neuro-muscular mechanisms of abdominal pumping...

J. Exp. Biol. (1973), 59, 149-168iWith 19 text-figuresPrinted in Great Britain

NEURO-MUSCULAR MECHANISMS OF ABDOMINALPUMPING IN THE LOCUST

BY G. W. LEWIS, P. L. MILLER and P. S. MILLSDepartment of Zoology, University of Oxford

(Received 11 January 1973)

INTRODUCTION

Much interest centres at present on the neural mechanisms which control centrallypatterned rhythmical activity in arthropods. Yet in only one example has identifi-cation of an underlying oscillator within the CNS been claimed - that driving thescaphognathite beat in crabs (Mendelson, 1971). Now that it is possible to recordsynaptic and spike activity from cell bodies within ganglia (Hoyle, 1970) and to knowsomething of the morphology of neurones within the CNS through the injection ofcobalt (Pitman, Tweedle & Cohen, 1972), it should soon be possible to discovermore about the motor neurones and interneurones responsible for some forms ofrhythmic activity in insects.

Locust ventilation continues to invite experiments because of the regularity andpersistence of the output under trauma. Before central analysis can be attempted,however, more must be known about the peripheral motor output and the functionsof abdominal muscles; this paper attempts to supply some of the needed information.

In locusts and dragonfly larvae pumping movements comprise alternate con-tractions of expiratory and inspiratory muscles (Mill & Pickard, 1972), whereas incockroaches and many other insects expiratory muscles alone are present, inspirationbeing achieved by cuticular elasticity (Farley, Case & Roeder, 1967). Recordings fromabdominal connectives in crickets (Huber, i960), locusts (Miller, 1966) and cock-roaches (Farley et al. 1967) have demonstrated the presence of bursts of impulsesphase-locked with expiration. Evidence is discussed here which supports the hypo-thesis of earlier workers that the bursts occur in interneurones which co-ordinate themotor output and thereby achieve a well synchronized ventilatory stroke in allabdominal segments.

MATERIAL AND METHODS

Schistocerca gregaria Forskal was obtained from the Centre for Overseas PestResearch in London and cultured under normal conditions.

Electromyograms were made from the muscles of an intact, restrained locust byinserting insulated copper wires through the cuticle into appropriate muscles. Forrecording from lateral nerve trunks the abdomen was cut down the mid-dorsal line,pinned out flat on a block, tissue-side uppermost, and perfused with Ringer's solution(Usherwood, 1968). Under these conditions rhythmical contractions in ventilatorymuscles continued for several hours. Suction electrodes or stainless-steel hookedwires surrounded with a paraffin and petroleum jelly mixture were used for recordingfrom nerve trunks which were usually cut distally to exclude sensory input. Records

G. W. LEWIS AND OTHERS

A

Fig. i. For legend see facing page.

Neuro-muscular mechanisms of abdominal pumping in the locust 151

ere also taken from abdominal connectives either intact or after they had beende-sheathed and split into bundles. Intracellular records were taken from musclefibres with micropipettes filled with o-6 M-K2SO4 and having a tip resistance of5-20 MXi. Some experiments were carried out in a chamber continuously ventilatedwith 5 % COa in air.

FUNCTIONAL ANATOMY

Muscles are numbered according to the system of Snodgrass (1935) and Albrecht(1953). There are several descriptions of the innervation of the pre-genital segmentsof Acrididae (references in Seabrook, 1968), but they are not based on the use ofelectrophysiological techniques to track the course of axons. However, Tyrer (1971)and Hinkle & Camhi (1972) have used this method to describe some features of theinnervation of the dorsal longitudinal muscles in the abdomen.

In the present study nerves have been traced using methylene blue and electro-physiological methods. Most attention has been paid to the fourth abdominal segment,but this is similar to the other pre-genital segments. The metathoracic ganglion fuseswith the first three abdominal ganglia during development. This ganglionic masssupplies nerves to the metathorax and to the first three abdominal segments. Thethoracic ganglia are labelled GI, Gi l and GUI: the separate abdominal ganglia arelabelled G4 to G8.

Each pre-genital abdominal ganglion gives rise to two pairs of lateral nerve trunks,an anterior dorsal trunk (labelled Ni) and a posterior ventral trunk (labelled N2).In addition a median nerve leaves posteriorly from the dorsal side of each ganglionand passes to the next posterior ganglion, giving rise to a pair of transverse nerves onthe way (Fig. 1). Cobalt chloride, introduced into the cut end of Ni and caused tomigrate up the axons and into the ganglion by electrophoresis (Pitman et al. 1972;lies & Mulloney, 1971) has indicated the positions of some cell bodies in the ganglionwhose axons run in N1, and has also shown that six of these axons have cell bodiesin the next anterior ganglion (Fig. 2D, E). Dorsal longitudinal muscles in the pro-thorax (Shepheard, i97o),"mesothorax and metathorax (Neville, 1963) receive muchof their innervation from the next anterior ganglion, and it may be that the abdominaldorsal longitudinals, innervated by Ni , also receive part of their innervation fromthe next anterior ganglion. The positions of some cell bodies in G4, whose axons runin N 2 and in the median nerve, are shown in Fig. 2 C. The median nerve has beenshown electrophysiologically to contain at least four motor axons each of whichdivides and runs into the two transverse nerves; four cell bodies appear ventrally inthe ganglion when cobalt is introduced into the median nerve (Fig. 2 B). In additionneurosecretory axons from the next posterior ganglion are believed to join the median

