Coupling of Efferent Neuromodulatory Neurons to Rhythmical Leg Motor Activity in the Locust
Sylvie Baudoux1,
Carsten Duch2, and
Oliver T. Morris1
1 Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom; and 2 Institut für Neurobiologie, Freie Universität Berlin, D-14195 Berlin, Germany
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ABSTRACT |
Baudoux, Sylvie, Carsten Duch, and Oliver T. Morris. Coupling of efferent neuromodulatory neurons to rhythmical leg motor activity in the locust. J. Neurophysiol. 79: 361-370, 1998. The spike activity of neuromodulatory dorsal unpaired median (DUM) neurons was analyzed during a pilocarpine-induced motor pattern in the locust. Paired intracellular recordings were made from these octopaminergic neurons during rhythmic activity in hindleg motor neurons evoked by applying pilocarpine to an isolated metathoracic ganglion. This motor pattern is characterized by two alternating phases: a levator phase, during which levator, flexor, and common inhibitor motor neurons spike, and a depressor phase, during which depressor and extensor motor neurons spike. Three different subpopulations of efferent DUM neurons could be distinguished during this rhythmical motor pattern according to their characteristic spike output. DUM 1 neurons, which in the intact animal do not innervate muscles involved in leg movements, showed no change apart from a general increase in spike frequency. DUM 3 and DUM 3,4 neurons produced the most variable activity but received frequent and sometimes pronounced hyperpolarizations that were often common to both recorded neurons. DUM 5 and DUM 3,4,5 neurons innervate muscles of the hindleg and showed rhythmical excitation leading to bursts of spikes during rhythmic activity of the motor neurons, which innervate these same muscles. Sometimes the motor output was coordinated across both sides of the ganglion so that there was alternating activity between levators of both sides. In these cases, the spikes of DUM 5 and DUM 3,4,5 neurons and the hyperpolarization of DUM 3 and DUM 3,4 neurons occurred at particular phases in the motor pattern. Our data demonstrate a central coupling of specific types of DUM neurons to a rhythmical motor pattern. Changes in the spike output of these particular efferent DUM neurons parallel changes in the motor output. The spike activity of DUM neurons thus may be controlled by the same circuits that determine the action of the motor neurons. Functional implications for real walking are discussed.
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INTRODUCTION |
Neuromodulation is essential for the adaptive regulation of an animal's neural and motor machinery because it allows the adjustment of motor patterns to fit different behavioral needs. Neuromodulators can influence profoundly all steps of information processing in neuronal pathways. They alter signaling by sensory neurons (Pasztor and Bush 1989
; Ramirez and Orchard 1990
) and the activity of central pattern generating networks (Chiel et al. 1990
; Harris-Warrick et al. 1992
; Katz and Harris-Warrick 1990
; Stevenson and Kutsch 1988
), and they have presynaptic effects on the terminals of the motor neurons and postsynaptic effects on muscle fibers (Evans and O'Shea 1977
, 1978
; O'Shea and Evans 1979
). Neuromodulators may be released locally in specific parts of the CNS, onto particular groups of muscle fibers or specific sensory organs, or they may be released more generally into the circulatory system.
The locust has a group of efferent neuromodulatory neurons, called the dorsal unpaired median (DUM) neurons, which contain (Evans and O'Shea 1978
) and release octopamine (Morton and Evans 1984
; Stevenson and Spörhase-Eichmann 1995
). Efferent DUM neurons have their somata at the dorsal midline of each segmental ganglion and project bilaterally to the periphery. The metathoracic ganglion of the locust contains 19 efferent DUM neurons (Campbell et al. 1995
) of different types that can be distinguished by their axonal projection patterns through the different lateral nerves (Watson 1984
). The peripheral effects of these neurons include the modulation of neurotransmitter release from motor neurons, the reduction of basal muscle tension, the increase of muscular twitch contraction force, the increase of muscle relaxation rate, and the suppression of myogenic rhythms (Evans and O'Shea 1977
, 1978
; Evans and Siegler 1982
; Malamud et al. 1988
; Whim and Evans 1988
). Neuromodulatory effects of octopamine on wing and leg proprioceptors have been demonstrated in the locust (Eder and Bräunig 1996
; Matheson 1997
; Ramirez and Orchard 1990
). So far there is no direct evidence that efferent DUM neurons also can influence central processing, and although DUM neurons have extensive branching patterns within the CNS, no conventional output synapses have been found (Watson 1984
).
