Ultrastructure of the Carotid Body in the Mammals

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INTERNATIONAL REVIEW OF CYTOLOGY VOL . 60

Ultrastructure of the Carotid Body in the Mammals ALAINVERNA Laboratory of Cytology. University of Bordeaux II. Talence. France I . Introduction . . . . . . . . . . . . . . . . . . . . I1 . Histological Features . . . . . . . . . . . . . . . . . A . Cell Clusters . . . . . . . . . . . . . . . . . . B . Blood Vessels . . . . . . . . . . . . . . . . . . C . Nerve Fibers . . . . . . . . . . . . . . . . . . D . Ganglion Cells . . . . . . . . . . . . . . . . . E . Other Cellular Elements . . . . . . . . . . . . . . 111. Ultrastructure of Type I and Type I1 Cells . . . . . . . . . A . Type I Cells . . . . . . . . . . . . . . . . . . B . TypeIICells . . . . . . . . . . . . . . . . . . IV . Type I Cell Innervation . . . . . . . . . . . . . . . . A . Origin of Nerve Endings on Type I Cells . . . . . . . . B . Ultrastructure of Nerve Endings on Type I Cells . . . . . C . Ultrastructure of Junctions between Nerve Endings and Type 1 Cells . . . . . . . . . . . . . . . . . . . . . D . Effects of Denervation upon Type ]/Type I1 Cells . . . . . E . Functional Interpretations of Type 1 Cell-Nerve Ending Relationships . . . . . . . . . . . . . . . . . . F . Noninnervated Type I Cells . . . . . . . . . . . . . V . Vascular Innervation and Efferent Inhibition . . . . . . . . A . Vasomotor Innervation . . . . . . . . . . . . . . . B . Barosensory Innervation . . . . . . . . . . . . . . C . The Problem of the Efferent Inhibition . . . . . . . . . . VI . Ultrastructural Changes after Stimulation of Chemoreceptor and Pathology . . . . . . . . . . . . . . . . . . . . . A . Changes in Type I Cells . . . . . . . . . . . . . . B . Changes in Nerve Endings . . . . . . . . . . . . . C . Pathology . . . . . . . . . . . . . . . . . . . VII . Embryology and Development . . . . . . . . . . . . . VIII . Concluding Remarks . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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I Introduction The carotid body of mammals is a very small organ that lies dorsal to the common carotid artery bifurcation . Its maximum dimension averages approximately 3 mm in man and 1.5 mm in the cat or dog . It receives arterial blood from a short vessel but its exact location varies greatly not only from one species to 27 1

Copyright 0 1979 by Academic Press. Inc . All rights of reproduction in any form reserved . ISBN 0-12-3643WO

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another but also from one individual of a given species to another (see Adams, 1958; Seidl, 1975). The carotid body is innervated by a branch of the glossopharyngeal nerve: Hering’s nerve or the sinus nerve. The latter denomination is due to the fact that the carotid body lies close to the carotid sinus and that both structures are innervated by the same nerve. The carotid body is also connected to the superior cervical ganglion by one or several nerves: the ganglioglomerular nerve(s) (Fig. 1). Since its discovery, the carotid body has been the subject of many controversies. It was considered as a gland by Luschka (1862) and then, as a vascular organ by Arnold (1865). The occurrence of chromaffin cells, described by Kohn (1900), led him to consider the carotid body as a paraganglion, a concept which was corroborated by the existence of a sympathetic innervation. However, some authors underlined that chromaffin cells are rather few in the carotid body (Monckeberg, 1905). What is more, de Castro (1926) demonstrated that carotid body cells are principally innervated by the glossopharyngeal nerve. Until then, this innervation was thought to be efferent (centrifugal), but in 1928, de Castro showed that transection of the glossopharyngeal nerve root (between its sensory ganglions and the medulla) does not affect the innervation of carotid body cells. This experiment demonstrated that this innervation is afferent (sensory) from IX

la

ica

SI

cca

cca

A

B

FIG.1. Schematic representation of carotid bifurcation in the (A) cat and the (B) rabbit (ventral

view of the left carotid). ap, ascending pharyngeal artery; cb, carotid body; cca, common carotid artery; cs, carotid sinus; eca, external carotid artery; ica, internal carotid artery; ggn, ganglioglomerular nerve; la, lingual artery; oa, occipital artery; scg, superior cervical ganglion; sla, superior laryngeal artery; sn, sinus nerve; IX, glossopharyngeal nerve.

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which de Castro (1928) deduced that the carotid body is a sensory organ which “tastes” the blood. This prophetical interpretation was confirmed physiologically by Heymans and co-workers (Heymans and Bouckaert, 1930; Heymans et al., 1933), who found that the carotid body is an arterial chemoreceptor which is sensitive to the chemical composition of the blood and which is particularly stimulated by hypoxia, hypercapnia, and acidemia. Its excitation leads to respiratory and cardiovascular reflexes (see Heymans and Neil, 1958; Angell-James and Daly, 1969; Biscoe, 1971; Howe and Neil, 1971). Arterial chemoreceptors are not only restricted to the carotid body. They are also present in the aortic arch region where there are several groups of cells referred to as “aortic bodies.” Their chemosensory function is well known (see Howe and Neil, 1971, for references) and it has been recently demonstrated that their ultrastructure is identical to that of the carotid body (Abbott and Howe, 1972). Moreover, “miniglomera’ ’ have been found in the carotid bifurcation region at some distance from the carotid body itself (de Castro, 1962) and even around the common carotid artery of the cat (Matsuura, 1973). In the latter case, both physiological and ultrastructural features of “miniglomera” were found to be identical to those of the carotid body. It seems, consequently, legitimate to extrapolate results concerning the carotid body to the whole arterial chemoreceptor system. The carotid body ultrastructure was studied as early as 1957 and the first works demonstrated the occurrence of osmiophilic vesicles in a category of cells (Lever and Boyd, 1957; Lever et al., 1959). The electron microscope also demonstrated nerve endings on these cells (type I cells) but, at the same time, showed that these terminals do not penetrate the type I cell cytoplasm as suggested by de Castro (1951). Furthermore, Lever et al. (1959) observed synaptic-like vesicles in these nerve endings. This observation was surprising since synaptic vesicles are, classically, located in the presynaptic part of the junctions, whereas, according to de Castro (1928), the nerve endings in question should be postsynaptic (the presynaptic element being the type I cell). The occurrence of synaptic-like vesicles in nerve endings on type I cells has been repeatedly confirmed by many authors and that is probably what led Biscoe and Stehbens (1967) to express doubts about de Castro’s conclusions and, then, Biscoe et al. (1970) to repeat his degeneration experiment. The results of Biscoe et al. (1970) were diametrically opposed to those of de Castro (1928) and, consequently, these authors asserted that type I cell innervation is efferent, a conclusion which implies that type I cells are not sensory. However, some authors did not accept this conclusion (Eyzaguirre et al., 1972); others postulated a double innervation of type I cells, efferent and afferent (Kobayashi, 1971b). Finally, more recent experiments have demonstrated that de Castro (1928) was right in asserting the afferent (sensory) nature of type I cell innervation (Fidone et al., 1975, 1977; Smith and Mills, 1976).

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However, the interpretation of the relations between type I cells and sensory nerve terminals is still in dispute and the transducer element cannot be considerbd as identified. Nevertheless, the ultrastructural descriptions are relatively concordant despite these different interpretations. This is not the case for the effects of experimental stimulation of chemoreceptors on carotid body ultrastructure. For example, some authors report that chronic hypoxia increases the number of osmiophilic vesicles in type I cells (Mgller et al., 1974), while others describe the inverse effect (Blessing and Kaldeweide, 1975; Laidler and Kay, 1978). The embryology of the carotid body also has been a subject of great controversy, particularly with respect to the origin of the type I cells, but this problem has been solved recently (Le Douarin et al., 1972; Pearse er al., 1973). Thus some very important results have been obtained in the course of the last 6 years. The object of this review is to present the current status of our knowledge concerning the structure of the carotid body and to discuss the possible functional implications of morphological observations. 11. Histological Features Histologically, the carotid body is characterized by an association between cell clusters and capillaries. These elements are invested by a collagenous connective tissue, more or less abundant according to the species. Many myelinated and unmyelinated nerve fibers travel in the connective tissue. Where the connective tissue is abundant the cell clusters are dispersed and the organ is described as “diffuse” or “disseminated” (Kohn, 1900; Watzka, 1943). This is the case in the rabbit, for example (Fig. 2). In other species, the interstitial tissue is minimal and the carotid body is described as “compact” (cat). However, this distinction seems to be of minor importance and, nevertheless, the amount of connecthe tissue increases with age (Watzka, 1943). Frequently, several cell clusters constitute a lobule which is supplied by one and the same arteriole (Adams, 1958). The cell cluster and its associated capillary are considered by Seidl(l975) as the basic functional unit or “glomoid. ”

