Similarities and differences in the structure of segmentally homologous neurons that control the hearts in the leech,Hirudo medicinalis

Summary The neural circuit that controls the hearts in the leech comprises an ensemble of synaptically interconnected cardiac motor neurons (HE cells) and cardiac interneurons (HN cells). Both the HE cells and the HN cells constitute segmentally homologous sets. We have investigated the structure of these neurons by iontophoretic injection of Lucifer Yellow dye.Bilateral pairs of HE cells have been identified in segmental ganglia 3–19 of the nerve cord. Their structure was found to be nearly identical from ganglion to ganglion and from animal to animal.Bilateral pairs of HN cells have been identified in segmental ganglia 1–7 of the nerve cord. Their dendritic structure was found to vary from ganglion to ganglion. These segmental differences among HN cells were observed consistently from animal to animal. Some of the segmental differences in HN cell structure correlate with previously described physiological differences.     

The segmentation of the gnathobdellid leeches with special reference to the Indian leech Hirudinaria and medicinal leech Hirudo

Large number of annuli in Hirudinea are not true segments, and in the absence of spacious bodycavity and septa in adult no decision was taken regarding limit of a somite, until Gratiolet 1862 recognised a segment by colour marking, repetition of nephridial openings, and especially by the presence of segmental receptors, distinguishing first annulus of a segment. Whitman 1884 gave precision to these determinations and analyzed morphology of leeches to logical completeness. He recognised that though Hirudinaria and Hirudo have 102 body annuli and posterior sucker, true segments are only 26 plus 7. Castle ('00) and Moore ('00) proposed a new scheme of segmentation, with segmental receptor bearing annulus, as central annulus of a complete somite, with nerve ganglion, like that of other annelids, in center of a segment. They orientated everything roundabout the ganglion without noticing distorted fate of organ system. In this paper both the views are compared. Morphological and embryological studies reveal that the annulus bearing the segmental receptors in uniformly first annulus of all segments, including incomplete segments at the two extremities, with nerve ganglion in first annulus of the segment. Clitellum occupies three natural segments, IX, X, XI; crop caeca, nephridia, testis sacs, haemocoelomic channels and “rhomboidal figures” formed by ventrolaterals, all make a complete unit, well integrated in such segment. Conclusive evidence comes from the presence of septa at the level of each nerve ganglion in embryos of Hirudinaria. These observations corroborate Gratiolet and Whitman's view.     

AN ANATOMICAL STUDY OF THE ABDOMINAL NERVOUS AND MUSCULAR SYSTEMS OF DRAGONFLY (AESHNIDAE) NYMPHS

The musculature of the fourth to eighth abdominal segments is typically composed of twenty pairs of segmental muscles associated with the body wall. In the first to third and ninth and tenth segments certain modifications to the basic plan occur in association with the abdominal-thoracic junction, the respiratory apparatus and the anal appendages. In some segments there are also paired muscles associated with the alimentary canal. Two large transverse muscles are present in the abdomen. There are eight abdominal ganglia, the first seven of which each give rise to three pairs of lateral nerves, the eighth to five pairs. In addition there are ten median abdominal nerves. The innervation fields of the various nerves are described. The first three pairs of lateral nerves of the last ganglion are homologous with the lateral nerves of the other abdominal ganglia; the fourth pair innervates most of segment nine; and the fifth pair innervates the remainder of segment nine, segment ten and the anal appendages. Certain of the abdominal muscles are innervated by branches from two different nerve roots. In segments six and seven the anterior point of attachment of the longitudinal stretch receptors is normally different from that in the other abdominal segments. This is discussed in the light of the types of movement which involve the abdomen and it seems apparent that these receptors are affected not only by swimming and abdominal flexion, as are the other longitudinal stretch receptors, but also by respiratory movements. Two distinct types of epidermal sensilla are present on the abdomen, spines and hairs. The former are the more numerous on the body, the latter on the anal appendages.     

