Search

Spontaneously active neurosecretory neurons in vertebrate and invertebrate nervous systems share similarities in firing frequencies, spike shapes, inhibition by the transmitters they themselves release and postactivation inhibition, an intensity-dependent period of suppressed spontaneous generation of action potentials following phases of high-frequency activity. High-frequency activation of spontaneously active serotonin-containing Retzius cells in isolated ganglia of the leech Hirudo medicinalis induced prolonged membrane hyperpolarisations causing periods of postactivation inhibition of up to 33 s. The duration of the inhibitory periods was directly related to both the number and rate of spikes during activation and was inversely proportional to a cell's spontaneous firing frequency. The periods of postactivation inhibition remained unaffected by both serotonin depletion through repeated injections of 5,7-dihydroxytryptamine and suppressing the afterhyperpolarisation following each action potential with tetraethylammonium (TEA), iberiotoxin or charybdotoxin, suggesting that neither autoinhibition by synaptic release of serotonin nor calcium-activated potassium channels contribute to the underlying mechanism. In contrast, the postactivation inhibitory period was significantly affected both by differential electrical stimulation of the same Retzius cells via microelectrodes filled with molar concentrations of either Na(+)-acetate or K(+)-acetate, and by partial inhibition of Na(+)/K(+)-ATPase with ouabain. Thus, postactivation inhibition in Retzius cells results from prolonged hyperpolarising activity of Na(+)/K(+)-ATPase stimulated by the accumulation of cytosolic Na(+ )during phases of high-frequency spike activity.

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.

During development, many embryos show electrical coupling among neurons that is spatially and temporally regulated. For example, in vertebrate embryos extensive dye coupling is seen during the period of circuit formation, suggesting that electrical connections could pre-figure circuits, but it has been difficult to identify which neuronal types are coupled. We have used the leech Hirudo medicinalis to follow the development of electrical connections within the circuit that produces local bending. This circuit consists of three layers of neurons: four mechanosensory neurons (P cells), 17 identified interneurons, and approximately 24 excitatory and inhibitory motor neurons. These neurons can be identified in embryos, and we followed the spatial and temporal dynamics as specific connections developed. Injecting Neurobiotin into identified cells of the circuit revealed that electrical connections were established within this circuit in a precise manner from the beginning. Connections first appeared between motor neurons; mechanosensory neurons and interneurons started to connect at least a day later. This timing correlates with the development of behaviors, so the pattern of emerging connectivity could explain the appearance first of spontaneous behaviors (driven by a electrically coupled motor network) and then of evoked behaviors (when sensory neurons and interneurons are added to the circuit).

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.

Postinhibitory rebound (PIR) is defined as membrane depolarization occurring at the offset of a hyperpolarizing stimulus and is one of several intrinsic properties that may promote rhythmic electrical activity. PIR can be produced by several mechanisms including hyperpolarization-activated cation current (I(h)) or de-inactivation of depolarization-activated inward currents. Excitatory swim motor neurons in the leech exhibit PIR in response to injected current pulses or inhibitory synaptic input. Serotonin, a potent modulator of leech swimming behavior, increases the peak amplitude of PIR and decreases its duration, effects consistent with supporting rhythmic activity. In this study, we performed current clamp experiments on dorsal excitatory cell 3 (DE-3) and ventral excitatory cell 4 (VE-4). We found a significant difference in the shape of PIR responses expressed by these two cell types in normal saline, with DE-3 exhibiting a larger prolonged component. Exposing motor neurons to serotonin eliminated this difference. Cs+ had no effect on PIR, suggesting that I(h) plays no role. PIR was suppressed completely when low Na+ solution was combined with Ca2+-channel blockers. Our data support the hypothesis that PIR in swim motor neurons is produced by a combination of low-threshold Na+ and Ca2+ currents that begin to activate near -60 mV.

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.

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.

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.

Sensitization of the local bending reflex of the medicinal leech Hirudo medicinalis was studied in a semi-intact preparation in which behavioral and electrophysiological recordings were made simultaneously. 1. Sensitization of local bending could be produced in two ways: by repeated stimulation of the mechanoreceptor sensitive to pressure (the P cell), and by stimutlation of the mechanoreceptor sensitive to noxious stimuli (the N cell). 2. Both forms of sensitization produced a central neuronal change, measured as an increase in the number of stimulus-evoked action potentials in cell 3 (an excitor of dorsal longitudinal muscles). 3. Intracellular stimulation of serotonin-containing neurons 21 and 61 mimicked the sensitizing stimuli, but stimulation of the Retzius cell, which also contains serotonin, did not. 4. Stimulation of the Leydig cell, which releases octopamine, decreased the strength of local bending.