A. CELLCLUSTERS

The cell clusters are made of a varying number of cells. The aspect of their nuclei enabled Gomez (1908) to describe two kinds of cells: type I and type 11. Type I cells exhibit a spherical or ovoid nucleus having little affinity for stains. Type I1 cells show a smaller nucleus which is frequently flattened or reniforrn and contains a condensed chromatin; these cells are less numerous than type I cells and are situated at the periphery of the clusters (Fig. 2). This classification

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FIG. 2. Semithin section of rabbit carotid body showing numerous capillaries (Ca), several cell clusters (arrows), and nerve fibers, either in small bundles (Bnf) or isolated (Nf). The collagenous connective tissue (Col) is abundant in this species. Bar = 20 p m . Inset: Higher magnification showing the aspect of the nuclei of type 1 cells (NI) and type I1 cells (NII). Bar = 5 pm.

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of carotid body cells was used again by de Kock and Dunn (1966) and also by Biscoe and Stehbens (1966) in the course of ultrastructural studies which revealed other important differences between these cells (see Section 111). However, other denominations are currently in use, e.g., type I and type I1 cells are called “glomus cells” and “sustentacular cells, respectively (Nishi and Stensaas, 1974), or “glomus cells” and “sheath cells” (McDonald and Mitchell, 1975a,b). Other names were used in the past (a list can be found in Biscoe, 1971). The cell islands are always close to a blood vessel and, by means of three-dimensional reconstructions, Seidl (1975) has shown that the cells are clustered on one side of the vessel. The distance between cell clusters and capillaries is variable and has been the subject of many contradictory estimations. This is not surprising since this distance is frequently below the resolving power of the light microscope, as shown later with the electron microscope. A statistical analysis of this problem has been recently undertaken (Lubbers et a l . , 1977). ”

B. BLOODVESSELS 1 . Arteries The carotid body is usually supplied by one artery originating from the carotid bifurcation area or from the occipitopharyngeal trunk in the cat (the internal carotid artery is vestigial in this species). However, considerable variations occur, even in a given species, e.g., Seidl (1975) has observed cat carotid bodies supplied by more than three arteries. Moreover, half of the carotid bodies studied by this author were supplied by arteries originating from the external carotid artery, occipital artery, ascendant pharyngeal artery, or common carotid artery. Frequently, the glomic artery (arteries), after providing branches to the carotid body, leaves the organ to supply neighboring structures such as the wall of the carotid sinus or the superior cervical ganglion (Chungcharoen et a l . , 1952). The histological structure of glomic arteries is unusual. These vessels have a very flexible wall, made of concentric elastic laminae surrounded by only a few layers of smooth muscle fibers (de Castro and Rubio, 1968; Heath and Edwards, 1971). According to de Castro (1940) the carotid body arteries are innervated by barosensory nerve fibers (see Section IV,B).

2. Capillaries The capillary network of the carotid body has been clearly demonstrated by means of gelatin injections (de Castro, 1940, 1951) and, more recently, by scanning electron micrographs of cast preparations (Keller et a l . , 1972; Seidl, 1975). It must be underlined that the vessels in question are not sinusoids but are true capillaries with a continuous (although fenestrated) endothelium. De Castro and Rubio (1968) have described two kinds of capillaries: Type I capillaries are

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very convoluted vessels 14 to 28 pm in diameter; type I1 capillaries are less numerous, have a diameter of 6 to 12 pm,and tend to constitute bridges between type I capillaries. This distinction has not been confirmed by other workers. On the other hand, de Castro and Rubio (1968) have noted that the communication between capillaries and venules is achieved through narrow postcapillary channels, and Seidl (1975) also described “vessels having a large lumen suddenly narrow prior to merging into the sinus. This situation may involve great variations in the blood velocity. Pericytes are usually present around carotid body capillaries. These cells have been frequently misinterpreted in the past (see Adams, 1958). ”

3. Arteriovenous Anastomoses The occurrence of arteriovenous anastomoses in the carotid body has been postulated by many authors (de Castro, 1940, 1951; de Boissezon, 1943, Celestino de Costa, 1944; Serafini-Fracassini and Volpin, 1966; de Castro and Rubio, 1968; Schafer et al., 1973; Seidl, 1975). According to de Castro and Rubio (1968), the arterial segment of these anastomoses is provided with baroreceptor nerve endings and these authors suggest that arteriovenous shunts regulate the blood flow through the capillaries by bypassing the blood directly to the veins under certain circumstances. Schafer et al. (1973) and Seidl(1975) also describe shunt vessels outside the carotid body, at its arterial pole. They distinguish two kinds of arteriovenous anastomoses (bridge anastomoses and spiral anastomoses). Unfortunately, in a more recent study, Seidl (1976) was unable to verify the presence of these structures. However, this author observed several cases where the artery and vein approached each other up to within a few micrometers. It must be added that other authors were not convinced that arteriovenous anastomoses actually exist in or around the carotid body (Hollinshead, 1942; Edwards, discussion on the paper of de Castro and Rubio, 1968). 4. Veins The carotid body venules form a superficial plexus from which several veins leave the organ to join neighboring venous trunks. The histology of this venous system has not been the object of special comments, except the possible occurrence of baroreceptor nerve endings in the wall of the veins described by Abraham (1 968). C. NERVEFIBERS The abundance of nerve fibers is the third histological characteristic of the carotid body. .These fibers originate from the sinus nerve, the ganglioglomerular nerve(s), or intrinsic ganglion cells.

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1 . Fiber Content of Nerves

a. The sinus nerve of the cat contains about 600 to 700 myelinated nerve fibers and a greater number of unmyelinated ones (de Castro, 1951). According to Eyzaguirre and Uchizono (1961) the ratio of unmyelinated to myelinated fibers varies along the nerve: It is greater near the carotid body than at the middle of the nerve. The authors suggest that a branch having a larger proportion of myelinated axons leaves the sinus nerve some distance before the carotid body to innervate the carotid sinus. Similar observations are made by Laurent and Banes (1964) who do not find unmyelinated fibers in the rabbit sinus nerve near its junction with the glossopharyngeal nerve, whereas such fibers appear numerous near the carotid body. However, these authors propose another explanation, namely, that unmyelinated fibers in the sinus nerve are terminal parts of myelinated ones. The diameters of the myelinated fibers range approximately from 1 to 10 pm in the cat and the rabbit (Eyzaguirre and Uchizono, 1961; Laurent and Banes, 1964). In the latter species, the diameter distribution curve is bimodal with a peak around 2 to 3 p m and another between 5 and 8 pm (Laurent and Barres, 1964; A. Verna, unpublished observations). Electrophysiological methods enabled Fidone and Sat0 (1969) to estimate that, among these myelinated fibers, approximately two-thirds are chemoreceptor afferents and one-third are baroreceptor afferents. The unmyelinated fibers of the sinus nerve have diameters ranging from 0.1 to 1.3 pm with a unimodal distribution (Eyzaguirre and Uchizono, 1961). According to Fidone and Sat0 (1969) about half of these fibers are sensory, two-thirds being baroreceptor afferents and one-third chemoreceptor afferents. The other half are efferent fibers of sympathetic origin or of central origin (see Section V). b. The ganglioglomerular nerve is essentially made of unmyelinated nerve fibers and contains only a few myelinated axons. The unmyelinated fibers have diameters between 0.1 and 2.0 p m and a possible bimodal distribution has been suggested (Eyzaguirre and Uchizono, 1961). 2. Nerve Fibers in the Carotid Body The nerve fibers are grouped in bundles at the organ periphery; they are more dispersed in the central regions and unmyelinated fibers are much more numerous than myelinated ones. Occasionally, myelinated nerve fibers loosing their myelin sheath have been observed in the carotid body (Kondo, 1971; Verna, 1975). The nerve fibers are accompanied by Schwann cells. However, when nerve fibers terminate on type I cells, Schwann cells are relayed by type I1 cells and it is frequently difficult to distinguish these two types of cells. With methylene blue or silver impregnation techniques, de Castro (1940) showed that a single fiber may innervate several cell clusters. Conversely, some type I cells receive terminals from different fibers (see Section IV). Other fibers go to the blood vessels and will be considered in Section V.

