Stacia B. Moffett - US grants

This research will investigate central nervous system regeneration following removal of a population of neurons. Brain damage of this sort is a severe challenge to those who must try to promote recovery in accident victims. A common response to neural trauma is widespread changes in neuron anatomy and connectivity, and some of these changes might promote recovery, if the proper conditions are provided. The rules that govern the regenerative responses of individual neurons can be studied in the relatively simple nervous system of some invertebrates. Knowledge of the neural strategy that is sufficient to restore normal behavior in these animals may point the way to chemical and anatomical manipulations that might promote recovery in the vertebrate nervous system. In the nervous sytem of the adult snail Melampus, removal of one of the two pedal ganglia causes loss of locomotory movements on the operated side. Repair of central tracts and growth of nerves into the denervated side of the foot are correlated with recovery. This research will relate behavioral recovery to the responses of identified neurons remaining in the CNS after pedal ganglion removal. Two mechanisms for reinnervation of the foot are hypothesized: 1) projections from neurons that normally extend out nerves of the missing ganglion from elsewhere in the nervous system could restore function, or 2) neurons in the remaining pedal ganglion and other ganglia that normally have no axons in the contralateral pedal nerves could innervate the periphery. Identified neurons in each category will be penetrated with intracellular recording electrodes and their behavioral role evaluated in semi-intact preparations before dye is introduced to reveal their anatomy. Inputs to the remaining pedal ganglion from the cerebral ganglion on the operated side will be explored with post-recovery lesions and at the level of identified neural connections to determine whether central pathways concerned with the initiation of locomotion have been reassigned during regeneration.

In contrast to the acid stomach of vertebrates, the anterior stomach of mosquito larvae is very alkaline (pH10). More acidic conditions are restored in the posterior stomach. These observations suggest that, while the animal is feeding, the stomach epithelium continuously recycles alkali between the gut of the animal and its physiological interior. Disturbance that causes the animal to stop feeding and initiate escape behavior interrupts gut alkalinization, suggesting a tight neural and/or endocrine control of the process. It is already clear that some transport proteins characteristic of other well-studied acid or alkali secreting cells are present in the cells of the mosquito larval stomach. Although this group of researchers has identified a vacuolar-type H+ ATPase and at least one anion exchanger, other transporters involved in this system are not known. The gut is innervated by axons that express immunoreactivity to the neurotransmitter serotonin and to nitric oxide synthase, the enzyme responsible for synthesis of the transmitter nitric oxide. The gut epithelium contains endocrine cells that show immunoreactivity to peptide hormones belonging to the FMRFamide family. The effects of serotonin on the anterior stomach have been partly described, and it appears that it, plus an additional messenger or messengers, constitute a sufficient stimulatory signal for gut alkalinization. It is probable that there are also inhibitory signals that come from the CNS and/or the gut itself. The major questions of the project are aimed at the cellular mechanisms that drive alkalinization of the anterior gut, reacidification in the posterior stomach, and the identity of the neural and endocrine control pathways. Development of isolated, perfused preparations of the stomach by this team of investigators was a critical basis for the projected studies, because they allow experiments that would be impossible in the whole animal: electrophysiological characterization of transport mechanisms by measurement of the transepithelial voltage of the tissue, the intracellular voltages and ionic concentrations using ion-specific intracellular microelectrodes, and the transepithelial ionic fluxes using isotopes. The perfused preparations also will be used to assay the activities of candidate control neuropeptides. In a parallel experimental approach, the presence and cellular locations of known transport proteins will be determined by fluorescence immunohistochemistry, a technique in which antibodies generated against defined molecular targets are localized by fluorescently tagged secondary antibodies. Mosquitoes are by far the world's most medically significant insects. They are potential vectors for approximately 100 arboviruses that cause human disease, including yellow fever, dengue and a number of forms of encephalitis. They also transmit nematodes that cause elephantiasis, and plasmodia that cause malaria. The larval mosquito is an aquatic form that feeds on detritus. Although the larval form does not transmit disease, it may be more vulnerable to 3rd generation control measures than adults, because larvae are generally concentrated in aquatic breeding sites whereas the winged adults disperse widely. Gut alkalinization is believed to protect the animal from infection by killing ingested microbes and viruses. Weakening of this mechanism by a specific attack on the cellular processes, or their control signals, could make the larvae more susceptible to endemic or applied microbial pathogens. This project could provide the knowledge base for such an approach.