Embryonic expression of the single Tribolium engrailed homolog

We have cloned and sequenced the single Tribolium homolog of the Drosophila engrailed gene. The predicted protein contains a homeobox and several domains conserved among all engrailed genes identified to date. In addition it contains several features specific to the invected homologs of Bombyx and Drosophila, indicating that these features most likely were present in the ancestral gene in the common ancestor of holometabolous insects. We used the cross-reacting monoclonal antibody, 4D9, to follow the expression of the Engrailed protein during segmentation in Tribolium embryos. As in other insects, Engrailed accumulates in the nuclei of cells along the posterior margin of each segment. The first Engrailed stripe appears as the embryonic rudiment condenses. Then as the rudiment elongates into a germ band, Engrailed stripes appear in an anterior to posterior progression, just prior to morphological evidence of the formation of each segment. As in Drosophila (a long germ insect), expression of engrailed in Tribolium (classified as a short germ insect) is preceeded by the expression of several homologous segmentation genes, suggesting that similar genetic regulatory mechanisms are shared by diverse developmental types. © 1994 Wiley-Liss, Inc.     

Twelve variable microsatellite loci for the North American medicinal leech,Macrobdella decora

We isolated and characterized 12 microsatellite loci for the North American medicinal leech, Macrobdella decora. Macrobdella decora is abundant in central and northern North American freshwater systems. The resulting microsatellite library, the first for any species of leech or any clitellate annelid, demonstrates the efficacy of this type of analysis on unexploited leech populations and should serve as a baseline for comparison with other species, notably the commercially important and threatened European medicinal leech, Hirudo medicinalis.     

Localization of the myomodulin-like immunoreactivity in the leech CNS

The distribution of myomodulinlike immunoreactivity in the leech CNS was determined using an antiserum raised against Aplysia myomodulin. Segmental ganglia contained approximately 60 immunoreactive neurons. In addition, numerous fibers containing immunoreactive varicosities were found throughout the neuropil. Using a combination of Lucifer Yellow injections and immunocytochemistry, we identified neurons including the anterior Pagodas (AP), annulus erector (AE), motor neurons, Leydig, longitudinal muscle motoneurons (L), S cells, and coupling interneurons, all of which are active during the touch-elicited shortening reflex. FMRF-amide-like immunoreactivity in three of these cells (L, AP, and AE) was previously demonstrated. Specific staining for myomodulin was abolished by preadsorption of the antiserum with synthetic myomodulin, but not with FMRF-amide. These results suggest a potential role for myomodulin in both intrinsic and extrinsic modulation of the leech touch-elicited shortening reflex. Further, it is possible that several neurons mediating this reflex contain multiple neuromodulatory peptides. © 1996 John Wiley & Sons, Inc.     

Formation and specification of neurons during the development of the leech central nervous system

In the leech embryo, neurogenesis takes place within the context of a stereotyped cell lineage. The prospective germ layers are formed during the early cleavage divisions by the reorganization and segregation of circumscribed domains within the cytoplasm of the fertilized egg. The majority of central neurons arise from the ectoderm, and central neuroblasts are distributed throughout both the length and width of each ectodermal hemisegment. Much of the segmental ganglion arises from medial neuroblasts, but there are also lateral ectodermal neuroblasts and mesodermal neuroblasts that migrate into the nascent ganglion from peripheral sites of origin. Some of these migratory cells are committed to neurogenesis prior to reaching their central destination. In addition, the leech embryo exhibits a secondary phase of neurogenesis that is restricted to the two sex segment ganglia. Secondary neurogenesis requires that a mitogenic or trophic signal be conveyed from the peripherally located male sex organ to a particular set of centrally located neuroblasts, apparently via already differentiated central neurons that innervate the sex organ. The differential specification of neuronal phenotypes within the leech central nervous system occurs in multiple steps. Some aspects of a neuron's identity are already specified at the time of its terminal cell division and would seem to involve the lineal inheritance of developmental commitments made by one of the neuron's progenitors. This lineage-based identity can then be modified by interactions between the postmitotic neuron and other neurons or non-neuronal target cells encountered during its terminal differentiation. © 1995 John Wiley & Sons, Inc.     