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D. GANGLION CELLS The occurrence of some ganglion cells in the carotid body has been known since the first histological studies (Kohn, 1900). These neurons are usually few and are located at the organ periphery. According to Biscoe and Silver (1966), there are three to five ganglion cells per carotid body in the cat. However, these cells seem to be more numerous in some mammals (as the hedgehog) than in others (Adams, 1958). They are generally multipolar neurons, of greater size than type I cells, frequently isolated or sometimes grouped in microganglia. Cell clusters made of type Ihype I1 cells and ganglion cells have been described in the “pilot whale” (de Kock, 1956) and in the rat (Kondo, 1976) but his arrangement appears rather exceptional. It has been suggested that carotid body ganglion cells may be of two kinds: orthosympathetic and parasympathetic (Smith, 1924; Watzka, 1943; McDonald and Mitchell, 1975b). Their function is probably related to the vasomotor innervation and will be considered in Section V.

E. OTHERCELLULAR ELEMENTS 1. Connective Cells Connective cells are numerous in the carotid body, particularly in its disseminated type. These cells form a thin capsule (sometimes inconspicuous) around the organ and its interstitial stroma. The latter contains many collagenous fibers but practically no elastic fibers. 2. Must Cells Mention must be made of the frequent occurrence of mast cells in the carotid body. These cells may be responsible, at least in part, for the serotonin content reported by some authors (Chiocchio et a l . , 1967, 1971a). 111. Ultrastructure of Type I and Type I1 Cells

The ultrastructure of carotid body-specific cells has been the object of many studies and a list of works prior to 1969 can be found in Biscoe (1971) and Kobayashi (1971b). Many species were used during these studies, in particular the cat. However, it seems there are no remarkable species differences with regard to the ultrastructure of type I and type I1 cells. A. TYPEI CELLS 1. General Description

Type I cells are enveloped by type I1 cells except in some places where the type I cell membrane is separated from extracellular spaces by a basement mem-

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brane only. Type I cells are of complex chape and a section in a cell cluster shows very intricate cytoplasmic portions (Fig. 3). These cells frequently send cytoplasmic processes toward other cell clusters or toward capillaries (Nishi, 1976). Because of this complex shape, the size of type I cells is difficult to determine; it can be said that the perikaryon measures about 10 pm. The nucleus is usually spherical or ovoid and contains a dispersed chromatin of low electron density. The nuclear envelope shows numerous pores. The cytoplasmic organelles are evenly distributed and, consequently, the cell shows no polarity. There are many mitochondria with transversal cristae. However, according to Seidl et al. (1977), the percentage of the cytoplasm volume occupied by mitochondria is considerably smaller in type I cells than in liver cells. This is surprising since the oxygen consumption of the carotid body is high (Daly et al., 1954; Leitner and Liaubet, 1971; h r v e s , 1970). The Golgi apparatus is well developed and different kinds of vesicles are present around it: clear smooth vesicles and coated vesicles, some of which contain an electrondense material (Fig. 4). The granular endoplasmic reticulum is usually dispersed in the cytoplasm but sometimes has an arrangement similar to that of the Nissl body of neurons. Free ribosomes, singly or associated in polysomes, are numerous. The centrioles show the usual structure and frequently give rise to a cilium of the 9 + 0 pattern. This kind of cilium has been observed not only in carotid body type I cells (Hess, 1968; Kobayashi, 1968; Kondo, 1971) and type I1 cells (Hess, 1968) but also in many other tissues. These cilia appear, consequently, nonspecific to carotid body cells and their function, if any, is probably not related to the chemoreception. Many microtubules are also present in the cytoplasm. They are intermingled in the perikaryon but show a parallel arrangement in the fingerlike processes; this often makes it difficult to distinguish between type I cell processes and nerve fibers. Other organelles and inclusions are occasionally visible in the cytoplasm: multivesicular bodies, lysosomes, pinocytotic vesicles, lipid droplets, a few glycogen particles. Mention must be made also of some small clear vesicles, similar to synaptic vesicles. These vesicles (about 60 nm in size) are preferentially located below the plasma membrane when the latter is exposed to extracellular spaces (without a type I1 cell cover) (Fig. 5 ) ; they are also involved in some junctions between type I cells and nerve endings (see Section IV,C,2). The content of these synaptic-like vesicles is unknown, but McDonald and Mitchell (1975a) have shown that about 40% of the synaptic-like vesicles in type I cells have a dense core after 5-hydroxydopamine administration. This result suggests that some of these vesicles may store a catecholamine. Finally, the most prominent feature of type I cells is the presence of numerous electron-dense membrane-bound granules, the so-called dense-cored vesicles. These vesicles, described for the first time in the carotid body by Lever and Boyd

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FIG. 3 . Electron micrograph of rabbit carotid body. This section of a cell cluster shows several type I cells (CI) and type I1 cells (CII). Dense-cored vesicles are abundant in certain regions of the type I cell cytoplasm (arrows). The cell cluster is very close to a capillary (Ca). Col, Collagen; Nf, unmyelinated nerve fiber; NE, nerve ending. Bar = 2 pm.

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FIG.4. Type I cell cytoplasm (rabbit carotid body). Section at the level of the Golgi apparatus ( G ) showing many cytoplasmic components: M, mitochondria; R, granular endoplasmic reticulum; ri, polyribosomes; mv, multivesicular bodies; ly , lysosome; dcv, dense-cored vesicles, mt, microtubules; PI,plasma membrane. Arrowheads indicate coated vesicles, either “empty” or containing a dense material. Bar = 1 pm.

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FIG.5 . A type I cell process (CI) is only separated from collagenous connective tissue (Col) by a basement membrane (Bm) (rabbit carotid body). At this level, numerous small synaptic-like vesicles (V) accumulate beneath the plasma membrane. Structures similar to presynaptic dense projections are also present (arrowheads). M, mitochondria. Bar = 0.5 p m .

(1957), are usually spherical, more rarely irregular, in shape; their size is very variable (from about 60 to 300 or 400 nm) not only from cell to cell but also within a given cell. There is always an electron-lucent space between the dense core and the membrane; this space is of variable size and, thus, the vesicles appear more or less “full.” The dense core is of variable electron opacity and, if sufficiently light, shows a faintly granular substructure. An extensive descriptive study of the dense-cored vesicle morphology has been published by Matthiessen et al. (1973) with respect to fixation conditions. The amount of dense-cored vesicles varies considerably from cell to cell. Usually, dense-cored vesicles are evenly distributed in the cytoplasm; however, they are sometimes (particularly in the mouse carotid body) more abundant just below the plasma membrane. Some authors have published exocytosis pictures (Bliimcke et al., 1967a; Bock et al., 1970; Bock and Gorgas, 1976b), but it seems that such pictures are very uncommon for the carotid body.

2 . The Content of Dense-Cored Vesicles As early as their discovery, type I cells dense-cored vesicles were compared to those of the adrenal medulla by Lever and Boyd (1957), and some years later,

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Lever et al. (1959) wrote: “possibly, the electron-dense membrane bound granular bodies. . . represent (as in the adrenal medulla) a stored form of some catecholamine. This conclusion was based on the demonstration (with the light microscope) of a faint positive chromaffin reaction in type I cells and on the effects of reserpine which causes the dense cores of vesicles to disappear. However, this result was, in part, an artifact since the aspect of dense-cored vesicles depends on the fixation method, as shown later by Chen et al. (1969): After reserpine, most dense-cored vesicles appear “empty ” after osmium tetroxide fixation (method used by Lever et al., 1959) but show a normal dense content after glutaraldehyde-osmium tetroxide fixation. Nevertheless, type I cells contain catecholamines andor indoleamines as shown unequivocally by the formolinduced fluorescence method (for references, see Kobayashi, 1971a; Bock and Gorgas, 1976b), but the intracellular localization of monoamines cannot be studied with the light microscope. The first evidence of the presence of catecholamines in type I cell dense-cored vesicles was given by Chen and Yates (1969). These authors demonstrated, by high-resolution autoradiography, that after administration of labeled catecholamine precursors there are many silver grains over type I cells, associated mainly with dense-cored vesicles. The same authors also demonstrated a positive reaction in dense cores after glutaraldehyde-potassium dichromate incubation (method of Wood and Barnett, 1964), i.e., a chromaffin reaction at the dense-cored vesicle level. A more direct argument has been given by Lishajko (1970) who has shown that isolated densecored vesicles (from a human carotid body tumor) release and take up dopamine. Finally, type I cell dense-cored vesicles seem to incorporate “false precursors of catecholamines such as 5-hydroxydopa (Hellstrom, 1975b) and 6-hydroxydopamine (Hess, 1976), and they undergo some shrinkage after catecholamine depletion by reserpine (Hess, 1977b). Obviously, these results do not prove that all type I cell dense-cored vesicles contain catecholamines. It is well known that many different secretion products are stored in similar dense-cored vesicles and it has been demonstrated that, even in a central monoaminergic neuron, some dense-cored vesicles contain substances other than monoamines, such as acid phosphatase for example (Sotelo, 1971). It is possible that, in the type I cells as well, some vesicles do not contain catecholamines. However, they, probably, are few according to the results of ultracytochemical studies. On the other hand, dense-cored vesicles contain not only catecholamines, but also proteins, ATP, and calcium. The proteins are probably responsible for the electron opacity of the dense cores; this explains why, after reserpine, catecholamine-depleted vesicles may have an electrondense content. The presence of proteins in type I cell vesicles has been demonstrated by means of Pronase digestion (Chen et al., 1969). This protein content may be considered as a binding substance for catecholamines but, for some authors, it is a secretion product per se (Capella and Solcia, 1971; Pearse, 1969). ”

