THE JOURNAL OF EXPERIMENTAL ZOOLOGY 278:119–132 (1997)

© 1997 WILEY-LISS, INC.

JEZ 780

Estimation of the Size and Directional Output ofFunctional Groups of Interneurons UnderlyingAbdominal Positioning Behaviors in Crayfish

LAWRENCE D. BREWER AND JAMES L. LARIMER*Department of Zoology, University of Texas at Austin, Austin, Texas

ABSTRACT Quantitative studies were made of a large population of interneurons that con-trols postural flexion and extension of the crayfish abdomen. The number of interneurons neededto produce a motor program was estimated by stimulating a single abdominal positioning inter-neuron and recording interneuronal activity that was evoked from rostral and caudal connectivesin an isolated abdominal nerve cord. We also examined the role that these functional groupshave in producing a stronger motor output in either a rostral or caudal direction and thus speci-fying various abdominal geometrics. The average number of interneurons responding to stimula-tion of a single abdominal positioning interneuron was 32 (range: 3–50; n = 27). The averagenumber of interneurons that decreased activity was 10 (range: 2–32). Of 653 activated interneu-rons from 20 preparations, approximately 43% fired between 2 and 5 Hz, 33% fired between 6and 15 Hz, and 25% fired >15 Hz. The size of a recruited group was usually but not alwayscorrelated with the strength of its motor response or with the direction of motor bias. Therefore,the contribution of a group may depend upon the number of active elements as well as synapticefficacy. J. Exp. Zool. 278:119–132, 1997. © 1997 Wiley-Liss, Inc.

Vertebrate as well as invertebrate movementsare encoded within the central nervous system(CNS) by populations of cells (Hensler, ’88; Lee etal., ’88; Zecevic et al., ’89; Churchland and Sej-nowski, ’92; Georgopoulos et al., ’92; Tsau et al.,’94). For example, in crayfish and lobsters groupsof synaptically interacting premotor interneuronscontrol postural movements of the segmented ab-domen (Miall and Larimer, ’82a; Jellies andLarimer, ’85, ’86; Jones and Page, ’86; Larimer’88; Murphy et al., ’89). Abdominal positioning isa relatively simple behavior involving extensionand flexion, however, the number of abdominalpositions ranging from full extension to full flex-ion is quite large. Many of these movements areproduced by different intensities of motor outputin different segments or directions of the abdomi-nal nerve cord, sometimes referred to as a motorbias (Kennedy et al., ’67; Jones and Page,’86;Larimer and Pease, ’88). A biased motor output isconsidered a major mechanism to produce the al-most infinite number of abdominal geometries rou-tinely seen in animals such as crayfish andlobsters.

In crayfish hundreds of premotor abdominal po-sitioning interneurons (APIs) have been describedboth physiologically and morphologically in ab-dominal ganglia one through six and many are

considered identified cells (Miall and Larimer,’82a, b; Larimer and Jellies, ’83; Larimer andMoore, ’84; Jellies and Larimer, ’85, ’86; Larimerand Pease, ’88; Murphy et al., ’89). An API mayoriginate in any of the six abdominal ganglia withone or more axons projecting in the rostral and/orcaudal directions. All axons that project caudallyterminate in the sixth abdominal ganglion (A6),and axons that project rostrally extend throughA1 into the thoracic ganglia and some terminatein the brain (see Larimer and Jellies, ’83: Fig. 7;Miall and Larimer, ’82b; Larimer and Moore, ’84).Furthermore, each API extends dendrites intomost if not all ganglia that it projects through.This type of organization has the potential for ex-tensive recruitment and other synaptic interac-tions among large groups of cells throughout theabdominal nerve cord. Numerous experimentshave indicated that APIs operate as members orelements of a group to produce abdominal posi-tioning movements (Miall and Larimer, ’82a; Jel-lies and Larimer, ’85, ’86; Murphy et al., ’89).

Using population statistics (Lincoln index and

*Correspondence to: James L. Larimer, Department of Zoology,University of Texas at Austin, Austin, TX 78712. E-mail: neuroserf@mail.utexas.edu

Received 7 November 1996; Revision accepted 23 December 1996

120 L.D. BREWER AND J.L. LARIMER

a maximum likelihood method) Larimer and Pease(’88) estimated that there are about 360 APIs inabdominal ganglia one through six. However, thenumber of APIs that are active during any givenbehavior is not known. We have designed amethod to estimate the number of interneuronsor APIs that are synaptically recruited when cur-rent is injected into a single API in an isolatedabdominal nerve cord. The number of recruitedinterneurons was estimated by recording from theconnectives both rostral and caudal to the gan-glion where the impaled API was located. Thenumber of interneurons or other APIs that areexcited can be determined since the axon(s) of eachAPI projects through the abdominal nerve cordinto the thoracic nerve cord and/or terminates inthe sixth and last abdominal ganglion. This studyalso addressed the question of whether interneu-ronal activity can be used to predict the directionof motor bias. Motor bias is defined as having astronger motor output in either the rostral or cau-dal ganglia. The direction of motor bias was com-pared to the size and firing frequency of therecruited interneuronal group.

The use of isolated nerve cords offers the ad-vantage of eliminating the confusion of sensoryfeedback in the system. For example, in an intactbehaving animal it would be almost impossible todistinguish APIs from sensory and other cells. Ourmethod, however, does not provide a direct mea-sure of the number of interneurons activated in afreely behaving animal; rather, these results rep-resent an estimate of the number of interneuronsrequired to produce a fictive behavior. The motoroutputs produced by this method are very similarif not identical to motor outputs observed in re-strained, semi-intact preparations (Jellies andLarimer, ’86; Murphy et al., ’89) (Larimer, per-sonal observation). This type of motor output hasalso been shown to produce abdominal movementsin behaving animals (Kennedy et al., ’67; Larimerand Eggleston, ’71).

The results reported here support the model pro-posed by Larimer (’88) that APIs operate withindistributed circuits and that functional groupsarise from synaptic recruitment. Evidence indi-cated that in addition to the size of an interneu-ronal group, firing frequency, synaptic efficacy, andlocal circuits also influence abdominal position-ing motor outputs including motor bias.

MATERIALS AND METHODSBoth male and female crayfish, Procambarus

clarkii, were used in these experiments. All indi-

viduals had a rostral-telson length of approxi-mately 8–15 cm. The animals were obtained fromWaubun Laboratories (Schriever, LA), maintainedin dechlorinated tap water at 18°C, and fed drycommercial cat food.