Parallel pathways coordinate crawling in the medicinal leech,Hirudo medicinalis

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.Abbreviations C1-C7 caudal segments 1 through 7 (comprise the tail sucker) - Circ. circular muscle(s) - CD circular element driver - CPG central pattern generator - ED elongation element driver - El elongation - El _init initiation of elongation - FN Faivre's nerve - fs + front sucker attachment - s— front sucker release - Long longitudinal muscle(s) - M1-M21 midbody segments 1 through 21 - R1-R4 rostral segments 1 through 4 (comprise the head) - rs + rear sucker attachment - rs rear sucker release - Sens sensory input - SR stretch receptors(s) - ti tonic inhibition     

Cell lineage and segmentation in the leech

Segments in the leech arise by the proliferation of longitudinally arrayed bandlets of blast cells derived from ten identifiable embryonic stem cells, two M, two N, four O/P and two Q teloblasts. In each bandlet, older blast cells lie ahead of those born later. By using microinjected cell lineage tracers it was shown previously that the teloblasts give rise to characteristic cell patterns made up of segmentally iterated complements of progeny designated as M, N, O, P and Q kinship groups. When a teloblast is injected after it has begun generating blast cells, a boundary is observed later in development between anterior, unlabelled progeny of blast cells produced before injection and posterior, labelled progeny of blast cells produced after injection. We have examined such boundaries in detail to establish the precise relationship between blast cell clones and segments, with the following conclusions: (i) in the M, O and P cell lines, one blast cell generates one segmental complement of progeny, but serially homologous blast clones intermix so that no segment boundaries can be defined based on primary blast cell clones; (ii) in the N and Q cell lines, two blast cells are required to generate a complete segmental complement of progeny; (iii) in the process of forming the germinal plate, cells derived from the N and Q teloblasts move past those derived from the M and O/P teloblasts, so that consegmental blast cell clones do not come into register until well after the establishment of segmentally iterated units within each bandlet.     

Dynamic filtration of the expression pattern variability of Drosophila zygotic segmentation genes

Analysis of the quantitative data obtained by processing the confocal images showed that the initial variability of the expression pattern of Drosophila zygotic segmentation genes was strongly reduced by the onset of gastrulation. The following variability components were studied: the range of gene expression intensity in different embryos, the time and succession of the formation of expression domain, types of formation, and domain positioning. At the level of zygotic genes, the positioning error proved to be dynamically filtered with time.     

Target tissues affect cellular properties of cocultured leech Retzius neurons

The development of many neurons, including the Retzius (Rz) neurons of the medicinal leech, is shaped in part by interactions with other cells in the environment. To explore the nature of the interaction between growing Rz processes and potential target tissues, adult Rz neurons were cultured directly in contact with some of the tissues that normally serve as their targets in vivo. The morphology of the regenerated processes of these neurons varied depending upon the identity of the target tissues, but other cellular properties remained unchanged. In particular, although during normal development contact with peripheral targets determines the sign of Rz neurons' response to acetylcholine (ACh) applied to the soma, these cultured neurons maintained their original response to ACh even after as long as 2 weeks in culture on novel targets. Hence, some features of cultured adult Rz neurons varied depending upon the conditions, whereas other features remained fixed. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 55–68, 1998     

Diversity and modulation of ionic conductances in leech neurons

A complete understanding of animal behavior at the cellular level requires detailed information on the intrinsic biophysical properties of neurons, muscles, and the synaptic connections they make. In the past 10 to 15 years, electrophysiological studies of leech neurons have revealed a diverse array of voltage-gated ionic conductances distinguished by their pharmacological sensitivity to classic ion channel blockers. Voltage-clamp studies have provided new information about the kinetics and voltage-dependence of Na⁺ conductances, several K⁺ currents, including I_A, I_K and I_K(Ca.)' and high- and low-voltage-gated Ca²⁺ conductances. These studies showed that the action potentials of most leech neurons result from the usual sequence of permeability changes to Na⁺, K⁺, and Ca²⁺ ions. They also added insight as to the role played by particular combinations of conductances in providing individual neurons with electrical properties appropriate for the particular information they encode. Evidence is accumulating on the modulatory actions of endogenous neurotransmitters such as FMRFamide, serotonin, and octopamine on motor behaviors in the animal. Parallel studies suggest that changes in behavior can be explained, at least in part, by the alteration of firing patterns of selected neurons and muscles resulting form modulation of multiple ion conductances. This makes the leech exceptionally attractive for neuroethological studies because it is one of the simplest organisms in which the methods of psychology and neurobiology can be combined. Information gathered from this animal will therefore increase our understanding regarding general principles underlying the cellular basis of behavior. © 1995 John Wiley & Sons, Inc.     

