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F. Schroedl, A. De Laet, M.J. Tassignon, P.P. Van Bogaert, A. Brehmer, W.L. Neuhuber, J.P. Timmermans; Intrinsic Choroidal Neurons (ICN) in the Human Eye: Projections, Targets and Basic Electrophysiological Data . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2798.
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Purpose: Intrinsic choroidal neurons (ICN) are supposed to represent a network for local autonomic regulation in the eye along with sympathetic, parasympathetic and sensory pathways. Chemical coding of ICN is also shared with extrinsic sources (eg the pterygopalatine ganglion) making it impossible to assess whether a nNOS/VIP nerve fiber is of intrinsic or extrinsic origin. Dye injections into single neurons allow the visualization of projections of these cells and are hence instrumental in defining the origin of target innervation. The aim of this investigation was to adopt this technique to the study of human choroids. Methods: Fifteen eyes of human donors (65-89 years; pm time 4-12 hrs) were obtained from the cornea bank of the University of Antwerp in accordance with the Declaration of Helsinki. Dissected choroids were stained with a fluorescent vital dye (Bergua et al., 1994), single ICN were identified and electrophysiological properties were determined by means of intracellular recordings. Subsequently, impaled ICN were iontophoretically filled with neurobiotin after recording and immunohistochemistry for nNOS, α-smooth muscle actin, and CGRP was applied. Intracellularly filled ICN were visualized with fluochrome-tagged streptavidin. Confocal laser scanning microscopy and mapping of processes (Neurolucida 3.0) were performed. Results: Processes of ICN could be traced over distances of up to 2612 µm. Some of them were found closely approaching both nNOS- positive and -negative ICN. Others were apposed to stromal non-vascular smooth muscle cells as well as vascular smooth muscle fibers. CGRP positive fibers forming boutons were closely apposed to ICN. Electrophysiological recording showed phasic firing without slow afterhyperpolarization, no spontaneous activity, an input resistance of 136 ±73 MΩ, a membrane time constant of 7±1 ms. Conclusions: Next to this first functional characterization of ICN, we were able to provide direct proof of reciprocal ICN-to-ICN contacts and innervation of both non-vascular and vascular smooth muscle as suggested hitherto on the basis of indirect evidence only. The present approach offers promising perspectives to further investigations on the function of ICN in ocular homeostasis.
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