Modeling a Neural Oscillator that Paces Heartbeat in the Medicinal Leech

SYNOPSIS. Heartbeat in the medicinal leech is paced by a neuraloscillator comprising two elemental oscillators whose activityis coordinated intersegmental coordinating fibers. The elementaloscillators each consist of a bilateral pair of heart interneuronslinked by reciprocal inhibitory synapses. The activity cycleof each elemental oscillator consists of alternating burstsof action potentials (plateau/burst phase) and periods inhibition(inactive phase). Oscillation ensues in the reciprocally inhibitorypairs because each neuron is able to escape from the inhibitionits contralateral partner and thus move on to the plateau/burstphase. We have identified and described membrane currents thatcontribute to oscillation and studied graded synaptic transmissionbetween the neurons, using discontinuous current clamp and switchingsingle electrode voltage clamp techniques. A hyperpolarization-activatedinward current, Ih, plays a major role in escape from inhibition,and Ca²⁺ currents produce plateau potentials that support burstformation and mediate graded synaptic transmission. To consolidate our knowledge and guide future research, we haveconstructed a first generation computer model of a neural oscillatorbased on reciprocal inhibition, using Hodgkin-Huxley equationsand a synaptic transfer model, derived from our biophysicalstudies, with Nodus software (De Schutter, 1989). This modelhas confirmed an important role for I_h in sustaining oscillationand has implicated a similarly important role for outward currents(particularly I_A), which remain to be studied. Neural oscillatorsbased on reciprocal inhibition appear to be ubiquitous, andour studies, biophysical and computational, provide insightsinto how they may operate.     

Spinal Mechanisms in the Control of Lamprey Swimming

SYNOPSIS. The lamprey, an anguilliform fish, swims using lateralundulatory movement; a transverse wave passes backward, fromhead to tail, the amplitude of the wave increasing as it movestailward. The wave of muscle activity producing this movementtravels down the body faster than the mechanical wave. The wayin which certain features of anguilliform movement contributeto its efficiency have been described. The neural activity underlyingswimming is characterized by: 1) rhythmical alternation betweenthe two sides of a single segment; 2) a burst duration thatremains a constant proportion of the cycle time and is independentof the cycle frequency; 3) rostrocaudal phase lag that is constantand also independent of the cycle frequency. Local circuitsin the lamprey spinal cord can generate this locomotory patternin the absence of sensory feedback following activation of excitatoryamino acid receptors; the pattern is centrally generated. Ithas been hypothesized that the spinal central pattern generatorfor locomotion consists of a series of segmental burst generatorscoupled together by an intersegmental coordinating system. Theintersegmental coordinating system functions to keep the frequenciesof the oscillators along the cord constant and to provide theappropriate rostrocaudal phase lag. Mechanosensitive units withinthe spinal cord are sensitive to movement of the spinal cord\notochordand movement of the spinal cord/notochord can entrain the burstpattern. Entrainment occurs through movement-related feedbackonto neurons at the local level. The possible roles this movement-relatedfeedback plays during locomotion are discussed.     

A Neural Network Model of Sensorimotor Transformations in the Local Bending Reflex of the Medicinal Leech

SYNOPSIS. In response to a moderate mechanical stimulus, theleech withdraws by forming a U-shaped local bend beneath thestimulus. Interneurons in the local bending reflex receive multiplesensory inputs, and have outputs to many motor neurons, suggestingthat stimulus location on the body is represented in a distributedfashion. However, it is possible that as yet unidentified interneuronsdedicated to the detection of specific spatial locations arenecessary for the full range of the local bending motor output.Using the backpropagation algorithm to optimize connectionsin a model of the reflex, we showed that the distributed processingmodel of sensorimotor integration was consistent with our knowledgeof the connection strengths between identified neurons in thereflex. The model further showed that the spatial and temporalconstraints on the linkage between sensory representations andmotor output can be satisfied by the appropriate connectivitybetween layers of interneurons and motor neurons in the leech,as well as in other systems with similar circuitry.     