Synapse formation and function: Insights from identified leech neurons in culture

Identified leech neurons in culture are providing novel insights to the signals underlying synapse formation and function. Identified neurons from the central nervous system of the leech can be removed individually and plated in culture, where they retain their characteristic physiological properties, grow neurites, and form specific synapses that are directly accessible by a variety of approaches. Synapses between cultured neurons can be chemical or electrical (either rectifying or not) or may not form, depending on the neuronal identities. Furthermore, the characteristics of these synapses depend on the regions of the cells that come into contact. The formation and physiology of synapses between the Retzius cell and its partners have been well characterized. Retzius cells form purely chemical, inhibitory synapses with pressuresensitive (P) cells where serotonin (5-HT) is the transmitter. Retzius cells synthesize 5-HT, which is stored in vesicles that recycle after 5-HT is secreted on stimulation. The release of 5-HT is quantal, calcium-dependent, and shows activity-dependent facilitation and depression. Anterograde and retrograde signals during synapse formation modify calcium currents, responses to 5-HT, and neurite outgrowth. The nature of these synaptogenic signals is being elucidated. For example, contact specifically with Retzius cells induces a localized selection of transmitter responses in postsynaptic P cells. This effect is signaled by tyrosine phosphorylation prior to synapse formation. © 1995 John Wiley & Sons, Inc.     

Kainate responses of leech Retzius neurons in situ and in vitro

Responses to the ionotropic glutamate receptor agonist kainate were measured in Retzius cells (RCs) of intact segmental ganglia (in situ), acutely isolated RCs, and cultured RCs (in vitro) of the leech Hirudo medicinalis. RCs in intact ganglia responded to kainate (5–20 μM) with depolarizations up to 30 mV or with an inward current under voltage-clamp that reversed near -10 mV. The membrane conductance increased by a factor of 2.5 at a holding potential of -70 mV in the presence of 20 μM kainate. In RCs in situ the membrane responses to 5 μM kainate increased when applied repeatedly 3-5 times. After this potentiation, the amplitude and time course of the membrane responses to 5 μM kainate were similar to the membrane response to 20 μM kainate. In current-clamp experiments kainate evoked an increase in intracellular calcium concentrations ([Ca²⁺]¡) only when the membrane depolarized beyond -40 mV. In voltage-clamped RCs at a holding potential of -70 mV, kainate caused no significant rise in [Ca²⁺]¡, indicating that the Ca²⁺ permeability of these kainate-gated ion channels appears to be negligible. The potentiation of the kainate-induced responses in RCs in situ was also present in voltage-clamped cells, where no or only small changes in [Ca²⁺]¡ occurred, suggesting that the underlying mechanism seemed to be independent of intracellular Ca²⁺ changes. In addition, the potentiation of the kainate-induced membrane responses was unaffected by cyclothiazide (100 μM), concanavalin A (0.5 mg/mL), and in the presence of extracellular low-Ca²⁺ and high-Mg²⁺ concentrations to suppress synaptic transmission in the ganglion. During whole-cell patch-clamp recordings (up to 50 min) potentiation remained the same indicating that small intracellular messenger molecules, which would be expected to dissipate, were not likely to be involved in mediating this potentiation. In acutely isolated RCs kainate induced no or only very small voltage responses. A potentiation of the kainate response was never observed in acutely isolated RCs. In cultured RCs (2–7 days in vitro) kainate evoked membrane responses with no apparent potentiation. Cultured RCs also responded with Ca²⁺ transients only when depolarized beyond -40 mV. The results show that RCs respond differently to kainate when kept isolated in culture compared to RCs in intact ganglia. The mechanism underlying the potentiation of the kainate response of RCs in situ, however, could not yet be identified. © 1996 John Wiley & Sons, Inc.     

Using reflexive behaviors of the medicinal leech to study information processing

The interneuronal network that produces local bending in the leech is distributed, in the sense that most of the interneurons involved are activated in all forms of local bending, even those in which their outputs would produce inappropriate movements. Such networks have been found to control a number of different behaviors in a variety of animals. This article reviews three issues: the physiological and modeling observations that led to the conclusion that local bending in leeches is controlled by a distributed system; what distributed processing means for this and other behaviors; and why the leech interneuronal network may have evolved to be distributed in the first place. © 1995 John Wiley & Sons, Inc.     