Animals were anesthetized on ice and then dis-sected in van Harreveld’s saline (van Harreveld,’36). Isolated abdominal nerve cords with motorroots attached were pinned ventral side up in aSylgard-lined glass Petri dish filled with fresh sa-line. The first and sixth abdominal ganglia wereremoved and the 1–2 (rostral) and 5–6 (caudal)connectives were desheathed and teased intobundles for extracellular recording (Fig. 1). Thethird abdominal ganglion was desheathed withfine forceps for intracellular impalement with mi-croelectrodes. All APIs were impaled in the thirdabdominal ganglion (A3). This ganglion was se-lected because a large number of abdominal posi-tioning elements were identified in A3 in earlierstudies.

A WPI 767 intracellular probe with bridge bal-ance (up to 1,000 MΩ) was coupled to microelec-trodes for stimulating and recording from impaledAPIs. Microelectrodes were sometimes filled with3 M KCl (20–40 MΩ). Alternatively, microelectrodetips were filled with an aqueous 3% Lucifer Yel-low CH solution (Sigma, St. Louis, MO), and theshanks were filled with 1 M lithium chloride (50–150 MΩ) (Stewart, ’78).

In most experiments, following physiological ex-amination, the fluorescent dye Lucifer Yellow CH(Sigma) was injected into impaled APIs by pass-ing hyperpolarizing pulses between 3 and 5 nAfor 500–750 ms every second or 1.5 s for 5–30 min.Nerve cords were then fixed in 4% paraformalde-hyde for 12–24 h, rinsed in Sorenson’s buffer (3 ×30 min washes), dehydrated in an ethanol seriesfrom 50 to 100%, and cleared in methyl salicylatefor 30 min. Dye-filled APIs were viewed with acompound microscope under near ultraviolet (UV)light (430 nm) and drawn using a camera lucida.

Suction electrodes were used to record sponta-neous and evoked interneuronal activity from axonbundles in the rostral and caudal connectives (Fig.1). Fictive tonic abdominal flexion motor activitywas usually recorded from the third superficialmotor roots of the second abdominal ganglion(A2F) and from the fourth abdominal ganglion(A4F). Extensor activity was recorded from thesecond motor root of the fourth abdominal ganglion(A4E) (Fig. 1). All extracellular electrical activitywas amplified using differential AC amplifiers (A-M Systems, model 1700; Everett, WA).

SIZE AND OUTPUT OF GROUPS OF INTERNEURONS 121

Any changes in interneuronal impulse activityin the ventral nerve cord in response to stimula-tion of an impaled API were recorded from therostral and caudal connectives (Fig. 1). This al-lowed us to determine the number of interneu-rons recruited during a fictive motor program (seebelow). Furthermore, stimulation of some APIsproduces a stronger motor output (bias) in the ros-tral or caudal direction. Hence, we were also ableto correlate this directional motor bias with in-terneuronal activity in the nerve cord.

Motor activity was displayed and photographedon a Tektronix 5111A storage oscilloscope (Beaver-ton, OR) or recorded directly with an Astro-MedDash IV chart recorder (West Warrick, RI). Inter-neuronal activity was stored on tape using a VetterFM VHS recorder (Rebersburg, PA) and later dis-played with the Dash IV using a pretriggered datacapture module. Waveforms (action potentials) weresampled at 10 kHz per channel with 64 kilosamplesof total memory per channel. This digital, high fre-quency record was used to identify individual wave-forms. Specific interneuronal records from eachanimal taken from the Dash IV were copied astransparencies. Each unique waveform (impulse) onthe transparency served as a template to identifysimilar waveshapes from the complete chart record-ing (Fig. 2A–C). Data were analyzed 1 s before, 1 sduring, and 1 s after API stimulation. Most stimu-lations were of 1 s duration.

Only enough current was injected to produce aficitive motor output typical of a naturally pro-duced and observable abdominal positioning move-ment. The following considerations led us to thisstimulus paradigm. First, stimulating differentAPIs with a standard current cannot be used be-cause many APIs require different amounts of cur-rent to give a motor output. Also, different APIswere impaled at different locations. Second, onecannot simply use a standard firing frequency foreach API because some APIs give a normal-look-ing motor output at 50 Hz while others require90 Hz. Third, it is generally known that with cray-fish and other motor systems, an increase instimulus intensity results in an increase in mo-tor neuronal recruitment and firing frequency(Atwood and Wiersma, ’67; Evoy and Kennedy, ’67;Davis and Kennedy, ’72). At extremely low stimu-lus intensities little or no motor output occurs,and at extremely high stimulus intensities a verystrong motor output is produced that is probablynot characteristic of behaving animals. Therefore,the best criterion was to set the stimulus strengthto give a normal-looking motor program that

Fig. 1. Diagram showing an isolated abdominal nerve cordpreparation with the first and sixth ganglia removed. A mi-croelectrode (IN) was used to stimulate, record, and dye-fillAPIs in the third abdominal ganglion (A3). Suction electrodeswere used to monitor flexor motor activity from the superfi-cial third roots (R3) of the second (A2F) and fourth (A4F)abdominal ganglia. Extensor motor activity was monitoredfrom the second root (R2) of the fourth abdominal ganglion(A4E). Suction electrodes were also used to record interneu-ronal activity from the rostral (RC) and caudal connectives(CC), which were teased into bundles.

122 L.D. BREWER AND J.L. LARIMER

would have produced an observable movement inan intact animal.

Occasionally coincidental impulses that occurredduring high neuronal activity caused distortedwaveforms; hence, these data could not be analyzed.While the visual overlay method is time consum-ing, it was found to be much more reliable thanone computer software package that we testedwhich was designed for this kind of analysis.

Interpretation of interneuronal datafrom the connectives

Interneuronal activity was characterized by thenumber of interneurons participating in a motorprogram as well as by their firing frequencies.These data are presented in histogram format.The contribution of the impaled cell is not includedin these figures even though they were partici-pating in these behaviors. Criteria for determin-ing if a neuron was participating in a behaviorusing this type of experimental paradigm are notwell established so we were forced to design ourown criteria. These criteria and their derivationsare discussed below.

Stimulation of each impaled API evoked activ-ity that can readily be divided into two groups:1) recruited interneurons that were silent be-fore API stimulation and 2) interneurons thatwere spontaneously active before API stimula-tion and increased their activity during thestimulus. Interneurons were further pooled intocategories according to their firing frequency:2–5, 6–10, 11–15, 16–20, and >20 Hz.