Fig. 1. Diagrams of the muscles and nerves of abdominal segments of Schutocerca gregaria.A. The segments have been cut down the mid-dorsal line and laid out flat. On the right,segments 4 and 5 show the branching of Ni , with the main dorsal longitudinal musclesremoved in segment 5. On the left, the muscles innervated by N j are shown,and those inner-vated by N1 have been removed. The median nerve is omitted. B. Dorsal view showing themuscles in segment 4 innervated by the posterior median nerve of GUI. The median nervepasses over G4 where it is joined by some neurosecretory axons and then runs to supply thedorse-ventral inspiratory muscle (192) and the spiracle closer (196). The median nerve ofG4 innervates the corresponding muscles in segment 5.


Fig. 2. Diagrams of ganglia of Schutocerca gregaria showing neuron cell bodies stained withcobalt after its introduction into axons peripherally. A, dorsal view of G4 of an adult withcobalt introduced into the anterior median nerve, which is believed to contain only neuro-secretory axons. Thirteen cell bodies are stained and most are near the midline of the ganglion.B, ventral view of G4 with cobalt introduced into the posterior median nerve which containstwo motor axons to the inspiratory muscles and two to the openers of the spiracles. Four cellbodies are stained. C, ventral view of G4 with cobalt introduced into Nz. Thirteen cellbodies are stained and most are ipsilateral or medial in position. D, ventral view of G4 withcobalt introduced into N i . About 32 cells are stained; those about which there is uncertaintyare put in outline only. In addition a bundle of axons runs anteriorly into GUI. E, ventralview of GUI of second instar Schutocerca. Cobalt has been introduced into the left N1 of G4and it has entered six cell bodies in the posterior region of GUI. Horizontal scales, 1 mm.

nerve at the point where it divides into the transverse nerves (cf. Chalaye, 1966;Smalley, 1970), and their cell bodies have a dorsal location (Fig. 2A). Probably onlya proportion of the neurones with wider axons in peripheral trunks have been stainedso far.

In quiescent locusts pumping takes place in the dorso-ventral plane. Dorso-ventralexpiratory muscles are innervated mainly by N 2 of the same segment (see Table 1),while the inspiratory muscles, which expand the sterna ventrally, are innervated bythe median nerve of the preceding segment. Thus the antagonistic muscles of onesegment are innervated by two ganglia. Records, to be described below, from aganglion therefore comprise expiratory activity to one segment and inspiratory activityto the next posterior segment. The dorso-ventral inspiratory muscles, which are atthe anterior end of the segment, may have evolved from intersegmental musclesarising in the preceding segment. Segments 1 and 2 lack inspiratory muscles, and thecorresponding axons in the median nerves supply spiracle muscles. Thus the GUIcomplex gives rise to four median nerves, the anterior two supplying two axons each


Table 1. Iimervation of muscles in the fourth abdominal segmentof the locust

Ventilatory function

Dorso-ventral expiratory

Dorso-ventral inspiratoryLongitudinal expiratory

Longitudinal inspiratorySpiracle openerSpiracle closer

Muscles

190191193 (no records)194

19a

182 (5 parts)183184185 (no records)186187188

189

195196

Innervation

NiN2NsNa

Median nerveNiNiNiNINININI

N2

Ni

Median nerve

8 10

Fig. 3. Diagram summarizing the median nerve supply to ventilatory and spiracle muscles inSMttocerca gregaria. Spiracles are shown below the nerve cord and other muscles above. Thenumber of arrowheads on each nerve indicates the number of motor axons. In abdominalsegments 3—8 the median nerve contains four motor axons, each of which splits into two wherethe nerve divides into right and left transverse nerves. Two axons supply the dorso-ventralinspiratory muscle on each side, and two go to the spiracle closer muscle. Spiracles 1, 3 and 4each receive four motor axons, two supplying the closer and two the opener. Opener muscles ofmore posterior spiracles are supplied by paired lateral nerves in N1.

