A central pattern generator underlies crawling in the medicinal leech

Crawling in the medicinal leech has previously been thought to require sensory feedback because the intact behavior is strongly modulated by sensory feedback and because semi-intact preparations will only crawl if they can move freely. Here we show that an isolated leech nerve cord can produce a crawling motor pattern similar to the one seen in semi-intact preparations, which consists of an anterior-to-posterior wave of alternating excitatory circular and longitudinal motor neuron bursts in each segment. The isolated cord also reproduces the patterns of activity seen in semi-intact preparations for several other kinds of cells: the dorsal inhibitor cell 1, the ventral excitor cell 4, and the annulus erector motor neuron. Because this correspondence is so strong, there must be a central pattern generator in the isolated cord that can produce the basic motor pattern for crawling without sensory feedback. A quantitative analysis of the isolated motor pattern, however, reveals that isolated and semi-intact preparations have longer periods than the intact behavior and that there are deficiencies in the timing of motor neuron bursts in the isolated pattern. These results suggest that sensory feedback modulates the isolated central pattern generator to help produce the normal motor pattern.     

Entrainment of leech swimming activity by the ventral stretch receptor

Rhythmic animal movements originate in CNS oscillator circuits; however, sensory inputs play an important role in shaping motor output. Our recent studies demonstrated that leeches with severed nerve cords swim with excellent coordination between the two ends, indicating that sensory inputs are sufficient for maintaining intersegmental coordination. In this study, we examined the neuronal substrates that underlie intersegmental coordination via sensory mechanisms. Among the identified sensory neurons in the leech, we found the ventral stretch receptor (VSR) to be the best candidate for our study because of its sensitivity to tension in longitudinal muscle. Our experiments demonstrate that (1) the membrane potential of the VSR is depolarized during swimming and oscillates with an amplitude of 1.5–5.0 mV, (2) rhythmic currents injected into the VSR can entrain ongoing swimming over a large frequency range (0.9–1.8 Hz), and (3) large current pulses injected into the VSR shift the phase of the swimming rhythm. These results suggest that VSRs play an important role in generating and modulating the swim rhythm. We propose that coordinated swimming in leech preparations with severed nerve cords results from mutual entrainment between the two ends of the leech mediated by stretch receptors.     

Respiratory nervous activity in the isolated nerve cord of the larval dragonfly, and location of the respiratory oscillator

ABSTRACT. Rhythmic respiratory nerve activity was recorded in the dragonfly larvae, Anax parthenope Julius Brauer (Anisoptera). Alternating expiratory and inspiratory bursts of spikes occurred in abdominal nerve cords isolated from all peripheral connections. These bursts are similar to the activity recorded in semi-intact preparations, suggesting that the respiratory rhythm can be generated without peripheral sensory feedback. Expiratory bursts started and ended at the same time in different segments of semi-intact preparations. When connectives were severed, the nerve cord separated from the last abdominal ganglion did not normally show rhythmic bursts; the last ganglion alone and the nerve cord connected to the last ganglion exhibited the rhythmic bursts. However, in a few cases the nerve cord separated from the last ganglion exhibited the rhythm. The results suggest that the last ganglion contains the main oscillator, but that other weak oscillators occur elsewhere.     

Neural correlates of swimming behavior in Melibe leonina

The nudibranch Melibe leonina swims by rhythmically bending from side to side at a frequency of 1 cycle every 2-4 s. The objective of this study was to locate putative swim motoneurons (pSMNs) that drive these lateral flexions and determine if swimming in this species is produced by a swim central pattern generator (sCPG). In the first set of experiments, intracellular recordings were obtained from pSMNs in semi-intact, swimming animals. About 10-14 pSMNs were identified on the dorsal surface of each pedal ganglion and 4-7 on the ventral side. In general, the pSMNs in a given pedal ganglion fired synchronously and caused the animal to flex in that direction, whereas the pSMNs in the opposite pedal ganglion fired in anti-phase. When swimming stopped, so did rhythmic pSMN bursting; when swimming commenced, pSMNs resumed bursting. In the second series of experiments, intracellular recordings were obtained from pSMNs in isolated brains that spontaneously expressed the swim motor program. The pattern of activity recorded from pSMNs in isolated brains was very similar to the bursting pattern obtained from the same pSMNs in semi-intact animals, indicating that the sCPG can produce the swim rhythm in the absence of sensory feedback. Exposing the brain to light or cutting the pedal-pedal connectives inhibited fictive swimming in the isolated brain. The pSMNs do not appear to participate in the sCPG. Rather, they received rhythmic excitatory and inhibitory synaptic input from interneurons that probably comprise the sCPG circuit.     