Spontaneously active interneurons were consid-ered as participating in a fictive positioning be-havior if they meet the following criteria. 1)Interneurons that increased activity to above 10Hz must have increased their firing rate by atleast 50% (e.g., prestimulus rate = 10 Hz and fir-ing rate during stimulus = 15 Hz); or interneu-rons that increased their firing rate by 40–49%were included if their poststimulus firing rate de-creased to their prestimulus rate. 2) Interneuronsthat fired below 11 Hz were only included if theiractivity increased by 100% (i.e., doubled), or iftheir firing rate increased by at least 60% andtheir poststimulus firing rate returned to their

Fig. 2. Chart record used to identify action potentials pro-duced by specific interneurons from the abdominal nerve cord.A: Electrophysiological records of interneuronal activity fromtwo rostral connective bundles (RC) and one caudal connec-tive bundle (CC) before, during, and after stimulation of anAPI (IN = intracellular electrode). The open arrow refers to aportion of the record shown in B (prestimulus) and the solidarrow refers to a portion of the record shown in C (stimulus= 6 nA). B, C: Chart record of interneuronal activity sampledat 10 kHz. B: The open arrowhead refers to a waveform thatwas used as a template. A transparency of the template isused to identify other waveforms which match the templateas shown in C. C: An example of comparing the template

with other waveforms. The open arrowhead refers to a wave-form that matches the template and is therefore consideredto be an action potential from the same neuron. The solidarrowheads refer to waveforms which do not match the tem-plate and are therefore considered to be action potentials fromdifferent neurons.

SIZE AND OUTPUT OF GROUPS OF INTERNEURONS 123

prestimulus rate. 3) Low activity cells that onlyincreased their rates from 1 to 2 Hz were ignored.4) Interneurons which were inhibited were alsoanalyzed. Interneurons that had a prestimulusrate below 11 Hz were counted as inhibited if theirrates decreased by at least 50%. Cells that had aprestimulus rate above 10 Hz were considered tobe inhibited if their firing rates decreased by atleast 40% during the stimulus.

These criteria were determined in part by ana-lyzing data from a single experiment in which thesame API was stimulated three times (in prepa-ration). By repetition we could observe how inter-neurons recorded from the connectives respondedor failed to respond to repeated stimulation. Thisalso allowed us to estimate the “noise” that waspresent in the abdominal nerve cord. While thesecriteria are not perfect (i.e., some cells that areincluded may not be part of the abdominal posi-tioning system and vice versa), we feel that theyare reasonable based on these observations.

In order to accurately determine the number ofAPIs participating during a fictive movement, ac-tivity from neurons that have bidirectional axonsshould only be counted once. However, this taskwas not always possible and we may have un-derestimated the number of bidirectional neu-rons due to several reasons: 1) branch pointfailure (Grossman et al., ’79a, b; Nicholls et al.,’92) (see Discussion for further explanation); 2)axonal inhibition in other parts of the nervecord; 3) waveform distortion; and 4) axonal dam-age during dissection.

In some experiments it was only possible torecord from one rostral and caudal hemicon-nective. Since APIs and nerve cord activity for flex-ion and extension are almost entirely bilaterallysymmetrical, counts of interneurons from theseexperiments were doubled.

RESULTSSingle APIs were impaled and depolarized to

give a motor output typical of those underlyingpostural movements. At the same time recordingswere made of evoked axonal activity from the ros-tral and caudal connectives. Several features re-garding the interneuronal circuitry controllingabdominal positioning were determined. First, weestimated the size of each interneuronal group andthe firing frequency of each unit. Second, we stud-ied the relationship between interneuronal groupactivity and the production of different motor out-puts in different segments of the nerve cord.

Evoked interneuronal activity was divided into

two groups: those that were recruited and thosethat showed increased activity. Interneuronsclassed as recruited were silent before API stimu-lation. Other interneurons were spontaneouslyactive before stimulation but increased their fir-ing rate during the stimulus. The average num-ber of interneurons recruited in response tostimulation of an API was 18 (range: 3–50; n =27 preparations). The average number of inter-neurons that increased activity was 14 (range: 3–32) and the average number of interneurons thatdecreased activity was 10 (range: 2–32). The num-ber of interneurons activated and inhibited dur-ing each of these API stimulations is summarizedin Table 1. Stimulation of most APIs produced aflexor motor output, one API produced an exten-sion motor program, and activation of two otherAPIs excited both flexor and extensor motor neu-rons. This type of mixed motor output has been

TABLE 1. Number of recruited interneurons, number ofinterneurons that increased (+) activity, and number ofinterneurons that decreased (–) activity in response to

intracellular stimulation of 27 APIs1

Comments Recruited +Activity –Activitya 30 8 8a 22 10 4a 8 3 3a, b 8 4 3a 6 6 6a 50 18 8a 26 32 18a 10 8 2

38 20 148 11 78 10 9

13 16 811 11 12

c 7 16 1012 17 9

3 11 1118 13 9

6 20 810 16 9

a 26 16 10a 28 22 10

34 20 10a, d 28 14 32c 22 15 10

17 20 1120 10 723 10 14

Average (SD) 18 (11.5) 14 (6.3) 10 (5.7)1All APIs produced fictive abdominal flexion unless otherwise indi-cated. a: Numbers were doubled since this represents a recording froma hemiconnective; b: flexion-producing ingibitor interneuron (this in-terneuron decreased motor activity); c: mixed motor output (excitatoryflexor and extensor motor neurons were activated); d: extension-pro-ducing interneuron.

124 L.D. BREWER AND J.L. LARIMER

observed previously (Murphy et al., ’89). Stimula-tion of another type of API activated a group ofinterneurons that decreased or inhibited flexormotor activity.

The number of interneurons recruited and thenumber that only increased activity were catego-rized according to their firing frequency(2–5, 6–10, 11–15, 16–20, and >20 Hz). Figure 3summarizes how interneurons responded duringstimulation from 20 experiments in which 653 in-terneurons were considered to be affected by APIstimulation. Forty-three percent of all interneu-rons that were recruited or that increased activ-ity fired between 2 and 5 Hz. Twenty-four percentof affected interneurons fired between 6–10 Hz.The total percentage of affected interneurons thatfired above 10 Hz was about 33%. Of this per-centage about half of these fired above 20 Hz.Only very weak motor outputs resulted unless atleast one or two interneurons fired at about 20Hz or more. Interneurons that increased their fir-ing frequencies usually did not exceed 35 Hz, buta few fired as high as 70 Hz. The ranges of firingfrequencies of recruited interneurons observed inthese experiments were similar to those observedin semi-intact animals (Jellies and Larimer, ’86;Murphy et al., ’89).

The latency from stimulus to firing of most re-cruited interneurons was between 20 and 75 ms.Those interneurons that showed longer latenciestended to fire at low frequencies, generally be-tween 2 and 5 Hz. Following API stimulation,most of the evoked interneuronal activity returnedto prestimulus firing rates within 20 ms; however,occasionally some interneurons continued to firehigher than their prestimulus rate.