to the opener and closer muscles of spiracles 3 and 4, while the third and fourthmedian nerves supply two large axons to each of the inspiratory muscles of segments3 and 4, and two small axons to the closer muscles of spiracles 5 and 6. The openermuscles of these spiracles are supplied by paired lateral axons running in N1. Moreposterior spiracles receive a similar pattern of innervation. Those spiracles whichnormally serve an expiratory function (nos. 5 to 10) may therefore be capable of someunilateral independence as is known to be the case in Blaberus (Miller, 1973). Anoutline of the median nerve supply to spiracles and ventilating muscles is given inFig- 3-

Auxiliary forms of pumping appear when the insect is stressed, and these include

154 G. W. LEWIS AND OTHERS

^ ^ 4. 4 *-«nU-l4-A-fr. H- W-ti- till U

250 msec

Fig. 4. Intracellular records from a fibre in muscle 192 (lower record) together with extra-cellular records from the most posterior transverse nerve of GUI (upper record), whichsupplies muscle 192, the doreoventral inspiratory muscle of segment 4. A burst is shown withthe two motor axons contributing at equal frequencies.

NIN2

med.n.

I

1 sec-Fig. 5. Simultaneous records of efferent activity in N i (top), N2 (middle) and the mediannerve (bottom) of G4. Regular expiratory bursts in N i and N2 are shown alternating withinspiratory bursts in the median nerve during normal ventilation in a locust with intact CNS.

longitudinal telescoping movements by the abdomen (Miller, i960). Longitudinalexpiratory muscles are innervated by Ni , and inspiratory muscles by N2 (Table 1).

Intracellular records from muscle fibres in segment 4, together with extracellularrecords from peripheral nerves, have shown that normally two, but sometimes three,excitatory axons contribute to ventilatory activity in each fibre. Two 'slow' axonsusually produce responses of 0-5-5 m ^ an<^ 2-10 mV respectively in each fibre anda third 'fast' axon produces responses 10-45 m ^ m size> but t n e ^ t ^ ^ n o t beenseen in muscles 186, 192, 194, 195 or 196 (Fig. 4) (Table 1). A complete inventory ofthe motor axons supplying pumping muscles has not been compiled, nor is the totalnumber of axons supplying each muscle known in most cases. In muscles 182, 183,187 and 190 small hyperpolarizations have at times been seen, probably coming fromactivity in inhibitory axons, but they do not make regular contributions to the pumpingcycle and are presumably important in other forms of activity.

PATTERNS OF FIRING IN THE INTACT LOCUST

Normal pumping is accompanied by discrete alternating motor bursts in N2 andthe median nerve. Bursts are also formed in some N1 axons synchronous with thosein N2 even when no apparent longitudinal movement occurs. They probably providetension in longitudinal muscles and thus prevent extension of the abdomen when thesterna are lifted during expiration. Well-formed bursts occur in Ni during hyper-ventilation. Most analyses have been carried out on the compound bursts of N2 andthe median nerve, or on intracellular records from selected muscles. The phasing ofactivity in different muscles during expiration has not been examined, but this andother aspects are the subjects of a current study by Mr R. Hustert at Cologne.

Bursts in N2 and the median nerve, recorded at about the same distance from G4,


45

40

35

30

25

20

)5

10

5

25

20

15

10

-100-80-60-40-20 0 20 40 60 80 100

Insp.-exp.

2=84-8S.D.=23-4

0 20 40 60 80 100 120 140 160 180Delay (msec)

Fig. 6. Histograms of the intervals between expiratory and the following inspiratory bursts (A),and between inspiratory and the following expiratory bursts (B), recorded from G4 in a locustwith intact CNS. Expiratory bursts were recorded in N2 and inspiratory bursts in the mediannerve, both cut distally. Negative values indicate overlap of the antagonistic bursts.

normally show no overlap. In one preparation the mean interval between expirationand inspiration was 12 msec, and between inspiration and expiration, 85 msec (Figs. 5and 6). Conduction delays probably do not materially alter these values at the peri-phery. Overlaps between antagonistic motor neurone bursts do occasionally occur,however, for example, when ventilation is slow and the firing frequencies of motorunits are lower than normal. These observations suggest that reciprocal inhibitorycoupling, if it occurs between the antagonists, does so mainly at the level of inter-neurones and not at that of motor neurones.

In intact locusts the duration of inspiratory activity in each cycle is usually lessvariable than that of expiratory activity, the latter showing a good correlation withcycle time (Fig. 7). When ventilation is slow, expiratory neurones may start to firetonically as soon as inspiration ceases and they then produce a burst at higher fre-quency with the expiratory movement some time later. Alternatively the expiratory3troke may be divided into two active phases separated by a plateau of tonic firing


30

a.2 20e3•ae

10

o oo

o o

oo o

oo

oo oo

o oo o

oo o o o

V10 20 30Cycle duration (sec)

40 50

Fig. 7. Plots of the duration of the expiratory bursts recorded in N 2 of G4 (O O), andof the inspiratory bursts recorded in the median nerve ( # # ) of G4 against the ventilatorycycle time in a locust with an intact CNS. Correlation coefficient for expiratory bursts is0-9942, and for the inspiratory bursts is 0-4869.

corresponding to a period of maintained compression (Miller, 1965). Other patternsmay occur, particularly when the ventilation frequency is low (Hustert, pers. com.).Muscle records show that expiratory bursts are normally initiated by 9I0W units whichaccelerate and then decelerate towards the end of the burst (Fig. 8A). Fast units, ifthey participate, usually fire later in the burst as has already been noticed in this(Hinkle & Camhi, 1972) and other arthropod systems (Davis, 1971). The two inspira-tory axons each fire at a similar and steady frequency throughout inspiration, some-times showing a slight acceleration (Fig. 8B).