Neural Generation of the Peristaltic and Non-Peristaltic Heartbeat Coordination Modes in the Leech, Hirudo Medicinalis

The bilateral paired heart tubes of the leech Hirudo medicinalisare controlled, via excitatory synapses, by a set of bilaterallypaired segmental heart motor neurons (HE cells) which are inturn controlled, via inhibitory synapses, by a set of bilaterallypaired segmental heart interneurons (HN cells). The HE cellsproduce rhythmic impulse bursts because their inherent steadydischarge is periodically inhibited by the HN cells, most ofwhich produce impulse bursts endogenously. The known synapticinteractions among the HN cells and HE cells can account wellfor the observed behavior of the hearts. The HE cells are coordinatedby the HN cells such that the segmental heart tube sectionson one side constrict in a caudorostral sequence (peristalsis),while the segmental heart tube sections on the other side constrictnearly synchronously (non-peristalsis). This difference in thecoordination modes of the two hearts is not permanent; reciprocalcoordination mode transitions occur every 10–50 heartbeatcycles. Only one member of HN(5) cell pair (the HN cells ofthe fifth segmental ganglion) is rhythmically active at a time,the other being completely inactive. By coordinating the frontand rear HN cells the active HN(5) cell produces non-peristalsisipsilaterally and peristalsis contralaterally. Reciprocal changesin the activity-inactivity pattern of the HN(5) cell pair areresponsible for the reciprocal changes in the coordination mode.     

Serotonergic Neural System not only Activates Swimming but also Inhibits Competing Neural Centers in a Pteropod Mollusc

Initiation of a particular behavior requires not only activationof the neural center directly involved in its control but alsoinhibition of the neural networks controlling competing behaviors.In the pteropod mollusc, Clione limacina, many identified serotonergicneurons activate or modulate different elements of the swimmingsystem resulting in the initiation or acceleration of the swimmingbehavior. Cerebral serotonergic neurons are described here,which produce excitatory inputs to the swimming system as wellas inhibitory inputs to the neural centers that control competingbehaviors. Whole-body withdrawal behavior is incompatible withswimming activity in Clione. The main characteristic of whole-bodywithdrawal is complete inhibition of swimming. Cerebral serotonergicneurons were found to produce a prominent inhibition of thepleural neurons that control whole-body withdrawal behavior.By inhibiting pleural withdrawal cells, serotonergic neuronseliminate its inhibitory influence on the swimming system andthus favor increased swimming speed. Serotonergic neurons alsoproduce a prominent inhibition of the Pleural White Cell, whichis presumably involved in reproductive or egg-laying behavior.Thus the serotonergic system directly activates swimming systemand, at the same time, alters a variety of other neural systemspreventing simultaneous initiation of incompatible behaviors.     

RFamide Peptides in the Leech, Hirudo medicinalis

RFamide peptides have been localized to a number of neuronsof the CNS of the leech, Hirudo medicinalis, using immunocytochemicaltechniques. The majority of this immunoreactivity appears tobe due to the peptide FMRFamide. Most of the identified RFamideimmunoreactive cells are cholinergic motor neurons, though someare interneurons. Superfused FMRFamide is active on the targetsof these identified neurons; in a few well studied cases, ithas been possible to show that FMRFamide mimics a specific physiologicalaction of an identified neuron on its target. In the leech as in other phyla where they occur, RFamide peptidesare widely distributed in neurons, and are neuromodulators withdiverse physiological effects.     

Electrical Interaction of Paired Ganglion Cells in the Leech

The two paired giant ganglion cells (PGC's) found in each ganglion of the leech central nervous system fire synchronously in response to certain sensory input. Polarizing current passed into either of these cells is seen to displace the membrane potentials of both cells, the voltage attenuation between the two somata ranging from 2 to 5 times. This attenuation factor remains unchanged when the direction of the polarizing current is reversed, and remains about the same when the other cell of the pair is directly polarized. When one of the PGC's is partially depolarized with outward current, a repetitive train of impulses is generated. Each spike is followed by a spike in the other cell. Occasionally, a small subspike potential is seen in place of a follower spike. This potential appears to differ in shape and time course from synaptic potentials elicited by afferent input to these cells, and appears rather to be an electrotonic potential derived from the prejunctional impulse in the stimulated PGC. It is proposed that transmission between these cells is electrical, being accomplished by a flow of local circuit current across a non-rectifying junction or connection to the spike-initiating region of the other PGC.     