The efferent DUM neurons were thought previously to act collectively during arousal (Hoyle and Dagan 1978
). In ballistic kicks of a locust hindleg, however, specific DUM neurons are activated in a distinctive and repeatable pattern that is linked closely to the motor pattern, whereas other DUM neurons are inhibited or unaffected (Burrows and Pflüger 1995
). These observations support the hypothesis that these neuromodulatory neurons act individually or in particular combinations in the control of movement. It is, however, not known if DUM neurons are coupled to central-pattern-generating networks or what roles they might play during a rhythmical motor behavior such as walking. If neuromodulators are to modify a specific behavior, then they should be released at the right time and in the appropriate location. This implies that the action of neuromodulatory neurons should constitute part the motor program. Such a link between motor patterns and neuromodulatory action could result from either sensory feedback acting on particular neuromodulatory neurons or from central coupling.
Both central and peripheral mechanisms seem to be responsible for the generation of walking movements in both vertebrates and invertebrates (Bässler 1993
; Delcomyn 1980
; Graham 1985
; Grillner 1985
; Pearson 1993
; Zill 1986
). Nevertheless, isolated CNSs treated pharmacologically can produce rhythmical motor patterns. In arthropods, isolated parts of the CNS have been induced to produce rhythmic motor activity by application of the muscarinic agonist pilocarpine (e.g., Chrachri and Clarac 1990
; Gorczyca et al. 1990; Johnston and Levine 1996
). In locusts and stick insects, pilocarpine application to an isolated thoracic ganglion results in coordinated rhythmical activity in the leg motor neuron pools (Büschges et al. 1995
; Ryckebusch and Laurent 1993
). Ryckebusch and Laurent concluded that pilocarpine activated a central pattern generator, which produced a pattern of motor neuron activity that would be appropriate for the movement of a single leg of a segmental pair of legs during walking. In a second study, using a chain of two or three thoracic ganglia treated with pilocarpine, they showed that the motor output of different segments is coupled centrally in a manner appropriate for the alternating tripod gait of walking (Ryckebusch and Laurent 1994
).
Here we investigate the activity of the efferent DUM neurons during this rhythmic activity appropriate for leg movements. Our study shows that efferent DUM neurons innervating the legs are rhythmically active during the motor rhythm, whereas DUM neurons innervating other targets are not. Furthermore, the spike activity of some DUM neurons is coupled to this centrally generated motor rhythm.
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METHODS |
Preparation
Experiments were carried out using 48 adult desert locusts, Schistocerca gregaria (Forksål), of either sex, taken from our crowded laboratory colony. We used an in vitro preparation as described by Ryckebusch and Laurent (1993)
. The metathoracic ganglion was dissected out of the animal, leaving the leg nerves long and the longitudinal tracheae intact, and pinned dorsal side uppermost in a silicone elastomer (Sylgard)-lined chamber filled with locust saline [composition (in mM): 140 NaCl, 10 KCl, 4 CaCl2, 4 NaHCO3, and 6 NaHPO4]. The tracheae were opened up at the surface to maintain a supply of air. Protease (SIGMA type XIV) was applied directly to the dorsal surface of the metathoracic ganglion as crystals and left to act for ~40 s before being rinsed off with fresh saline. This facilitated penetration with microelectrodes and enabled pilocarpine to pass through the sheath of the ganglion. A stock solution of 0.8 mM pilocarpine hydrochloride (Sigma) in saline was prepared and added to the bath to give final concentrations in the range 0.2-0.4 mM.
Electrophysiology
The rhythmical motor output evoked by pilocarpine under these conditions has been well described and characterized by Ryckebusch and Laurent (1993
, 1994)
. We used polyethylene suction electrodes to record the motor output of the ganglion in leg nerves 3B and 5A because these contain the main units used by Ryckebusch and Laurent to characterize the rhythmical motor pattern. Levator trochanteris and slow extensor tibiae (SETi) spikes could be observed in N3B, and the activity of two depressor trochanteris motor neurons (1 fast, Df; 1 slow, Ds) and the common inhibitor motor neuron 1 (CI1) were recorded in N5A.
Paired intracellular recordings were made from DUM neurons with glass microelectrodes filled with 6% hexaminecobaltic chloride with resistances of 80-150 M
. DUM neurons were identified by their soma position (dorsal midline), characteristic soma spikes (long-duration, large-amplitude spikes with a prominent afterhyperpolarization) and by their characteristic axonal branch patterns after intracellular staining with cobalt (injected by 5-10 nA depolarizing current pulses of 500-ms duration at 1 Hz for 10-20 min). After cobalt injection, 15 min was allowed for diffusion of ions into the lateral nerves and fine processes. The cobalt then was precipitated with ammonium sulphide and the ganglion washed in saline before fixation in formaldehyde. Silver intensification was carried out following the technique of Bacon and Altman (1977)
.