”

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However, this hypothetical secretory polypeptide (called ‘‘glomin by Pearse, 1969) has not been identified yet. Furthermore, an autoradiographic study using tritiated leucine, monoamines, and ATP has shown that the turnover of these products in type I cells is slow by comparison with endocrine cells of the adrenal medulla or of the gut (Kobayashi, 1976, 1977). Another problem is to specify what kind of monoamine is present in a given type I cell. Unfortunately, the cytochemical identification of monoamines is not easy and species differences are an additional confusing factor. To summarize, both cytochemical and biochemical studies have demonstrated the presence of large amounts of dopamine and, to a lesser extent, of noradrenaline in the carotid body of many mammals (Dearnaley et al., 1968; Knoche et al., 1969; Zapata et al., 1969; Lishajko, 1970; Chiocchio et al., 1971a,b; Hellstrom and Koslow, 1976). On the other hand, results concerning adrenaline and serotonin are more conflicting. Thus, we question if these different monoamines are stored in different cells, and this leads us to consider a possible classification of type I cells. ”

3 . Different Kinds of Type I Cells Until now, we have considered type I cells as a homogenous population. However, several authors have attempted to classify these cells on the basis of ultrastructural or cytochemical features or both. a. Light and Dark Cells. In early studies (Garner and Duncan, 1958; Lever et al., 1959) light-dark variations of the electron opacity of the type I cell cytoplasm were noticed in the rabbit and, to a lesser extent, in the cat carotid body. This observation has been confirmed for other species such as man (Grimley and Glenner, 1968), horse (Hoglund, 1967), monkey (Al-Lami and Murray, 1968b), and, again, cat (Morita et al., 1969) and rabbit (A. Verna, unpublished observations). However, only electron-dense cells have been found in rat and mouse carotid bodies (Bock and Gorgas, 1976b). It must be added that the ‘‘light’’ or “dark” appearance of cytoplasms is not restricted to carotid body cells but has also been observed in many other cells, e.g., the adrenal medulla. However, the significance of this background density variation is obscure. For many authors it is a fixation artifact (Benedeczky and Smith, 1972; Wacker and Forssmann, 1972); for others it corresponds to different levels of activity (Garner and Duncan, 1958). Nevertheless, this criterion seems to be insufficient to subdivide type I cells. b. ChromafSin and Nonchromaffin Type I Cells. Chromaffinity in the carotid body has been a matter of controversy for years because of the contradictory results reported by many authors (see Adams, 1958; Kobayashi, 1971a). Nowadays, these controversies are only of historical interest since there is no doubt that type I cells contain catecholamines, despite the fact that they are generally chromaffin negative. However, it appears that a few cells react positively to the chromaffin reaction, at least in the carotid body of the dog (Kobayashi, 1968)

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and man (Kobayashi, 1971a). At first sight, it would seem that nonchromaffin cells contain monoamines in amounts too small to react positively; in this interpretation it is not necessary to suppose that chromaffin and nonchromaffin cells are distinct cell types. But it is also possible that chromaffin and nonchromaffin cells contain different monoamines and, in this case, they may be considered to be different cells. This interpretation has been postulated by Kobayashi (1968) who described, in the dog carotid body, very distinct chromaffin and nonchromaffin cells and supposed that (but without experimental support) they contain noradrenaline and adrenaline, respectively. These cells also showed other ultrastructural differences, particularly with respect to their dense-cored vesicles: Those of nonchromaffin cells were of moderate electron density and measured about 100 to 200 nm, whereas chromaffin cell dense-cored vesicles were very electron opaque and measured up to 300 nm. It must be mentioned here that, in the rabbit carotid body as well, a few cells are characterized by very large and very electron-opaque dense-cored vesicles; now these cells take up tritiated noradrenaline whereas the other cells do not (A. Verna, unpublished observations). This observation is therefore consistent with the suggestion of Kobayashi on the possible presence of noradrenaline in chromaffin type I cells. Furthermore, dopamine P-hydroxylase (the enzyme that catalyzes the conversion of dopamine to noradrenaline) has been identified in the denervated cat carotid body by Belmonte et al. (1977). This result is consistent with the observations of Morita et al. (1969) who have described, in the cat carotid body, a few cells characterized by their very large (170 to 400 nm) and very opaque dense-cored vesicles; the possibility that these cells may contain noradrenaline was evoked by these authors and confirmed later by Bock and Gorgas (1976b). The scarcity of such cells is consistent with the relatively low levels of dopamine P-hydroxylase found by Belmonte et al. (1977). Thus, it may be considered, as a working hypothesis, that most type I cells contain dopamine (which does not give a positive chromaffin reaction under the light microscope), whereas a few, characterized by larger dense-cored vesicles, contain noradrenaline; the latter cells are chromaffin positive but their irregular occurrence would explain the contradictory results obtained by different authors on different species. Are chromaffin and nonchromaffin cells really distinct cell types? It is difficult to answer this question since it has been suggested, on cytochemical grounds, that intermediate forms (containing a mixture of noradrenaline and dopamine) also exist in the cat carotid body (Chiocchio et al., 1971b; Bock and Gorgas, 1976b). It may be useful to consider here the morphometric analysis of dense-cored vesicles since chromaffin and nonchromaffin cells also differ by the size of their dense-cored vesicles. c. Morphometric Analysis of Dense-Cored Vesicles. The first attempt was that of Morita et al. (1969) who have described, in the cat carotid body, “light” and “dark” type I cells, the latter group being further divided into three sub-

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types, according to the mean diameter of their dense-cored vesicles. Unfortunately, the authors give neither the number of cells nor the number of vesicles which have been used to calculate each mean. The results of Hellstrom (1975a) and McDonald and Mitchell (1975a,b) appear more convincing at fiist sight. They describe, in the rat carotid body, two kinds of type I cells which differ significantly in the mean diameter of their dense-cored vesicles. According to Hellstrom (1975a) “small-vesicle cells” and “large-vesicle cells” have densecored vesicles of 47.4 and 63.0 nm mean profile, respectively. The author gives the frequency histograms of the mean diameters for 33 cells from one carotid body and 30 cells from another: These histograms are bimodal but, here again, we do not know the exact number of measured vesicles in each cell. Once distinguished by the size of their dense-cored vesicles, the two kinds of cells were found to have other differences: The dense-cored vesicles are almost twice as abundant in large-vesicle cells as in small-vesicle cells; the volume density of the mitochondria is slightly larger and the volume density of the nuclei is slightly smaller in large-vesicle cells than in small-vesicle cells. Finally, large-vesicle cells are about 1.5 times as numerous as the other cells. McDonald and Mitchell (1975a,b) also described two kinds of type I cells which differ in the mean diameter of their dense-cored vesicles: 90 versus 116 nm. These numbers are very different from those of Hellstrom (1975a) although they concern the same species, rat (probably owing to some methodological factor). McDonald and Mitchell (1975a,b) also remarked, as Hellstrom (1975a) did, that dense-cored vesicles are more abundant in cells with larger vesicles but found the two types of cells in nearly equal proportions. Furthermore, they added that only a few small-vesicle cells (called “B cells” by these authors) are in contact with nerve fibers. McDonald and Mitchell (1975a,b) give the number of investigated cells and the total number of measured vesicles. It should be pointed out that an average of only 34 measured vesicles per cell has been considered as sufficient to characterize 10 cells as “B cells.” One may wonder if such a small sample is representative of a population of several thousands of vesicles. Another critical factor is the way in which cells are selected to be studied. It is absolutely necessary to avoid an a priori selection and it is regrettable that McDonald and Mitchell (1975a,b) did not say if the cells were selected at random or not. In a preliminary study, I considered the dense-cored vesicles in 30 type I cells, selected at random, from a rabbit carotid body (Verna, 1977). An average of 250 vesicles (140 < n < 434) were measured for each cell. The results showed, first, great variations in the size of dense-cored vesicles (extreme values: 50 to 250 nm) not only from cell to cell, but also within a given cell. The mean diameter was calculated for each cell, and the extreme values were 93 and 146 nm. Thus, there were small- and large-vesicle cells in the rabbit carotid body as well but these cells were the extremes of a continuous series; this was shown by the unimodal distribution of the 30 mean diameters, most cells having dense-cored