How group size and firing frequencyaffected motor output

Representative examples of the morphology andmotor output of some impaled APIs are shown be-low (Figs. 4 and 5). As indicated in the model pro-posed by Larimer (’88), stimulation of each APIactivated a set of interneurons. As might be ex-pected, larger interneuronal groups producedstronger motor outputs than smaller interneu-ronal groups, as defined by more motor neuronsfiring at high frequency. However, in a few in-stances (n = 4 of 27), the size and firing frequencyof the interneuronal population were not alwayscorrelated with the strength of motor output; somelarge groups produced a weaker motor output thansome smaller groups.

Flexion-producing interneuronalpopulation

Stimulation of the API shown in Figure 4A pro-duced a strong flexor motor output (Fig. 4B). ThisAPI is one of the most commonly encounteredflexion-producing APIs (Jellies and Larimer, ’85;Larimer and Jellies, ’83; Larimer and Moore, ’84;Larimer and Pease, ’88). Analysis of nerve cordactivity identified 64 interneurons that were af-fected by API stimulation. Of these interneurons34 were recruited (i.e., neurons that were previ-ously silent), 20 increased activity, and 10 wereinhibited. The distribution according to firingfrequency of interneurons that were recruitedand increased activity is presented in Figure4C. Most interneurons (n = 27) fired at low fre-quencies (2–5 Hz); however, a relatively largepopulation of APIs (n = 16) fired at higher fre-quencies (>15 Hz). This group appeared to havea high level of synaptic interactions that pro-duced a strong motor response. This API maybe important in evoking natural flexion due toits extensive ability to recruit other neurons.Stimulation of other APIs recruited very few ifany interneurons at higher frequencies (i.e., Fig.5C, column A).

Fig. 3. Percentage of interneurons that were recruited(lined bars) or increased (+) activity (open bars) according tofiring frequency (Hz) in response to stimulation of an API. Atotal of 653 interneurons from 20 preparations were consid-ered to be affected by stimulation of an API. Approximately43% of all interneurons fired at very low frequencies (2–5Hz) and 15% fired above 20 Hz. Usually only very weak mo-tor outputs were produced unless a group contained at leastone or two cells that fired at about 20 Hz.

SIZE AND OUTPUT OF GROUPS OF INTERNEURONS 125

Different flexion-producing interneuronalgroups provide different and

unexpected motor outputsWhile the strength of motor output was usu-

ally correlated with the size and firing frequencyof the interneuronal group, the examples pre-sented in Figure 5 show that these criteria didnot always indicate the strength of motor ac-tivity. The interneuronal groups and the motoroutputs produced by stimulating two differentflexion APIs were compared. Each API had twoaxons, one that projected in the rostral direc-tion and one that projected in the caudal direc-tion (Fig. 5A1, B1).

During current injection the firing frequenciesof the impaled APIs shown in Figures 5A1, B1 were50 and 60 Hz, respectively. Stimulation of the APIshown in Figure 5A1 produced a strong flexor mo-tor output in the fourth abdominal ganglion (A4F)and an even more robust motor output in the sec-ond abdominal ganglion (A2F) (Fig. 5A2). Stimu-lation of the API shown in Figure 5B1 produced aweaker motor response, especially in the secondabdominal ganglion (A2F) (Fig. 5B2). Stimulationof the API (Fig. 5B1) that produced the weakermotor output activated more than six times asmany interneurons at frequencies above 10 Hz(Fig. 5C, column B) than stimulation of the APIthat produced the stronger motor output (Fig. 5C,column A). Thus the size of an interneuronal groupdid not always indicate the strength of a motorresponse.

Fig. 4. Interneuronal group activated in response to stimu-lation of an identified flexion-producing API. A: Morphologyof a flexion-producing API. This API was located entirely onone side of the midline and had an axon that projected inboth the rostral and caudal directions. Dashed line repre-sents the midline of the ganglion. Rostral is toward the top.B: Flexor motor output (A4F and A2F) produced during stimu-lation of this API. The motor neuron with the largest actionpotential firing in trace A4E is the extensor peripheral in-hibitor. I = current; IN = intracellular electrode; A4F = flexoroutput from fourth abdominal ganglion; A4E = extensor out-put from fourth abdominal ganglion; A2F = flexor output fromsecond abdominal ganglion. C: Frequency histogram of thenumber of interneurons, according to firing frequency, thatwere recruited or increased (+) activity in response to stimu-lation of the API. Each column represents the combined num-ber of interneurons that were recruited (lined bar) andincreased activity (open bar) at a particular frequency. Hence,the total number of affected interneurons as shown in col-umn A at 2–5 Hz was 27. Twenty of these interneuronswere recruited and seven showed increased activity. A rela-tively large number of interneurons (n = 16) were firedabove 15 Hz.Figure 4.

126 L.D. BREWER AND J.L. LARIMER

Comparison between the strength anddirection of interneuronal activity

and motor outputInterneuronal group activity in the rostral and

caudal connectives was compared with the direc-tion and strength of motor output in an effortto explain motor bias. A motor output that isstronger in either the more rostral or caudalmotor roots is defined as biased. Rostral motoroutput refers to motor activity anterior to thesite of intracellular impalement (A3) and cau-dal motor output refers to motor activity poste-rior to A3 (see Fig. 1). The activity (firingfrequency) and the axonal direction of the im-paled API were considered when assessing thestrength of group activity; however, the contri-bution of the impaled API itself was not in-cluded in the following frequency histograms.Only the number of recruited interneurons andthe number of interneurons that increased ac-tivity are included in these figures.

In 8 of 12 experiments the direction andstrength of interneuronal activity were closelycorrelated with the direction and strength ofmotor output (see Table 2). Interneuronal ac-tivity resulting from stimulation of the remain-ing four APIs did not reflect the strength ofmotor output (see Table 2). However, in 10 of12 experiments the strength and direction ofmotor output were correlated with the directionof the stimulated APIs axon (Table 2).

Fig. 5. Comparison of two interneuronal groups activatedby different flexion-producing interneurons. A1, B1: Morphol-ogy of two flexion-producing APIs. Each API had an axonthat projected in both the rostral and caudal directions.Dashed line represents the midline of the ganglion. Rostralis toward the top. A2B2: Flexor motor outputs produced dur-ing stimulation of the command elements in A1 and B1, re-spectively. Stimulation of the API shown in A1 produced aslightly stronger flexor motor output (A2) than stimulation ofthe API shown in B1 (B2). The motor output in root A2F wasmuch stronger in A2 than in B2. During current injection thefiring frequencies of the impaled APIs shown in A1 and B1were 50 and 60 Hz, respectively. The intracellular trace (IN)was not bridge balanced in A2. See Figure 4 for explanationof the abbreviations. C: Frequency histogram of the numberof interneurons, according to spiking frequency, that were re-cruited or increased (+) activity in response to API stimula-tion. Data from columns A and B are counts of interneuronsresulting from stimulation of the APIs shown in A1 and B1,respectively. Very few interneurons were active above 10 Hzduring stimulation of the API shown in A1 (column A), but alarge population of interneurons was activated above 10 Hzduring stimulation of B1 (column B). The population that con-tained a larger number of interneurons that fired at frequen-cies above 10 Hz (column B) produced a weaker motor outputthan the smaller population (column A). This figure followsthe same format as explained in Figure 4.