Rickettsia Infection in Natural Leech Populations

Field-collected specimens of glossiphoniid leeches, Torix tagoi, Torix tukubana, Hemiclepsis marginata, and Hemiclepsis japonica, were surveyed for Rickettsia infection by using a diagnostic PCR assay. Rickettsia was detected in 96% (69/72) of T. tagoi, 83% (24/29) of T. tukubana, 29% (33/113) of H. marginata, and 0% (0/30) of H. japonica. The frequencies of Rickettsia infection were stably maintained in different seasons. In H. marginata and T. tukubana, distant local populations exhibited remarkably different frequencies of Rickettsia infection. Eggs carried by infected females of T. tagoi and H. marginata were all Rickettsia-positive, indicating nearly 100% vertical transmission. Analysis of 16S rDNA sequences revealed that phylogenetic relationship of the leech-associated Rickettsia reflected the specific and populational divisions of the host leeches. However, circumstantial lines of evidence strongly suggested that horizontal transmission of Rickettsia must have occurred in the ancestors of these leeches. In T. tagoi and T. tukubana, infected individuals were remarkably larger in size than uninfected individuals, wheras in H. marginata, infected and uninfected individuals were almost comparable in size. This study first provides information on ecological aspects of leech-bone endocellular bacteria of the genus Rickettsia. On the basis of these data, we discuss possible mechanisms whereby Rickettsia infection is maintained in natural populations of these leeches in the freshwater ecosystem.     

Functional Organization of the Neural Control of Circulation in Aplysia

The abdominal ganglion of Aplysia provides a useful model forstudying the functional organization of motor systems. Herewe review studies of the neural network controlling circulation,emphasizing the organizational features it may share with othermotor systems controlled by the abdominal ganglion. We identifiedseven motor neurons to the heart and vascular system. Motorneurons having similar motor effects (e.g. the two heart inhibitors,or the three vasoconstrictors), together with cells of unknownmotor function located near them, make up distinct homogeneouscell groups. The members of each group appear to be nearly identicalwith respect to biophysical and neurochemical properties, sizeand effectiveness of synaplie inputs, and firing patterns. Thereare no interconnections between the members of the groups, butfive interneurons innervate the homogeneous groups in variouscombinations, exciting some groups and inhibiting others. Twoof the interneurons, Interneuron I (cell I₁₀) and InterneuronII, are command cells which produce centrally generated motorprograms in the absence of sensory feedback. Eacli command apparentlycodes for a specific homeostatic function, such as increasedcardiac output. Coordination of the two commands is achievedby mutual inhibitory connections between them, ensuring thatthe motor neurons of the system receive only one command ata time. Some synaptic connections made by the command interneuronsappear to be functionally ineffective; the possible significanceof them is discussed. Available evidence suggests that manyfeatures of the network controlling circulation may be characteristicof other visceromotor systems of the abdominal ganglion.     

Multiple CheY Homologs Control Swimming Reversals and Transient Pauses in Azospirillum brasilense

Chemotaxis, together with motility, helps bacteria foraging in their habitat. Motile bacteria exhibit a variety of motility patterns, often controlled by chemotaxis, to promote dispersal. Motility in many bacteria is powered by a bidirectional flagellar motor. The flagellar motor has been known to briefly pause during rotation because of incomplete reversals or stator detachment. Transient pauses were previously observed in bacterial strains lacking CheY, and these events could not be explained by incomplete motor reversals or stator detachment. Here, we systematically analyzed swimming trajectories of various chemotaxis mutants of the monotrichous soil bacterium, Azospirillum brasilense. Like other polar flagellated bacterium, the main swimming pattern in A. brasilense is run and reverse. A. brasilense also uses run-pauses and putative run-reverse-flick-like swimming patterns, although these are rare events. A. brasilense mutant derivatives lacking the chemotaxis master histidine kinase, CheA4, or the central response regulator, CheY7, also showed transient pauses. Strikingly, the frequency of transient pauses increased dramatically in the absence of CheY4. Our findings collectively suggest that reversals and pauses are controlled through signaling by distinct CheY homologs, and thus are likely to be functionally important in the lifestyle of this soil organism.     

Swimming in mantids

The initiation, form of leg strokes and minimal requirements for swimming in Sphodromantis lineola are described.     