LESION EXPERIMENTS

After de-afferentation of the abdomen, or isolation of the thoracic and abdominalnerve cord, the frequency of ventilatory bursting declines but the pattern of firingremains essentially unchanged. In intact locusts sensory discharges in phase withventilation have been recorded from N1 and N 2 similar to those seen in cockroachesby Farley & Case (1968); their contribution is unimportant for the basic patterningof the motor output, but they may play a role in controlling some aspects of theoutput both tonically and phasically (cf. Davis, 1969; Pearson, 1972).

Brain removal also results in a considerable reduction of pumping frequency,whereas the subsequent removal of other parts of the CNS anterior to GUI has lesseffect (Fig. 9). Low-threshold COa receptors are believed to lie in or near the brain


A

!•ai>

.aV

&u3

HO100908070605040302010

n = 6 71=3 n=10 n=22 r,5=78-9 5=65-7 5=72-2 5=69-5 5=64-6j=6 n=3 J -8 s=9 J=10

«=105=55-2

I I I • I I I I I I I I I I I I I I I I I I I I I 1 I

2 4 2 4 2 4 6 2 4 6 2 4 6 8 2 4 6 8 2 4 6 8 1 0

4 0

I 30§, 20

10

GUI G5

Fig. 8 A. Mean temporal structure of the expiratory burst in the larger slow unit of the dorso-ventral expiratory muscle 191 of segment 4. Bursts have been grouped according to the numberof impulses in them, and the mean inter-impulse intervals have been plotted sequentially,n = number of bursts sampled; x = mean interspace interval; t = number of spikes in theburst.

B. Sequential inter-impulse intervals from one motor axon in the median nerves of GUI,G4, G5 and G6. Each plot represents one complete inspiratory burst.

DO

50

40

30

20

10

-

-

<

X

1 1

[ I1

II I

i i X

CNSintact

Brain Sub.-o.g. GI Gil GUI Isolatedremoved removed removed removed removed G4

Fig. 9. Diagram illustrating the effects on ventilatory frequency of removal of parts of theCNS. Results are based on recordings from Nz of G4 from four locusts which were perfusedwith s % CO| throughout. The vertical bars indicate two standard deviations.


GIHN2 "GIII med.n."G4N2 *G4 med.n. "

1 soc

Fig. io. Extracellular records of efferent activity in GIII-N2, GUI median nerve, G4~Naand G4 median nerve (from top to bottom). Both the GII-GIII and GIII-G4 connectiveshave been cut. Thus the top two traces represent activity from an isolated GIII and theyshow long expiratory and short inspiratory bursts as in the intact locust. The lower tworecords from G4 comprise short expiratory and long inspiratory bursts - a pattern not nor-mally seen in intact locusts.

Gill IG4G5

' itlillVli J 1 .

1 sec

1 sec

Fig. n A. Extracellular records of efferent activity in N2 of GIII , G4 and G$ in a locustwith intact CNS. Synchronized expiratory bursts appear in the nerves.

B. Records of activity in the median nerves of GIII (posterior median nerve to segment 4),G4, G5 and G6 during the production of two inspiratory motor bursts.

(Miller, i960) and their activity may maintain the ventilatory frequency. Continuedventilatory bursting can be recorded from a completely isolated GIII-G8 preparation,or from any single ganglion removed from such a preparation. However, while anisolated GIII continues to produce short inspiratory bursts and longer more variableexpiratory firing, as in the intact locust, a single abdominal ganglion, or the G4-G8chain, produces cycles of activity at a much reduced frequency (sometimes only iftreated with C02 or submerged in Ringer) with short expiratory bursts and longintervening periods of inspiratory firing (Fig. 10).

The bursting frequency in an intact locust or in the isolated cord can be increasedby continuous electrical stimulation of the anterior thoracic connectives. In intactlocusts it can be raised to 3 Hz by this means. The response may be produced throughthe action of command interneurones running between the head and the metathoracicganglion (Miller, 1966).

