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.     

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.     

Monoamines in the nervous system of the tube-wormChaetopterus variopedatus (Polychaeta): Biochemical detection and serotonin immunoreactivity

Summary The presence and distribution of biogenic monoamines in the tube-wormChaetopterus variopedatus were investigated by a radioenzymatic method and HPLC with electrochemical detection, and the cellular localization of serotonin by peroxidase-antiperoxidase (PAP) immunohistochemistry with an antibody against serotonin-formaldehyde-protein conjugate. Dopamine, norepinephrine, epinephrine, serotonin (5-HT) and some of their metabolites were detectable, dopamine and norepinephrine being present in substantially larger amounts than 5-HT and epinephrine. With few exceptions, the largest amounts of amines were localized in the most nerve-rich tissues such as tentacles, and those containing cerebral ganglia and the ventral nerve cord. Serotonin-immunoreactive unipolar neurons were widely distributed in the dorso-lateral cerebral ganglia, the neurosecretory pharyngeal ganglion and the segmental ganglia of the anterior (dorsolateral) and posterior (medioventral) nerve cords. Some nerve-fiber tracts stained in the cerebral ganglia, but the neuropiles of segmental ganglia were the most intensely reactive CNS structures. Numerous reactive fibers were also present in connectives, commissures and segmental nerves. All peripheral sensory structures included serotonin-immunoreactive cells and neurites, especially the parapodial cirri and the bristle receptors of the setae. Trunk and parapodial muscles contained reactive varicose fibers and neuronal somata. These results suggest that monoamines are abundant and widespread in these worms and that 5-HT appears to have a key sensory role.     

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.     

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.     

Functionally heterogeneous segmental oscillators generate swimming in the medicinal leech

Swimming behavior in the leech Hirudo medicinalis arises from neuronal circuits within the ventral nerve cord. Although the ventral nerve cord comprises a series of homologous segmental ganglia, it remains unresolved whether the swim oscillator circuits within individual ganglia are functionally equivalent. We have extended previous studies on pairs of ganglia to test whether individual ganglia throughout the nerve cord are capable of generating swim oscillations and to measure the cycle periods of local oscillations. We found that the swim-generating function of individual ganglia is broadly distributed, but not uniform. The swim-like oscillations in isolated ganglia from the anterior ganglia nerve cord were less robust than those from mid-cord. Swimming activity in posterior cord ganglia is even weaker we were unable to obtain swim-like oscillations from individual ganglia of the nerve cord posterior to segment 12. Swim-cycle periods exhibited a U-shaped function: those recorded in the most anterior individual ganglia (2.3 s for ganglion M2) and short chains of posterior ganglia (up to 4.0 s) were two to four times longer than those obtained from mid-cord ganglia (near 1.0 s). We conclude that the leech swim system comprises a functionally heterogeneous set of local oscillator units.     

Injury-induced expression of endothelial nitric oxide synthase by glial and microglial cells in the leech central nervous system within minutes after injury.

It is known that nitric oxide (NO) is produced by injured tissues of the mammalian central nervous system (CNS) within days of injury. The aim of the present experiments was to determine the cellular synthesis of NO in the CNS immediately after injury, using the CNS of the leech which is capable of synapse regeneration, as a step towards understanding the role of NO in nerve repair. We report that within minutes after crushing the nerve cord of the leech, the region of damage stained histochemically for NADPH diaphorase, which is indicative of nitric oxide synthase (NOS) activity, and was immunoreactive for endothelial NOS (eNOS). On immunoblots of leech CNS extract, the same antibody detected a band with a relative molecular mass of 140,000, which is approximately the size of vertebrate eNOS. Cells expressing eNOS immunoreactivity as a result of injury were identified after freezing nerve cords, a procedure that produced less tissue distortion than mechanical crushing. Immunoreactive cells included connective glia and some microglia. Calmodulin was necessary for the eNOS immunoreactivity: it was blocked by calmodulin antagonist W7 (25 microM), but not by similar concentrations of the less potent calmodulin antagonist W12. Thus in the leech CNS, in which axon and synapse regeneration is successful, an increase in NOS activity at lesions appears to be among the earliest responses to injury and may be important for repair of axons.     

Serotonin patterns locomotor network activity in the developing zebrafish by modulating quiescent periods

Developing neural networks follow common trends such as expression of spontaneous, recurring activity patterns, and appearance of neuromodulation. How these processes integrate to yield mature, behaviorally relevant activity patterns is largely unknown. We examined the integration of serotonergic neuromodulation and its role in the functional organization of the accessible locomotor network in developing zebrafish at behavioral and cellular levels. Locally restricted populations of serotonergic neurons and their projections appeared in the hindbrain and spinal cord of larvae after hatching (approximately day 2). However, 5-HT affected the swimming pattern only from day 4 on, when sustained spontaneous swimming appeared. 5-HT and its agonist quipazine increased motor output by reducing intervals of inactivity, observed behaviorally (by high-speed video) and in recordings from spinal neurons during fictive swimming (by whole-cell current clamp). 5-HT and quipazine had little effect on the properties of the activity periods, such as the duration of swim episodes and swim frequency. Further, neuronal input resistance, rheobasic current, and resting potential were not affected significantly. The 5-HT antagonists methysergide and ketanserin decreased motor output by prolonging the periods of inactivity with little effect on the active swim episode or neuronal properties. Our results suggest that 5-HT neuromodulation is integrated early in development of the locomotor network to increase its output by reducing periods of inactivity with little effect on the activity periods, which in contrast are the main targets of 5-HT neuromodulation in neonatal and adult preparations.     