DUM neurons project bilaterally to innervate target organs and muscles on both sides of the body and are characterized morphologically according to the lateral nerves that contain their axons (see Table 1). Three neurons could be identified as individuals using morphological criteria alone: DUMDL and DUM1b could be distinguished by the fact that only DUM1b has an axon in N1A. DUMETi could be distinguished from other DUM 5 neurons by its larger diameter soma and axon and its more extensive fine branches.
Analysis
Data were stored on a Racal four-channel FM tape recorder and analyzed off-line on a computer running Spike2 software (Cambridge Electronic Design) after digitization through a CED 1401plus interface. Spikes in the DUM neurons were converted into separate spike event channels using a hardware window discriminator, whereas extracellular motor nerve recordings were digitized at 10 kHz. Sorting of the extracellular recordings into different motor units was carried out manually with assistance from a spike-sorting option within Spike2. This was necessary because the precise spike waveform of particular motor units varied from preparation to preparation and even within preparations due to changes in the gain of the recordings over time. In addition, some levator trochanteris motor neuron spikes are so similar in shape and size to SETi spikes that they cannot be distinguished by these criteria alone. However, because SETi is the only unit that spikes in bursts out of phase with the levators in N3B (see Ryckebusch and Laurent 1993
), it was possible to separate these units on the basis of burst timing. Differences in spike height of the three motor units in N5A were sufficiently consistent for the computer to use this as a reliable criterion for discrimination. Once units had been separated into different spike event channels, the correspondingtime series were used to construct phase histograms and event auto-/cross-correlograms.

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| FIG. 1.
Establishment of the motor pattern after bath application of pilocarpine to an isolated metathoracic ganglion. Motor output was monitored by extracellular recordings of nerves 5A and 3B of the same side and simultaneous intracellular recordings were made from a DUM 5 and a DUM 3,4 neuron. A: before pilocarpine application, the only active motor unit was the slow depressor trochanteris (Ds) in nerve 5A. Both DUM neurons received synaptic inputs but did not spike: the DUM 5 received both excitatory and inhibitory PSPs, whereas the DUM 3,4 received predominantly inhibitory PSPs. B: 5 min after pilocarpine application the motor rhythm consisted of alternate spiking of the levator trochanteris motor neurons (lev) and the common inhibitor 1 (CI1) with the slow depressor trochanteris (Ds). Slow extensor tibiae motor neuron (SETi) was not active. Both DUM neurons spiked but without any clear pattern. C: after 10 min, the motor pattern became fully established, and the lev spiked in phase with CI1 and in antiphase with the slow (Ds) and the fast (Df) depressors trochanteris. DUM 5 neuron now produced rhythmical bursts of spikes, whereas the DUM 3,4 neuron spiked sporadically.
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| FIG. 2.
Synaptic inputs to a DUM 3,4 neuron were linked closely to onset of the motor pattern. During the 1st 2 levator bursts the DUM 3,4 neuron produced 2 spikes superimposed on a hyperpolarization of the baseline membrane potential (shown at higher gain in the bottom set of traces). This characteristic waveform ( ) differed from spikes occurring out of phase with the levator bursts ( ).
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TABLE 2.
Activity recorded in 79 different DUM cells from 48 preparations of a metathoracic ganglion treated with pilocarpine
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Phase histograms were plotted in relation to the levator burst cycle, with each onset of levator bursts in N3B corresponding to phase 0 of one cycle and phase 1 of the next. Each phase histogram was divided into 25 bins of equal width. The spike counts for each bin were divided by the total number of cycles analysed to give a per cycle mean value.
Event auto- and cross-correlograms were constructed using a built-in function of Spike2 as follows: a histogram was defined that contained 200 bins each 200 ms wide to cover time lags from
20 to +20 s. The histogram was centered on the first spike event of the reference time series. The occurrence of spikes at different time lags in relation to this reference spike was represented by increasing the count of the relevant bins. The histogram then was centered on the next spike and the relevant bin counts increased. This process was repeated until the histogram had been centered on every spike of the reference time series. The histogram was not normalized but instead presented total event counts. In the autocorrelograms, the correlation of each event with itself at time 0 in the histogram is ignored, and periodic peaks and troughs with a large peak centered at lag = 0 indicated periodic clustering of spikes.
Similarly, a large peak at lag = 0 in a cross-correlogram indicated that reference and sample spikes tended to occur at the same time as each other.
