Axon extension and retraction by leech neurons: severing early projections to peripheral targets prevents normal retraction of other projections.

During leech embryogenesis, interactions between homologous neurons in neighboring segments lead to the selective retraction of longitudinal axonal projections by midbody AP and AE neurons, which maintain lateral axonal projections to the periphery. Results of experiments reported here show that disconnecting the lateral projections from the periphery rescues the projections normally fated to retract. We propose that these neurons normally progress through two states during early development, one in which they are insensitive to interactions with their homologs (state A) and a second in which they are sensitive (state B). Establishment of lateral connections with their targets triggers the switch from state A to state B; cutting these projections puts neurons back to state A.

Modulation of the pattern of axonal projections of a leech motor neuron by ablation or transplantation of its target.

The sixth segmental ganglion in the ventral nerve cord of the leech H. medicinalis contains a bilateral pair of rostral penile evertor motor neurons (RPEs) that in the adult innervate the male genitalia. During embryogenesis, the RPEs extend numerous extraganglionic projections. Only two of these innervate the target and are normally retained in the adult, while the others retract. Early, but not late, removal of the male genitalia results in the indefinite retention and continued growth of projections that would normally retract. Any of these projections can innervate targets transplanted to ectopic locations. We conclude that an RPE motor neuron requires a signal, provided by its interaction with the target organ during a critical period, in order to stop extending axons, stabilize those axons that contact the target, and retract those that do not.

RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse.

Receptor protein tyrosine phosphatases (RPTPs) are important for growth-cone migration [1-5], but their specific roles have yet to be defined. Previously, we showed that the growth cones of the Comb cell, an embryonic cell in the leech, express high levels of an RPTP called HmLAR2 [6,7]. Here, we report the use of RNA interference (RNAi) to block expression of HmLAR2 in individual Comb cells in the developing embryo. HmLAR2 mRNA levels were reduced in the soma, processes and growth cones of Comb cells injected with double-stranded RNA (dsRNA) for HmLAR2, but no decrease was detected when control dsRNAs were injected. Consistent with this observation, the level of phosphotyrosine increased significantly in the growth cones of Comb cells injected with HmLAR2 dsRNA. Within 24 hours, the growth cones of treated cells showed a distinct collapsed phenotype, with sharp reductions in lamellipodial surface area and in numbers of filopodia. These experiments indicate a key role for LAR-like RPTPs in maintaining the integrity of the growth cone.

Number and distribution of neurons in leech segmental ganglia.

The number of neurons and their distribution were determined for specific segmental ganglia from the nerve cord of four different species of leech. Quantitative data were obtained by using computer-aided techniques for the analysis of nerve structure from serially sectioned or whole-mounted tissue. The species studied were Hirudo medicinalis, Macrobdella decora, Haemopis marmorata (family Hirudinidae), and Haementeria ghilianii (family Glossophoniidae). Two sets of ganglia were studied in each species: middle ganglia (9, 10, and 11) and sex ganglia (5 and 6). The middle ganglia, as well as the rest of the 21 segmental ganglia, except 5 and 6, are thought to be quite similar. The sex ganglia are associated with the sexual organs and appear to have more neurons. The data reported here indicate that: a) the number of neurons in a specified ganglion varies by one to two percent from animal to animal of a given species; b) the middle ganglia of a particular leech each have approximately the same number of neurons, with a variation also within two percent; c) the middle ganglia of Hirudo, Macrobdella, and Haemopis have nearly the same number of neurons (about 400), but those of Haementeria have some 20 fewer (about 380); d) the sex ganglia of Hirudo, Macrobdella, and Haemopis have a few hundred more neurons than their middle ganglia, with the exact number varying according to the species, but the sex ganglia in Haementeria have only about 20 more neurons than their middle ganglia; e) the distribution of neuronal somata among glial packets is not symmetric about the midsaggital plane of the animal, and the number of somata in each packet is variable; and f) the geometry of the glial packets is generally invariant, but occasionally packets are found in abnormal positions.

Computer reconstruction of all the neurons in the optic ganglion of Daphnia magna.

The cellular architecture of the Daphnia compound eye visual system was studied by using computer-aided techniques. All the neurons in one half of the bilaterally symmetric optic ganglion (OG) were reconstructed in three dimensions from serial electron micrographs. The techniques employed were those developed by Levinthal and collaborators (Macagno, Levinthal, and Sobel, Ann. Rev. Biophys. Bioeng. 8:323-351, 1979). The approximately 200 neurons reconstructed were classified according to where they branch in the OG (the lamina and/or the medulla) and whether they send processes to the supraesophageal ganglion and/or across the midplane. Within each class, neurons were further characterized according to cell body location and size and location of their branching fields. Centrifugal processes from neurons with cell bodies not in the OG were also identified. These results provide the bases for a detailed examination of the synaptic connectivity of the identified neurons and for hypotheses concerning their functional roles in visually evoked behaviors.

