Rhythmic swimming activity in neurones of the isolated nerve cord of the leech.

1. Repeating bursts of motor neurone impulses have been recorded from the nerves of completely isolated nerve cords of the medicinal leech. The salient features of this burst rhythm are similar to those obtained in the semi-intact preparation during swimming. Hence the basic swimming rhythm is generated by a central oscillator. 2. Quantitative comparisons between the impulse patterns obtained from the isolated nerve cord and those obtained from a semi-intact preparation show that the variation in both dorsal to ventral motor neurone phasing and burst duration with swim cycle period differ in these two preparations. 3. The increase of intersegmental delay with period, which is a prominent feature of swimming behaviour of the intact animal, is not seen in either the semi-intact or isolated cord preparations. 4. In the semi-intact preparation, stretching the body wall or depolarizing an inhibitory motor neurone changes the burst duration of excitatory motor neurones in the same segment. In the isolated nerve cord, these manipulations also change the period of the swim cycle in the entire cord. 5. These comparisons suggest that sensory input stabilizes the centrally generated swimming rhythm, determines the phasing of the bursts of impulses from dorsal and ventral motor neurones, and matches the intersegmental delay to the cycle period so as to maintain a constant body shape at all rates of swimming.     

Sensory modification of leech swimming: interactions between ventral stretch receptors and swim-related neurons

The neuronal circuits that generate the leech swimming rhythm comprise oscillatory interneurons that provide appropriately phased output to drive swim-related motoneurons. Within ganglia, these interneurons express three phases; between ganglia there exists a phase delay between homologs. Our earlier experiments revealed that stretch receptors embedded in the body wall participate in intersegmental coordination and setting intersegmental phases. To identify the basis for these sensory effects, we mapped interactions between a ventral stretch receptor and swim-related neurons. Connections between this receptor and motoneurons are weak and variable in quiescent preparations, but during fictive swimming stretch receptor activation modulates motoneuron oscillations, hence, these effects are polysynaptic, mediated by interneurons. We identified a strong, nonrectifying, and apparently direct electrical connection between the stretch receptor and oscillator neuron 33. The ventral stretch receptor also interacts with most of the other oscillatory interneurons, including inhibitory inputs to cells 28 and 208, excitatory input to the contralateral cell 115, and mixed input to the ipsilateral cell 115. These direct and indirect interactions can account for previously described effects of body-wall stretch on motoneuron activity. They also could mediate the previously described modification of intersegmental phase relationships by appropriately phased stretch receptor activation.     

Modulation of swimming behavior in the medicinal leech

The effects of serotonin on the electrical properties of swim-gating neurons (cell 204) were examined in leech (Hirudo medicinalis) nerve cords. Exposure to serotonin decreased the threshold current required to elicit swim episodes by prolonged depolarization of an individual cell 204 in isolated nerve cords. This effect was correlated with a more rapid depolarization and an increased impulse frequency of cell 204 in the first second of stimulation. In normal leech saline, brief depolarizing current pulses (1 s) injected into cell 204 failed to elicit swim episodes. Following exposure to serotonin, however, identical pulses consistently evoked swim episodes. Thus, serotonin appears to transform cell 204 from a gating to a trigger cell.Serotonin had little effect on the steady-state currentvoltage relation of cell 204. However, serotonin altered the membrane potential trajectories in response to injected current pulses and increased the amplitude of rebound responses occurring at the offset of current pulses. These changes suggest that serotonin modulates one or more voltage dependent conductances in cell 204, resulting in a more rapid depolarization and greater firing rate in response to injected currents. Thus, modulation of intrinsic ionic conductances in cell 204 may account in part for the increased probability of swimming behavior induced by serotonin in intact leeches.Abbreviations AHP afterhyperpolarizing potential - DCC discontinuous current clamp - DP dorsal posterior nerve - G2 segmental ganglion 2 - PIR postinhibitory rebound - RMP resting membrane potential     

Development of swimming in the medicinal leech,the gradual acquisition of a behavior

Observing the development of behavior provides an assay for the developmental state of an embryo’s nervous system. We have previously described the development of behaviors that were largely confined to one or a few segments. We now extend the work to a kinematic analysis of the development of swimming, a behavior that requires coordination of the entire body. When leech embryos first begin to swim they make little forward progress, but within several days they swim as effectively as adults. This increase in efficacy depends on changes in body shape and on improved intersegmental coordination of the swim central pattern generator. These kinematic details suggest how the swim central pattern generating circuit is assembled during embryogenesis.     