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RESULTS |
Pilocarpine-induced motor pattern
The pilocarpine-induced motor rhythm described by Ryckebusch and Laurent (1993)
was characterized by two phases: a levator phase, during which the anterior coxal rotator, levator trochanteris, common inhibitor and flexor tibiae motor neurons spiked, and a depressor phase, during which the depressors trochanteris, SETi, and depressor tarsus motor neurons spiked.

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| FIG. 3.
Spike event autocorrelograms demonstrate the difference between bursting and unpatterned spike activity for 3 different types of DUM neuron. Autocorrelograms for DUMDL (top) and DUM 3 (middle) show no clear structuring. DUM 3,4,5 neuron (bottom) produced clearly separated bursts of spikes resulting in an autocorrelogram that had a prominent peak around lag 0. Period of bursting was fairly regular (~7 s), resulting in periodic peaks and troughs in the autocorrelogram. All DUM types have a narrow trough in their autocorrelograms at lag 0 because of their low spike frequencies (i.e., few spikes occur within 200 ms of each other). Data for DUM 3 and DUM 3,4,5 comes from a 170-s continuous recording from the same preparation, data for DUMDL from a 10-min continuous recording from another preparation. Bin width for autocorrelograms was 200 ms.
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The general phase relationships of these units were found to be fixed over time and from preparation to preparation, making it unnecessary to monitor the activity of all of them in the present study. The motor output we recorded in N3B and N5A was consistent with Ryckebusch and Laurent's findings (1993): activity of the levator trochanteris motor neurons in N3B and common inhibitor motor neuron 1 in N5A defined the levator phase of the motor pattern which alternated with activity in SETi (N3B) and the two depressor trochanteris motor neurons (N5A) during the depressor phase. The period of the rhythm varied from cycle to cycle (ranging from 4 to 10 s), and the depressor phase was more variable in duration than the levator phase.
Buildup of rhythmic motor activity followed a characteristic course. Before application of pilocarpine, none of the motor neurons in lateral nerve 3B and only the slow depressor trochanteris (Ds) in N5A spiked (Fig. 1A). The first signs of rhythmic activity appeared within 1-5 min of pilocarpine application and consisted of silent periods in the tonic spiking of Ds during which the CI1 and the levator trochanteris motor neurons (lev) spiked (Fig. 1B). The final rhythm often took 5-10 min to stabilize and included bursts of spikes produced by the fast depressor trochanteris motor neuron (Fig. 1C). In many preparations, the slow extensor tibiae motor neuron (SETi) was the last to be recruited to the rhythm (Fig. 4).

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| FIG. 4.
A: DUMDL spikes sporadically during the motor rhythm. A single levator burst cycle is marked (lev cycle). B: phase histograms of levator spikes (top) and DUMDL spikes (bottom) plotted in relation to the levator burst cycle. Levator spikes peak in the 1st half of the levator cycle, but DUMDL spikes occur throughout the cycle with no clear preferred phase. Data from 108 cycles over 10-min recording. Same preparation as Fig. 3, top.
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| FIG. 5.
Activity of DUM 3 and DUM 3,4 neurons during the motor pattern. Both neurons spike sporadically and share many common inputs, especially hyperpolarizing ones (dotted lines).
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In most preparations (41 of 48), we only recorded the motor output from one side of the ganglion. Rhythmic activity was observed in all 48 ganglia, with reciprocal patterns of activity in levator and depressor phase units. In seven preparations, we recorded the motor output from both sides of the ganglion, and in four of these preparations, the rhythmical motor output was well coordinated across the ganglion, with each levator phase in one hemiganglion coinciding with a depressor phase in the other. This coordinated motor pattern recorded in N5A and N3B resembled that seen in these nerves in a fictive walking preparation (see Ryckebusch and Laurent 1993
).

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| FIG. 6.
DUM 3,4,5 and DUMETi (DUM 5 innervating the extensor tibiae muscle) produce rhythmical bursts of spikes during a well-structured motor pattern, monitored by recordings of nerve 5A and nerve 3B of the same side. Each burst of spikes is superimposed on a wave of depolarization.
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| FIG. 7.
A: 2 DUM 5 neurons burst rhythmically and in phase with each other during the motor rhythm, monitored by extracellular recordings of nerve 5A (top) and nerve 3B (middle) of the same side. Lower DUM neuron generates more spikes despite being injected with some hyperpolarising current. B: autocorrelogram of the DUM 5* spike train has a pronounced peak at lag 0 bounded by 2 smaller troughs, indicating that this neuron produced bursts of spikes separated by gaps. DUM 5/DUM 5* cross-correlogram has a similar shape around lag 0, indicating that both DUM 5 neurons were bursting in phase with each other. Data from 12-min continuous recording.