Segmental differentiation in the leech nervous system: the genesis of cell number in the segmental ganglia of Haemopis marmorata.

In hirudinid leeches, the segmental ganglia associated with the sexual organs contain several hundred more neurons than other midbody ganglia. To determine whether this difference arises by differential cell addition or by differential cell death, cell counts were made in several segmental ganglia during the course of embryonic and postembryonic development. The results show that all ganglia behave equally in early development. In each case, at least 10-20% more cells than will make up the adult complement of about 400 neurons is generated, and by about 20 days of embryonic development cell loss brings the number down to about 400 cells. By about 30 days, when animals emerge from their cocoons, additional cells have begun to appear in the sex ganglia. The number of extra cells continue to increase gradually over the next several months, until the adult number of 600-700 neurons is attained. These observations indicate that at least some segmental differences in the size of neuronal populations are due to differential cell proliferation and that these differences can arise quite late in the maturation of an animal.

Segregation of afferent projections in the central nervous system of the leech Hirudo medicinalis.

Sensory axons originating in peripheral tissues converge onto each segmental ganglion in the central nervous system (CNS) of the leech, where they segregate into well-defined regions of the synaptic neuropil. Here we report on several aspects of the molecular and anatomical organizations of these afferent projections that bear upon the hypothesis that surface markers are involved in organizing these axons as they grow into the CNS. First, we show that the distribution of some surface markers in the adult is restricted to axons of peripheral origin and is not present on the neighboring axons of central neurons. Second, we demonstrate that the number of afferents increases postembryonically as the leech increases in size, suggesting that at least some of the cues employed by afferent axons to grow to appropriate central targets must be present throughout the life of the animal. We then show, using anterograde axonal tracing and immunohistochemistry, that there is both convergence and divergence of afferent axons into highly specific regions of the neuropil. Lastly, we examine the distribution of surface markers present on different subsets of afferents and show that axons having one type of marker segregate from those having the second type. Our results, considered together with previous observations in this system, provide new clues about the organization of afferent projections in the nervous system of the leech. They also suggest how a relatively small number of molecular markers might mediate fiber-fiber interactions to organize afferent axons as they grow into the CNS.

Segmental differentiation in the leech central nervous system: proposed segmental homologs of the heart accessory neurons.

As part of an on-going study of segmental differentiation in the central nervous system (CNS) of the leech Hirudo medicinalis, a search was made for putative segmental homologs of the heart accessory (HA) neurons, which exist exclusively as a bilateral pair in the ganglia of the fifth and sixth body segments. As it is not yet feasible to obtain adequate cell lineage information in H. medicinalis, potential homologs of the HA neurons were determined using morphological, immunohistochemical, and electrophysiological criteria. Among cells in other body ganglia with somata in the same locations as HA neurons, a pair was found having extensive morphological and physiological similarities to HA neurons. These we have called HA-like (HAL) neurons. Adult HA and HAL neurons have closely related patterns of primary branching, in terms of shape, intraganglionic pathways taken, and extraganglionic projections. The number, location, and relative thickness of branches are also similar among these cells. In embryos 10 to 11 days old, HA and HAL neurons have virtually identical branching patterns, with primary and secondary branches of nearly uniform caliber. Differences in branch thickness develop gradually; by embryonic day 20, they resemble those found in adult neurons. Two features found to differ between HA and HAL neurons were the cell body diameter (larger for the HA cells) and the expression of antigens recognized by the monoclonal antibody Laz1-1 (absent at a detectable level in the HA neurons). At a physiological level, the HA and HAL neurons showed action potentials of similar size and shape, as well as inhibitory synaptic inputs from a common source, the heart interneurons (HN). The observations presented here suggest that there is a common developmental origin for the HA and HAL neurons, and hence that their fates are positionally determined by as yet unknown factors.

Genesis of segmental identity in the leech nervous system.

The Antennapedia-class homeobox genes are likely to play a role in the specification of neuronal identities in invertebrates. The leech Hirudo medicinalis, a species well-suited for the analysis of these genes at the level of identified neurons, contains homologs of many Antennapedia-class and related genes. The expression pattern in the central nervous system of four leech homebox genes was examined in detail. Lox1 is expressed during early gangliogenesis in one pair of transient neurons present in every segment and, at later stages, in 15-20 pairs of neurons per segment. Lox2 is expressed in 25-30 pairs of neurons repeated in the posterior two-thirds of the midbody. Lox4 is present in 20-30 pairs of iterated neurons in the posterior half of the midbody, and in smaller subset of them in more anterior ganglia. Lox6 is expressed in 15-20 pairs of neurons of the third subesophageal neuromere and in fewer cells of more posterior ganglia. The subsets of neurons that express these homeobox genes are different but overlapping. Combinations of Lox genes could in theory generate enough variability to specify all central neurons in a leech ganglion.

