Pentylenetetrazol-induced seizure-like behavior and neural hyperactivity in the medicinal leech.

This study examined the capacity of a known pro-epileptic drug, pentylenetetrazol (PTZ), to elicit seizure-like activity in the medicinal leech, Hirudo verbana. During in vivo experiments, PTZ elicited increased motor activity in a concentration-dependent manner with the highest concentration (10 mM) eliciting episodes of highly uncoordinated exploratory and swimming behavior. Co-application of the anti-epileptic drug, phenytoin, failed to reduce the absolute amount of PTZ-induced motor behavior, but was able to prevent expression of abnormal exploratory and swimming behaviors. During in vitro experiments in which extracellular recordings of connective nerve activity were made, bath application of 1 µM PTZ in Mg(2+)-free saline elicited a significant increase in spontaneous activity. This PTZ-induced increase in activity was completely inhibited by phenytoin. Interestingly, PTZ-induced hyperactivity was also blocked by co-application of the endocannabinoid 2-arachidonoyl glycerol and the selective serotonin re-uptake inhibitor (SSRI) fluoxetine. These findings suggest that the leech can be a useful system in which to study potential anti-epileptic treatments.

The medicinal leech genome encodes 21 innexin genes: different combinations are expressed by identified central neurons.

Gap junctional proteins are important components of signaling pathways required for the development and ongoing functions of all animal tissues, particularly the nervous system, where they function in the intracellular and extracellular exchange of small signaling factors and ions. In animals whose genomes have been sufficiently sequenced, large families of these proteins, connexins, pannexins, and innexins, have been found, with 25 innexins in the nematode Caenorhabditis elegans Starich et al. (Cell Commun Adhes 8: 311-314, 2001) and at least 37 connexins in the zebrafish Danio rerio Cruciani and Mikalsen (Biol Chem 388:253-264, 2009). Having recently sequenced the medicinal leech Hirudo verbana genome, we now report the presence of 21 innexin genes in this species, nine more than we had previously reported from the analysis of an EST-derived transcriptomic database Dykes and Macagno (Dev Genes Evol 216: 185-97, 2006); Macagno et al. (BMC Genomics 25:407, 2010). Gene structure analyses show that, depending on the leech innexin gene, they can contain from 0 to 6 introns, with closely related paralogs showing the same number of introns. Phylogenetic trees comparing Hirudo to another distantly related leech species, Helobdella robusta, shows a high degree of orthology, whereas comparison to other annelids shows a relatively low level. Comparisons with other Lophotrochozoans, Ecdyzozoans and with vertebrate pannexins suggest a low number (one to two) of ancestral innexin/pannexins at the protostome/deuterostome split. Whole-mount in situ hybridization for individual genes in early embryos shows that ∼50% of the expressed innexins are detectable in multiple tissues. Expression analyses using quantitative PCR show that ∼70% of the Hirudo innexins are expressed in the nervous system, with most of these detected in early development. Finally, quantitative PCR analysis of several identified adult neurons detects the presence of different combinations of innexin genes, a property that may underlie the participation of these neurons in different adult coupling circuits.

Activation of two forms of locomotion by a previously identified trigger interneuron for swimming in the medicinal leech.

Higher-order projection interneurons that function in more than one behavior have been identified in a number of preparations. In this study, we document that stimulation of cell Tr1, a previously identified trigger interneuron for swimming in the medicinal leech, can also elicit the motor program for crawling in isolated nerve cords. We also show that motor choice is independent of the firing frequency of Tr1 and amount of spiking activity recorded extracellularly at three locations along the ventral nerve cord prior to Tr1 stimulation. On the other hand, during Tr1 stimulation there is a significant difference in the amount of activity elicited in the ventral nerve cord that correlates with the motor program activated. On average, Tr1 stimulation trials that lead to crawling elicit greater amounts of activity than in trials that lead to swimming.

Reduced axon sprouting after treatment that diminishes microglia accumulation at lesions in the leech CNS.

The role of mammalian microglia in central nervous system (CNS) repair is controversial. Microglia accumulate at lesions where they act as immune cells and phagocytize debris, and they may secrete neurotrophins, but they also produce molecules that can be cytotoxic, like nitric oxide (NO). To determine the importance of microglial accumulation at lesions on growth of severed CNS axons in the leech (Hirudo medicinalis), in which axon and synapse regeneration are notably successful even when isolated in tissue culture medium, microglial migration to lesions was reduced. Pressure (P) sensory neurons were injected with biocytin to reveal the extent of their sprouting 24 hours after lesioning. To reduce microglia accumulation at lesions, cords were treated for 3.5 hours with 3 mM ATP or 2 mM N(omega)-nitro-L-arginine methyl ester (L-NAME) or 50 microM Reactive blue-2 (RB2) beginning 30 minutes before injury. Lesioned controls were either not treated with drug or treated 3 hours later with one of the drugs, after the migration and subsequent accumulation of most microglia had occurred, but before the onset of axon sprouting, for a total of seven separate conditions. There was a significant reduction in total sprout lengths compared with controls when microglial accumulation was reduced. The results suggest that microglial cells are necessary for the usual sprouting of injured axons.