The role of sensory input in maintaining output from the locust oviposition digging central pattern generator

Summary A quantitative EMG analysis is presented of the effects of deafferentation on the motor program for oviposition digging in the locust Locusta migratoria. We examined the activity of two groups of antagonistic muscles, the opener and closer muscles of the ventral ovipositor valves, in terms of the cycle frequency, burst duration, and relative burst onset times. There were no significant differences between the pattern frequency produced in intact, semi-intact, or deafferented animals within 10 min of the onset of the pattern. Over time, however, the pattern in deafferented animals showed a significant decrease in frequency, which it did not do in intact or semi-intact animals. Seven out of 10 deafferented preparations ceased producing the digging rhythm within 35 min of onset, but none of the semi-intact preparations did so. Mechanosensory hairs cover the ovipositor valves, and are in a position to supply sensory input to the digging pattern generator during the natural behaviour. When nerves carrying sensory axons from these hairs were electrically-stimulated tonically, the motor pattern was restored in deafferented animals. The effects of the stimulation outlasted the stimulation itself for several minutes, and could be repeated several times. We suggest that tonic input is necessary for the maintenance of the digging rhythm, possibly by maintaining levels of some modulatory substance(s) within the CNS.Abbreviations CPG central pattern generator - DUM dorsal unpaired median neuron - EMG electromyogram - LC left ovipositor ventral closer muscle - LCDUR duration of activity of LC - LCFREQ frequency of activity bursts in LC - LCONSET onset of activity in LC relative to LO - LO left ovipositor ventral opener muscle - LODUR duration of activity of LO - LOFREQ frequency of activity bursts of LO - RO right ovipositor ventral opener muscle - RODUR duration of activity in RO - ROFREQ frequency of activityb bursts of RO     

Neuronal control of heartbeat in the medicinal leech

Summary The leech heartbeat consists of a constriction-dilation rhythm of two lateral heart tubes extending over the length of the body. The beats of the segmental sections of these two tubes are coordinated in such a manner that the heart tube of one body side produces a frontward peristaltic wave while the heart tube on the other body side produces nearly concerted constrictions. This rhythm is metastable, in that left and right heart tubes alternate between peristaltic and concerted constriction modes, with a given mode lasting for tens or hundreds of beat cycles.The constriction-dilation cycles of the segmental heart tube sections are controlled by a set of rhythmically active motor neurons, the heart excitors, or HE cells. A bilateral pair of HE cells is located in all but the two frontmost and the two rearmost segmental ganglia of the ventral nerve cord. Each HE cell innervates via excitatory synapses the circular muscle fibers in the wall of the ipsilateral heart tube section. The activity cycle of the HE cells consists of an active phase, during which they are depolarized and produce a burst of impulses, and an inactive phase during which they are repolarized by a burst of inhibitory synaptic potentials. The intersegmentally coordinated activity cycles of the HE cell set are maintained in an isolated ventral nerve cord. Hence the generation of the heart excitor rhythm does not require sensory feedback.We are indepted to Amy Kelly and King-Wai Yau for advice on the use of the intracellular staining technique and to John Kretz for calling to our attention the existence of an afferent impulse burst rhythm emanating from denervated heart tube preparations. We thank Georgia Harper for excellent technical assistance. This research was supported by Grant GB 31933X from the National Science Foundation and NIH research grants GM17866 and Training Grant GM 01389 from the Institute of General Medical Sciences.     

The post-embryonic development of cell properties and synaptic drive underlying locomotor rhythm generation in Xenopus larvae.