1. The maximum force exerted against an isometric force transducer by 6 leeches weighing 2.6–3.7 g, as they squeezed through apertures of different widths varied inversely with aperture width. 2. T cells in the leech skin code for velocity of indentation, not pressure or displacement. The frequency with which T cells fire is best described by two log functions, one for low, another for fast indentations. T cells responded to indentation velocities down to 10 μms−1. 3. The average threshold pressure for 5 P cells was 150 kPa and for 5 N cells was 521 kPa. 4. We conclude from these data that when leeches explore their mechanical environment and initiate contact with external objects, the threshold pressure for N cells is rarely crossed. Of the three classes of mechanoreceptor, T cells are the main modality through which leeches obtain contact information, though P cells may occasionally be recruited for local pressure peaks.

Cell Tr2 is a neuron in the subesophageal ganglion of the leech that can trigger swim episodes. In this report, we describe the ability of Tr2 to terminate ongoing swim episodes as well as to trigger swimming. Stimulation of Tr2 terminated ongoing swim episodes in nearly every preparation tested, while Tr2 stimulation triggered swim episodes in only a minority of the preparations. We suggest that the primary role of Tr2 is in the termination rather than the initiation of swimming activity. The swim trigger neuron Tr3 and a swim-gating neuron, cell 21, hyperpolarized during Tr2-induced swim termination. Another swim-gating neuron, cell 204 was sometimes slightly excited, but more often, hyperpolarized during Tr2-induced swim termination. In contrast to these cells, Tr2 stimulation excited another swim-gating neuron, cell 61. The responses of the swim-gating cells were variable in amplitude and sometimes not evident during Tr2-induced swim termination. Hence, the effects of Tr2 stimulation on swim-gating neurons seem unlikely to be the direct cause of swim termination. Oscillator cells examined during Tr2-induced swim termination include: 27, 28, 33, 60, 115, and 208. The largest effect seen in an oscillator neuron was in cell 208, which was repolarized by up to 10 mV during Tr2 stimulation. Tr2 stimulation did not produce any obvious synaptic effects in motor neurons DI-1, VI-1, and DE-3. Our findings indicate that other, yet undiscovered, connections are likely to be important in Tr2-induced swim termination. Therefore, we propose that cell Tr2 is probably a member of a distributed neural network involved in swim termination.

Two types of potassium channels of identified (p-) neurones of the leech (Hirudo medicinalis) were investigated by using the patch-clamp technique. The open-state probability of these channels in cell-attached patches can be reduced by addition of 5-hydroxytryptamine to the bath solution. After excising the patches the application of alkaline phosphatase to the cytosolic face of the patch increases the open probability. The 5-HT1A-receptor agonist buspirone mimics the effect of 5-HT. Our experiments show that the effect of 5-HT might be due to a channel phosphorylation via a 5-HT1A-receptor subtype.

The membrane potential of identified nerve (Retzius) cells and neuropil glial cells from 11 (±1) day-old embryos of the leechHirudo medicinalis was recorded using conventional intracellular microelectrodes. At this stage all ganglia of the segmental nervous system are formed. The membrane potential of Retzius cells was −68±4 mV (±SD,n=8), and showed a slope of 42 mV between 10 mM and 100 mM external K concentration. Retzius cells were able to fire action potentials which had a fast Na-dependent component, and, under appropriate conditions, also generated slow Ca (Ba) action potentials. The mean membrane potential of the neuropil glial cell at physiological K concentration (4 mM) was −83±5 mV (±SD,n=10), and showed a dependence of 56 mV for a tenfold change in the external K concentration (> 4mM). Neuropil glial cells showed no signs of voltage-activated excitability, but they repeatedly depolarized in the presence of 0.1 mM 5-HT.

1. A system of coupled neuronal oscillators, located in nearly all ganglia of the nerve cord, generates the rhythmic activity expressed as undulations in swimming medicinal leeches. Excitatory and inhibitory outputs from these segmental oscillators are conveyed to the dorsal and ventral longitudinal muscles of the body wall via a set of inhibitory and excitatory motor neurons. Contrary to published reports that electrical interactions alone interconnect the inhibitory motor neurons, the experiments described here reveal that chemical synaptic inhibition connects the dorsal inhibitory (DI) motor neurons to their ventral inhibitory (VI) counterparts. 2. These inhibitory interactions apparently result from monosynaptic, non-spike-mediated connections. The DI-VI connections are functionally important, for membrane potential oscillations in VI motor neurons are greatly attenuated when phasic DI input is removed. 3. Unlike previously described inhibition between the DI motor neurons and the dorsal excitatory (DE) motor neurons, the DI-VI inhibitory connections extend across the midline and hence provide synaptic links that insure that the left and right sides of the leech body make swimming movements in concert. 4. Depolarizing current pulses injected into DI motor neurons during the expression of swimming activity delay the cycle phase; hence these inhibitory motor neurons are candidates for membership in the oscillator circuit that generates swimming movements in the leech.