The embryonic development of peripheral neurons in the body wall of the leech Haemopis marmorata.

The appearance of peripheral neurons within the skin during embryonic development of the leech is described. These neurons were labeled using a monoclonal antibody, Lan3-6, which recognizes antigens in both the cell body and the axons of these cells. Within the 5 annuli that are found in each midbody segment, peripheral neurons first label in the middle and last in the most anterior and posterior ones. In each annulus, the number of cells labeled is initially 4 and increases as development proceeds. By the end of embryogenesis, all annuli show approximately equal numbers of Lan3-6 labeled neurons. The development of peripheral neurons in the skin of the rear sucker is also described.

Regeneration of afferent axons into discrete tracts within peripheral nerves in the leech.

We have analyzed the pathway followed by regenerating afferent axons in peripheral nerves of the leech Hirudo medicinalis by anterograde labeling with horseradish peroxidase. We show that axons are able to reestablish appropriate pathways following a lesion (crush) which greatly disrupts the organization of the nerves. Our results are consistent with a pathway selection mechanism involving axon surface markers which are retained on the distal stumps of crushed axons.

A group of related surface glycoproteins distinguish sets and subsets of sensory afferents in the leech nervous system.

The distribution of 4 surface glycoproteins on axons of peripheral neurons was studied in the leech Hirudo medicinalis through monoclonal antibodies. All 4 glycoproteins have a similar molecular weight of 130 kDa. Immunohistochemical localization of these glycoproteins on tissue sections of nerves and neuropil reveals tracts of afferent axons organized as nested sets. Their distribution suggests a possible role for these molecules in mediating axon fasciculation.

The number and size of neurons in the CNS of gastropod molluscs and their suitability for optical recording of activity.

Several factors that affect the suitability of opticaL methods for monitoring neuron activity were evaluated in several species of gastropod molluscs. The mean cell body diameter and the total number of cells in the central nervous system were determined In 6 preparations and qualitative evaluations were made for an additional 25 species. There was a factor of 10 difference in mean diameter between species with the smallest cells (prosobranchs) and those with the largest (certain opisthobranchs). Several opisthobranchs had about 5000 central neurons; we estimate that the prosobranchs and pulmonates had at least 5 times as many neurons. When the opacity to transmitted light was measured the percent transmissions of circumesophageal ganglia were between 4% and 40%. We attempted to measure optical signals associated with spike activity in 20 gastropod species; in most species signals were readily detected in single trials.

Mapping of neuronal contacts with intracellular injection of horseradish peroxidase and Lucifer yellow in combination.

A technique for the simultaneous visualization in the light microscope of processes of neurons filled with horseradish peroxidase and Lucifer yellow in combination has been developed. The technique is applied to determine the location, number and distribution of presumptive synaptic sites between neurons in the leech central nervous system.

Morphological evidence that regenerating axons can fuse with severed axon segments.

Regenerating axons of sensory neurons in the leech nerve cord usually reconnect with their normal targets by growing the entire distance from the site of lesion to the target. However, in less than 1% to nearly 10% of cases a rapid restoration of the normal arborization occurs when the regenerating axon connects with the severed distal segment of the same cell or another cell of the same modality. The passage of horseradish peroxidase (mol. wt approximately 40,000 daltons) from the regenerating axon selectively into the axon or cell with which it has connected indicates that the two have joined or fused, rather than become linked by an electrical synapse, as sometimes occurs for other neurons in the leech. These results support the conclusions, based largely on physiological data from regenerating motor axons in crayfish, that unusually rapid and complete regeneration can occur when a growing axon fuses with its severed distal segment.

Cellular interactions and pattern formation in the development of the visual system of Daphnia magna (Crustacea, Branchiopoda). II. Induced retardation of optic axon ingrowth results in a delay in laminar neuron differentiation.