Biogenic monoamines in Hirudo medicinalis

Summary The distribution of certain catecholamines and indoleamines in the ventral nerve cord and the body segments of the medical leech, Hirudo medicinalis, was studied with the fluorescence microscope technique of Falck and Hillarp, with microspectrofluorometry, and with chemical determinations of the amines. The six cells of the segmental ganglia previously shown to be chromaffin were found to contain an amine, most probably 5-hydroxytryptamine. In the two giant cells, the amine was found on the surface of coarse intracellular granules, lying mainly at the cell membrane, and at the nucleus. The two giant cells send their axons to the body muscles, which thus seem to have a 5-hydroxytryptaminergic innervation. The four smaller amine-containing cells of the segmental ganglia send their axons to the neuropil of the ganglion.The only cell type found to contain a catecholamine (probably noradrenaline) was situated in the anterior segmental nerve in the cell cluster anterior of the nephridial duct, one cell in each nerve. The axon of this cell terminates in two or more segmental ganglia; thus these neurons seem to be afferent.This work was supported by grants from the Swedish Natural Science Research Council (project no. 99-35) and the Swedish Medical Research Council (projects no. B 68-12 X-712-03 B and B 68-14 X-56-04 B).     

Embryonic cell lineages in the nervous system of the Glossiphoniid leech Helobdella triserialis

The lines of descent of cells of the nervous system of the leech Helobdella triserialis have been ascertained by injection of horseradish peroxidase (HRP) as a tracer into identified cells of early embryos. Such experiments show that the nervous system of the leech has several discrete embryological origins. Some of the neurons on one side of each of the segmental ganglia derive from a single cell, the ipsilateral N ectoteloblast. Other neurons derive from a different precursor cell, the ipsilateral OPQ cell that gives rise to the O, P, and Q ectoteloblasts. The positions within the ganglion of neuronal populations derived from each of these sources are relatively invariant from segment to segment and from specimen to specimen. Other nerve cord cells derive from the mesoteloblast M; of these four per segment appear to be the precursors of the muscle cells of the connective. The A, B, or C macromeres contribute cells to the supraesophageal ganglion. In preparations in which an N ectoteloblast was injected with HRP after production of its bandlet of n stem cells had begun, the boundary between unstained (rostral) and stained (caudal) tissues can fall within a ganglion or between ganglia. This suggests that each hemiganglion contains the descendants of more than one, and probably two, n stem cells.     

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.     

Gangliogenesis in leech: morphogenetic processes leading to segmentation in the central nervous system

 Using intracellular lineage tracers to study the main neurogenic lineage (N lineage) of the glossiphoniid leech embryo, we have characterized events leading from continuous columns of segmental founder cells (nf and ns primary blast cells) to discrete, segmentally iterated ganglia. The separation between prospective ganglia was first evident as a fissure between the posterior boundary of nf- and the anterior boundary of ns-derived progeny. We also identified the sublineages of nf-derived cells that contribute parallel stripes of cells to each segment. These stripes of cells project ventrolaterally from the dorsolateral margin of each nascent ganglion to the ventral body wall. The position and orientation of the stripes suggests that they play a role in forming the posterior segmental nerve; they are not coincident with the ganglionic boundary, and they form well after the separation of ganglionic primordia. Previous work has shown that cells in the anterior stripe express the leech engrailed-class gene. Thus, in contrast to the role of cells expressing engrailed in Drosophila, the stripes of N-derived cells expressing an engrailed-class gene in leech do not seem to play a direct role in segmentation or segment polarity. Received: 10 October 1997 / Accepted: 12 December 1997     

Glutamate-like immunoreactivity in the leech central nervous system

Summary Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of GINs consistently reacted with the glutamate antibody. Two medial pairs of GINs were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of GINs in the first segmental ganglion. An additional lateral pair of GINs was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of GINs in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.     

Glutamate-like immunoreactivity in the leech central nervous system.

Using a monoclonal antibody for glutamate the distribution was determined of glutamate-like immunoreactive neurons in the leech central nervous system (CNS). Glutamate-like immunoreactive neurons (GINs) were found to be localized to the anterior portion of the leech CNS: in the first segmental ganglion and in the subesophageal ganglion. Exactly five pairs of GINs consistently reacted with the glutamate antibody. Two medial pairs of GINs were located in the subesophageal ganglion and shared several morphological characteristics with two medial pairs of GINs in the first segmental ganglion. An additional lateral pair of GINs was also located in segmental ganglion 1. A pair of glutamate-like immunoreactive neurons, which are potential homologs of the lateral pair of GINs in segmental ganglion 1, were occasionally observed in more posterior segmental ganglia along with a selective group of neuronal processes. Thus only a small, localized population of neurons in the leech CNS appears to use glutamate as their neurotransmitter.     