INTERSEGMENTAL CO-ORDINATION

During slow ventilation the abdominal segments may sometimes be seen to contractin sequence, either with anterior or with posterior segments leading, but as ventilationaccelerates contractions become apparently synchronous in all segments. A reductionof intersegmental delays at higher cycling frequencies has been reported in the gillbeat of Limulus (Fourtner, Drewes & Pax, 1971) and it also characterizes locustpumping.


Table 2. Delays between the production of expiratory bursts recordedin nerve 2 from GUI and G4

Negative delay values indicate that the posterior ganglion fires earlier than the anterior one.Results are from thirteen preparations. CNS intact.

Preparationnumber

1

2

3456789

1 0

11

12

13

Number ofbursts

196

i°578614616

5295568892

13169

Mean delay(msec)

41052-40

12-74— 409

-16-8511-87

7-3o2103

-O-541634

-37511-7039-38

8.D.

27-49I3-OOIS'2011-56

S.E.

1-96[-27[-72I-48

18-03 a-6617-60 4-4214-3110-628-93

14-iS10-50

98•09

[-19[•50[•09

9-96 08713-39 :t'2I

Simultaneous recordings have been made from homologous nerve trunks of up tofour segments in a ventilating locust in order to determine the order in which theabdominal ganglia produce motor bursts. Most attention has been paid to recordsfrom N2 and the median nerve of GUI, G4, G5 and G6 (Fig. 11). Table 2 showsthe results of a comparison of the timing of expiratory bursts from GUI and G4,and histograms of the delays between the different ganglia are shown in Fig. 12.

Although there is much variation in the order in which the ganglia produce expira-tory or inspiratory bursts, anterior ganglia tend to produce motor bursts earlier thanposterior ones and GUI is usually in the lead. Variations may occur from cycle tocycle within one preparation, as well as between preparations. During fast ventilationdelays of about 10-15 msec occur between adjacent ganglia and a total delay of50-75 msec exists between GUI and G8. However, because of the large amount ofvariation, not much significance can be attached to these figures. When ventilationis slow, or when parts of the anterior cord have been removed, intersegmental delaysmay be in the order of hundreds of milliseconds.

Extracellular records from abdominal connectives show bursts of spikes phase-locked to, but slightly in advance of, the expiratory motor bursts (Fig. 13). They canbe recorded in the connective at any point between GUI and G8, but the spikes arelarger nearer the anterior end. Connective bursts continue after de-afferentation andcan be recorded in an isolated GIII-G8 chain. They also appear in the posteriorconnectives of an isolated GUI. Single spikes have been tracked between GUI andG8 propagating at I - I - 3 5 m/sec (at 21 CC) with no detectable reduction in velocityas they pass through intervening ganglia. Left and right connective units seem to actindependently. They cannot be recorded in the absence of GUI. It seems thereforethat they occur in a single interneurone in each connective which extends throughoutthe abdominal nerve cord without intervening synapses and is activated in GUI.

Sometimes the latter part of a connective burst can be seen to be formed by moreone unit. Some of the additional activity may be in the axons of the six motorhan

i6o G. W. LEWIS AND OTHERS

fr - 2 0 6 20 40a - 1 0 10 30 50

'40 i

-40 -20 0 20 40-30 -10 10 30

= 141.v=22-8S.D. = 10-2

-20 6 20 40 60 80-10 10 30 50 70

Delay (msec)

B

1 I

-40 -20 6 20 40 60 80-30 -10 10 30 50 70 90

Delay (msec)

Fig. 12 A. Histograms of the intervals between the production of expiratory motor bursts inN2 of GUI and Nz of G4 (upper), and between N2 of GUI and Nz of G5 (lower).

B. Histograms of the duration of the intervals between the production of inspiratory burstsby the median nerve of GUI and that of G4 (top); by the median nerve of GUI and thatof Gs (middle); and by the median nerve of GUI and that of G6 (bottom). Negative valuesindicate that bursts appear in a posterior ganglion earlier than in an anterior one. Results areall from one locust.

cells whose somata are in the ganglion anterior to that from which their axons emerge,as already described. Additional bursts of spikes propagated anteriorly and phase-locked with expiration can sometimes be seen in intact locusts and they are probablyin interneurones with afferent connexions.

Posteriorly propagated bursts in each connective usually start at a low frequency,and then accelerate to a steady firing of 50-10x3 spikes/sec, at about which time theexpiratory motor burst starts (Fig. 14). The durations of connective and motor burstsare correlated even though the motor burst often starts ca. 100 msec later (Fig. 15);but there is no relation between the detailed structure of connective and motor bursts.The delay between the onset of connective and expiratory motor bursts sometime


100 msec

Fig. 13. Extracellular records of an expiratory burst in N i of G4 (upper) and an associatedburst in the connective between GUI and G4. The arrowhead indicates the first spike in theconnective burst.

0

5

10

•a1203'I255.g 30

i35

40

45500 750 1000 1250

msecFig. 14. Plot of a single connective burst. Separate interspike intervals are plotted against time.