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Changes in the synaptic drive and spiking of DUM neurons coincided with generation of the rhythmic
motor pattern
Before pilocarpine application, DUM neurons usually spiked infrequently and never in patterned bursts. All types of DUM neurons received postsynaptic potentials (Fig. 1A) and sometimes spiked at low frequencies (0.02-0.1 Hz). Pilocarpine caused an increase in the frequency of spikes (Fig. 1B). As the motor output became established, the spike activity of DUM neurons settled into patterns that were characteristic for the different types (see next section). Injection of hyperpolarizing and depolarizing current into individual DUM neurons did not alter the frequency of the rhythm nor did it add new motor units to the rhythm. Patterned DUM neuron activity never preceded the occurrence of coordinated motor output. Instead, changes in DUM activity often coincided with the first visible changes in motor output. For example, changes in synaptic inputs to a DUM 3,4 neuron coincided precisely with the first burst of levator and CI1 spikes (Fig. 2). During this first levator burst, the DUM 3,4 neuron produced a pair of spikes that was superimposed on a marked hyperpolarization (Fig. 2,
). This suggests simultaneous excitation, giving rise to the pair of spikes, and inhibition, producing the hyperpolarization. The spikes do not seem to result from postinhibitory rebound because the neuron spikes during the hyperpolarization not after it. The resulting waveform was very different from that of other spikes immediately preceding and following this first levator burst (Fig. 2,
). Similar inputs were observed during the second levator burst, but by the third levator burst the DUM 3,4 cell showed only a hyperpolarization.

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| FIG. 8.
A: Activity of a DUM 3,4,5 and a DUM 3,4 neuron during a pilocarpine rhythm in which only 1 hemiganglion produced a structured output. Within this hemiganglion, the levators spiked synchronously with the common inhibitor 1 and alternately with the slow and fast depressor trochanteris motor neurons, whereas the other hemiganglion produced almost no activity (C). DUM 3,4,5 was depolarized and produced a burst of spikes during the levator phase ( ), whereas DUM 3,4 stopped spiking during this phase. B: motor spikes and DUM spikes are plotted as phase histograms relative to the levator cycle of the rhythmically active hemiganglion. Slow depressor trochanteris motor neuron (DS) peaks shortly after the levator burst ends. Majority of DUM 3,4,5 spikes occurred during the levator burst, but there was also an increase from phase 0.5 onward, leading up to the next levator burst. DUM 3,4 shows the opposite spike pattern with few spikes during the levator bursts and frequent spiking during the rest of the cycle period. Data from 114 cycles during 15 min of continuous recording. C: recording of N3B on both sides following that in A showing a noncoordinated motor activity in which the levators do not strictly alternate on a cycle to cycle basis.
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| FIG. 9.
A: a DUM 3,4,5 neuron generates bursts of spikes around the levator phases ( ) of both sides of the ganglion during a motor pattern that was well coordinated across the ganglion. Extracellular recordings of nerves 3B, on the left and on the right, showed alternating levator and depressor phases within each hemiganglion. Levator bursts (lev) alternated from side to side and coincided with SETi bursts from the opposite side, as would be required for tripod walking. Phase histograms in B confirm that the DUM 3,4,5 neuron is more likely to spike during the levator bursts of either side. Left: occurrence of spikes plotted in relation to the levator cycle of the left side. Right: same data plotted in relation to the right levator cycle. Ipsilateral levators spike during the 1st half of the levator cycle of that side, contralateral levators spike out of phase during the 2nd half of the levator cycle. Because the DUM 3,4,5 neuron produces a burst of spikes around each levator burst from either side, the histogram of DUM spikes has 2 peaks that coincide with the ispilateral and contralateral levator peaks. Data from 74 cycles (left side) and 72 cycles (right side) during 13 min of continuous recording.
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Spike activity was characteristic for the different types of DUM neuron
The pattern of spikes observed in DUM neurons during established motor patterns were characteristic for different morphological types. A broad distinction could be made between those DUM types that produced rhythmic bursts of spikes and those that did not (Table 2 and Fig. 3). DUM neurons that produced spikes in bursts resulted in autocorrelograms with a peak at lag 0 surrounded by two troughs, and if the period of bursting was regular, there were further positive peaks at intervals corresponding to the cycle period (Fig. 3, 3rd trace). DUM neurons that spiked sporadically did not have a peak at lag 0 in their auto-correlograms (Fig. 3, top 2 traces): such activity was classified as "unpatterned" for the purposes of Table 2.
DUM 1 NEURONS.