Association of LAR-like receptor protein tyrosine phosphatases with an enabled homolog in Hirudo medicinalis.

Receptor protein tyrosine phosphatases (RPTPs) are thought to play important functions in pathfinding and target recognition by growing neuronal processes. The leech RPTPs HmLAR1 and HmLAR2 are expressed selectively by central neurons, Comb cells, and peripheral muscle tissues in the Hirudo medicinalis embryo. To explore the functions of HmLARs, we have sought to determine their physiological substrates. We report here the cloning and embryonic expression of Lena, the leech homolog of Enabled, a cytosolic protein implicated in actin-based cell motility. Lena is expressed in embryonic central neurons and in the Comb cell. We present experimental evidences indicating that Lena associates selectively with the intracellular domain of HmLAR1 and HmLAR2. Additionally, RNA interference (RNAi) of HmLAR1 in intact leech embryos leads to the hyperphosphorylation of Lena. We propose, therefore, that Lena is an in vivo substrate of HmLAR1 in neurons and perhaps of HmLAR2 in the Comb cells.

Distribution and development of dopamine- and octopamine-synthesizing neurons in the medicinal leech.

Although the medicinal leech is a well-studied system in which many neurons and circuits have been identified with precision, descriptions of the distributions of some of the major biogenic amines, such as dopamine (DA) and octopamine (OA), have yet to be completed. In the European medicinal leech Hirudo medicinalis and the American medicinal leech Macrobdella decora,we have presented the first immunohistochemical study of DA neurons in the entire central nervous system, and of OA-immunoreactive (ir) neurons in the head and tail brains. Dopaminergic neurons were identified using the glyoxylic acid method and antisera to DA and its rate-limiting synthetic enzyme tyrosine hydroxylase (TH). Octopaminergic neurons were recognized using a highly specific antiserum raised against OA. An antibody raised against DA-beta-hydroxylase (DbetaH), the mammalian enzyme that converts DA to norepinephrine (NE), was found to immunostain OA-ir neurons. This antibody appears to cross-react with the closely related invertebrate enzyme tyramine-beta-hydroxylase, which converts tyramine to OA, suggesting that the OA-ir cells are indeed octopaminergic, capable of synthesizing OA. Because the DbetaH antiserum selectively immunostained the OA-ir neurons, but not the DA-synthesizing cells, our results also indicate that the DA-ir neurons synthesize DA and not NE as their end product. The expression of TH immunoreactivity was found to emerge relatively early in development, on embryonic day 9 (47-48% of development). In contrast, OA expression remained absent as late as embryonic day 20. Higher order processes of some of the dopaminergic and octopaminergic neurons in the adult brain were observed to project to a region previously described as a neurohemal complex. Several TH-ir processes were also seen in the stomatogastric nerve ring, suggesting that DA may play a role in the regulation of biting behavior. By mapping the distributions and developmental expression pattern of DA and OA neurons in the leech, we aim to gain a better understanding of the functional roles of aminergic neurons and how they influence behavior.

Chemical and thermal stimuli have short-lived effects on the retzius cell in the medicinal leech.

During the appetitive phase of feeding, hungry leeches detect a prey by the integration of signals perceived by different sensory systems. Earlier reports suggested that chemical or thermal sensory stimulation of the lip was associated with increased afferent activity in cephalic nerves connecting the lip to the central nervous system. These authors further suggested that this activity was relayed to Retzius cells in segmental ganglia, which then released serotonin to initiate and control all aspects of feeding behavior. In this study, we show that chemosensory or thermal activation of the lip lasting for at least 5 min produces a distinct signal in the cephalic nerves consisting of action potentials of low amplitude. These small amplitude signals are clearly distinguishable from the large action potentials evoked by mechanosensory stimuli applied to the same area of the lip. Both types of sensory stimuli also evoke an increase in the firing frequency of the Retzius cells in segmental ganglia. However, the response recorded in the nerves and the Retzius cells during a maintained stimulus is not constant but decreases with an exponential time course. These results agree with our earlier observations on a semi-intact feeding preparation in which we showed that the firing frequency of the Retzius cell decreased as soon as the leech began to ingest its meal. Therefore, our data provide further evidence suggesting that it is unlikely that heat or chemical cues maintain the Retzius cell in an active state throughout the consummatory phase of feeding.

Development of neuronal circuits and behaviors in the medicinal leech.

We are studying the neuronal mechanisms responsible for establishing circuitry underlying the local bending response in the medicinal leech. Local bending replaces an embryonic behavior, circumferential indentation, during the time of initial chemical synaptogenesis in leech embryos. We found that the electrical connections among the motor neurons are established first, about 5% of embryonic time (almost 2 full days) before chemical connections form. The inhibitory connections from muscle inhibitors to muscle excitors are, we hypothesize, responsible for the emergence of local bending. We have also found that the central processes of the excitors--but not the inhibitors--have much longer central processes when their peripheral processes are kept from contacting their target muscles. This system should allow us to test ideas about how individual neurons find their appropriate targets to form functional neuronal circuits.

Regulation of pharyngeal motility by FMRFamide and related peptides in the medicinal leech, Hirudo medicinalis.