In the first 24 h of post-embryonic development, the motor rhythm underlying swimming in Xenopus laevis tadpoles changes from brief (ca. 7 ms) ventral root discharge in each cycle to bursts of activity lasting around 20 ms (Sillar et al. 1991). Because individual motoneurons in the spinal cord of newly hatched embryos normally fire only a single impulse per cycle, two possible changes underly the transition to motor bursts seen in larval ventral roots; desynchronization of neurons in a given ventral root which continue to fire once per cycle, or the developmental acquisition of a multiple spike capability in individual motoneurons. Here we have recorded intracellularly from ventrally positioned spinal neurons, presumed to be myotomal motoneurons, in stage 37/38 embryos and 24 h later in development in stage 42 larvae. We find that (i) larval neurons are able to fire more than one impulse per cycle of fictive swimming activity; (ii) unlike in the embryo, they generally will fire multiple impulses in response to injected depolarizing current; (iii) the synaptic drive to motoneurons during swimming increases dramatically in complexity, although it still consists of alternating phases of synaptic excitation and chloride-dependent inhibition, superimposed upon tonic synaptic depolarization. The results therefore suggest a developmental change in the membrane properties of rhythmically active neurons as a major factor in the post-embryonic development of swimming in Xenopus larvae. This change appears to occur in premotor rhythm generating interneurons as well as in the motoneurons themselves and may satisfy a demand for behavioural flexibility that allows larvae to survive in a complex and changing environment.     

Modulation of swimming rhythmicity by 5-hydroxytryptamine during post-embryonic development in Xenopus laevis.

During the first 24 h of post-embryonic development in Xenopus laevis, a rapid change in the neural activity underlying swimming occurs in which the duration of ventral root discharge on each cycle increases from a single compound impulse to discrete bursts of activity. Moreover, this change in motor output progresses rostrocaudally, suggesting that it could result from the influence of a descending neural pathway upon the spinal rhythm-generating circuitry during early post-embryonic development. To begin to examine whether serotonergic neurons of brainstem raphe nuclei might have a role in this swimming development, we have studied the effects of 5-hydroxytryptamine (5HT) on fictive swimming in embryonic and larval animals. As previously demonstrated for other vertebrate locomotor rhythms, we find that bath-applied 5HT enhances the duration of motor activity on each cycle of larval fictive swimming. In addition, our results show that the sensitivity of the swimming rhythm to exogenous 5HT follows a strict rostrocaudal gradient. In young embryos (stages 32-36) 5HT does not affect the duration of ventral root impulses per cycle; by the time of hatching (stage 37/38), rostral but not caudal discharge is enhanced, and by stage 42 (24 h post-hatching) 5HT can increase motor burst durations along most of the length of the animal. These reversible changes induced by bath-applied 5HT closely resemble the normal rostrocaudal development of burst discharge during swimming in animals some 12 h older.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specialized brain regions and sensory inputs that control locomotion in leeches

Locomotor systems are often controlled by specialized cephalic neurons and undergo modulation by sensory inputs. In many species, dedicated brain regions initiate and maintain behavior and set the duration and frequency of the locomotor episode. In the leech, removing the entire head brain enhances swimming, but the individual roles of its components, the supra- and subesophageal ganglia, in the control of locomotion are unknown. Here we describe the influence of these two structures and that of the tail brain on rhythmic swimming in isolated nerve cord preparations and in nearly intact leeches suspended in an aqueous, “swim-enhancing” environment. We found that, in isolated preparations, swim episode duration and swim burst frequency are greatly increased when the supraesophageal ganglion is removed, but the subesophageal ganglion is intact. The prolonged swim durations observed with the anterior-most ganglion removed were abolished by removal of the tail ganglion. Experiments on the nearly intact leeches show that, in these preparations, the subesophageal ganglion acts to decrease cycle period but, unexpectedly, also decreases swim duration. These results suggest that the supraesophageal ganglion is the primary structure that constrains leech swimming; however, the control of swim duration in the leech is complex, especially in the intact animal.     

The development of swimming rhythmicity in post-embryonic Xenopus laevis.