External sensilla of the locust abdomen provide the central nervous system with an interganglionic network

External mechanoreceptors and contact chemoreceptors on the cuticle of the sixth abdominal segment of locusts have divergent primary projections of their sensory neurons that form arbours in the segmental and anterior abdominal ganglia. Homologous interganglionic projections from adjacent segments converge in the neuropile of each abdominal ganglion. Of the contributing types of sensilla, three were previously unknown for locust pregenital segments: tactile mechanosensory hairs with dual innervation, external proprioceptors of the hairplate type covered by intersegmental membranes and single campaniform sensilla that monitor cuticular strain in sternites and tergites. In general, interdependence of motor coordination in the abdominal segments is based on a neural network that relies heavily on intersegmental primary afferents that cooperate to identify the location, parameters and strength of external stimuli.     

Sensory innervation in the rim of the octopus sucker

Anatomical components of afferent innervation in the rim of the octopus sucker are described. In the sensory epithelium under the smooth cuticle two associated ciliated receptor cell-types (presumably chemosensitive) occur in clusters. A third ciliated receptor cell-type under the toothed cuticle may be a mechanoreceptor. A non-ciliated receptor cell-type of unknown function, under the toothed cuticle, is characterized by a microvillus-lined apical canal containing dense granular material. The axons of the latter two receptors go directly into large nerve tracts which nm through the infundibular muscle and on to the ganglion of the sucker. The axons of the first cell-types terminate on interneurons either in the base of the epithelium or below the epithelium. All the interneurons of the basal region of the epithelium migrate centripetally and develop into encapsulated interneurons. Within the epithelium, fine fibers provide collateral contact among cluster receptors. Collateral interaction among basal and encapsulated interneurons occur in the infundibular plexus. The microanatomy of the rim of the sucker suggests that chemosensory cues are funneled into the interneurons where they are concentrated into integrated signals, while other sensory input is probably sent directly to the ganglia of the sucker and/or arm.     

Extension and retraction of axonal projections by some developing neurons in the leech depends upon the existence of neighboring homologues. II. The AP and AE neurons

To assess the generality of our previous finding (Gao and Macagno, 1987) that segmental homologues play a role in the establishment of the pattern of axonal projections of the heart accessory HA neurons, we have extended our studies to two other identified leech neurons: the anterior pagoda (AP) neurons and the annulus erector (AE) motor neurons. Bilateral pairs of AP neurons are found in the first through the twentieth segmental ganglia (SG1 through SG20) of the leech ventral nerve cord. All AP neurons initially extend axonal projections to the contralateral periphery as well as longitudinal projections along the contralateral interganglionic connective nerves toward anterior and posterior neighboring ganglia. Although the peripheral projections are maintained by all AP neurons throughout the life of the animal, the longitudinal projections disappear in all but two segments: the AP neurons in SG1 maintain their anterior projections and extend them into the head ganglion, and those in SG20 maintain their posterior projections and extend them into SG21 and the tail ganglion. When single AP neurons are deleted anywhere along the nerve cord before processes begin to atrophy, however, the longitudinal projections are retained by their ipsilateral homologues in adjacent ganglia. The rescued processes appear to take over the projections of the deleted neurons. In cases where two or more AP neurons on the same side of the nerve cord are deleted from adjacent ganglia, a contralateral homologue sometimes extends projections to the periphery ipsilaterally or on both sides. We obtained similar results when we deleted single AE neurons from midbody ganglia. Thus, our experiments with three different identified neurons consistently show that the initial pattern of projections is the same in all ganglia, but that the existence of homologues in adjacent ganglia leads to the pruning of some of the initial projections. A consequence of this homologue-dependent process retraction is that neurons normally lacking neighboring homologues will have patterns of projections different from those neurons that do have such neighbors. Process loss by the HA, AP, and AE neurons may be the result either of competition for targets, inputs, or growth factors or of direct interactions among homologous cells.     