Cosexuality in the Leech,Nephelopsis obscura (Erpobdellidae)

Summary

Hermaphroditism of the erpobdellid leach Nephelopsis obscura is described on the basis of macroscopic and histological studies of the gonads. Perivitellogenic cells (female primordial germ cells) proliferate from the septa of segments XII and XIII in individuals of 26–30 mg and spermatogenic cells from segments XII to XIII of 20–25 mg specimens. The majority of hatch- lings develop functional testisacs when they reach 170 mg and functional ovisacs when they reach 250 mg. Oocytes proliferate only after the formation of stage 5–6 spermatozoa. Thus, N. obscura is a sequential cosexual hermaphrodite with spermatogenesis and mature spermatozoa development commencing before oogenesis. During the second cycle of gametogenesis observed in larger animals, both mature spermatozoa and ova co-occur and N. obscura exhibits simultaneous hermaphroditism.     

The External Structure and Distribution of Sensilla in the Medicinal Leech

Abstract The body of Hirudo medicinalis consists of 32 segments. The quinquannulate midbody segments 3–18 bear 14 sensilla on the central (neural) annulus. Elsewhere segments are represented by fewer than five annuli but sensilla are retained on the neural annulus. From neural sensilla protrude S hairs (cilia from uniciliate cells) which are thought to detect water currents. Two categories of multiciliate cell, of unknown function, are also present within neural sensilla; (i) grouped cilia extending beyond the cuticle (G hairs), and (ii) cilia which radiate out beneath the cuticle. Studies of the entire external surface of leech body wall with a scanning electron microscope revealed the presence of large numbers of sensilla on every annulus including the neural annuli. These sensilla lack S hairs. Our results show: (i) annular sensilla have a significantly smaller surface area than neural sensilla (p < 0.001 Mann-Whitney test, 2 tailed), (ii) The position and number of the small sensilla varies from annulus to annulus, segment to segment and individual to individual, (iii) Significantly higher numbers of small sensilla were found where non-neural annuli remain single and have not undergone a further division during development as happens in the quinquannulate segments. The data suggest that small sensilla continue to be added during the adult life of the leech.     

Sodium-dependent Potassium Channels in Leech P Neurons

In leech P neurons the inhibition of the Na⁺-K⁺ pump by ouabain or omission of bath K⁺ leaves the membrane potential unaffected for a prolonged period or even induces a marked membrane hyperpolarization, although the concentration gradients for K⁺ and Na⁺ are attenuated substantially. As shown previously, this stabilization of the membrane potential is caused by an increase in the K⁺ conductance of the plasma membrane, which compensates for the reduction of the K⁺ gradient. The data presented here strongly suggest that the increased K⁺ conductance is due to Na⁺-activated K⁺ (K_Na) channels. Specifically, an increase in the cytosolic Na⁺ concentration ([Na⁺]_i) was paralleled by a membrane hyperpolarization, a decrease in the input resistance (R_in) of the cells, and by the occurrence of an outwardly directed membrane current. The relationship between R_in and [Na⁺]_i followed a simple model in which the R_in decrease was attributed to K⁺ channels that are activated by the binding of three Na⁺ ions, with half-maximal activation at [Na⁺]_i between 45 and 70 mM. At maximum channel activation, R_in was reduced by more than 90%, suggesting a significant contribution of the K_Na channels to the physiological functioning of the cells, although evidence for such a contribution is still lacking. Injection experiments showed that the K_Na channels in leech P neurons are also activated by Li⁺.     

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 µm and extends over ~15 µm around the mean penetration depth, which is adjustable between 0 and 50 µm. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.Download video file.^{(116M, mov)}     

Neural Control of Muscle Androgen Receptors

The number of cytosolic androgen receptors in rat skeletal muscle increases following denervation and disuse. This increase was postulated to represent altered intracellular distribution and consequent diminished sensitivity of skeletal muscle to androgens. To test this hypothesis, we measured total (homogenate) androgen receptor levels after denervation. Total (homogenate) androgen receptor binding did not change in response to denervation of leg muscles from adult male rats. An increase in cytosolic receptor number with no increase in total (homogenate) receptor levels supports the hypothesis of altered intracellular distribution of androgen receptors in denervated muscle. Cytosolic androgen receptor binding in muscle from male rats increased by 40% after denervation, whereas in females the increase was 17%. These increases could not be altered by endocrine manipulations of males or females.