TABLE 2. Direction of the stimulated APIs axon, anddirection and strength of both the interneuronal

and motor activity1

Axonal Nerve cord Motordirection activity activityR and C2 R = C R = CR and C2 R = C R =CR and C2 R < C R < CC2 R < C C onlyC2 R < C R < CC2 R < C R < CR2 R = C R = CR2 R > C R > CR and C3 R < C R = CR and C3 R < C R = CR and C3 R > C R = CR3 R = C R > C1All 3 variables are presented in relation to each other from 12 ex-periments. All impaled APIs were flexion-producing. R = rostral; c =caudal; > = stronger than; < = weaker than; = indicates no bias ob-served.2Direction and strength of activity in nerve cord were reflected inmotor output.3Direction and strength of activity in verve cord did not reflect motoroutput.

SIZE AND OUTPUT OF GROUPS OF INTERNEURONS 127

Specific example of interneuronal activityand motor output that was correlated

Stimulation of an API with a single axon thatprojected in the caudal direction (Fig. 6A) resulted

Fig. 6. Stimulation of a flexion-producing interneuron thatresulted in both interneuronal and motor output activitiesthat were biased in the caudal direction. A: Morphology of aflexion-producing API that projected an axon in the caudaldirection. Dashed line represents the midline of the ganglion.Rostral is toward the top. B: Stimulation of this API resultedin a stronger caudal (A4F) than rostral (A2F) flexor motoroutput. The intracellular trace (IN) was not bridge balanced.See Figure 4 for explanation of the abbreviations. C: Fre-quency histogram of the number of interneurons, accordingto spiking frequency, that were recruited or increased (+)activity in the rostral (column A) and caudal (column B)connectives as a result of intracellular stimulation of theAPI. Each column represents the combined number of in-terneurons that were recruited and increased activity ata particular frequency. Hence, the total number of affectedinterneurons as shown in column A at 2–5 Hz was six.Two of these interneurons were recruited and four showedincreased activity.

in a stronger output from the caudal flexor motorroots (Fig. 6B, trace A4F), while rostral motor out-put was considerably weaker (Fig. 6B, trace A2F).The number of interneurons responding to APIstimulation from the rostral and caudal con-nectives was about equal, 12 and 14, respectively,but the number of interneurons firing above 10Hz was four times greater in the caudal direction(Fig. 6C). Eight axons from the caudal connectivesfired above 10 Hz, but only two axons from therostral connectives fired at higher frequencies.Hence, both interneuronal and motor activity werebiased (stronger) in the caudal direction.

Specific example of interneuronal activityand motor output that was not correlated

Stimulation of an API with axons that projectedin both the rostral and caudal directions (Fig.7A) resulted in an equally robust output fromthe rostral and caudal flexor motor roots (Fig.7B). However, interneuronal activity in the cau-dal connectives was stronger than that in the ros-tral connectives (Fig. 7C). Six axons fired above20 Hz in the caudal connectives, while only oneaxon fired above 20 Hz in the rostral con-nectives. Hence, the stronger interneuronal ac-tivity in the caudal connectives might be expectedto produce a stronger caudal motor output. How-ever, this was not observed; instead, motor outputwas not biased in either direction. Inconsistenciesof this kind suggest that factors such as synap-tic efficacy and the type of interneurons re-cruited are of importance in explaining bias aswell as the impulse traffic traveling in a par-ticular direction.

128 L.D. BREWER AND J.L. LARIMER

DISCUSSIONThe number of interneurons that comprised a

functional group controlling the fictive abdominalpositioning movements examined here rangedfrom 11 to 68. A total of 360 APIs have been esti-mated in abdominal ganglia one through six byLarimer and Pease (’88); therefore, only a subsetof this total is active during a fictive behavior.Only APIs in ganglia two through five were ex-amined in this study, and from these gangliaLarimer and Pease (’88) estimated that 214 APIsare present. Since the APIs from ganglia one andsix were not included, the number of APIs par-ticipating in an abdominal positioning behavioris greater than that reported here. However, wefeel our sample gives us some indication of theorganization and functional activity of a group ofinterneurons participating in a specific behavior.While we cannot be sure that every interneuroncounted participated in a fictive behavior, previ-ous evidence suggests that most of these inter-neurons are APIs that are synaptically recruitedinto functional groups (Jellies and Larimer, ’86;Murphy et al., ’89).

Usually interneuronal groups that containedrelatively large numbers of interneurons firing athigh frequencies (>15 Hz) produced strong motoroutputs. Thus coding for abdominal positioningmovements appears to be determined by the to-tal spike frequency emanating from a recruitedgroup of APIs. This type of coding also controlsthe direction of cockroach turning during escape(Liebenthal et al., ’94). Little is known about howAPIs are recruited, but it is probable that thestrength of sensory or descending inputs influ-ences the number and firing frequencies of theAPIs that form a functional group.

However, the total number of spikes did not al-

Fig. 7. Stimulation of a flexion-producing interneuron thatresulted in an interneuronal activity that was stronger inthe caudal connectives, but the motor output was equallystrong in the rostral and caudal directions. A: Morphology ofa flexion-producing API that projected an axon in the rostraldirection. Dashed line represents the midline of the ganglion.Rostral is toward the top. B: Stimulation of this API resultedin a flexor motor output that was equally strong in the cau-dal (A4F) and rostral (A2F) motor roots. See Figure 4 forexplanation of the abbreviations. C: Frequency histogram ofthe number of interneurons, according to spiking frequency,that were recruited or increased (+) activity in the rostral(column A) and caudal (column B) connectives as a result ofintracellular stimulation of the API. Interneuronal activitywas stronger in the caudal connectives, but the motor out-puts were equally strong in the rostral and caudal directions.This figure follows the same format as explained in Figure 6.Figure 7.