Arrow indicates the onset of the expiratory motor burst in N2.

shows a correlation with total ventilatory cycle time, but this is not always so (Fig. 16).The start of the connective burst overlaps the end of the preceding inspiratory burst,sometimes by more than 100 msec, but there is usually a pause between the end ofthe connective burst and the start of the next inspiratory burst. The connective unitfires tonically throughout temporary pauses in ventilation until the next stroke isinitiated.

Section of both GII-III connectives leads to slower and sometimes less wellco-ordinated ventilation. The spike frequency in each connective unit may be lowerand the ' burst', phase-locked with expiration, may now comprise a period of uniformtonic firing. Most expiratory bursts are still represented in every ganglion, butoccasionally they appear in GUI alone; they are always accompanied by connectivebursts and the failures therefore seem to occur more posteriorly within abdominalganglia (Fig. 17). Even when there is no G 4 expiratory burst, G4 inspiratory firingmay show a temporary drop in frequency which coincides with the connective burst.This suggests that the connective unit weakly inhibits inspiratory firing. At othertimes bursts may appear in abdominal ganglia with no accompanying GUI burst,but there is then no connective burst.

II KXB 59


1100

Ii 900

700 -

700 900Connective burst length (msec)

1100

Fig. 15. Plot of the duration of expiratory bursts in the N1 of G III, which supplies segment 3,against the duration of corresponding bursts recorded in the G I I I - G 4 connective. Correlationcoefficient = 0-93.

With one connective cut between GUI and G4 normal coordination usuallycontinues posteriorly if pumping is strong, with a symmetrical output in the twoN2 of G4. However, abdominal ganglia may again occasionally fail to match GUIexpiratory bursts. At other times they have been seen to form bursts at twice thefrequency of GUI (Fig. 18). Frequency doubling of this sort occurs when the GUIbursts are long and G4 fires only at the beginning and end of them, the two shortexpiratory bursts thus formed being separated by a period of inspiratory firing inthe G4 median nerve. Activity in the G4 median nerve is therefore not incompatiblewith connective-unit firing. Thus while the inspiratory motor neurones apparentlyreceive weak inhibition from the connective unit, they are strongly inhibited duringexpiratory activity within their own ganglion.

Electrical stimulation of abdominal connectives in an intact or isolated GIII-G8preparation produces immediate excitatory responses in N2 expiratory motor nervesin several segments, and cessation of activity in inspiratory nerves. Stimulation mayinitiate a ventilatory cycle, and it is possible to pace ventilation in this way (cf. Mill,1970). Such stimulation may affect a co-ordinating system which enables more orless synchronized activity to appear in several segments, as will be discussed below.

Electrical stimulation of the lateral nerves is without effect on the median nerves.However, stimulation of the median nerves, at least in isolated ganglia, re-sets thephase of spikes in the train and produces a temporary inhibition of excitatory activityin N2. The effect is probably brought about by antidromic stimulation of the motornerves since sensory axons are believed to be absent from the median nerves. Theresult suggests that the median nerves form collateral inhibitory synapses on the N2motor axons, or an antecedent interneurones.


300

200

100

• —m ••• •• •• • • • 4

200

100

700 800 900 1000 1100 1200Cycle time (msec)

:: A • •

600 1200 1800Cycle time (msec)

2400

Fig 16. Two plots of the interval between the start of bursts in the GITI-G4 connective andthe start of corresponding expiratory bursts in N 3 of G4 against ventilatory cycle time. In A,in which the CNS is intact but the abdominal cord has been de-afferented, there is a positivecorrelation (correlation coefficient = 0-9279) between them. In B, in which the CNS is intactand only the N2 recorded from has been cut peripherally, there is no such correlation.

DISCUSSION

Each locust abdominal ganglion when isolated is capable of producing burst cyclesin ventilatory nerves. We can therefore consider four possible ways in which suchactivity may be co-ordinated in the intact insect to produce a well synchronizedventilatory output.

1. Fast-acting intersegmental proprioceptive loops may co-ordinate the output,ventilatory stroke in one segment rapidly initiating one in an adjacent segment.

164

GIUG4G5G6


1 secFig. 17. Extracellular records of expiratory motor bursts from N j of G M , G.+, G5 and G6(top to bottom) in a locust in which the G I I - n i connectives have been sectioned and the nervesrecorded from have been cut peripherally. The G H I - N 2 recorded supplies segment 3. Therecords are continuous and they show cycle-to-cycle variations in the output. Note thedifferent times at which bursts commence in the four nerves, and the occasional failure ofG4, G5 and G6 to match a burst in GITI.

GIIIN2GUI med.nG4N2G4 med.n.