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.     

Modulation of swimming behavior in the medicinal leech

Expression of swimming in the medicinal leech (Hirudo medicinalis) is modulated by serotonin, a naturally occurring neurohormone. Exogenous application of serotonin engenders spontaneous swimming activity in nerve-cord preparations. We examined whether this activity is due to enhanced participation of swim motor neurons (MNs) in generating the swimming rhythm. We found that depolarizing current injections into MNs during fictive swimming are more effective in shifting cycle phase in nerve cords following serotonin exposure. In such preparations, the dynamics of membrane potential excursions following current injection into neuronal somata are substantially altered. We observed: 1) a delayed outward rectification (relaxation) during depolarizing current injection, most marked in inhibitory MNs; and 2) in excitor MNs, an enhancement of postinhibitory rebound (PIR) and afterhyperpolarizing potentials (AHPs) following hyperpolarizing and depolarizing current pulses, respectively. In contrast, we found little alteration in MN properties in leech nerve cords depleted of amines. We propose that enhanced expression of swimming activity in leeches exposed to elevated serotonin is due, partly, to enhancement of relaxation, PIR and AHP in MNs. We believe that as a consequence of alterations in cellular properties and synaptic interactions (subsequent paper) by serotonin, MNs are reconfigured to more effectively participate in generating and expressing the leech swimming rhythm.Abbreviations AHP Afterhyperpolarizing potential - DCC Discontinuous current clamp - DE Dorsal excitor motor neuron - DI Dorsal inhibitor motor neuron - IPSP Inhibitory postsynaptic potential - MN Motor neuron - PIR Postinhibitory rebound - VE Ventral excitor motor neuron - VI Ventral inhibitor motor neuron     

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.     

Promotion of regeneration and axon growth following injury in an invertebrate nervous system by the use of three-dimensional collagen gels.

We describe the application of three-dimensional collagen matrices to the study of nerve cord repair in the leech. Our experiments show that ganglia and connectives of the leech ventral nerve cord can be maintained for up to four weeks embedded in 3D gels constructed from mammalian type I collagen. Severed nerve cords embedded in the collagen gel reliably repaired within a few days of culture. The gel was penetrable by cells emigrating from the cut ends of nerves and connectives, and we consistently saw regenerative outgrowth of severed peripheral and central axons into the gel matrix. Thus, 3D gels provide an in vitro system in which we can reliably obtain repair of severed nerve cords in the dish, and visualize cell behaviour underlying regenerative growth at the damage site: and which offers the possibility of manipulating the regenerating cells and their extracellular environment in various ways at stages during repair. Using this system it should be possible to test the effect on the repair process of altering expression of selected genes in identified nerve cells.     

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.     

Intersegmental coordination in invertebrates and vertebrates

How does the CNS coordinate muscle contractions between different body segments during normal locomotion? Work on several preparations has shown that this coordination relies on excitability gradients and on differences between ascending and descending intersegmental coupling. Abstract models involving chains of coupled oscillators have defined properties of coordinating circuits that would permit them to establish a constant intersegmental phase in the face of changing periods. Analyses that combine computational and experimental strategies have led to new insights into the cellular organization of intersegmental coordinating circuits and the neural control of swimming in lamprey, tadpole, crayfish and leech.     