The medicinal leech possesses FMRFamide-like immunoreactivity in neural processes and somata associated with the pharynx and pharyngeal ganglia. The pharynx possessed about 25 immunoreactive somata; about half of the approximately 20 neurons of the pharyngeal ganglia were immunoreactive. We provide brief descriptions of several neurons located in the first neuromere of the subesophageal ganglion involved in controlling pharyngeal motility. Double-labeling experiments indicate that one of these cells, named Swallow neuron 1 (SW1), contains a FMRFamide-like peptide. Stimulation of SW1 caused the mouth to open and the pharynx to dilate. Upon termination of SW1 stimulation, the mouth closed, and a peristaltic wave progressed from the mouth down the length of the pharynx. Stimulation of SW1 did not produce 1:1 postsynaptic potentials in pharyngeal muscle cells. Thus, SW1 is apparently not a motor neuron. The pharynx responded to application of FMRFamide and related peptides by producing a series of 20- to 35-s phasic contractions superimposed upon an increase in basal tonus. The peptide-induced response was quantified by measuring increases in basal tonus, peak tension, and integrated area. Although there were some differences in the order of potency depending upon which parameter was considered, the approximate order of potency of RFamide peptides tested was: pQDPFLRFamide > or = FMRFamide approximately YGGFMRFamide > or = YMRFamide approximately FLRFamide approximately GGKYMRFamide approximately YLRFamide > leucomyosuppressin approximately perisulfakinin. Except for differences in potency, each of the RFamide peptides produced similar contractile waveforms. FMRFamide-induced responses were reduced by the protein kinase C inhibitor bisindolylmaleimide I (10 microM), the nonspecific protein kinase inhibitor H-7 (50 microM), and were increased by the protein phosphatase inhibitor okadaic acid (1 microM). However, the FMRFamide-induced response was unaffected by the protein kinase A inhibitor H-89 (1 microM), the phosphodiesterase inhibitor theophylline (1 mM), the phospholipase A(2) inhibitor OBAA (0.1 microM) or the cation channel blocker amiloride (100 microM).

Embryonic assembly of a complex muscle is directed by a single identified cell in the medicinal leech.

The present study examines the morphological development of a highly organized muscle layer in the leech Hirudo medicinalis, in an effort to characterize those factors that are important in directing its assembly. The tubular body wall of the leech contains 3 major muscle layers that are anatomically distinct: an inner layer of longitudinal muscle, an outer layer of circular muscle, and a grid of oblique muscle sandwiched between them. The oblique muscle layer appears later in development than the other 2 and is preceded by several days by the development of a single, complex cell (here called the comb, or C-cell) whose shape strongly resembles the organization of the oblique muscle grid. There is a bilateral, mirror-image pair of C-cells in each segment. The C-cell has a central, longitudinally oriented soma and projects about 35 fine, parallel processes both medially and laterally at approximately 45 degrees to the long axis. Using a combination of intracellular and antibody labels, it was found that individual muscle cells align themselves with these processes to form correctly oriented fascicles during development. Photoablation of the C-cell at early stages resulted in the complete absence of all oblique muscle fascicles that would have corresponded to that cell; therefore, this discrete muscle-associated cell is considered to be an identified "muscle organizer." Such cellular organizers may direct muscular and neuromuscular assembly in many species.

Identification of motor neurons that contain a FMRFamidelike peptide and the effects of FMRFamide on longitudinal muscle in the medicinal leech, Hirudo medicinalis.

Excitatory motor neurons in the leech are cholinergic. By using a combination of intracellular Lucifer yellow injection and indirect immunofluorescence, we localized FMRFamidelike immunoreactivity to a number of the motor neurons innervating longitudinal and dorsoventral muscle in the leech. All excitatory motor neurons innervating longitudinal muscle (cells 3, 4, 5, 6, 8, L, 106, 107, 108) were labeled with an antiserum to FMRFamide, while the inhibitory motor neurons innervating longitudinal muscle (cells, 1, 2, 7, 9, 102) were not. The excitatory motor neuron innervating medial dorsoventral muscle (cell 117) was labeled, while the excitatory motor neuron innervating lateral dorsoventral muscle (cell 109) was not. The inhibitory motor neuron innervating dorsoventral muscle (cell 101) was also labeled. Nerve terminals along dorsoventral muscle were also labeled with the antiserum. FMRFamide was bath applied to strips of longitudinal muscle while recording tension, and the muscle's response was compared to its response to the previously identified neuromuscular transmitter ACh. Brief applications of FMRFamide caused a contraction approximately one-tenth as large as that caused by an equimolar amount of ACh. The muscle response to FMRFamide was unaffected by curare. During extended exposures, FMRFamide caused a maintained contraction in longitudinal muscle without any apparent desensitization of the FMRFamide receptors and occasionally triggered an irregular myogenic rhythm. This extended exposure to FMRFamide caused a post-exposure potentiation of the longitudinal muscle's response to ACh that shorter applications of FMRFamide did not. Thus FMRFamide may act as a transmitter or modulator in cholinergic motor neurons innervating longitudinal and dorsoventral muscles in the leech.