Groups of embryonic photoreceptors in one side of the compound eye of Daphnia were irradiated with an ultraviolet microbeam at a stage when the cells were postmitotic but had yet to elaborate axons. Immediately after irradiation, the embryos were placed under fluorescent illumination. On the average, 16 of the irradiated photoreceptors were killed by the exposure. Previous observations suggest that an approximately equal number were rescued by the post-irradiation fluorescent illumination. The schedule of differentiation of the rescued photoreceptors was affected such that their axons arrived at the target region in the optic lamina from 2 to 10 hr after they would have normally. Serial section electron microscopic analysis showed that differentiation of laminar neurons contacted by the delayed axons also was delayed by a length of time corresponding to the delay in axon arrival. These and previous observations indicate that the differentiation of laminar neurons is triggered by contact with optic axons and can be initiated over a period of several hours after these cells become postmitotic.

Interactions between segmental homologs and between isoneuronal branches guide the formation of sensory terminal fields.

Process outgrowth and peripheral field innervation by an identified mechanosensory neuron were examined in the intact embryonic leech. The dorsal pressure-sensitive (PD) neurons of the leech CNS are found as bilateral pairs in every segmental ganglion, and are amenable to study at early ages in intact embryos. Each PD has one major axonal projection that putatively pioneers the nerve to the dorsal body wall and branches extensively in its own segment, and two minor projections that innervate, via neighboring ganglia, smaller areas in adjacent segments. We found that adjacent embryonic PD cells form overlapping terminal fields in the body wall, but that the extent of overlap was governed by inhibitory interactions among these fields. When one PD neuron was ablated, the adjacent PD cell changed its peripheral arborization by (1) its major axon producing more filopodia and extending longer side branches toward the ablated cell and (2) its minor axon producing a large arbor in the operated segment. Interestingly, although growth was biased toward the side of the ablated neuron, reduced outgrowth of the PD cell was found on the side away from the ablation, while the total extent of arborization of the PD cell kept relatively constant. Further, we found that axotomy of the major PD projection resulted in extensive outgrowth of its minor projections. These results suggest that a single PD neuron has a limited capacity for growth, each of its branches growing at the expense of the others, and that inhibitory interactions between neighboring PD neurons influence the extent and direction of that growth.

Developing neurons use a putative pioneer's peripheral arbor to establish their terminal fields.

Pioneer neurons are known to guide later developing neurons during the initial phases of axonal outgrowth. To determine whether they are also important in the formation of terminal fields by the follower cells, we studied the role of a putative leech pioneer neuron, the pressure-sensitive (PD) neuron, in the establishment of other neurons' peripheral arbors. The PD neuron has a major axon that exits from its segmental ganglion to grow along the dorsal-posterior (DP) nerve to the dorsal body wall, where it arborizes extensively mainly in its own segment. It also has two minor axons that project to the two adjacent segments but branch to a lesser degree. We found that the peripheral projections of several later developing neurons, including the AP motor neuron and the TD sensory neuron, followed, with great precision, the major axon and peripheral arbor of the consegmental PD neuron, up to its fourth-order branches. When a PD neuron was ablated before it had grown to the body wall, the AP and TD axons grew normally toward and reached the target area, but then formed terminal arbors that were greatly reduced in size and abnormal in morphology. Further, if the ablation of a PD neuron was accompanied by the induction, in the same segment, of greater outgrowth of the minor axon of a PD neuron from the adjacent segment, the arbors of the same AP neurons grew along these novel PD neuron branches. These results demonstrate that the peripheral arbor of a PD neuron is a both necessary and sufficient template for the formation of normal terminal fields by certain later growing follower neurons.

Cell- and tissue-specific expression of putative protein kinase mRNAs in the embryonic leech, Hirudo medicinalis.

Protein kinases play important roles in various cellular interactions underlying metazoan development. To complement existing analyses of protein kinase function in the development of members of the three phyla, Chordata, Arthropoda, and Nematoda, we have begun to examine the cell- and tissue-specific localization of protein kinases in another metazoan phylum, the Annelida. For this purpose, we used the polymerase chain reaction to amplify putative protein kinase catalytic domain cDNAs from the medicinal leech, Hirudo medicinalis. This strategy allowed us to identify 11 cytoplasmic and receptor tyrosine kinase catalytic domains, and 2 cytoplasmic serine/threonine kinase catalytic domains. Using these cDNAs as probes for nonradioactive whole-mount in situ hybridization, we examined the embryonic expression pattern of each of the corresponding putative kinase mRNAs. As has been found in other species, most of the Hirudo protein kinase mRNAs were expressed in a highly specific manner in certain embryonic cells and tissues. We found both neuron- and glia-specific kinases within the nervous system, as well as kinases expressed in non-nervous tissues, such as the haemocoelomic, muscular, and excretory systems. These kinase cDNAs encode proteins likely to be critical for proper development, and can be used as cell- and tissue-specific histological probes for the analysis of Hirudo embryogenesis.