None of the DUM 1 neurons recorded showed rhythmical bursting during the pilocarpine-induced motor pattern, but five recordings revealed sporadic spiking. Figure 4 shows an example where DUMDL received many synaptic potentials, but there was no clear patterning in its spike output. Furthermore, DUMDL did not produce spikes at any preferred phase of the motor pattern (Fig. 4B). In the other two recordings made of DUM 1 neurons, they did not spike at all.
DUM 3 AND DUM 3,4 NEURONS.
These neurons had similar spike activity during pilocarpine-evoked motor rhythms. In most preparations (32/42), they produced unpatterned activity of the type shown in Fig. 5. In 10 preparations where 2 of these neurons were recorded simultaneously for periods
30 min, they showed many simultaneous changes in membrane potential that might have resulted from common synaptic inputs. In some cases (10/42), DUM 3 and DUM 3,4 neurons were observed to produce rhythmical bursts of spikes, although hyperpolarizing inputs were still present.
DUM 3,4,5 AND DUM 5 NEURONS.
Sixteen of 17 DUM 3,4,5 and all 13 DUM 5 neurons recorded during pilocarpine-induced motor patterns produced rhythmical bursts of spikes. An example is shown in Fig. 6, where a DUM 3,4,5 and DUMETi (the DUM 5 neuron that innervates the extensor tibia muscle) showed rhythmical bursts at different phases of the cycle. The frequency of DUM neuron bursting was approximately twice that of the cycle period of one hemiganglion. On other occasions, the two recorded DUM 5 neurons produced bursts during the same phases of the cycle (Fig. 7). The cross-correlogram in Fig. 7B has a similar shape to the autocorrelogram of the upper DUM 5 neuron, indicating that both neurons produced bursts of spikes during the same phases of the cycle.
Activity of individual DUM neurons is coupled to the motor pattern
DUM neurons project bilaterally, so it is possible that they might receive synaptic drive linked to the motor output of both sides of the ganglion. However, in most pilocarpine preparations, the output of the ganglion is not coordinated across the two sides, i.e., levator bursts do not alternate strictly from side to side and the intrinsic cycle frequencies are different for the two sides (Ryckebusch and Laurent 1993
). This would tend to mask any coupling between DUM spiking patterns and motor output of just one side. Coupling between DUM spike activity and the motor pattern was observed in two types of preparation. In the first, and rarer kind, one side of the ganglion produced a well-structured motor pattern, whereas the other side produced a motor pattern with a less stable structure. Although variations did appear over long stretches of time, on a cycle-by-cycle basis there was no strict alternating activity with between the levators of both sides of the ganglion (Fig. 8C) In the second type, the motor output was well coordinated across both sides of the ganglion (Fig. 9) with levator activity in one hemiganglion either preceding or following levator activity of the other side with a short latency of 1-3 s, and never occurring at the same time.
Figure 8 presents an example where only one side of the ganglion produced a well-structured motor output (see Fig. 8C). The spike activity of the two DUM neurons in this preparation occurred during the phase of the motor pattern produced by the structured side. A DUM 3,4,5 neuron was depolarized rhythmically and produced bursts of spikes during each levator burst (highlighted by shaded bars in Fig. 8A), whereas a DUM 3,4 did not spike during this phase of the motor pattern. When DUM spikes were plotted as a phase histogram in relation to the levator cycle, a single peak occurred for the DUM 3,4,5 neuron during the levator burst, whereas the DUM 3,4 neuron produced the fewest spikes during the time when the levator motor neurons were most active (Fig. 8B).
Figure 9 presents an example of a preparation where the motor pattern was well coordinated across the ganglion. The recording of a DUM 3,4,5 neuron in this preparation revealed bursts of spikes around the levator phase of either side (Fig. 9A). This was confirmed by double peaks in the phase histograms relative to the cycle period of the left and right sides (Fig. 9B). Because the DUM 3,4,5 neuron appeared to be coupled to the motor output of both sides of the ganglion, a new reference cycle was created to represent the joint output of both hemiganglia. Here each cycle was defined as from the start of one levator burst to the start of the next levator burst, irrespective from which side the bursts originated. This enabled DUM neuron activity to be compared with the levator bursts of both sides of the ganglion (Fig. 10). Only a single peak was apparent in the DUM 3,4,5 spikes, and this occurred during the onset of the levator bursts. Similarly DUM 3,4,5 and DUM 5 spike activity was coupled to particular phases of the motor pattern in the other three preparations, where the motor pattern was well coordinated across the ganglion.

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| FIG. 10.