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vesicles about 115 nm in mean diameter. In conclusion, it seems impossible to distinguish clear-cut categories of type I cells in the rabbit carotid body from a morphometric study of their dense-cored vesicles. d. Significance of the Various Aspects of Type I Cells. As we have seen, there exist very different type I cells, either from a cytochemical or from a cytological point of view, and there probably exist intermediate forms as well. Unfortunately, there has been no functional interpretation of these findings until now. Does this diversity reflect different stages of evolution of a single cell type? We must not forget that the electron microscope gives us a static picture. However, it is possible that type I cells undergo some changes during their life. Mitotic type I cells have been observed, although rarely (Kondo, 1971; Verna, 1977; see also Adams, 1958); some degenerating type I cells, characterized by very large and opaque dense-cored vesicles and pycnotic nuclei, are often present in the rabbit carotid body (Verna, 1977). So an evolution of type I cells with correlative morphological (and probably biochemical) changes is not inconceivable. It may be noteworthy to recall here that Celestino da Costa (1944) considered type I cells as undifferentiated or immature cells (“metaneurogonia”) of sympathetic origin, like chormaffin cells of the adrenal medula. This interpretation is compatible with the recent demonstration of the embryological origin of type I cells (see Section VII) and has received additional support from the work of Korkala er al. (1973): These authors have shown that glucocorticoids increase the storage of catecholamines in type I cells of the adult rat, an effect which is usually observed only in the catecholamine-storing cells (i.e., SIF cells) of newborn animals (Eranko and Eranko, 1972). Korkala er al. (1973) suggested that carotid body type I cells remain, in adult rats, at a relatively primitive stage. From this point of view, carotid body chromaffin cells, which look like adrenal chromaffin cells, may be considered as aberrantly differentiated type I cells. It may be recalled here that a small adrenal medulla has been observed inside the carotid body by de Castro (1926). It would be interesting to see if carotid body chromaffin cells are innervated by glossopharyngeal afferent nerve fibers (as any type I cell; see Section IV) or by sympathetic preganglionic fibers. McDonald and Mitchell (1975b) say they have unpublished data in favor of the latter possibility. 4. Connections between Type I Cells

Type I cells in a cluster are separated from each other (and from type I1 cells and nerve endings) by an intercellular space of about 20 nm. It has been shown that all these intercellular spaces are accessible to vascularly injected horseradish peroxidase (Woods, 1975). Adjacent type I cells sometimes present symmetrical junctions of the zonula adherens type. These junctions have been observed by many authors working on different species (Biscoe and Stehbens, 1966; Hess, 1968; Kondo, 1971) but seem to be more frequent in the rat carotid body (Hess, 1975a; McDonald and Mitchell, 1975b; Morgan et a l . , 1975). Another kind of

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junction seems to be frequent in the latter species; it is characterized by an asymmetrical structure with an accumulation of dense-cored vesicles and, sometimes, of synaptic-like vesicles on only one side of the junction (Hess, 1975a; McDonald and Mitchell, 1975b; Morgan et al., 1975). These junctions are considered as “synapses” by McDonald and Mitchell (1975b) but without any evidence other than a morphological one. A junction of this kind has been reported between a type I and a type I1 cell (Hess, 1975a); similar accumulations of vesicles also occur at the level of membrane differentiations facing interstitial connective spaces (see Section IV,E,l, and Fig. 5). In the latter case, it is difficult to consider this structure as a synapse. Finally, a few narrower junctions (tight junctions?) have been reported between type I cells (Al-Lami and Murray, 1968a; Hess, 1975a) but we do not know their exact nature for lack of tracer studies. There has been no physiological evidence until now (because of technical difficulties) concerning a possible coupling between type I cells. B. TYPEI1 CELLS Type I1 cells are much less numerous than type I cells: According to Biscoe and Pallot (1972) there are about four or five times as many type I cells as type I1 cells in the cat carotid body; in the rat, type I cells outnumber type I1 cells by a factor of 3 to 5 (McDonald and Mitchell, 1975b). Type I1 cells are located at the periphery of type I cell clusters (Fig. 3) and surround the major part of these cells with a thin cytoplasmic layer, sometimes only 0.2 p m thick. Type I1 cells also send cytoplasmic sheets between neighboring type I cells, toward the cluster center, but this occurs more frequently in the cat than in the rat carotid body (Hess, 1975a). The type I1 cell nucleus is generally flattened, sometimes lobulated, and more electron dense than the type I cell nucleus. The cytoplasm contains the usual organelles: few mitochondria, a Golgi apparatus, endoplasmic reticulum, centrioles, sometimes a cilium of the 9 + 0 pattern, microtubules, and microfilaments. Type I1 cells do not contain dense-cored vesicles; they do not take up tritiated monoamines. Type I1 cells envelop nerve fibers and nerve endings like Schwann cells but, as for type I cells, a certain proportion of nerve ending membrane is not covered and, thus, is exposed to surrounding connective spaces through a basement membrane. Type I1 cells also have a basement membrane.

IV. Type I Cell Innervation As pointed out in the Introduction, the interpretation of the relationship between type I cells and nerve fibers is one of the most disputed problems concerning the carotid body. Between 1970 and 1975, the great question was whether

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type I cell innervation is sensory in nature or not. Now it has been demonstrated that many, if not all, nerve endings on type I cells are afferent (sensory) as previously suggested by de Castro (1928). However, there is still a controversy about the part played by the type I cells in the sensory process: For some authors, these cells are the transducing elements which stimulate the nerve endings by release of an excitatory transmitter; for others, the transducing element is the nerve ending itself, but its activity is modulated by type I cells by release of an inhibitory transmitter. In fact, these very different conceptions arise principally from contradictory results of physiological experiments or from speculative interpretations of morphological observations. However, the latter are relatively concordant. We shall first consider the origin of nerve endings on type I cells, then their cytological features and the ultrastructure of their junctions with type I cells. Finally, current hypotheses as to the meaning of these structures will be briefly considered. A.

ORIGIN OF

NERVEENDINGS ON TYPEI CELLS

Two methods have been used up to now to determine the source of nerve endings in the carotid body: One of them is the well-known degeneration method; the other is the more recent tracer method which uses the axoplasmic flow. That it does not provoke pathological changes which are, sometimes, difficult to interpret is the great advantage of this method. 1.. Degeneration Studies The nerve fibers ending on type I cells may originate from (1) intracranial neurons, (2) neurons located in the sensory ganglions of the glossopharyngeal nerve, (3) neurons located in the sympathetic superior cervical ganglion, and (4) neurons located in the carotid body itself. In 1926, de Castro showed, with the light microscope, that transection of the glossopharyngeal nerve leads to degenerative changes in nerve fibers and nerve endings associated with type I cells. This result was confirmed in electron microscopy by Biscoe and Stehbens (1967), Hess (1968), and Hess and Zapata (1972). However, it is more difficult to determine if all the nerve endings on type I cells arise from glossopharyngeal nerve fibers (via the sinus nerve). In fact, Biscoe and Stehbens (1967) have observed a few apparently normal nerve endings which persist 3 months after cutting the sinus nerve. Hess and Zapata (1972) also admit that 20 days after severance of the IXth nerve (in cats) “an occasional synapse, perhaps normal in appearance, can be found,” and more recently, McDonald and Mitchell (1975a,b) assert that, in the rat carotid body, about 5% of the nerve endings on type I cells remain unaffected 25 days after glossopharyngeal nerve section. From results of further degeneration studies (after