Temporal correlation between neuronal tail ganglion activity and locomotion in the leech,Hirudo medicinalis

Intracellular and extracellular recordings were performed in the posterior ventral nerve cord of restrained crawling preparations of the medicinal leech,Hirudo medicinalis. Short-latency neuronal activities in the tail ganglion nerves correlated with different phases of crawling behavior. Eight neurons with characteristic activation patterns during crawling were identified morphologically and physiologically in the tail ganglia of 23 preparations. The axons of four of these neurons projected through posterior tail brain nerves; four ascending interneurons had projections in the connectives or in Faivre's nerve. These interneurons are suitable candidates for carrying information between the front end and the tail end of the animal to coordinate the behavioral components during a crawling step.     

Neuronal responses to purinoceptor agonists in the leech central nervous system

Exracellular nucleotides like ATP and its derivatives are possible chemical messengers in vertebrate nervous systems. In invertebrate nervous systems, however, little is known about their role in neurotransmission. We have studied the reponse of identified neurones of the leech Hirudo medicinalis to the purinoceptor agonist ATP, ADP, AMP, and adenosine using conventional intracellular microelectrodes and whole-cell patch-clamp recording. Bath application of the agoinsts depolarized the different neurons, but not neuropil glial cells. The most effective responses (up to 10 mV) were observed with ATP (100 μM) or ADP (100 μM) in the noxious and touch cells. In most neurons the nonhydrolyzable ATP derivative ATP-γ-S (5 μM) induced larger depolarizations that 100 μM ATP, indicating that most of the potency of ATP is lost presumably due to its degradation by ectonucleotidases. In medial noxios cells, ATP (100 μM) induced an inward current of 1.7 ± 1.1 nA at a holding potential of ?60 mV. The ATP-induced current-voltage relationship showed an inward rectification and a reversal potential close to 0 m V. In a Na⁺-free extracellular solution, the ATP-induced inward current decreased and in a Na⁺- and Ca²⁺-free saline only a small residual current persisted. The possible P₂ purinoceptor antagonist suramin did not antagonize the ATP-induced current, but itself evoked an inward current and a conductance increase. We conclude that ATP activates nonselective cation channels in medial noxious cells of the leech with the order of potency of purinoceptor agonists ATP ≥ ADP > AMP. The results suggest that these cells express purinoceptors of the P₂ type. 1994 John Wiley & Sons, Inc.     

Steroids,histamine, and serotonin in the medicinal leech salivary gland secretion

Lipids represent 20% of the total weight of the dried pool of medicinal leech salivary gland secretion (SGS) obtained from about 50 individual animals. SGS lacks phospholipids, but contains steroids. Immunochemiluminescent analysis of SGS revealed the presence of free steroid hormones: cortisol, progesterone, testosterone, estradiol, and dehydroepiandrosterone. Micro-chromatographic-mass spectrometric analysis of SGS and its low molecular weight fraction (LMW) (molecular masses ranged from 220 to 850 Da) has shown the multicomponent nature of the LMW fraction. Using standard preparations as the reference steroid hormones (cortisol, dehydroepiandrosterone, androstenedione, and testosterone) and histamine and serotonine have been identified in SGS.     

The role of glutamate in swim initiation in the medicinal leech

Antagonists were used to investigate the role of the excitatory amino acid,l-glutamate, in the swim motor program ofHirudo medicinalis. In previous experiments, focal application ofl-glutamate or its non-NMDA agonists onto either the segmental swim-gating interneuron (cell 204) or the serotonergic Retzius cell resulted in prolonged excitation of the two cells and often in fictive swimming. Since brief stimulation of the subesophageal trigger interneuron (cell Tr1) evoked a similar response, we investigated the role of glutamate at these synapses. Kynurenic acid and two non-NMDA antagonists, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and Joro spider toxin, effectively suppressed (1) the sustained activation of cell 204 and the Retzius cell following cell Tr1 stimulation and (2) the monosynaptic connection from cell Tr1 to cell 204 and the Retzius cell, but did not block spontaneous or DP nerve-activated swimming. Other glutamate blockers, including -d-glutamylaminomethyl sulfonic acid,l(+)-2-amino-3-phosphonoproprionic acid and 2-amino-5-phosphonopentanoic acid, were ineffective. DNQX also blocked both indirect excitation of cell 204 and direct depolarization of cell Tr1 in response to mechanosensory P cell stimulation. Our findings show the involvement of non-NMDA receptors in activating the swim motor program at two levels: (1) P cell input to cell Tr1 and (2) cell Tr1 input to cell 204, and reveal an essential role for glutamate in swim initiation via the cell Tr1 pathway.