Data from Fig. 7 plotted as phase histograms using a reference cycle that represents the joint output of both hemiganglia. Here a single cycle is defined as from the start of 1 levator burst to the start of the next, irrespective of the side from which the bursts originated. When plotted in this way, the levator spikes of both sides (both lev) fall within the 1st half of the cycle, indicating that there is always a gap between a levator burst from 1 side and the next levator burst from the other side, and that this gap is roughly equal to the levator burst duration. Double peak in DUM 3,4,5 spikes observed in Fig. 7B is converted into a single peak that coincides with the peak in levator spikes, confirming the link between the DUM 3,4,5 and the levator phases of either side. Data from 139 cycles during a 13-min recording.
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DISCUSSION |
We have shown that the pattern of spike production in specific efferent neuromodulatory neurons is linked to the rhythmical activity in the leg motor neurons. Our study demonstrates that the spike activity of particular efferent DUM neurons is coupled centrally to a rhythmical motor pattern and that these DUM neurons are not activated as a homogeneous group but as individuals. In our locust preparation, a stable and long-lasting rhythmic activity was induced in leg motor neurons of isolated metathoracic ganglia by the application of pilocarpine. Such rhythms were first described in detail by Ryckebusch and Laurent (1993)
and are characterized by two phases: a levator phase and a depressor phase, which alternate within each hemiganglia. Our results show that these locomotor-like rhythms expressed in leg motor neurons also involve modulation of the activity of efferent DUM neurons and that this rhythmical activation involves only a specific subset of DUM neurons.
In Tritonia, neuromodulatory neurons form part of a central pattern generator and their rhythmic activity is the result of synaptic interactions within the central-pattern-generating networks. In this system, brief hyperpolarization of several neuromodulatory neurons can reset the swim rhythm, whereas depolarization increases the amplitude and duration of the postsynaptic potentials in neurons intrinsic to this circuit (for review, see Katz and Frost 1996
). By contrast, in our study, the neuromodulators were efferent components and changes in the spike output of efferent DUM neurons proceeded in parallel with changes in the motor output. In fact, current injection into individual DUM neurons did not have any obvious effects on the centrally generated motor patterns: it did not change the frequency of the rhythms nor did it introduce new motor units into the rhythms.
Different subsets of DUM neurons were linked differently to the pilocarpine-induced motor rhythm
Efferent DUM neurons were activated differently according to their morphological type, and this finding points to close links between the action of a specific locomotory circuit and particular subsets of DUM neurons. Moreover, it suggests that DUM neurons may be allocated more precisely to specific motor acts than previously anticipated by Hoyle and Dagan (1978)
. This strengthens the hypothesis that different DUM types have specific roles in particular motor behaviors (Burrows and Pflüger 1995
). In all DUM neuron types, a general increase in spike activity could be observed. Such an increase could be induced either by muscarinic receptors on the DUM neurons or by the activation of presynaptic sources that provide a nonspecific excitatory drive. However, there were clear differences in the spiking activity of different DUM neuron types during pilocarpine-evoked motor patterns; this suggests that particular DUM neuron types are activated in parallel to the generation of activity in central-rhythm-generating networks. Thus our results provide clear evidence for a specific excitatory drive of efferent DUM neurons.
Recordings of DUM 1 neurons never showed rhythmical excitation or bursts of spikes during the motor pattern. DUM1b innervates the salivary glands, whereas DUMDL innervates the dorsal longitudinal flight muscle, neither of which are involved directly in producing movements of the legs. Therefore there would appear to be little point in altering their spike output during rhythmical motor programs of the legs.
DUM 3 and DUM 3,4 had the most variable spike activity during pilocarpine-evoked motor patterns. They usually spiked sporadically, but occasionally their activity showed bursts of spikes. The most characteristic feature of these neurons was the presence of frequent and sometimes pronounced hyperpolarizing inputs. Depolarizing inputs also were observed, but in most preparations, they resulted in an apparently unstructured spike output. DUM 3,4 neurons innervate major flight muscles via nerve 4 (Kutsch and Schneider 1987
), but none of the target muscles for DUM 3 or DUM 3,4 neurons via nerve 3 are known (for review, see Stevenson and Spörhase-Eichmann 1995
), making interpretation of their activity difficult. However, it is interesting to note that inhibition of DUM 3 and DUM 3,4 neurons also has been observed during the motor program underlying kicking of a hindleg (Burrows and Pflüger 1995
).