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removal of the superior cervical ganglion or section of the sympathetic trunk) it was concluded by McDonald and Mitchell (1975a,b) that the few endings which did not degenerate after sinus nerve section were terminals from sympathetic preganglionic fibers which reach the carotid body by the ganglioglomerular nerves. In a serial ultrathin section analysis of the rat carotid body, Kondo (1976) also described a preganglionic efferent fiber in direct contact with a type I cell but he added: “The latter pathway seems to be of minor importance because of its rare occurrence.” This opinion is shared by Hess (1977a) who, studying chronically denervated rat carotid bodies, observed no nerve terminal on type I cells after section of the glossopharyngeal nerve and concluded: “The glomus cells do not receive any significant autonomic innervation. ” Furthermore, Hess (1977a) called attention to the risk of misinterpreting a type I cell process as a nerve fiber. On the other hand, it is also possible that a few type I cells, located at the organ periphery, may be related to the dendrites of intrinsic ganglion cells as demonstrated by Kondo (1976). However, these junctions are obviously of low frequency due to the well-known paucity of ganglion cells in the carotid body. To summarize, and without minimizing the functional importance of the abovementioned unusual autonomic fibers, it can be said that most nerve endings on type I cells are sinus nerve fiber terminals. The problem now is to determine if this innervation is sensory or not. One way to do this is to localize the neuronal cell bodies: If they are in the glossopharyngeal sensory ganglions it will be possible to admit their sensory nature. This attempt was first made by de Castro (1928) by way of intracranial section of the glossopharyngeal nerve roots, above (central to) the sensory ganglions. He reported that no change could be detected in type I cell innervation 12 days after operation. This study was, of course, a light microscope study after silver staining but the results were confirmed later in electron microscopy by de Castro and Rubio (1968), who concluded that the fibers innervating type I cells have their cell bodies in the glossopharyngeal nerve sensory ganglions and, consequently, are afferent (sensory) in nature. Unfortunately, the same experiment was repeated by Biscoe et al. (1970) but with diametrically opposed conclusions. These authors reported that about 60% of nerve endings on type I cells degenerate 128 days after cutting the glossopharyngeal nerve intracranially and they concluded in favor of the efferent nature of the type I innervation. This interpretation was also supported by the occurrence of synaptic-like microvesicles in the nerve endings which make them similar to efferent or motor terminals in other areas. To resolve the apparent discrepancy between de Castro’s and Biscoe ’s works, the same experiment was repeated by Hess and Zapata (1972), Nishi and Stensaas (1974), and McDonald and Mitchell (1975a,b) but with results supporting the original conclusions of de Castro (1928) and not those of Biscoe’s group. It is

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possible, as pointed out by Hess and Zapata (1972), that Biscoe et al. (1970) performed their operation of intracranial severance of the IXth nerve too closely to the petrous ganglion, causing injury and retrograde degeneration of the sensory neurons, This would also explain the slow rate of degeneration they reported. Finally, the problem was investigated using more physiological methods. 2. Autoradiographic Studies It has been well demonstrated that after perikaryal uptake of a tracer, it migrates with the axoplasmic flow and accumulates in the axon terminals. This phenomenon makes it possible to trace neuronal projections (Lasek et al., 1968) and has been used recently by Fidone et al. (1975, 1977) and also by Smith and Mills (1976). After administration of tritiated amino acids to the petrosal ganglion these authors observed autoradiographically that most nerve endings on type I cells were labeled. Consequently, it appears that these nerve endings arise from neurons in the petrosal ganglion. Since this ganglion seems to consist entirely of sensory unipolar neurons (Stensaas and Fidone, 1977) we can consider the sensory nature of most terminals on type I cells as demonstrated. It must be added that many of the labeled sensory nerve endings observed by Fidone et al, (1 975, 1977) and by Smith and Mills (1976) exhibit clear synaptic-like microvesicles. This point will be discussed later. Finally, the use of this new method leads to the same conclusions as those expressed by de Castro (1928) nearly half a century ago. B. ULTRASTRUCTURE OF NERVE ENDINGS ON TYPEI CELLS 1 . Identification The nerve endings on type I cells are characterized by a local accumulation of organelles such as mitochondria and clear microvesicles. They may also contain some dense-cored vesicles and glycogen particles but are often devoid of neurotubules and microfilaments. Their mitochondria are generally of smaller diameter and have a more electron-dense matrix than those of type I cells. However, this distinction is impossible in the case of type I cell processes whose mitochondria are frequently similar to nervous mitochondria. It should be emphasized that it is sometimes difficult to distinguish between nerve terminals and type I cell processes without the help of serial sections: As previously mentioned, type I cell processes may also contain clear microvesicles and a few dense-cored vesicles. A good criterion to identify type I cell processes is the presence of ribosomes. Unfortunately, ribosomes are scarcely distributed in type I cell extensions, so the probability of their occurring in a single section is very low.

2 . Size and Shape The size of nerve endings on type I cells varies considerably (roughly from 1 to 10 pm), but this notion of size is of very little significance because of the very

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complicated shape of many terminals (see three-dimensional reconstructions in Nishi and Stensaas, 1974). It would be better to quantify the surface of contact between nerve endings and type I cells or the volume of the terminals. Based on morphological criteria, several attempts have been carried out in the past to separate various kinds of nerve terminals, such as “basket” and “bulbous endings” (Al-Lami and Murray, 1968a), “calyciform” endings (Eyzaguirre et al., 1972; Nishi and Stensaas, 1974), “bouton-like” endings (Eyzaguirre et al., 1972; Vema, 1971), and “en passant” endings (Hoglund, 1967; Abbot et al., 1972; Vema, 1971), etc. In fact, later studies have shown that nerve terminals on type I cells are very polymorphous: So, one and the same nerve fiber may innervate several type I cells, after branching, by means of terminals of very different size and shape. This conclusion results from both light microscope studies (examination of de Castro’s original slides by Eyzaguirre and Gallego, 1975) and serial ultrathin section analysis (Biscoe and Pallot, 1972; Nishi and Stensaas, 1974; Kondo, 1976). This polymorphism explains the very different aspects previously described in single ultrathin sections and is consistent with the great variation in size observed along the course of one terminal thanks to a particularly favorable plane of section (Vema, 1973). In conclusion, it is very difficult to distinguish different kinds of nerve terminals on type I cells based on morphological criteria, especially in single section studies. However it must be added that Nishi and Stensaas (1974) describe both large and small calyciform endings after three-dimensional reconstruction from serial sections. The significance of this distinction is obscure, particularly because Nishi and Stensaas (1974) demonstrate that both kinds of terminals are glossopharyngeal afferents which do not degenerate after intracranial section of nerve roots. Another interesting result given by the same authors is the fact that large calyciform endings frequently send short processes to neighboring type I cells. 3. Organelle Content

a. Mitochondria. Mitochondria are abundant in nerve endings on type I cells. They frequently show longitudinally oriented cristae and a very electrondense matrix. However, these features depend on the fixation conditions and also on the species. The mitochondria are sometimes so numerous that they completely fill the neuroplasm (Fig. 6), leading Bock et al. (1970) to call these nerve endings “Mitochondriensacke. These aspects have been observed not only in different mammal species such as man (Bock et al., 1970), guinea pig (Kondo, 1971), and rabbit (Vema, 1971, 1973), but also in the avian carotid body (King et al., 1975) and the amphibian carotid labyrinth (Kobayashi, 1971b). It may be fascinating to observe a great amount of mitochondria in the sensory nerve endings of an organ which respond to the level of arterial oxygen partial pressure. However, it must be added that accumulation of mitochondria in nerve terminals is not a feature of the carotid body. It has been observed also in nerve terminals ”

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FIG.6 . Nerve ending (NE) showing an unusual accumulation of slender mitochrondria (rabbit carotid body). CI, Type I cell; CII, type I1 cell. Bar = 1 p m .

or fibers in the carotid sinus (Rees, 1967a; Chiba, 1972; Knoche and Addicks, 1976), in the heart (Chiba and Yamauchi, 1970), in the brain (Sotelo and Palay, 1968; Bouchaud, 1974), and in the spinal cord (Leonhardt, 1976). The problem is to determine the significance of these accumulations of mitochondria in nerve endings. For some authors it has been considered as a characteristic feature of sensory nerve endings (Munger, 1971; King et al., 1974) but for others it is a pathological change, perhaps linked to a process of aging (Seitelberger, 1971; Bouchaud, 1974; Leonhardt, 1976). In favor of this interpretation is the fact that many mitochondria-rich nerve endings exhibit a noticeable proportion of altered mitochondria, lamellar bodies and dense bodies. On the other hand, large dendritic varicosities observed in the brain and filled with mitochondria have been interpreted as growing dendritic tips by Sotelo and Palay (1968). Maybe both phenomena (degeneration and regeneration) occur in the carotid body, leading to some renewal of nerve endings as suggested for the carotid sinus (Knoche and Addicks, 1976) and the olfactory system (Graziadei, 1973). It must be underlined that mitochondria accumulation does not characterize a special kind of nerve terminal in the carotid body: Both serial section (Biscoe and Pallot, 1972) and single section studies (Verna, 1973) have shown that the amount of mitochondria varies greatly from one region of a given nerve ending to another.

