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.

Seasonal differences and protection by creatine or arginine pretreatment in ischemia of mammalian and molluscan neurons in vitro.

We investigated the dose-response relationship of protection by creatine against ischemic damage, and we asked whether or not such protection may be observed in invertebrate neurons that might provide a simpler experimental model. Rat isolated pyramidal neurons from the CA3 region of hippocampus subjected to ischemia ("in vitro ischemia") showed anoxic depolarization (AD) after 3-7 min of incubation in anoxic medium. Membrane potential (MP) was reduced 25-78% from preanoxic value. Inward current was decreased by an average 49%. Supplementation with creatine protected against these changes, with 1 mM being the minimal effective concentration, 2 mM providing a near-maximal protection, a maximal effect being obtained with 5 mM creatine. No additional protection was provided by up to 20 mM creatine. Isolated giant neurons of Lymnaea stagnalis showed a similar response to in vitro ischemia. However, a clear seasonal dependence of sensitivity of these cells was detected. In cells obtained during summertime (May-August), AD latency ranged from 3 to 10 min; during wintertime (December-March), this response did not occur even after 25-50 min. The addition of creatine to the medium did not cause changes in AD latency, probably because these neurons rely on a phosphoarginine/arginine energy system. However, treatment of the cells, harvested during summertime, with 2 mM arginine did provide clear protection against anoxic-aglycaemic changes. Summing up, besides confirming previous findings on creatine protection in mammalian neurons, we (1) better characterized their dose-response relationship and extended the findings to the CA3 region and to isolated neurons, (2) found that invertebrate neurons are not protected by creatine but by arginine supplementation and (3) reported a novel mechanism of seasonal dependence in sensitivity of in vitro ischemia by invertebrate neurons.