All DUM 5 and DUM 3,4,5 neurons produced bursts of spikes during the pilocarpine motor pattern. These bursts appeared to be caused by rhythmical excitatory inputs, with each burst of spikes usually superimposed on a visible depolarization of membrane potential. Such bursting activity is not seen in isolated DUM somata treated with pilocarpine in vitro (Tuschick 1994
). Moreover, the period of bursting was irregular and, in some cases, synchronized between the two recorded neurons. These observations are not consistent with the hypothesis that DUM cells become endogenous oscillators under pilocarpine treatment. Instead it seems as if their bursting activity results from the action of specific circuits presynaptic to the DUM neurons because some DUM neurons spiked tonically, whereas others showed rhythmical bursting activity.
In some preparations, the rhythmic motor patterns were well coordinated across the ganglion, and the rhythmical output of DUM 5 and DUM 3,4,5 neurons was coupled to the levator phase of either side. Thus these subsets of DUM neurons appear to receive central inputs from local circuits controlling the hindlegs. DUM 5 and DUM 3,4,5 neurons both have axons in nerve 5, which innervates the hindlegs, and are known to project to target muscles involved in moving the legs. These types of DUM neuron also are activated and spike during the motor program, which leads up to ballistic kicking movements of the hindleg (Burrows and Pflüger 1995
).
What effects might DUM neurons have during real walking?
Because the motor output of an isolated ganglion treated with pilocarpine reliably produces a well-structured and long-lasting motor output that shows similarities to walking (Ryckebusch and Laurent 1993
, 1994
), it is worth considering the functional implications of the observed patterns of DUM spike activity.
The spike output of DUM 5 and DUM 3,4,5 neurons was linked to the levator phases of both sides of the ganglion, as was apparent in those preparations where the output was coordinated across the ganglion so that there was alternating activity between levators of both sides. If this were to occur during real walking, DUM neuron spikes would reach similar muscles on both sides of the body simultaneously, with one leg in the swing phase and the opposite one in the stance phase. How might this be interpreted? One possible hypothesis would be that octopamine released by DUM neurons acts on muscles to assist walking movements during both phases, independent of the precise timing of its release. During the stance phase, the force of muscle contraction has to be increased, both to enhance joint stiffness via cocontraction of antagonistic muscles and to provide power for the actual forward movement of the animal. During the swing phase, muscle tonus must be reduced quickly for the rapid forward movement of the leg relative to the body (Duch and Pflüger 1995
). Efferent DUM neurons can fulfill both these functions (Evans and O'Shea 1978
; O'Shea and Evans 1979
). According to this hypothesis, the precise timing of spikes in DUM 3,4,5 and DUM 5 neurons may not have functional significance. Instead, it may simply reflect the available mechanism of central coupling between these efferent neuromodulatory neurons and the rhythmical motor networks that at least ensures increased activity.
If restriction of DUM 5 and DUM 3,4,5 neuron activity to the levator phase is of functional relevance, then it raises the question what such neuromodulatory neurons might do over the short time scale necessitated by rhythmical activation during each step taken by the legs. One possibility is that DUM neurons may have effects in the periphery over a shorter time scale than has been shown to date, e.g., phasic effects on sensory structures. Such effects still would occur simultaneously on both sides of the body, unless it is possible for the DUM spikes to be directed preferentially down one or the other of the bilateral axons. This may be possible because DUM neurons have independent axonal spike-initiating sites distal to the bifurcation of the primary neurite within the ganglion (Heitler and Goodman 1978
). During kicks of the hindleg, however, DUM neurons produced the same pattern of spikes regardless of whether a single leg or both legs kicked (Burrows and Pflüger 1995
). Alternatively, we may need to consider central effects when interpreting patterns of DUM activity. Parker (1996)
has shown that DUM neurons may have central effects on tibial motor neurons, although these effects appear variable. The final answer to the role of neuromodulation during specific motor patterns can only be addressed by recordings made during real behavior. Nevertheless, our study clearly demonstrates that DUM neurons can be activated individually in the control of movement and that such neuromodulatory processes can be coupled centrally to motor programs.
 |
ACKNOWLEDGEMENTS |
We thank M. Burrows, P. D. Evans, T. Matheson, P. L. Newland, H.-J. Pflüger, and two anonymous referees for helpful comments on the manuscript.
This work was supported by a studentship from the Wellcome Trust to S. Baudoux, National Institute of Neurological Disorders and Stroke Grant NS-16058 and a Wellcome Trust Grant to M. Burrows and a Deutsche Forschungsgesellschaft Grant (Graduiertenkolleg) to C. Duch.
 |
FOOTNOTES |
Address for reprint requests: S. Baudoux, Dept. of Zoology, University of Cambridge, Downing St., Cambridge CB2 3EJ, United Kingdom.
Received 3 March 1997; accepted in final form 16 September 1997.
 |
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