Multiple changes in peptide and lipid expression associated with regeneration in the nervous system of the medicinal leech.

BACKGROUND: The adult medicinal leech central nervous system (CNS) is capable of regenerating specific synaptic circuitry after a mechanical lesion, displaying evidence of anatomical repair within a few days and functional recovery within a few weeks. In the present work, spatiotemporal changes in molecular distributions during this phenomenon are explored. Moreover, the hypothesis that neural regeneration involves some molecular factors initially employed during embryonic neural development is tested. RESULTS: Imaging mass spectrometry coupled to peptidomic and lipidomic methodologies allowed the selection of molecules whose spatiotemporal pattern of expression was of potential interest. The identification of peptides was aided by comparing MS/MS spectra obtained for the peptidome extracted from embryonic and adult tissues to leech transcriptome and genome databases. Through the parallel use of a classical lipidomic approach and secondary ion mass spectrometry, specific lipids, including cannabinoids, gangliosides and several other types, were detected in adult ganglia following mechanical damage to connected nerves. These observations motivated a search for possible effects of cannabinoids on neurite outgrowth. Exposing nervous tissues to Transient Receptor Potential Vanilloid (TRPV) receptor agonists resulted in enhanced neurite outgrowth from a cut nerve, while exposure to antagonists blocked such outgrowth. CONCLUSION: The experiments on the regenerating adult leech CNS reported here provide direct evidence of increased titers of proteins that are thought to play important roles in early stages of neural development. Our data further suggest that endocannabinoids also play key roles in CNS regeneration, mediated through the activation of leech TRPVs, as a thorough search of leech genome databases failed to reveal any leech orthologs of the mammalian cannabinoid receptors but revealed putative TRPVs. In sum, our observations identify a number of lipids and proteins that may contribute to different aspects of the complex phenomenon of leech nerve regeneration, establishing an important base for future functional assays.

Construction of a medicinal leech transcriptome database and its application to the identification of leech homologs of neural and innate immune genes.

BACKGROUND: The medicinal leech, Hirudo medicinalis, is an important model system for the study of nervous system structure, function, development, regeneration and repair. It is also a unique species in being presently approved for use in medical procedures, such as clearing of pooled blood following certain surgical procedures. It is a current, and potentially also future, source of medically useful molecular factors, such as anticoagulants and antibacterial peptides, which may have evolved as a result of its parasitizing large mammals, including humans. Despite the broad focus of research on this system, little has been done at the genomic or transcriptomic levels and there is a paucity of openly available sequence data. To begin to address this problem, we constructed whole embryo and adult central nervous system (CNS) EST libraries and created a clustered sequence database of the Hirudo transcriptome that is available to the scientific community. RESULTS: A total of approximately 133,000 EST clones from two directionally-cloned cDNA libraries, one constructed from mRNA derived from whole embryos at several developmental stages and the other from adult CNS cords, were sequenced in one or both directions by three different groups: Genoscope (French National Sequencing Center), the University of Iowa Sequencing Facility and the DOE Joint Genome Institute. These were assembled using the phrap software package into 31,232 unique contigs and singletons, with an average length of 827 nt. The assembled transcripts were then translated in all six frames and compared to proteins in NCBI's non-redundant (NR) and to the Gene Ontology (GO) protein sequence databases, resulting in 15,565 matches to 11,236 proteins in NR and 13,935 matches to 8,073 proteins in GO. Searching the database for transcripts of genes homologous to those thought to be involved in the innate immune responses of vertebrates and other invertebrates yielded a set of nearly one hundred evolutionarily conserved sequences, representing all known pathways involved in these important functions. CONCLUSIONS: The sequences obtained for Hirudo transcripts represent the first major database of genes expressed in this important model system. Comparison of translated open reading frames (ORFs) with the other openly available leech datasets, the genome and transcriptome of Helobdella robusta, shows an average identity at the amino acid level of 58% in matched sequences. Interestingly, comparison with other available Lophotrochozoans shows similar high levels of amino acid identity, where sequences match, for example, 64% with Capitella capitata (a polychaete) and 56% with Aplysia californica (a mollusk), as well as 58% with Schistosoma mansoni (a platyhelminth). Phylogenetic comparisons of putative Hirudo innate immune response genes present within the Hirudo transcriptome database herein described show a strong resemblance to the corresponding mammalian genes, indicating that this important physiological response may have older origins than what has been previously proposed.

Functions of the subesophageal ganglion in the medicinal leech revealed by ablation of neuromeres in embryos.

Two general trends in the evolution of the nervous system have been toward centralization of neuronal somata and cephalization of the central nervous system (CNS). These organizational trends are apparent in the nervous system of annelid worms, including leeches. To determine if the anterior brain of the leech serves functions similar to those of the brains of more complex organisms, including vertebrates, we ablated one of the two major regions of the cephalic brain--the subesophageal ganglion (SubEG). For anatomical reasons, ablations were performed in embryos, rather than in adults. At the end of embryonic development, we observed the leeches' spontaneous behaviour and their responses to moderate touch. We observed that, although the midbody ganglia of the leech CNS display a high degree of local autonomy, the cephalic brain provides generalized excitation to the rest of the CNS, is a source of selective inhibition that modulates behaviour, integrates sensory information from the head with signals from the rest of the body, and plays an important role in organizing at least some complicated whole-body behaviours. These roles of the leech cephalic brain are common features of brain function in many organisms, and our results are consistent with the hypothesis that they arose early in evolution and have been conserved in complex nervous systems.

Development of swimming in the medicinal leech, the gradual acquisition of a behavior.

Observing the development of behavior provides an assay for the developmental state of an embryo's nervous system. We have previously described the development of behaviors that were largely confined to one or a few segments. We now extend the work to a kinematic analysis of the development of swimming, a behavior that requires coordination of the entire body. When leech embryos first begin to swim they make little forward progress, but within several days they swim as effectively as adults. This increase in efficacy depends on changes in body shape and on improved intersegmental coordination of the swim central pattern generator. These kinematic details suggest how the swim central pattern generating circuit is assembled during embryogenesis.

Sequential development of electrical and chemical synaptic connections generates a specific behavioral circuit in the leech.

Neuronal circuits form during embryonic life, even before synapses are completely mature. Developmental changes can be quantitative (e.g., connections become stronger and more reliable) or qualitative (e.g., synapses form, are lost, or switch from electrical to chemical or from excitatory to inhibitory). To explore how these synaptic events contribute to behavioral circuits, we have studied the formation of a circuit that produces local bending (LB) behavior in leech embryos. This circuit is composed of three layers of neurons: mechanosensory neurons, interneurons, and motor neurons. The only inhibition in this circuit is in the motor neuron layer; it allows the animal to contract on one side while relaxing the opposite side. LB develops in two stages: initially touching the body wall causes circumferential indentation (CI), an embryonic behavior in which contraction takes place around the whole perimeter of the segment touched; one or 2 d later, the same touch elicits adult-like LB. Application of bicuculline methiodide in embryos capable of LB switched the behavior back into CI, indicating that the development of GABAergic connections turns CI into LB. Using voltage-sensitive dyes and electrophysiological recordings, we found that electrical synapses were present early and produced CI. Inhibition appeared later, shaping the circuit that was already connected by electrical synapses and producing the adult behavior, LB.