SIZE AND OUTPUT OF GROUPS OF INTERNEURONS 129

ways indicate the strength of motor output. In afew instances strong motor outputs occurred eventhough relatively few interneurons fired at highfrequencies and vice versa (Fig. 5). Several expla-nations can be offered to account for these results.Strong synaptic inputs from only a few interneu-rons could have produced a robust motor output.The large number of interneurons firing at lowfrequencies could also have contributed toward astrong motor output through temporal and/orspatial summation. The relatively weak motor out-puts may have occurred even though interneu-ronal group activity was strong because very fewof these interneurons may have had synaptic in-puts onto the motor neurons, or the connectionswere comparatively weak (Fig. 5). There is alsothe possibility that inhibitory inputs onto the in-terneurons or the motor neurons resulted in aweaker motor output. Thus other factors in addi-tion to the size of the recruited group may influ-ence motor neuron firing. These and other possibleexplanations are further discussed in the follow-ing section.

Interneuronal activity and motor biasKennedy et al. (’67) found that stimulation of cer-

tain APIs, then called command neurons, produceda stronger motor output in either the rostral or cau-dal direction (motor bias). The production of motorbias programs is believed to be an importantmechanism of achieving a wide variety of abdomi-nal movements. Larimer and Pease (’88) found astrong correlation between the direction of motorbias and the axonal projection of a stimulated API;however, there were exceptions to this rule. Theresults reported here confirm and extend the find-ings of Larimer and Pease (’88). We have added anew dimension to the analysis of bias by compar-ing the direction and magnitude of bias with thedirection, size, and firing frequencies of the inter-neuronal groups that underlie fictive behaviors.

The strength and direction of interneuronal ac-tivity were compared with the strength and thedirection of motor output from interneuronalgroups recruited by stimulation of 12 differentAPIs (Table 2). Six of these 12 groups exhibited amotor bias, and in 5 of these 6 the strength anddirection of interneuronal activity reflected the di-rection of motor bias. The most likely mechanismsthat could produce a motor bias would include alarger population of APIs firing in one directionthan another, stronger synaptic inputs in one di-rection, or inhibitory inputs onto the APIs or ontomotor neurons, in particular ganglia. For example,

in one experiment there was a rostral motor biasbut the nerve cord activity was equally robust inthe rostral and caudal directions (Table 2). In thiscase we would predict that there were strongersynaptic inputs onto the motor neurons in themore rostral ganglia, or inhibitory inputs ontothe interneurons or motor neurons in the morecaudal ganglia resulted in a weaker caudal mo-tor output. However, we cannot rule out the pos-sibility that some of the interneuronal activityin the caudal direction may have been prima-rily concerned with other motor systems (Bur-dohan and Larimer, ’95).

Some of the data from Table 2 can be inter-preted in another manner. Six of the 12 APIsstimulated had bidirectional axons. Stimulationof three of these APIs resulted in a correlationbetween interneuronal and motor activity. Stimu-lation of the remaining three APIs produced astronger interneuronal activity in the rostral orcaudal direction, but the motor output was notbiased (Table 2). Therefore, we must conclude thatthe direction of interneuronal activity does not al-ways indicate the direction of a motor bias.

Several explanations can be offered to accountfor these discrepancies. First, APIs are involvedin coordinating with several motor systems (Mur-chison and Larimer, ’90, ’92; Chrachri et al., ’94;Burdohan and Larimer, ’95); therefore, some ofthese interneurons may have been involved pri-marily with motor systems other than abdominalpositioning. Second, the synaptic efficacy of APIscould greatly determine the strength of motor out-put. Third, flexor inhibitory interneurons are fre-quently encountered and some APIs frequentlyreceive inhibitory postsynaptic potentials (per-sonal observation). Therefore, an API providingexcitatory inputs onto motor neurons in one gan-glion may be inhibited in another ganglion. Oneinhibitory interneuron was analyzed (Table 1), andstimulation of this flexor inhibitor evoked activ-ity in 12 interneurons that fired at frequenciesthat ranged from 3 to 33 Hz. Little is known abouthow these inhibitory interneurons interact withinthe abdominal positioning system. Fourth,neuromodulators have been shown to affectswimmeret and abdominal positioning motorsystems (Livingstone et al., ’80; Harris-Warrickand Kravitz, ’84; Kravitz, ’88; Ma et al., ’92;Barthe et al., ’93; Chrachri and Neil, ’93). It isconceivable that a modulator could be affectingsome ganglia more than others. Fifth, local inter-neurons may be important modulators within theabdominal positioning system. At least two iden-

130 L.D. BREWER AND J.L. LARIMER

tified local nonspiking interneurons have beenshown to inhibit local excitatory flexor motor ac-tivity in P. clarkii (Jellies and Larimer, ’85; Togaet al., ’90). Sixth, interneurons with bidirectionalaxons could also be important in producing a mo-tor bias. Based on previous morphological data,about one third of all APIs have bidirectionalaxons, thus about two thirds of the remainingAPIs have axons that course only in the rostralor caudal direction (Miall and Larimer, ’82a, b;Larimer and Jellies, ’83; Larimer and Moore, ’84;Jellies and Larimer, ’85, ’86). Only occasionallywas it possible to show that a given recruited APIhas a bidirectional axon based on analyzing fir-ing frequency in the rostral and caudal con-nectives. Therefore, the number of recruited APIswith bidirectional axons was probably underesti-mated. We can speculate that the inability to de-tect these recruited APIs may have been due toseveral factors: 1) axonal inhibition in other partsof the nerve cord, 2) axonal damage during dis-section, 3) waveform distortion, or 4) branch pointfailure (Grossman et al., ’79a, b; Nicholls et al.,’92). Conduction block or branch point failurecould produce a motor bias since one axon firingat a high frequency could produce a vigorous mo-tor response in one direction while the other axonfrom the same interneuron firing at a lower fre-quency would produce a weaker response in theother direction. In this manner the parts of theone neuron could function as two separate units.

Organization of the abdominalpositioning system

Why are so many interneurons required for thisbehavior? At least two explanations can be offered.First, APIs are known to participate in severalbehaviors, including swimmeret (Murchison andLarimer, ’90, ’92) and uropod movements (Taka-hata and Hisada, ’85, ’86a, b; Burdohan andLarimer, ’95). Since many APIs have axonal pro-jections that course through the thoracic ganglia(Larimer and Jellies, ’83; Larimer and Moore, ’84)they probably interact with other neural centersas well (Barthe et al., ’93; Chrachri and Neil, ’93;Chrachri et al., ’94). This type of distributed or-ganization has been observed in a wide variety ofanimals (Cleary and Byrne, ’93; Otto and Hennig,’94; Tsau et al., ’94; Wu et al., ’94).