The post-embryonic development of 'fictive' swimming in immobilized Xenopus laevis tadpoles has been examined during the first day of larval life. In Xenopus embryos (stage 37-38; Nieuwkoop & Faber 1956), the rhythmic ventral root activity underlying swimming occurs as single brief (ca. 7 ms) compound impulses on each cycle. However, by stage 42 (about 24 h after hatching), ventral root discharge consists of bursts lasting around 20 ms per cycle. In addition to increased burst duration in each cycle of larval swimming, the range of cycle periods within an episode increases, although mean period values (ca. 70-80 ms) remain similar to those of the younger animal. Consequently, motoneurons at developmental stage 42 are active during swimming for a greater percentage (ca. 25%) of cycle time than at stage 37-38 (ca. 10%). Developmental stage 40 (ca. 12 h post-hatching) is an intermediate stage in rhythm development. Ventral root discharge varies from bursts of 10-20 ms at the start of an episode to embryonic (ca. 7 ms) spikes at the end of an episode. Furthermore, discharge varies from bursts of activity in rostral segments of stage 40 larvae to 7 ms spikes more caudally, as in embryos. The data thus suggest that Xenopus swimming rhythmicity develops relatively rapidly, along a rostrocaudal gradient, and may involve acquisition of multiple spiking in spinal neurons.     

Control of leech swimming activity by the cephalic ganglia

We investigated the role played by the cephalic nervous system in the control of swimming activity in the leech, Hirudo medicinalis, by comparing swimming activity in isolated leech nerve cords that included the head ganglia (supra- and subesophageal ganglia) with swimming activity in nerve cords from which these ganglia were removed. We found that the presence of these cephalic ganglia had an inhibitory influence on the reliability with which stimulation of peripheral (DP) nerves and intracellular stimulation of swim-initiating neurons initiated and maintained swimming activity. In addition, swimming activity recorded from both oscillator and motor neurons in preparations that included head ganglia frequently exhibited irregular bursting patterns consisting of missed, weak, or sustained bursts. Removal of the two head ganglia as well as the first segmental ganglion eliminated this irregular activity pattern. We also identified a pair of rhythmically active interneurons, SRN1, in the subesophageal ganglion that, when depolarized, could reset the swimming rhythm. Thus the cephalic ganglia and first segmental ganglion of the leech nerve cord are capable of exerting a tonic inhibitory influence as well as a modulatory effect on swimming activity in the segmental nerve cord.     

The structure of the ventral nerve cord of Caenorhabditis elegans.

The nervous system of Caenorhabditis elegans is arranged as a series of fibre bundles which run along internal hypodermal ridges. Most of the sensory integration takes place in a ring of nerve fibres which is wrapped round the pharynx in the head. The body muscles in the head are innervated by motor neurones in this nerve ring while those in the lower part of the body are innervated by a set of motor neurones in a longitudinal fibre bundle which joins the nerve ring, the ventral cord. These motor neurones can be put into five classes on the basis of their morphology and synaptic input. At any one point along the cord only one member from each class has neuromuscular junctions. Members of a given class are arranged in a regular linear sequence in the cord and have non-overlapping fields of motor synaptic activity, the transition between fields of adjacent neurones being sharp and well defined. Members of a given class form gap junctions with neighbouring members of the same class but never to motor neurones of another class. Three of the motor neurone classes receive their synaptic input from a set of interneurones coming from the nerve ring. These interneurones can in turn be grouped into four classes and each of three motor neurone classes receives its synaptic input from a unique combination of interneurone classes. The possible developmental and functional significance of these observations is discussed.     

Neural Control of Swimming in the Leech

Leeches swim by undulating; they alternately form crests thentroughs at their anterior end and move Them backward, therebyproducing forward thrust. These movements are accomplished byalternating contractions of dorsal and ventral longitudinalmuscles in each of the 21 body segments. These contractionsare caused by bursts of impulses in groups of excitatory andinhibitory motor neurons. Connections among motor neurons helpto coordinate these bursts: synergistic muscle excitors areelectrotonically coupled, which aids in keeping their burstsnearly synchronous; muscle inhibitors also inhibit the excitorsto the same muscles, and it is this inhibition which keeps theexcitors from being tonically active during swimming. Neuronssensitive to either dorsal or ventral body wall stretch producereciprocal stretch reflexes to the muscle excitors, probablyvia the inhibitors. That these stretch reflexes may be involvedin generating the periodic bursts is supported by the resultsof both behavioral and electrophysiological experiments.     