] sec

Fig. 18. Extracellular records from G I I I - N a (supplying segment 3), G i n median nerve(supplying segment 4), G4-N2 and G4 median nerve. Both GIT-GITI connectives and oneG I I I - G 4 connective have been cut. Note that short expiratory bursts in G4-N2 may occur atthe end, or at the beginning and the end of a long G n i - N 2 expiratory burst, and that periodsof G4-median nerve firing separate the short G4-N2 expiratory bursts. G4 produces ven-tilatory cycles at twice the frequency of GIIT and its pattern of firing resembles that of theisolated G4-G8 chain. G4-median nerve inspiratory firing coincides with G I I I - N 2 expira-tory firing. Moreover low-frequency firing in the G Ill-median nerve overlaps with the weakG I I I - N 2 expiratory burst. These are not features seen in the intact locust during normalventilation.

However, although proprioceptors are active during pumping in intact locusts,isolated cords maintain a co-ordinated rhythm and sensory input cannot thereforebe the only co-ordinating mechanism.

2. Each segmental oscillator may trigger activity in the next ganglion as it becomesactive. In the crayfish and lobster swimmeret systems a cycle in one segment isbelieved to cause the next to fire through the activity of co-ordinating interneuroneswhose bursts are phase-locked to the motor bursts, but start slightly after them(Stein, 1971)- While such a system may co-ordinate locust ventilation when GUIis absent, it does not account for the very short intersegmental delays nor explain theconnective bursts which are propagated all the way down the cord from GUI inadvance of motor bursts.

3. Each segmental oscillator may be triggered by activity in interneurones whichrun the length of the abdominal cord. Once triggered the pattern of the burst cyclemay be determined by each segmental ganglion. A separate set of command inter-


lieurones running throughout the abdominal cord may serve to establish a commonlevel of excitability in all ganglia, as in the swimmeret system (Stein, 1971). Sucha system does not, however, explain the correlation between the durations of connec-tive and expiratory motor bursts and again fails to account for the form of theconnective bursts.

4. Each set of segmental expiratory motor neurones may be driven by an inter-neurone which determines the initiation, duration and possibly the intensity of theiractivity. Such a unit, in relaying patterned information, would qualify as a co-ordi-nating interneurone and be comparable to those postulated in other ventilatorysystems (Farley et al. 1967) and in swimmerets (Stein, 1971). It might by-pass orsuppress the activity of segmental oscillators as do the interneurones which supplythe stomatogastric ganglion of crayfish and which when stimulated at more than 2 Hzsuppress local oscillations and drive motor neurones directly (Dando & Selverston,1972).

With a conduction velocity of 1-1-35 m / s e c the postulated co-ordinating inter-neurone in locusts would be expected to excite G4 7-10 msec after GUI, and toexcite G8 25-35 msec after GUI. The measured mean delays are somewhat greater,but the large variability probably means that factors other than conduction delaysaccount for much of the difference. Fluctuating thresholds, or states of excitability,varying independently within ganglia may be more important in determining thefiring order than the timing of instructions delivered by the co-ordinating inter-neurone. The long latency (ca. 100 msec) between the arrival of the first connectivespike and the start of the expiratory motor burst suggests that temporal summation,perhaps also affected by local conditions, is important. Locust ventilation apparentlyprovides an example where burst cycles executed by the motor neurones of one seg-ment are organized by neurones in another. Similarly the activity of anterior spiracles,and head pumping, is thought to be organized from the metathoracic ganglion(Miller, 1967), while abdominal motor neurones may respond to a flight oscillatorseveral segments away (Waldron, 1967; Hinkle & Camhi, 1972).

Bentley (1969 a) recorded from the neuropile of the cricket mesothoracic ganglionand found motor units which fired bursts in phase with ventilation. During theinterburst they were hyperpolarized by IPSPs. He detected other units firing duringthe interburst which may have supplied the IPSPs. His results suggest that ventilatoryand other motor neurones receive much inhibitory as well as excitatory input.

A model to explain locust ventilation on the lines of alternative 4 above was putforward by Miller (1966). This must now be modified to account for more recentresults. The model (Fig. 19) comprises a burst-forming pacemaker situated in themetathoracic ganglion. The ' discharge' or relaxation phase of the pacemaker, whichis of relatively constant duration, determines the onset and duration of inspiration.It does so by inhibiting the firing of two autoactive units - the co-ordinating inter-neurones which run the length of the abdominal cord. The duration of the ' charge'phase of the pacemaker, which corresponds to expiration and intervening pauses, iscontrolled extrinsically by command fibres which respond to CO2, oxygen and otherfactors elsewhere in the CNS. (An oscillator with similar properties was postulatedby Bentley (19696) to account for the chirp intervals in stridulating crickets.) Theco-ordinating interneurones fire throughout this period; the patterning of their