A second reason for the large number of APIsmay be that no single API codes for an entire be-havior. Rather, positioning behaviors are encodedby ensembles of interneurons (APIs) organizedinto functional groups that act in concert (Larimer,

’88). During any one movement, a subset (group)of the total population of APIs is activated. Withina group each API makes only a fractional contri-bution toward a behavior (Larimer, ’88); however,some APIs probably make larger contributionsthan others based on firing frequency and theirsynaptic efficacy with motor neurons (discussedbelow). Each API may belong to more than onegroup, and as a result the contribution of any oneAPI during different behaviors may be quite vari-able. For example, an API may fire at a high fre-quency while acting as a member of one group,but as part of another group the same API mayfire at a lower frequency. The combination of manyinterneurons or APIs into a seemingly infinitenumber of groups provides the variety requiredto account for the large repertoire of abdominalpositioning behaviors.

While the number of groups formed are ex-tremely large, we have recently completed stud-ies that indicate that at least some of these groupsmay have some degree of cohesion (Brewer andLarimer, ’94). For example, repetitively stimulat-ing the same API with a microelectrode in thesame animal consistently activates the samegroup of interneurons provided synaptic fatiguedoes not occur. Furthermore, stimulating the sameAPI in different animals activates a similar butnot identical number of interneurons (Brewer andLarimer, ’94).

Comparison of abdominal positioningsystem with other motor systems

Several large populations of neurons that con-trol behavior in invertebrates as well as verte-brates are now being studied. As with theabdominal positioning system, other systemsappear to use subsets from the larger interneu-ronal population to perform particular tasks.

During cockroach escape behavior a large popu-lation of thoracic interneurons (over 100) controlsleg movements. These thoracic interneuronsproject through several ganglia to affect leg mo-tor neurons and are organized into parallel andserial groups (Ritzmann and Pollack, ’86, ’90;Casagrand and Ritzmann, ’91). Furthermore,some interneurons or groups affect motor neuronaloutput more strongly than other groups (Ritzmannand Pollack, ’90). Several other systems are alsoorganized similarly, including the interneuronsthat control the head movements of crickets dur-ing eye cleaning (Hensler, ’88), the local bendingreflex of leeches (Lockery and Kristan, ’90), andthe descending propriospinal neurons that control

SIZE AND OUTPUT OF GROUPS OF INTERNEURONS 131

fictive scratching in turtles (Berkowitz and Stein,’94a, b). Hence, the interneuronal organizationthat controls these behaviors is very similar tothe abdominal positioning system.

In summary, abdominal positioning movementsare encoded by a synaptically recruited functiongroup of interneurons that represents a subsetformed from a larger population of APIs. Our ob-servations show that some APIs recruited largegroups of interneurons while others recruitedmuch smaller groups. Usually, large groups withinterneurons firing at high frequencies producedstrong motor outputs while smaller groups withinterneurons firing at lower frequencies producedweaker motor outputs. However, on occasion somelarge groups with interneurons firing at high fre-quencies produced relatively weak motor outputs,and some smaller groups produced relativelystrong motor outputs. The contribution of a grouptherefore may depend on such factors as groupsize, firing frequency, synaptic efficacy, the pres-ence of inhibitory inputs, neuromodulation, andthe role of local circuits.

ACKNOWLEDGMENTSFigures were prepared by Gwen Gage, Janet

Young, and Kristina Schlegel. We thank Drs.Wesly Thompson and John Burdohan for readingan early version of the manuscript. This researchwas supported by NIH grant NS05423, a JacobJavits award to J.L.L.

LITERATURE CITEDAtwood, H.L., and C.A. Wiersma (1967) Command inter-

neurons in the crayfish nervous system. J. Exp. Biol.,46:249–261.

Barthe, J.-Y., M. Bevengut, and F. Clarac (1993) In vitro,proctolin and serotonin induced modulations of the ab-dominal motor system activities in crayfish. Brain Res.,623:101–109.

Berkowitz, A., and P.S.G. Stein (1994a) Activity of descend-ing propriospinal axons in the turtle hindlimb enlargementduring two forms of fictive scratching: Broad tuning to re-gions of the body surface. J. Neurosci., 14:5089–5104.

Berkowitz, A., and P.S.G. Stein (1994b) Activity of descend-ing propriospinal axons in the turtle hindlimb enlargementduring two forms of fictive scratching: Phase analyses. J.Neurosci., 14:5105–5119.

Brewer, L.D., and J.L. Larimer (1994) Estimation of identi-fied interneurons forming a functional group controlling ab-dominal positioning in the crayfish. Soc. Neurosci. Abstr.,20:1407.

Burdohan, J.A., and J.L. Larimer (1995) Interneurons in-volved in the control of multiple motor centers in crayfish.J. Exp. Zool., 273:204–215.

Casagrand, J.L., and R.E. Ritzmann (1991) Localization ofventral giant interneuron connections to the ventral me-

dian branch of thoracic interneurons in the cockroach. J.Neurobiol., 22:643–658.

Chrachri, A., and D.M. Neil (1993) Interaction and synchro-nization between two abdominal motor systems in crayfish.J. Neurophysiol., 69:1373–1383.

Chrachri, A., D.M. Neil, and B. Mulloney (1994) State-dependent responses of two motor systems in the crayfish,Pacifastacus leniusculus. J. Comp. Physiol. A., 175:371–380.

Churchland, P.S., and T.J. Sejnowski (1992) The Computa-tional Brain. MIT Press, Cambridge, MA.

Cleary, L.J., and J.H. Byrne (1993) Identification and char-acterization of a multifunction neuron contributing to de-fensive arousal in Aplysia. J. Neurophysiol., 70:1767–1776.

Davis, W.J., and D. Kennedy (1972) Command interneu-rons controlling swimmeret movements in the lobster.II. Interactions of effects on motoneurons. J. Neuro-physiol., 35:13–19.

Evoy, W.H., and D. Kennedy (1967) Central nervous orga-nization underlying control of antagonistic muscles inthe crayfish. I. Types of command fibers. J. Exp. Zool.,165:223–238.

Georgopoulos, A.P., J. Ashe, N. Smyrnis, and M. Taira(1992) The motor cortex and the coding of force. Sci-ence, 256:1692–1695.

Grossman, Y., I. Parnas, and M.E. Spira (1979a) Differentialconduction block in branches of a bifurcating axon. J.Physiol., 295:283–305.

Grossman, Y., I. Parnas, and M.E. Spira (1979b) Ionic mecha-nisms involved in differential conduction of action poten-tials at high frequency in a branching axon. J. Physiol.,295:307–322.

Harris-Warrick, R.M., and E.A. Kravitz (1984) Cellularmechanisms for modulation of posture by octopamine andserotonin in the lobster. J. Neurosci., 4:1976–1993.

Hensler, K. (1988) Intersegmental interneurons involved inthe control of head movements in crickets. J. Comp. Physiol.A., 162:111–126.