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b. Dense-Cored Vesicles. Nerve endings terminating on type I cells usually contain a few dense-cored vesicles (about 90 to 110 nm in diameter) but these vesicles are never associated with the synaptic-like membrane differentiations. From the work of McDonald and Mitchell (1975a,b) it appears that dense-cored vesicles are less numerous in the sinus nerve afferent terminals than in the preganglionic sympathetic efferent endings. In both cases, we do not know the content of these vesicles. c. Synaptic-like Microvesicles. Electron-lucent vesicles about 60 nm in diameter are almost always present in nerve endings on type I cells but are very irregularly distributed: They are often dispersed in the neuroplasm, sometimes densely packed in clusters. It has been suggested by Nishi and Stensaas (1974) and McDonald (1977b) that synaptic-like vesicles are more abundant in small than in large nerve endings. According to McDonald (1977b) the number and distribution of these vesicles vary with the activity of nerve terminals (see Section VI,B). In a quantitative study of the rat carotid body, McDonald and Mitchell (1975a,b) measured the clear vesicles and found a mean diameter of 61 nm in sinus nerve afferent terminals and of 53 nm in the few preganglionic sympathetic efferent terminals (previously identified by degeneration experiments). The significance of synaptic-like vesicles in sensory terminals will be discussed below, but it already can be said that we have no evidence about their content. We presume they contain no acetylcholine, since the acetylcholine contents of normal and denervated carotid bodies are not significantly different from each other (Fidone et a l . , 1976). d. Other Nerve Terminal Components. The nerve terminals on type I cells, like terminals anywhere in the nervous system, occasionally show pinocytotic vesicles, lysosomes, and smooth reticulum profiles. Glycogen particles are also frequently present and sometimes accumulate in very large amounts (Verna, 1973). This is not peculiar to the carotid body and has been observed in many other nerve endings such as, for example, the carotid sinus barosensory terminals (Chiba, 1972; Bock and Gorgas, 1976a; Knoche and Addicks, 1976). C. ULTRASTRUCTURE OF JUNCTIONS BETWEEN NERVEENDINGS A N D TYPEI CELLS Nerve terminals and type I cells are always separated by an extracellular space approximately 20 nm wide. From place to place, the juxtaposed membranes show local differentiations characterized by an increased electron density. At this level, an electron-dense material is present in both the nerve terminal and the type I cell adjoining cytoplasm. The intercellular gap also contains a material of slight electron density appearing in cross section as a dark line equidistant from the membranes. Generally these specialized zones do not exceed 0.5 p m in length but several of them may follow one another along the area of apposition

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between nerve ending and type I cell. The junctional zones may be symmetrical without any associated vesicles; they are consequently similar to the “puncta adherentia” well known in the nervous system (see Peters et al., 1970). In other cases, different kinds of vesicles are clearly related to the junctional dense material: These junctions are therefore comparable to chemical synapses and the association between the vesicles and the junctional dense material may be referred to as a “synaptic complex” by analogy with the structure of the nervous system synapses (Palay, 1958). However, the junctions between carotid body type I cells and nerve endings are characterized by their polymorphism since different kinds of vesicles may be observed in variable proportions and, moreover, vesicles may be present on one side of the junction or on the other and sometimes on both. In this review, the term junction will be used in preference to synapse because the synaptic nature of the relationship between type I cells and nerve endings cannot be considered as having been demonstrated. 1 . Junctions Characterized by an Accumulation of Vesicles on the Neural Side The synaptic-like clear vesicles which are numerous in the nerve endings on type I cells sometimes accumulate at the level of a membrane area characterized by increased electron density and the presence of some cone-shaped patches of opaque cytoplasmic material next to it. These structures are identical to the “dense projections” described by Gray (1963) in the central nervous system synapses. On the type I cell side, the facing membrane also shows an increased electron density and an associated cytoplasmic dense material but the latter is of more uniform thickness and sometimes is inconspicuous. There is no accumulation of vesicles (Fig. 7). This kind of junction was the first to be described in the ultrastructural study of the carotid body (Biscoe and Stehbens, 1966) and has been observed regularly since those early studies, involving nerve endings of very different aspects such as “knobs” of medium size and large terminals filled with mitochondria. Comparative studies have demonstrated its occurrence in the carotid body (or homologous organ) of many vertebrate species (see Kobayashi, 197 1 b). However, if these descriptions were concordant, it would be surprising to find such junctions in a sensory receptor since the structure of these junctions suggests a transmission from nerve endings toward type I cells: Synaptic vesicles and dense projections are indeed classically thought to be presynaptic in the nervous system synapses (see Peters et al., 1970; Akert et a l . , 1972). This is one of the reasons which has lead some authors to express doubts about the afferent nature of the type I cell innervation (an edifying list of papers and author’s conclusions on this problem can be found in Kobayashi, 197 lb). Fortunately, the above-mentioned autoradiographic studies have demonstrated that efferent-like junctions actually occur between sensory sinus nerve afferent terminals and type I cells (see Fig. 2 in Smith and Mills, 1976, for example). One consequence of this is the difficulty

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FIG.7. Junction between a type I cell (CI) and a nerve ending (NE) (rabbit carotid body). Synaptic-like vesicles (V) accumulate in the nerve ending at the level of membrane differentiations whereas there is no vesicle on the type I cell side. Note the nerve ending area (arrow) which is not covered by the type I1 cell (CII). Col, Collagen; M, mitochondria; Nu, nucleus. Bar = 0.5 pm.

to distinguish morphologically efferent-like ‘‘synaptic complexes” in sensory terminals from true efferent junctions involving possible autonomic fibers on type I cells. However, McDonald and Mitchell (1975a,b) assert that it is possible in the rat carotid body. They specify that in sympathetic efferent terminals, clear vesicles are slightly smaller and more closely packed and dense-cored vesicles more numerous than in sinus nerve afferent terminals. 2. Junctions Characterized by an Accumulation of Vesicles on the Type I Cell Side In these junctions, different kinds of vesicles accumulate near the type I cell junctional membrane which shows associated dense projections, whereas, in the nerve ending, clear vesicles are located away from the junctional zone. The neuroplasmic junctional material is very marked and shows a uniform thickness and a filamentous structure. This disposition is therefore compatible with the “classical” interpretation of de Castro (1928) since it suggests a transmission from type I cell to nerve ending. However, it was only in 1970 that the occurrence of vesicles associated with the junctional zones of the type I cell membrane were first noticed in the mouse carotid body (Kobayashi and Uehara, 1970). It seems that this kind of junction is more frequent in the mouse and rat than in the