Experiments on the central pattern generator for swimming in amphibian embryos

The central nervous system of paralysed Xenopus laevis embryos can generate a motor output pattern suitable for swimming locomotion. By recording motor root activity in paralysed embryos with transected nervous systems we have shown that: (a) the spinal cord is capable of swimming pattern generation; (b) swimming pattern generator capability in the hindbrain and spinal cord is distributed; (c) caudal hindbrain is necessary for sustained swimming output after discrete stimulation. By recording similarly from embryos whose central nervous system was divided longitudinally into left and right sides, we have shown that: (a) each side can generate rhythmic motor output with cycle periods like those in swimming; (b) during this activity cycle period increases within an episode, and there is the usual rostrocaudal delay found in swimming; (c) this activity is influenced by sensory stimuli in the same way as swimming activity; (d) normal phase coupling of the left and right sides can be established by the ventral commissure in the spinal cord. We conclude that interactions between the antagonistic (left and right) motor systems are not necessary for swimming rhythm generation and present a model for swimming pattern generation where autonomous rhythm generators on each side of the nervous system drive the motoneurons. Alternation is achieved by reciprocal inhibition, and activity is initiated and maintained by tonic excitation from the hindbrain.     

Activation of two forms of locomotion by a previously identified trigger interneuron for swimming in the medicinal leech

Higher-order projection interneurons that function in more than one behavior have been identified in a number of preparations. In this study, we document that stimulation of cell Tr1, a previously identified trigger interneuron for swimming in the medicinal leech, can also elicit the motor program for crawling in isolated nerve cords. We also show that motor choice is independent of the firing frequency of Tr1 and amount of spiking activity recorded extracellularly at three locations along the ventral nerve cord prior to Tr1 stimulation. On the other hand, during Tr1 stimulation there is a significant difference in the amount of activity elicited in the ventral nerve cord that correlates with the motor program activated. On average, Tr1 stimulation trials that lead to crawling elicit greater amounts of activity than in trials that lead to swimming.     

A model for intersegmental coordination in the leech nerve cord

The neuronal circuits that generate swimming movements in the leech were simulated by a chain of coupled harmonic oscillators. Our model incorporates a gradient of rostrocaudally decreasing cycle periods along the oscillator chain, a finite conduction delay for coupling signals, and multiple coupling channels connecting each pair of oscillators. The interactions mediated by these channels are characterized by sinusoidal phase response curves. Investigations of this model were carried out with the aid of a digital computer and the results of a variety of manipulations were compared with data from analogous physiological experiments. The simulations reproduced many aspects of intersegmental coordination in the leech, including the findings that: 1) phase lags between adjacent ganglia are larger near the caudal than the rostral end of the leech nerve cord; 2) intersegmental phase lags increase as the number of ganglia in nervecord preparations is reduced; 3) severing one of the paired lateral connective nerves can reverse the phase lag across the lesion and 4) blocking synaptic transmission in midganglia of the ventral nerve cord reduces phase lags across the block.     

Activity of neuromodulatory neurones during stepping of a single insect leg

Octopamine plays a major role in insect motor control and is released from dorsal unpaired median (DUM) neurones, a group of cells located on the dorsal midline of each ganglion. We were interested whether and how these neurones are activated during walking and chose the semi-intact walking preparation of stick insects that offers to investigate single leg-stepping movements. DUM neurones were characterized in the thoracic nerve cord by backfilling lateral nerves. These backfills revealed a population of 6-8 efferent DUM cells per thoracic segment. Mesothoracic DUM cells were subsequently recorded during middle leg stepping and characterized by intracellular staining. Seven out of eight identified individual different types of DUM neurones were efferent. Seven types except the DUMna nl2 were tonically depolarized during middle leg stepping and additional phasic depolarizations in membrane potential linked to the stance phase of the middle leg were observed. These DUM neurones were all multimodal and received depolarizing synaptic drive when the abdomen, antennae or different parts of the leg were mechanically stimulated. We never observed hyperpolarising synaptic inputs to DUM neurones. Only one type of DUM neurone, DUMna, exhibited spontaneous rhythmic activity and was unaffected by different stimuli or walking movements.     

Development of spinal motor networks in the chick embryo.