Excitatory synapseInhibitory synapse

Fig. 19. A model is shown which can account for some features of the co-ordinated ventilatoryactivity in the abdomen of an intact locust. Only a few of the motor neurones of one segmentare shown. Cell 1 is a burst-producing interneurone, or group of interneurones, in the meta-thoracic ganglion which receives excitatory and inhibitory input from anterior centres. Itproduces bursts of impulses of relatively constant duration and these establish the inspiratoryphase; interbursts of variable duration establish the expiratory phase. Bursts of impulsesproduced by cell 1 inhibit the auto-activity of two co-ordinating interneurones, one in eachconnective (2). These cells run the length of the abdomen and in each ganglion they synapsewith a small interneurone (3) which sums their activity and distributes it to expiratory motorneurones of both sides (4). Thus cells 2 drive cells 4 via cell 3 which also strongly inhibits theinspiratory motor neurones (5). Cells 2 also directly but weakly inhibit the inspiratory motorneurones. When inhibition by cell 3 is lifted, the inspiratory motor neurones fire as a result oftheir own endogenous activity and there is weak positive coupling between them. Simul-taneously their activity inhibits the expiratory motor neurones via an interneurone (6). Theexpiratory motor neurones too may be positively coupled to each other but this has not beenindicated. The pattern is repeated in each segment. In addition further connexions betweenneighbouring cells 3 are shown and these account for the coordination of ventilation whencells 1 and 2 are inactive (e.g. after removal of GUI). When not driven by cells 2 they arecapable of endogenous burst formation. Their short bursts determine the expiratory strokeswhich are separated by long periods of firing by the inspiratory motor neurones.

activity may be determined by intervening cells or by the direct action of the pace-maker on them. Bursts in the co-ordinating interneurones always precede expiratorymotor bursts from GUI and abdominal ganglia. Action by the interneurones on Otherneurones within GUI may be looked upon as similar to that in more posteriorganglia. In each ganglion their input is summed by a further interneurone whichthen distributes excitatory activity to the expiratory motor neurones through synapseswith appropriate thresholds. Activity in the co-ordinating interneurones also weaklyinhibits the inspiratory motor neurones. These neurones fire at a more or less steadyfrequency which is usually independent of the cycle time, but they may show a slightacceleration during the burst. The phase of their spikes in a train can be re-set byantidromic stimulation. These features suggest that the motor neurones are auto-active and that they weakly excite each other. They may also inhibit the expiratorymotor neurones. No direct evidence for excitatory coupling between synergists hasbeen obtained but there is some evidence from antidromic stimulation for inhibitorycoupling between antagonists in one direction, although it cannot yet be said whetherthis is direct or via an interneurone.


The intraganglionic intemeuronal distributor may itself be capable of initiatingburst cycles when the co-ordinating interneurone is inactive. Its bursts are short andthey determine the expiratory stroke. They are separated by longer periods of endo-genous firing by the inspiratory motor neurones. Bursts are simultaneously trans-mitted through long collaterals to neighbouring ganglia where they initiate furtherexpiratory bursts. Such a mechanism accounts for co-ordinated activity in theabsence of GUI. However, in the intact system the endogenous activity of segmentaloscillators is normally suppressed by the co-ordinating interneurone.

Many features of this model are hypothetical and unsupported by evidence, butit bears some resemblance to the models put forward by Pearson (1972) to accountfor cockroach walking and by Bentley (1969ft) to explain cricket stridulation. It ishoped that intracellular recordings which are now being made from some of the unitsinvolved will provide more direct evidence.

SUMMARY

1. The muscles involved in dorso-ventral and longitudinal ventilation in the pre-genital segments of Schistocerca gregaria are described. Expiratory muscles are shownto be innervated by paired lateral nerves whereas the dorso-ventral inspiratory musclesare innervated by the unpaired median nerve system.

2. Normal pumping activity is brought about by alternating bursts of impulses inexpiratory and inspiratory motor nerves. Inspiratory bursts are relatively invariant,whereas expiratory bursts show a positive correlation with ventilatory cycle length.The firing patterns of some units within the bursts are described.

3. In general anterior segments fire motor bursts earlier than posterior segmentsduring well synchronized active ventilation, the metathoracic ganglion firing first.However, much variation is seen both within one locust and between different locusts.Burst-formation continues in isolated nerve cords.

4. Activity, phase-locked with expiration, has been recorded in the connectives.The evidence suggests that it occurs in a pair of co-ordinating interneurones whichrun from the metathoracic ganglion to the last abdominal ganglion and determinethe initiation, duration and possibly the intensity of the expiratory motor bursts ineach segment. A second parallel system may co-ordinate activity when the meta-thoracic co-ordinating interneurones are inactive. Inspiratory motor neurones areprobably autoactive and the duration of their firing may normally be determinedby the discharge phase of a metathoracic oscillator which acts by inhibiting theco-ordinating interneurones.

We are indebted to our colleagues Dr Caroline Pond and Dr Malcolm Burrows forassistance and valuable discussions, and to the Science Research Council for financialsupport.

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