Jellies, J., and J.L. Larimer (1985) Synaptic interactions be-tween neurons involved in the production of abdominal pos-ture in the crayfish. J. Comp. Physiol. A, 156:861–873.

Jellies, J., and J.L. Larimer (1986) Activity of crayfish ab-dominal positioning interneurons during spontaneous andsensory-evoked movements. J. Exp. Biol., 120:173–188.

Jones, K.A., and C.H. Page (1986) Postural interneurons inthe abdominal nervous system of lobster. II. Evidence forneurons having both command and driver roles. J. Comp.Physiol. A, 158:273–280.

Kennedy, D., W.H. Evoy, B. Dan, and J.T. Hanawalt (1967)The central nervous organization underlying control of an-tagonistic muscles in the crayfish. II. Coding of position bycommand fibers. J. Exp. Zool., 165:239–248.

Kravitz, E.A. (1988) Hormonal control of behavior: Aminesand the biasing of behavioral output in lobsters. Science,241:1775–1781.

Larimer, J.L. (1988) The command hypothesis: A new viewusing an old example. Trends Neurosci., 11:506–510.

Larimer, J.L., and A. Eggleston (1971) Motor programsfor abdominal positioning in crayfish. Z. Vgl. Physiol.,74:388–402.

Larimer, J.L., and J. Jellies (1983) The organization offlexion-evoking interneurons in the abdominal nervecord of the crayfish, Procambarus clarkii. J. Exp. Zool.,226:341–351.

Larimer, J.L., and D. Moore (1984) Abdominal positioning in-

132 L.D. BREWER AND J.L. LARIMER

terneurons in crayfish: Projections to and synaptic activa-tion by higher CNS centers. J. Exp. Zool., 230:1–10.

Larimer, J.L., and C.M. Pease (1988) A quantitative study ofcommand elements for abdominal positioning behavior inthe crayfish, Procambarus clarkii. J. Exp. Zool., 247:45–55.

Lee, C., W.H. Rohrer, and D.L. Sparks (1988) Population cod-ing of saccadic eye movements by neurons in the superiorcolliculus. Nature, 332:357–360.

Liebenthal, E., O. Uhlmann, and J.M. Camhi (1994) Criticalparameters of the spike trains in a cell assembly: Coding ofturn direction by the giant interneurons of the cockroach.J. Comp. Physiol. A, 174:281–296.

Livingstone, M., R.M. Harris-Warrick, and E.A. Kravitz (1980)Serotonin and octopamine produce opposite postures in lob-sters. Science, 208:76–79.

Lockery, S.R., and W.B. Kristan (1990) Distributed process-ing of sensory information in the leech. II. Identification ofinterneurons contributing to the local bending reflex. J.Neurosci., 10:1816–1829.

Ma, P.M., B.S. Beltz, and E.A. Kravitz (1992) Serotonin-con-taining neurons in lobsters: Their role as “gain-setters” inpostural control mechanisms. J. Neurophysiol., 68:36–54.

Miall, R.C., and J.L. Larimer (1982a) Interneurons in-volved in abdominal posture in crayfish: Structure, func-tion and command fiber responses. J. Comp. Physiol. A,148:159–173.

Miall, R.C., and J.L. Larimer (1982b) Central organization ofcrustacean abdominal posture motoneurons: Connectivityand command fiber inputs. J. Exp. Zool., 224:45–56.

Murchison, D., and J.L. Larimer (1990) Dual motor out-put interneurons in the abdominal ganglia of the cray-fish Procambrus clarkii: Synaptic activation of motoroutputs in both the swimmeret and abdominal position-ing systems by single interneurons. J. Exp. Biol.,150:269–293.

Murchison, D., and J.L. Larimer (1992) Synaptic interac-tions among neurons that coordinate swimmeret and ab-dominal movements in the crayfish. J. Comp. Physiol. A,170:739–747.

Murphy, B.F., M.L. McAnelly, and J.L. Larimer (1989) Ab-dominal positioning interneurons in crayfish: Participationin behavioral acts. J. Comp. Physiol. A, 165:461–470.

Nicholls, J.N., A.R. Martin, and B.G. Wallace (1992) From

Neuron to Brain, Ed. 3. Sinaurer Associates, Inc., Sunder-land, MA, pp. 140, 450.

Otto, D., and R.M. Hennig (1993) Interneurons descendingfrom the cricket subesophageal ganglion control stridula-tion and ventilation. Naturwissenschaften, 80:36–38.

Ritzmann, R.E., and A.L. Pollack (1986) Identification of tho-racic interneurons that mediate giant interneuron-to-mo-tor pathways in the cockroach. J. Comp. Physiol. A,159:639–654.

Ritzmann, R.E., and A.L. Pollack (1990) Parallel motor path-ways from thoracic interneurons of the ventral giant inter-neuron system of the cockroach, Periplaneta americana. JNeurobiol., 21:1219–1235.

Stewart, W.W. (1978) Functional connections between cellsas revealed by dye-coupling with a highly fluorescentnapthalamide tracer. Cell, 14:741–759.

Takahata, M., and M. Hisada (1985) Interactions betweenthe motor systems controlling uropod steering and abdomi-nal posture in crayfish. J. Comp. Physiol. A, 157:547–554.

Takahata, M., and M. Hisada (1986a) Local nonspiking in-terneurons involved in gating of the descending motor path-way in crayfish. J. Neurophysiol., 56:718–731.

Takahata, M., and M. Hisada (1986b) Sustained membranepotential change of uropod motor neurons during the fic-tive abdominal posture movement in crayfish. J. Neuro-physiol., 56:702–717.

Toga, T., M. Takahata, and M. Hisada (1990) An identifiedset of local nonspiking interneurons which control the ac-tivity of abdominal postural motoneurones in crayfish. J.Exp. Biol., 148:477–482.

Tsau, Y., J.-Y. Wu, H.P. Hopp, L.B. Cohen, D. Schiminovich,and C.X. Falk (1994) Distributed aspects of the response tosiphon touch in Aplysia: Spread of stimulus information andcross correlation analysis. J. Neurosci., 14:4167–4184.

van Harreveld, A. (1936) A physiological solution for fresh-water crustaceans. Proc. Soc. Exp. Biol., 34:428–432.

Wu, J.-Y., L.B. Cohen, and C.X. Falk (1994) Neuronal activ-ity during different behaviors in Aplysia: A distributed or-ganization? Science, 263:820–823.

Zecevic, D., J.-Y. Wu, L.B. Cohen, J.A. London, H.P. Hopp,and C.X. Falk (1989) Hundreds of neurons in the Aplysiaabdominal ganglion are active during the gill-withdrawalreflex. J. Neurosci., 9:3681–3689.