~ ) Pergamon 0306-4522(94)00430-7 Neuroscience Vol. 64, No. 3, pp. 777-785, 1995 Elsevier ScienceLtd Copyright @~ 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00 ULTRASTRUCTURE OF CALCITONIN GENE-RELATED PEPTIDE-IMMUNOREACTIVE, UNMYELINATED AFFERENTS TO THE CAT CAROTID BODY: A CASE OF VOLUME TRANSMISSION F. TORREALBA* and R. CORREA Departamento de Ciencias Fisiol6gicas, Facultad de Ciencias Biol6gicas, Pontificia Universidad Cat61ica de Chile, Casilla I14-D, Santiago 1, Chile Abstract--To relate the ultrastructure of unmyelinated afferents to the cat carotid body with the known electrophysiological properties of cat chemosensory C-fibers, we took advantage of the fact that the calcitonin gene-related peptide is exclusively present in a population of sparsely branched afferents to the carotid body. They have a morphology identical to the afferents originating from carotid sinus nerve unmyelinated axons. Immunoreactive axons were stained using pre-embedding protocols and horseradish peroxidase-labeled secondary antibody. Labeling was present only in unmyelinated axons and boutons distributed in the interstitial and parenchymal tissue. The varicosities had an average diameter of 0.7 pm, and contained both small, clear vesicles and larger dense-core vesicles. No labeled axons were ever seen to contact glomus cells, but could be observed as close as 0.2/~m to a glomus cell, always with an interposed glial process. With a very sensitive protocol, that used tungstate-stabilized tetramethylbenzidine as the chromogen, amorphous deposits of reaction product were often detected in the extracellular space around a labeled bouton. We interpret these findings as indicating that the reciprocal chemical transmission between the oxygen-sensitive glomus cells and the unmyelinated afferents takes place through non-synaptic trans- mission, via the rather large extracellular space of the carotid body. In addition, the larger distances between glomus cells and unmyelinated afferents could explain the lowered sensitivity and sluggishness of chemosensory C-fibers, compared to the A-fibers. Two classes of chemoreceptor afferents, with conduc- tion velocities corresponding to those of A- and C-fibers, have been found in the cat carotid (sinus) nerve, 2'12 a branch of the glossopharyngeal nerve that contains the afferents to the carotid body. A-fiber chemoreceptors have lower thresholds, higher dis- charge frequencies and a more rapid acceleration of discharge, as compared to the C-fiber chemorecep- tors/ 2 A- and C-fiber chemoreceptors also differ in the morphology of their axonal arborizations in the carotid body, 29 and probably in the central distri- bution of their terminals in the brainstem/ Specifi- cally, myelinated carotid nerve afferents originate larger and complex axonal arborizations in the carotid body, 29 and project to the lateral subnuclei of the nucleus of the tractus solitarius, s while unmyeli- nated afferents produce sparsely branched terminal arbors in lhe carotid body, 29 and project to medial regions of the nucleus of the tractus solitariusY As part of a more general study on the anatomical, neurochemical and functional differences between A- and C-fiber chemoreceptors that innervate the cat carotid body, we examined the fine structure of *To whom correspondence should be addressed. Abbreviations: CGRP, calcitonin gene-related peptide; DAB, diaminobenzidine; IR, immunoreactive; TMB, tet ramethylbenzidine. calcitonin gene-related peptide (CGRP)-immuno- reactive (IR) chemosensory afferents in the carotid body, focusing on the nature of the possible contacts of these axons and the glomus cells. To better define the structural relationships between the C-fiber chemoreceptors and the glomus (principal) cells of the cat carotid body, we took advantage of the fact that CGRP exclusively labels a population of very fine afferents to the cat carotid body. Under the light microscope, these CGRP-IR axons are identical to the sparsely branched afferents that originate from unmyelinated axons of the carotid n e r v e . 2s'29 Further- more, sympathetic efferents to the carotid body are not immunoreactive to this peptide. 28 The use of a specific label for the unmyelinated afferents, such as CGRP immunoreactivity, makes it easier to study their ultrastructure, than if routinely stained carotid bodies were serially reconstructed, 26 since in that case the sympathetic efferents would confuse the analysis. EXPERIMENTAL PROCEDURES Adult mongrel cats of either sex were anesthetized with thiopental (60mg/kg, i.p.), and perfused through the abdominal aorta with a saline flush, followed by 1 1 of 4% paraformaldehyde, 0.5% glutaraldehyde in 0.1 M phos- phate buffer (pH 7.4). The carotid bodies and sinus nerves were removed and left overnight in the fixative. They were 777