Intracellular stimulation of sensory cells elicits swimming activity in the medicinal leech

Intracellular stimulation of each of three different types of mechanoreceptors, the T, P and N cells, evokes swimming behavior in leech preparations. Stimulation of an individual N cell or P cell evoked swimming in 75% and 53% respectively, of the preparations tested. Stimulation of an individual T cell was ineffective in eliciting swimming; however, simultaneous stimulation of two T cells evoked swimming in 59% of our preparations. Stimulation of mechanosensory neurons elicited swimming activity for a limited number of trials; i.e. the response habituated. The number of swim episodes evoked before habituation to criterion did not differ significantly for the different types of mechanoreceptors. The duration of swim episodes declined significantly over the course of N cell stimulation. The tendency for swim length to decline with repeated stimulation was present as well for swim episodes elicited by P or T cell stimulation. Swim initiation recovered spontaneously following habituation resulting from T cell stimulation. Spontaneous recovery following N cell stimulation was not demonstrated. However, N cell stimulation evoked swimming again after DP nerve shock or to a limited extent, after cell 204 stimulation. Spontaneous recovery of swim initiation to P cell stimulation was not investigated. A previous study detailed habituation of swimming activity to mechanical stimulation of the body wall (Debski and Friesen 1985). Only the T cells are activated significantly by this stimulus. Stimulation of sensory receptors other than mechanoreceptors was not effective in eliciting swimming in our preparation. We conclude that T cells mediate swim initiation elicited by stroking of the body wall and that the cessation of swimming to this stimulus is not due to sensory adaptation.     

Generation of a locomotory rhythm by a neural network with recurrent cyclic inhibition

An oscillatory intersegmental neuronal network drives the swimming rhythm of the leech. This network consists of interneurons joined via inhibitory connections to form a series of segmentally iterated, concatenated rings. Recurrent cyclic inhibition in these rings produces a multiphasic activity rhythm. By theoretical analysis of such concatenated interneuronal rings and construction of their electronic analogs it is shown that the interneural network identified in the central nervous system of the leech has properties appropriate for generating the observed motor output.     

Modulation of swimming activity in the medicinal leech by serotonin and octopamine

1. The monoamines serotonin (5-HT) and octopamine (OA) enhance the expression of swimming activity in the medicinal leech (Willard, 1981; Belanger and Orchard, 1988). We explored further the effects of these monoamines and related agents on swimming activity observed in isolated leech nerve cords. 2. We confirmed that swimming activity is induced reversibly following exposure of the nerve cord to 5-HT (50 microM); the half-maximal rate of swimming activity develops in about 15 min. Swimming activity returns to control levels about 30 min after drug washout. 3. Swim-induction by 5-HT is blocked by the presence of 10 microM cyproheptadine (a 5-HT antagonist). 4. Although apparently less effective than 5-HT, OA application to nerve cords also induced swimming activity. 5. Depletion of endogenous amines from nerve cords by acute exposure to reserpine (10-150 microM) blocked stimulus-evoked swimming activity within 4 hr. 6. Subsequent application of 5-HT (50 microM) or OA (100 microM) reinstated stimulus-evoked swimming and induced repeated episodes of non-triggered swimming activity. 7. Application of cAMP and cAMP analogs, as well as phosphodiesterase inhibitors (theophylline and IBMX), mimicked the effects of the monoamines, suggesting that 5-HT and OA may activate swimming activity by increasing neuronal cAMP. 8. We obtained episodes of swim-like activity from individual, isolated ganglia exposed to 5-HT or OA. Such episodes were usually brief, with variable cycle period. 9. We conclude that individual nerve cord ganglia contain the complete neuronal circuitry required to generate the rudiments of swimming activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Positive feedback loops sustain repeating bursts in neuronal circuits

