, the preparation describedis of potential value for research in several fields: neuromuscular transmission,muscular contraction, central integration and the physiology of learning. Its use in thelatter field results from its spontaneous activity, which may be modifiable by simpleconditioning. By some criteria this kind of physiological alteration can be regarded asconstituting a form of learning. The physiological changes underlying the process mustbe located in the small piece of isolated—and therefore accessible—nervous system con-stituting the metathoracic ganglion.

Learning changes occurring in this preparation may arise as a result of an inherenttendency of the motorneuron pacemaker system to adjust its frequency of firing auto-matically following the receipt of certain sorts of input, provided these are timed withrespect to its own output in a regular and characteristic way. The association of theinput with the output may be provided for by inherent neuronal connexions forminga coupled feed-back relationship serving as a regulatory device in relation to posture.This regulatory system could, perhaps, account for the remarkably rapid adjustmentsmade by insects to loss of, or damage to, limbs, which is evident so quickly in theirlocomotory patterns. An understanding of the physiological basis of this adjustmentmechanism will be of intrinsic interest, whether or not it is eventually found to becomparable to other forms of learning.

Of some interest in connexion with general neuromuscular physiology is the strongsuggestion afforded by the present work that an insect muscle can have a hetero-geneous composition, comprising a mixture of muscle fibres of different types, havinglarge ranges in membrane electrical, mechanical contractile, and excitation-contractioncoupling properties. That this is true of some crustacean muscles has recently beenconclusively shown by measuring various parameters of single muscle fibres of thesame muscles (Atwood et al. 1965). A comparable analysis of insect muscle would bemuch more difficult to achieve on account of the smaller diameter of the fibres, butshould be technically feasible.

SUMMARY

1. A preparation is described which consists of an isolated locust metathoracicganglion, together with one motor nerve and the skeletal muscle which it supplies(the anterior coxal adductor) in a state suitable for tension recording.

2. Mechanical responses were recorded from the whole muscle, or bundles of fibres

Insect ganglion-nerve-muscle preparation 427

and electrical responses of single fibres were recorded intracellularly. Some fibreswere found in the muscle which have unusual properties. A single excitatory axonsupplies the muscle.

3. Preganglionic stimulation applied to cut nerve trunks may excite an inhibitory-conditioning axon supplying the same muscle.

4. Direct stimulation of the motor nerve was combined with preganglionicstimulation in order to excite the two axons, and their interaction in relation tocontraction of the muscle was studied.

5. The preparation shows spontaneous activity in the single excitatory axon sup-plying the muscle.

6. Various preganglionic stimulations were found to cause prolonged changes inthe spontaneous motor output. By correlating the stimuli to the output in certain ways,long-lasting changes in mean output frequency were obtained. These may be regardedas a simple form of learning.

REFERENCES

ATWOOD, H. L. & DORAI-RAJ, B. S. (1964). Tension development and membrane responses in phasicand tonic muscle fibers of a crab. J. Cell. Comp. Phytiol. 64, 55—72

ATWOOD, H. L., HOYLE, G. & SMYTH, T. (1965). Electrical and mechanical responses in single in-nervated crab muscle fibres. J. Phystol. 180, 449-482.

CERF, J. A. GRUNDFEST, H. HOYLE, G. & MCCANN, F. V. (1959). The mechanism of dual responsivenessin muscle fibers of the grasshopper, Romalea mwroptera. jf. Gen. Pkysiol. 43, 377-95.

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