Developmental origin of segmental differences in the leech ectoderm: survival and differentiation of the distal tubule cell is determined by the host segment

The body plan of the adult leech is metameric, with each hemisegmental complement of ectodermal and mesodermal tissues being produced from a set of seven serially repeated embryonic blast cells. Previous studies have shown that homologous o blast cells give rise to an almost identical complement of descendant cells in each of the 21 abdominal segments, but that one o blast cell derivative--the distalmost cell of the nephridial tubule--is only present in 15 abdominal segments in the mature leech. Here we show that all o blast cells generate a presumptive distal tubule cell and that this cell migrates to its normal position in all abdominal segments. However, in segments which normally do not contain the mesodermal portion of the nephridium, the distal tubule cell dies before undergoing its terminal morphological differentiation. To ascertain whether the fate of the distal tubule cell is determined by its lineage history or by the segmental environment into which it is born, we utilized a previously described procedure for altering the segmental register between different embryonic cell lines. This procedure allowed us to effectively transplant o blast cells into more posterior segments prior to the cell divisions which generate their descendant clones. The results indicate that the survival or death of the distal tubule cell is determined by the identity of the host segment and that a given distal tubule cell could be effectively murdered or rescued by slipping its blast cell precursor into an appropriate segment. These findings suggest that the segment-specific pattern of distal tubule cell survival is not inherent to the O cell line, but arises from interactions with surrounding tissues.     

Neuronal competition determines the spatial pattern of neuropeptide expression by identified neurons of the leech.

Staining adult and embryonic leech ventral nerve cords with antibodies raised against the molluscan neuropeptides small cardioactive peptide B (SCP) and FMRFamide results in segment-specific and bilaterally asymmetric patterns of cell staining. One immunoreactive neuron, the RAS interneuron, is present in only four rostral segmental ganglia, while another, the CAS interneuron, is restricted to the four most caudal abdominal ganglia and tail. In addition to their segment-specific distributions, only one RAS or CAS cell is found in each segmental ganglion, and they alternate sides between adjacent ganglia (either L-R-L-R or R-L-R-L) with a fidelity of about 95%. This paper utilizes cell deletion techniques to investigate the determination of the asymmetric and alternating pattern of RAS and CAS neurons. We show that developmentally equivalent RAS and CAS homologs are present on both sides of the appropriate ganglia, and that within each ganglion one of the initially paired homologs loses the ability to assume the immunoreactive RAS or CAS fate 2-3 days after axonogenesis has begun. These experiments suggest that there is a competitive interaction between bilateral homologs which ensures that only one mature RAS/CAS neuron is formed per ganglion, and that contralateral RAS/CAS neurons are not required in the same or adjacent ganglia for the determination of the RAS or CAS developmental pathways. Nerve cord transections between ganglia in the CAS domain can alter the spatial pattern of CAS neuron determination, confirming that both bilateral homologs retain the ability to express neuropeptide until late embryonic stages, and suggesting that the alternating pattern of RAS/CAS cells requires communication between adjacent ganglia through the longitudinal connectives.     

Sensory innervation of the canine esophagus, stomach, and duodenum.

The sensory innervation of the postpharyngeal foregut was investigated by injecting the enzyme horseradish peroxidase (HRP) into the walls of the esophagus, stomach, or duodenum. The transported HRP was identified histochemically, labeled neurons in the spinal and vagal ganglia were counted, and the results were plotted using an SAS statistical program. The spinal sensory fields of each viscus were defined using three determinations: craniocaudal extent, principal innervation field, and peak innervation field. The data revealed that innervation fields are craniocaudally extensive, the sensory field of each viscus overlaps significantly with its neighbor, yet each viscus can be characterized by a field of peak innervation density. Craniocaudal innervation of the esophagus spans as many as 22-23 paired spinal ganglia (C1-L2). There are two peak innervation fields for the cervical (C2-C6 and T2-T4) and for the thoracic (T2-T4 and T8-T12) sectors of the esophagus. The sensory innervation of the stomach extends craniocaudally over as many as 25 paired spinal ganglia (C2-L5). The peak innervation field of the stomach spans a large area comprising the cranial, middle, and the immediately adjoining caudal thoracic ganglia (T2-T10). The duodenum is innervated craniocaudally by as many as 15 paired thoracolumbar ganglia (T2-L3). Peak innervation originates in the middle and caudal thoracic ganglia and cranial lumbar (T6-L1) ganglia. There is a recognizable viscerotopic organization in the sensory innervation of the postpharyngeal foregut; successively more caudal sectors of this region of the alimentary canal are supplied with sensory fibers from successively more caudal spinal dorsal root ganglia. Vagal afferent innervation of the esophagus, stomach, and duodenum is bilateral and originates predominantly, but not exclusively, from vast numbers of neurons in the nodose (distal) ganglia. The esophagus is innervated bilaterally and more abundantly by jugular (proximal) ganglia neurons than is either the stomach or duodenum. The physiological significance of the findings are discussed in relation to the phenomena of visceral pain and referred pain.     