Voluntary movements in animals are often episodic, with abrupt onset and termination. Elevated neuronal excitation is required to drive the neuronal circuits underlying such movements; however, the mechanisms that sustain this increased excitation are largely unknown. In the medicinal leech, an identified cascade of excitation has been traced from mechanosensory neurons to the swim oscillator circuit. Although this cascade explains the initiation of excitatory drive (and hence swim initiation), it cannot account for the prolonged excitation (10–100 s) that underlies swim episodes. We present results of physiological and theoretical investigations into the mechanisms that maintain swimming activity in the leech. Although intrasegmental mechanisms can prolong stimulus-evoked excitation for more than one second, maintained excitation and sustained swimming activity requires chains of several ganglia. Experimental and modeling studies suggest that mutually excitatory intersegmental interactions can drive bouts of swimming activity in leeches. Our model neuronal circuits, which incorporated mutually excitatory neurons whose activity was limited by impulse adaptation, also replicated the following major experimental findings: (1) swimming can be initiated and terminated by a single neuron, (2) swim duration decreases with experimental reduction in nerve cord length, and (3) swim duration decreases as the interval between swim episodes is reduced.     

Seasonal variation of serotonin content and nonassociative learning of swim induction in the leech Hirudo medicinalis

Summary It is possible to obtain habituation of swim induction by stimulating the leech with repetitive light electrical trains. After obtaining this simple form of nonassociative learning, it is also possible to potentiate its response by a series of nociceptive skin brushings (dishabituation). Serotonin applied to the animal is the only neurotransmitter found to mimick dishabituation. We have observed that in the period April–June most animals did not exhibit potentiation of the swimming response after nociceptive stimulation while injection of serotonin mimicked dishabituation as in the animals treated in the period October–March. We have seen correlation between the changes in nonassociative learning and the seasonal variation of serotonin levels in segmental ganglia. This finding strengthens the hypothesis of serotonin as the neurotransmitter mediating dishabituation in swim induction of the leech.Abbreviations AHP afterhyperpolarization - HPLC high pressure liquid chromatography     

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.     

Parallel pathways coordinate crawling in the medicinal leech,Hirudo medicinalis

Changes in the behavior of crawling leeches were investigated after various kinds of manipulations, including selective transection or inactivation of body parts, as well as partial or complete transection of the central nerve cord, using a frame-by-frame analysis of video tapes of the crawling animals. From these studies, we found that: 1. Leeches made rhythmic crawling cycles even after their suckers were prevented from contacting the substrate by covering them over with glue. Hence, engagement and disengagement of the suckers are not necessary links in the crawling cycle. 2. Cutting the small, medial connective (Faivre's nerve) had no influence on crawling, but contraction during the whole-body shortening reflex was interrupted. Thus two behaviors which use the same motor output (i.e., whole-body shortening and the contraction phase of crawling) are mediated by two different pathways. 3. Cutting all the connectives between two ganglia in the middle of the leech resulted in a loss of coordination between the parts of the animal on either side of the cut. Therefore, temporally coordinated sucker activity must be mediated through these connectives. 4. Pieces of leech bodies produced by complete transection produced rhythmic crawling cycles as long as the pieces included the head or tail plus 2–4 adjacent midbody segments. In all cases, the crawling movements progressed without delays as the movements reached the cut ends. Pieces of animals that included only midbody segments did not produce crawling movements. 5. These results can be explained by a model composed of intersegmental pathways for both elongation and contraction, circuits in the head and tail brains that switch between elongation and contraction, and both ascending and descending inhibitory influences that determine when the cycle switches from elongation to contraction and back again.Abbreviations C1-C7 caudal segments 1 through 7 (comprise the tail sucker) - Circ. circular muscle(s) - CD circular element driver - CPG central pattern generator - ED elongation element driver - El elongation - El _init initiation of elongation - FN Faivre's nerve - fs + front sucker attachment - s— front sucker release - Long longitudinal muscle(s) - M1-M21 midbody segments 1 through 21 - R1-R4 rostral segments 1 through 4 (comprise the head) - rs + rear sucker attachment - rs rear sucker release - Sens sensory input - SR stretch receptors(s) - ti tonic inhibition     

To swim or not to swim: regional effects of serotonin, octopamine and amine mixtures in the medicinal leech