1. Bilateral pairs of heart accessory neurons (HA cells) have been identified on the ventral surface of the fifth and sixth segmental ganglia ofHirudo medicinalis. These neurons are rhythmically active and fire in a metastable phase relation to the heartbeat central pattern generator and heart activity. 2. The HA neurons project into the contralateral roots of their own ganglion and innervate the contralateral heart. The HA neurons of the fifth ganglion (HA (5) cells) send axons into the contralateral anterior connectives and branch in several more anterior ganglia to form axons that project into the contralateral roots. The HA(6) neurons are similar but their intersegmental projection is directed posteriorly. 3. The HA neurons receive rhythmic inhibitory synaptic input directly from the neurons of the heartbeat central pattern generator (HN interneurons). The HA cells also receive excitatory synaptic input, which can be spontaneous or driven by discharge in N mechanosensory cells. 4. The HA neurons can induce myogenic activity in quiescent hearts and accelerate the rhythm of hearts that are already myogenic. The HA neurons can also increase the beat tension in hearts that are entrained by phasic excitatory drive from heart motor neurons (i.e. the activity of the heartbeat central pattern generator). These influences, are mediated by tonic activity in the HA neurons. 5. The HA neurons appear to modulate the myogenic properties and contractile strength of the hearts.

Decouverte d'un nouveau membre du circuit oscillatoire de la nage chez la sangsue, un interneurone pair intersegmentaire appele cellule 115. Description detaillee de cette cellule 115 et mise en evidence de l'interaction inhibitrice reciproque de celle-ci avec les motoneurones DI

1. We have investigated the organization of feeding behavior by the medicinal leech,Hirudo medicinalis L. Hungry leeches position themselves at the water surface when they are in a resting state, and they are alerted by either mechanical or photic stimuli. 2. Alerted leeches orient into water waves and swim toward their apparent source with accuracy. They also exhibit an optokinetic orientation by swimming into moving bars of light. 3. Swimming ceases whenHirudo contact any surface which they explore by crawling, and upon finding a warm region, they bite. Biting has a thigmotactic component and a temperature preference of 37–40 °C. 4. Hirudo ingest blood meals averaging 8.9 times their initial weight. After feeding, leeches seek deeper water by crawling and they avoid warm surfaces. 5. Feeding by the medicinal leech has several behavioral alternatives which can be employed once the leech is alerted. Further, the act of feeding, itself, substantially alters the behavior of this sanguivorous annelid.

1. We have investigated functional roles of serotonin (5-hydroxytryptamine, 5-HT) in the feeding behavior of the medicinal leech,Hirudo medicinalis. Bathing intact leeches in serotonin (3×10−5 mol/l, 20 min) significantly alters three components of their feeding behavior. Leeches exposed to serotonin initiate swimming toward a vibrating point with a shorter latency, bite a warm surface more frequently, and ingest more blood than control animals. 2. Thermal stimulation of the prostomium evokes pharyngeal peristalsis, jaw movements and an obvious salivation by semidissected preparations. The application of serotonin (10−8 to 10−6 mol/l) directly activates and increases the rates of pharyngeal peristalsis and salivary secretion in these preparations. Serotonin also produces jaw movements and a constriction of the crop when centrally applied. 3. Vibrational stimuli, which evoke the swimming phase of feeding behavior, synaptically excite Retzius cells within segmental ganglia. Thermal stimuli, which evoke the biting phase, synaptically excite 5-HT-containing neurons within the subesophageal ganglia. Intracellular stimulation of identified 5-HT-containing neurons produces pharyngeal peristalsis reliably and enhances salivation. 4. Pharmacological lesions of particular 5-HT neurons abolishes feeding behavior. However, a brief exposure to exogenous 5-HT restores biting behavior. Well-fed leeches do not normally bite, but a brief bath in 5-HT will evoke biting behavior by them as well. 5. Serotonin is localized to a limited population of identifiable neurons in the C.N.S., and plays a pivotal and mandatory role in organizing the feeding behavior of the medicinal leech.

The potassium concentration was measured in the cytoplasm, perimicrovillar extracellular space (=‘vacuole’) and intercellular space of leech photoreceptors with double-barrelled potassium-sensitive microelectrodes in darkness and upon photostimulation. The mean intracellular potassium concentration in cells with membrane potentials >50 mV was 100±34 mmol/l. Photostimulation with 90 saturating 20 ms light flashes (1/s) evoked a potassium loss of 10.6±7.6 mmol/l. In the dark, there was no potassium concentration gradient between vacuole and intercellular space (K VAC + =4.5±0.9 mmol/l, K ECS + =4.5±0.5 mmol/l). In both compartments the potassium concentration increased upon repetitive photostimulation. Thus, the potassium loss from the cell is due to potassium movements across both the receptive and the non-receptive membrane domains.