Descending course of primary afferent collaterals in the cat's spinal cord.

After injecting horseradish-peroxidase into the lower thoracic, lumbar and sacral spinal cord segments of cats, labelled perikarya were found in several spinal ganglia localized cranially to the site of injection. The segmental distance between the injection site and the rostralmost localized ganglion with labelled cells varied depending on the medio-lateral localization of the injection. The longest distance (10 segments) was achieved by medial injections. Primary sensory neurones with long descending collaterals belong to large ganglion cells.     

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.     

Transplantation of neurons reveals processing areas and rules for synaptic connectivity in the cricket nervous system

In order to assess the nature of spatial cues in determining the characteristic projection sites of sensory neurons in the CNS, we have transplanted sensory neurons of the cricket Acheta domesticus to ectopic locations. Thoracic campaniform sensilla (CS) function as proprioceptors and project to an intermediate layer of neuropil in thoracic ganglia while cercal CS transduce tactile information and project into a ventral layer in the terminal abdominal ganglion (TAG). When transplanted to ectopic locations, these afferents retain their modality-specific projection in the host ganglion and terminate in the layer of neuropil homologous to that of their ganglion of origin. Thus, thoracic CS neurons project to intermediate neuropil when transplanted to the abdomen and cercal CS neurons project to a ventral layer of neuropil when transplanted to the thorax. We conclude that CS can be separated into two classes based on their characteristic axonal projections within each segmental ganglion. We also found that the sensory neurons innervating tactile hairs project to ventral neuropil in any ganglion they encounter after transplantation. Ectopic sensory neurons can form functional synaptic connections with identified interneurons located within the host ganglia. The new contacts formed by these ectopic sensory neurons can be with normal targets, which arborize within the same layer of neuropil in each segmental ganglion, or with novel targets, which lack dendrites in the normal ganglion and are thus normally unavailable for synaptogenesis. These observations suggest that a limited set of molecular markers are utilized for cell–cell recognition in each segmentally homologous ganglion. Regenerating sensory neurons can recognize novel postsynaptic neurons if they have dendrites in the appropriate layer of neuropil. We suggest that spatial constraints produced by the segmentation and the modality-specific layering of the nervous system have a pivotal role in determining synaptic specificity. © 1993 John Wiley & Sons, Inc.     

Segmental specialization of neuronal connectivity in the leech

1. Every segmental ganglion of the leech Hirudo medicinalis contains two serotonergic Retzius cells. However, Retzius cells in the two segmental ganglia associated with reproductive function are morphologically distinct from Retzius cells elsewhere. This suggested that these Retzius cells might be physiologically distinct as well. 2. The degree of electrical coupling between Retzius cells distinguishes the reproductive Retzius cells; all Retzius cells are coupled in a non-rectifying manner, but reproductive Retzius cells are less strongly coupled. 3. Retzius cells in standard ganglia depolarize following swim motor pattern initiation or mechanosensory stimulation while Retzius cells in reproductive ganglia either do not respond or hyperpolarize. 4. In standard Retzius cells the depolarizing response caused by pressure mechanosensory neurons has fixed latency and one-to-one correspondence between the mechanosensory neuron action potentials and Retzius cell EPSPs. However, the latency is longer than for most known monosynaptic connections in the leech. 5. Raising the concentration of divalent cations in the bathing solution to increase thresholds abolishes the mechanosensory neuron-evoked EPSP in standard Retzius cells. This suggests that generation of action potentials in an interneuron is required for production of the EPSP, and therefore that the pathway from mechanosensory neuron to Retzius cell is polysynaptic. 6. P cells in reproductive segments have opposite effects on reproductive Retzius cells and standard Retzius cells in adjacent ganglia. Thus the difference in the pathway from P to Retzius is not localized specifically in the P cell, but elsewhere in the pathway, possibly in the type of receptor expressed by the Retzius cells.