Focally treating the head brain of the medicinal leech Hirudo medicinalis with various biogenic amines affected the initiation, termination and maintenance of fictive swimming (i.e., the neural correlate of swimming). Application of serotonin to saline surrounding only the head brain inhibited fictive swimming, whereas removing serotonin induced swimming. This contrasts sharply with previous observations that serotonin applied to the nerve cord induces swimming. Although application of octopamine to the brain activated swimming, a mixture of octopamine and serotonin inhibited swimming. Subsequent removal of this mixture from the brain activated robust swimming and was more potent for activating swimming than either the removal of serotonin or the application of octopamine. Swim episodes induced by brain-specific manipulations of octopamine had more swim bursts per episode than those induced by serotonin. These brain-specific effects of the amines on fictive swimming are probably due to the modulation of higher-order circuits that control locomotion in the leech. We observed that serotonin or a mixture of serotonin and octopamine hyperpolarized an identified descending brain interneuron known as Tr2. Removal of the mixture caused Tr2 to exhibit membrane potential depolarizations that correlated in time with the expression of swim episodes.     

Initiation of swimming activity by trigger neurons in the leech subesophageal ganglion

The aim of this study was to identify neurons in the subesophageal ganglion of the medicinal leech which initiate swimming activity and to determine their output connections. We found two bilaterally symmetrical pairs of interneurons, Tr1 and Tr2, located in the first division of the subesophageal ganglion which initiate swimming activity in the isolated nervous system when depolarized with brief (1-3 s) current pulses. Tr1 and Tr2 are considered trigger neurons because elicited swimming episodes outlast the stimulus duration, and because the length of elicited swim episodes is nearly independent of the intensity with which Tr1 and Tr2 are stimulated. Tr1 and Tr2 have similar morphologies. The neurites of both cells cross contralaterally in the subesophageal ganglion, project posteriorly, and exit the subesophageal ganglion in the contralateral connective. The axons of Tr1 and Tr2 extend as far posterior as segmental ganglion 18 of the ventral nerve cord. Tr1 provides direct excitatory drive to three groups of segmental neurons which are capable of initiating swimming: swim-initiating interneurons (cells 204 and 205), serotonin-containing interneurons (cells 61 and 21), and the serotonergic Retzius cells. In addition, all Retzius cells in the subesophageal ganglion are excited directly by Tr1. These three groups of neurons are excited even if Tr1 stimulation is subthreshold for swim initiation. In contrast to Tr1, Tr2 stimulation evokes transient inhibition in swim-initiating and serotonin-containing interneurons, and has little immediate effect on Retzius cells. In addition, Tr2 indirectly inhibits several oscillator neurons, including cells 208, 33, and 60. When Tr1 is stimulated during a swimming episode the swim period decreases for several cycles, while stimulation of Tr2 during swimming episodes reliably resets the ongoing swimming rhythm. Our findings indicate that Tr1 and Tr2 are trigger neurons which initiate swimming activity by different pathways. These neurons also have functional interactions with the swim oscillator network since either Tr1 or Tr2 stimulation during swimming can modulate the ongoing swimming rhythm.     

Modification of leech behavior following foraging for artificial blood

In this study we examined whether the foraging for artificial blood affected the behavioral responsiveness of leeches to electrical stimulation of the body wall. After foraging for artificial blood, electrical stimulation of the posterior end of the leech significantly increased the percentage of stimulation trials that elicited locomotory activity—swimming and crawling—compared to the behaviors elicited when leeches did not forage or foraged for normal saline. On the other hand, shortening always dominated the behavioral profile of the leech to anterior stimulation even after foraging for artificial blood. In intact anterior end-isolated nerve cord preparations, we also found that application of artificial blood to the intact anterior end was sufficient to modify motor responsiveness to DP nerve stimulation. Full strength artificial blood had an overall negative effect on the likelihood of DP nerve stimulation initiating swimming and on the average length of elicited swim episodes compared to when pond water surrounded the anterior end. Application of a 10% solution of artificial blood to the anterior end led to an increase in the likelihood of DP nerve stimulation eliciting swimming.