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.     

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     

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.     

Pharmacological Evidence for the Modulation of Monoamine Release by Adenosine in the Invertebrate Nervous System

An in vitro preparation from the pedal ganglia of the marine bivalve, Mytilus edulis, was used to examine the modulation of transmitter release by adenosine and its analogs from invertebrate nervous tissue. The ganglia of this organism contain the monoamines dopamine (DA), serotonin (5-HT), and norepinephrine (NE), and the presynaptic release of these substances is known to be calcium-dependent. This organism also contains a DA-sensitive adenylate cyclase system which resembles that seen in mammals. Neural tissue from the pedal ganglia was incubated with labeled monoamines, and release studies were then conducted in superfusion chambers; release of monoamines was evoked by the addition of 50 mM KCl. Addition to the superfusion medium of the adenosine analog, 5'-N-ethylcarboxamidoadenosine (NECA; 10 nM), inhibited the release of 5-HT and DA, and to a lesser extent NE, whereas 100-fold higher concentrations of adenosine itself and the adenosine analog, R-N6-phenylisopropyladenosine, were required to achieve comparable levels of inhibition. The inhibitory effects of NECA on neurotransmitter release were blocked by the adenosine receptor antagonist, theophylline (IC50 = 10-14 microM). The results from this study indicate for the first time the possible role of adenosine as a modulator of neurotransmitter release in the invertebrate nervous system.     

A new approach to the analysis of complex dose-response relationships: a multivariate study of octopamine action on the leech Leydig neurone

Octopamine (OA) hyperpolarizes leech Leydig neurones but response amplitudes and thresholds vary. Associations between response, OA dose and other variables [initial resting potential, action potential (AP) frequency and amplitude, temperature, microelectrode resistance, position of ganglion along nerve cord, animal, treatment order] were examined using canonical correlation. A complex response was expressed by summing changes in membrane potential and AP frequency over 2 min of OA treatment. Only dose and ganglion position influenced this response. One significant canonical variate related dose to immediate changes in membrane potential and summed changes in AP frequency throughout the OA effect. Response to OA was greater in cells in posterior ganglia.     

Neuropeptides modulate the transmitter-induced glycogenolysis and gluconeogenesis in leech segmental ganglia

The effects of four neuropeptides (AKH, FMRFamide, proctolin, VIP) on the alterations in glycogen levels induced by other transmitters were studied in isolated leech segmental ganglia. With the exception of FMRFamide, which produced a small increase (14%) in glycogen, the peptides were by themselves without effect on the glycogen levels. Proctolin abolished the glycogenolytic effects of 5-HT, dopamine and octopamine but not histamine. AKH in combination with ACh produced glycogenolysis although each by themselves were ineffective. AKH modified the effects of other transmitters in different ways i.e. by reduction or reversal of effect. VIP and noradrenaline produced an increase in glycogen (cf. noradrenaline alone which decreased glycogen), but did not modify the effects of the other transmitters. FMRFamide produced a complex variety of modulatory effects on the other transmitters. It is concluded that the glycogen stores in leech ganglia, which are localized principally in the glial cells, may be controlled in complex ways by the different combinations of monoamines, amino acids and neuropeptides.     

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.     

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.     

IDENTIFICATION OF SEROTONIN WITHIN VITAL-STAINED NEURONS FROM LEECH GANGLIA

Abstract— We have measured serotonin (5-HT) within large and small neurosomata which are vitally stained by Neutral Red dye. A micro-radioenzymatic technique which is sensitive to 50fmol of 5-HT was employed on intact ganglia, 75 μm Retzius Cells (RZ) and a 10 μm ventro-lateral cell (VL) taken from the leech Macrobdella decora. The stain does not affect the levels of 5-HT in either ganglia or RZ. The VL cell body contains 5-HT at concentrations of at least 100 m m . Microspectrofluorometry of all the ganglionic neurosomata which fluoresce following the Falck-Hillarp formaldehyde condensation reaction detected rapidly-fading emission peaks of 509–523 nanometers. We conclude that all seven fluorescent neurons in the leech ganglion very probably contain serotonin.     

Serotonin modulates muscle function in the medicinal leech Hirudo verbana

The body wall muscles of sanguivorous leeches power mechanically diverse behaviours: suction feeding, crawling and swimming. These require longitudinal muscle to exert force over an extremely large length range, from 145 to 46 per cent of the mean segmental swimming length. Previous data, however, suggest that leech body wall muscle has limited capacity for force production when elongated. Serotonin (5-HT) alters the passive properties of the body wall and stimulates feeding. We hypothesized that 5-HT may also have a role in allowing force production in elongated muscle by changing the shape of the length-tension relationship (LTR). LTRs were measured from longitudinal muscle strips in vitro in physiological saline with and without the presence of 10 μM 5-HT. The LTR was much broader than previously measured for leech muscle. Rather than shifting the LTR, 5-HT reduced passive muscle tonus and increased active stress at all lengths. In addition to modulating leech behaviour and passive mechanical properties, 5-HT probably enhances muscle force and work production during locomotion and feeding.     

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.