Compensatory Regeneration in the Auditory Interneuron AN2 of the Cricket Gryllus Bimaculatus
Professor Hadley Wilson Horch
From the daily cortical alterations of learning and memory formation to the regeneration and reorganization that occur in response to injury, neuronal plasticity is essential to the functioning of an animal. While the peripheral nervous system regenerates after injury, the central nervous system generally does not, and depriving a neuron of input from its afferent usually leads to debilitating consequences such as shrinkage or even cell death. When a cricket loses an ear on one of its forelegs, however, the dendrites of the auditory interneurons cross to seek input from the other ear and re-establish functional connections with input of the correct sound frequencies (Hoy et al. 1985, Schildberger et al. 1986). Because this regeneration is unique and is not seen even in other sensory systems within the cricket, the cricket auditory system is an ideal model system for neuronal regeneration.
We chose AN2, an auditory interneuron sensitive to 20KHz sound, for this study because it can consistently be backfilled and visualized with fluorescent dye. We hypothesized that when the dendrites ipsilateral to the original afferent regenerate, the membrane within the dendritic arbor is conserved and the new growth is accompanied by a reduction of old arbor.
First instar Gryllus Bimaculatus were unilaterally deafferented by removal of their right forelegs above the tympanal membranes and deafferented again once a week thereafter till adulthood to prevent the ear from regenerating. AN2 in the adult prothoracic ganglia were prepared for visualization by either backfill or ionophoresis. Backfills were done by allowing biocytin to be taken up the cut neck connective on the deafferented side and into the rest of the cell by retrograde transport and then be immunohistochemically reacted with fluorescent streptavadin. We also attempted to identify AN2 by electrophysiology using an intracellular electrode filled with electrically charged fluorescent dye and then directly inject the dye by passing a current through the electrode.
Three-dimensional representations of the ganglia were created using a Zeiss LSM 510 Meta confocal microscope and analyzed with Improvision Volocity software, with the main focus upon changes in the dendritic arbor of the ipsilateral tuft. Although not enough data have been collected to permit statistical analysis, preliminary results show that the more medial branches of the ipsilateral tuft cross over the midline to seek contralateral input after ear removal while a lateral branch seem to extend further towards the ipsilateral edge of the ganglion, perhaps in search of the lost afferent.
Since the backfill method has thus far yielded images with a high level of noise from extraneous cells, refining the techniques to produce cleaner and more usable images will be a priority as the project continues into the academic year. It is paramount to further reduce the number of axons that take up the biocytin and to develop a system for canceling out unwanted signals using the analysis software so that accurate measurements can be made. Achieving consistent results with ionophoresis will also be helpful since the dye would be directly injected into the single appropriate cell.
By systematically quantifying the changes that occur in AN2, we will be able to better understand what functional regeneration entails.