We have examined the cellular and synaptic mechanisms underlying the genesis of alternating motor activity in the developing spinal cord of the chick embryo. Experiments were performed on the isolated lumbosacral cord maintained in vitro. Intracellular and whole cell patch clamp recordings obtained from sartorius (primarily a hip flexor) and femorotibialis (a knee extensor) motoneurons showed that both classes of cell are depolarized simultaneously during each cycle of motor activity. Sartorius motoneurons generally fire two bursts/cycle, whereas femorotibialis motoneurons discharge throughout their depolarization, with peak activity between the sartorius bursts. Voltage clamp recordings revealed that inhibitory and excitatory synaptic currents are responsible for the depolarization of sartorius motoneurons, whereas femorotibialis motoneurons are activated principally by excitatory currents. Early in development, the dominant synaptic currents in rhythmically active sartorius motoneurons appear to be inhibitory so that firing is restricted to a single, brief burst at the beginning of each cycle. In E7-E13 embryos, lumbosacral motor activity could be evoked following stimulation in the brainstem, even when the brachial and cervical cord was bathed in a reduced calcium solution to block chemical synaptic transmission. These findings suggest that functional descending connections from the brainstem to the lumbar cord are present by E7, although activation of ascending axons or electrical synapses cannot be eliminated. Ablation, optical, and immunocytochemical experiments were performed to characterize the interneuronal network responsible for the synaptic activation of motoneurons. Ablation experiments were used to show that the essential interneuronal elements required for the rhythmic alternation are in the ventral part of the cord. This observation was supported by real-time Fura-2 imaging of the neuronal calcium transients accompanying motor activity, which revealed that a high proportion of rhythmically active cells are located in the ventrolateral part of the cord and that activity could begin in this region. The fluorescence transients in the majority of neurons, including motoneurons, occurred in phase with ventral root or muscle nerve activity, implying synchronized neuronal action in the rhythm generating network. Immunocytochemical experiments were performed in E14-E16 embryos to localize putative inhibitory interneurons that might be involved in the genesis or patterning of motor activity. The results revealed a pattern similar to that seen in other vertebrates with the dorsal horn containing neurons with gamma-aminobutyric acid (GABA)-like immunoreactivity and the ventral and intermediate regions containing neurons with glycine-like immunoreactivity.     

The entrainment of rhythmically discharging reticulospinal neurons of the eel by sensory nerve stimulation

Recordings made from decerebrated, paralyzed eels (Anguilla anguilla) producing rhythmical spinal motoneuronal activity showed that around 65% of identified reticulospinal units, belonging to the inferior reticular division, discharged rhythmically. The reticulospinal bursts, lasting from 300 up to 3000 ms, were in time with spinal motoneuronal bursting activity. In different fish the modal cycle period varied between 2 to 4 s and burst duration and firing frequency of each neuron showed large changes from cycle to cycle. Burst responses similar in form to those occurring spontaneously were evoked from reticular neurons when the ophthalmic nerve was stimulated regularly (intervals of 1 to 10 s) but the cycle period, firing frequency and burst duration were now more predictable. For stimulation intervals between 2 and 5 s, each ophthalmic nerve stimulus was normally followed by a burst from the reticulospinal neuron. The cycle period of the reticular rhythm then became equal to the interstimulus interval and the reticulospinal unit was entrained by the stimulus. Beyond this range of interstimulus intervals, complete entrainment was lost. We suggest that regular sensory input provides a powerful stabilising influence to rhythmically active motor systems in the brainstem and spinal cord.     

Motor patterns in the stomatogastric ganglion of the lobster Panulirus argus.

1. Acitivity patterns arising from the thirty cells of the stomatogastric ganglion of Panulirus argus are described for both a semi-intact preparation and an isolated one. 2. The thirty or so cells can be divided so far into two functional groupings: the gastric mill group, with at least ten motor elements, and the pyloric group with at least fourteen. There is some, but not extensive, interaction between groups. 3. The main gastric mill activity is arranged in two sets of elements, each of which is composed of reciprocating elements innervating antagonistic muscles. Thus alternation in activity between the single LC and the two LG neurones results in alternate closing and opening of the lateral teeth; alternation between the four GM and single CP units results in alternate protraction and retraction of the medial tooth. 4. The two sets are phased to each other in such a way that they cause gastric mill teeth to operate effectively to masticate food. 5. The main pyloric activity is arranged in a three-part cycle with each of three sets of units active in sequence. Activity in two PD and one AB unit is followed by bursts in IC and LP units followed in turn by activity in up to seven PY units. Activity in a single VD neurone is locked to this cycle in a more complex pattern.