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cat carotid body. This could explain why these junctions were discovered so late since the cat was the most frequently used species in carotid body studies. Later, similar observations were made on other mammals such as the rabbit (Verna, 1971, 1973), the guinea pig (Kobayashi, 1971b), the rat (Hess, 1975a; McDonald and Mitchell, 1975a,b; Morgan et al., 1975; Kondo, 1976), and, finally, the cat (Smith and Mills, 1976). Identical junctions also occur in the carotid body of birds, such as the swallow (Kobayashi, 1971b), the domestic fowl (King et al., 1975), and the duck (Osborne and Butler, 1975). However, these descriptions revealed some minor differences possibly related to the species considered. In the mouse carotid body, the “afferent synaptic complexes” described in type I cells by Kobayashi and Uehara (1970) are characterized by small vesicles about 30 to 40 nm in diameter and filled with an electron-dense granular substance. In the rabbit carotid body different kinds of vesicles may be observed in type I cell “synaptic complexes,” namely: small vesicles (about 60 nm), occasionally small granular vesicles (about 70 nm), and dense-cored vesicles (about 90 nm) (Verna, 1973, 1975). The latter are consequently somewhat smaller than the mean diameter of the whole dense-cored vesicle population (about 115 nm in this species; Verna, 1977). In some junctions only small vesicles (the majority of them, if not all, having a clear content) take part in the synaptic complex; in others, there are only a few large dense-cored vesicles (Fig. 8), but the most frequent occurrence is a mixture of the different kinds of vesicles. In the rat carotid body, both small clear vesicles and large dense-cored vesicles participate in the junctions with nerve endings (McDonald and Mitchell, 1975a,b; Morgan et al., 1975; Kondo, 1976). According to McDonald and Mitchell (1975b) the ratio of small clear vesicles to large dense-cored vesicles is about 111 except at the junctions between nerve endings and type I cell processes where small clear vesicles are much more numerous than large dense-cored vesicles (average ratio: 25/1). Both McDonald and Mitchell (1975b) and Kondo (1976) assert that large dense-cored vesicles involved in the junctions are not different from those located elsewhere in the cell. 3 . So-called “Reciprocal Synapses”

The two kinds of junctions described above may occur side by side at the level of one and the same nerve terminal. This arrangement has been described in the rat carotid body as “reciprocal synapse” by McDonald and Mitchell (1975a,b). It has also been observed by Morgan et al. (1975) in the same species and by Smith and Mills (1976) in the cat. The rabbit carotid body also shows similar pictures but they are exceptional in this species (Fig. 9). Lastly, “reciprocal synapses” have also been described in the domestic fowl (King et al., 1975) and the duck (Osborne and Butler, 1975) carotid body. According to McDonald and Mitchell (1975a,b) only 5% of sinus nerve afferent terminals show “reciprocal synapses” but this evaluation may be underestimated since it is based on single

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FIG.8. Junctions between type I cells (CI) and nerve endings (NE)(rabbit carotid body). (A) In this junction, synaptic-like vesicles accumulate on the type I cell side. Most of these vesicles have clear contents but a few exhibit a dense granule (arrow). Note the unsymmetric arrangement of the junctional dense material. Nu, Nucleus. Bar = 0.5 p m . (B) In this case, large dense-cored vesicles (arrow) accumulate in the type I cell at the level of the junctional dense material. CII, Type I1 cell. Bar = 0.5 p m .

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FIG.9. Junctions similar to those in Fig. 7 (fine arrow) and Fig. 8B (thick arrow) sometimes occur side by side, as in this example (rabbit carotid body). This arrangement is described as “reciprocal synapse” by some authors (see text). CI, Type I cell; M, mitochondria; NE, nerve ending; Nu, nucleus. Bar = 0.5 pm.

section studies. The interval between both members of a “reciprocal synapse” may be as small as 100 nm. It must be added that “reciprocal synapses” are described as paired junctions of opposite polarity. However, we do not know (for lack of statistical studies on serial sections) if this association in pairs occurs at random or not.

D. EFFECTSOF DENERVATION UPON TYPEI/TYPE11 CELLS It is well known that taste buds which, like the carotid body, are innervated by the IXth nerve, degenerate rapidly after denervation (see Guth, 1971). It was consequently legitimate to expect similar behaviour in carotid body cells: Therefore, short-term and long-term consequences of denervation upon carotid body ultrastructure have been explored by several authors. Biscoe and Stehbens (1967) did not find any alteration in the ultrastructure of type I cell nor of type I/type I1 cell relations up to 3 months postoperatively . Hess (1968) has noted that, 7 days after denervation, type I1 cells show proliferative cytoplasmic extensions whereas type I cells do not exhibit any striking changes. Abbott et al. (1972) have studied more precisely the early ultrastructural changes after sinus nerve section. These authors write: “The majority of glomus cells appear to be unaffected by loss of their nerve supply. However, they add ”

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that lysosomes and lipid bodies are slightly more numerous in type I and type I1 cells between 1 and 10 days after denervation. They also describe, at the 21-day stage, some hypertrophy of type I cells which leads to cytoplasmic processes filling the spaces presumably left by the disappearance of nerve endings. In a more recent study, Hess (1977a) underlined that, “after denervation, the early changes seen in the carotid body reside in the capsule cell.” According to this author, these cells (type I1 cells), like Schwann cells, hypertrophy and ensheath type I cells with more numerous layers and in a more irregular manner for about 2 or 3 months. On the contrary, type I cells do not show any morphological change even up to 13 months after denervation. It is consequently clear that carotid body type I cells, contrary to gustatory cells, are not morphologically affected by denervation. However, this conclusion does not mean that denervation does not elicit any effect at all on type I cells: It has been demonstrated that, after degeneration of their sensory terminals, these cells become hyposensitive to reserpine which can no longer totally deplete their catecholamine stores (Hess, 1975b). From this observation Hess deduced that intact sensory nerve endings exert a retrograde effect on type I cells, which he considered as a trophic influence. It is not known if the carotid body development is dependent on its sensory nerve supply as suggested for other receptors such as avian Herbst and Grandry corpuscules (Saxod, 1972) and mammal mechanoreceptors (Zelena, 1976).

E. FUNCTIONAL INTERPRETATIONS OF TYPEI CELL-NERVE ENDING RELATIONSHIPS In summary, the studies reported in this chapter have demonstrated that:

i. Type I cells are innervated by sensory nerve fibers from the glossopharyngeal nerve. ii. A few efferent autonomic fibers may also end on some type I cells. iii. The structure of the junctions between type I cells and nerve endings is similar to the structure of chemical synapses in the nervous system. iv. Two kinds of junction may be distinguished according to the situation of vesicles (on type I cell side or on nerve ending side). v. These two kinds of junction may coexist side by side at the level of the same nerve ending. Several attempts have been made to conciliate ultrastructural and physiological findings. Some of them, based on the assumption of a type I cell efferenr innervation (Biscoe, 1971), are no longer defensible. However, among the hypotheses which take the sensory nature of type I cells into account, a controversy still remains as to the identity of the transducer structure. Basically, there are two opposite views: For some authors the type I cell is the receptor, for others it is not, the transducer being the sensory nerve terminal itself.

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1 . Type I Cell as a Transducer In this interpretation the type I cell is responsible for the transduction of stimuli and the subsequent depolarization of the afferent nerve terminals, possibly by release of a chemical transmitter. This is the “classical” interpretation of de Castro (1928), more recently defended by Eyzaguirre et al. (1972). The structure of the junctions characterized by the occurrence of vesicles on the type I cell side supports this view. Furthermore, the participation of a chemical transmitter is also supported by the experiments of Eyzaguirre e f al. (1965): These authors have shown that stimulation in vitro of a superfused carotid body induces the release of a substance which increases the sensory discharge of a second carotid body, situated downstream (Loewi effect). Some years later, Eyzaguirre and Zapata (1968) demonstrated that this Loewi effect persists even if the stimulated carotid body is totally denervated 4 days previously. This result suggests that the presumed transmitter is located in carotid body cells and not in nerve terminals. Unfortunately, as we have seen, different kinds of vesicles are involved in junctions, and we know nothing about their content. It is possible that the large dense-cored vesicles (diameter about 90 nm) contain a catecholamine as do the other dense-cored vesicles located elsewhere in the cell, but we have no information about the small-vesicle contents. Do these different vesicles contain different substances? This possibility cannot be ruled out. Furthermore, the accumulation of small vesicles near a particular zone of the type I cell membrane is not restricted to the occurrence of a nerve ending: Small vesicles (most of them having a clear content) may accumulate at the level of a membrane area covered by a basement membrane only and facing the extracellular spaces without any interposed type I1 cell cytoplasm. At these sites, a dense material similar to the synaptic dense projections occurs beneath the type I cell membrane (Fig. 5). This observation suggests the release of the contents of the microvesicles at nonsynaptic sites, since the target element is lacking. It must be recalled that similar observations have been made by Taxi er al. (1969) describing nonsynaptic accumulation of vesicles in ganglion cell dendrites and also in chromaffin cells (SIF cells) of the rat sympathetic superior cervical ganglion. Several interpretations could be proposed for these observations: Small vesicles actually contain a chemical transmitter which is released not only at the nerve ending level but also at other sites around the cell. In this way, we can presume like Torrance (1968) that nerve endings “respond to a general concentration of a transmitter rather than to a localized high concentration within a narrow synaptic cleft. This interpretation could also explain why, during reinnervation experiments, it is possible to record a chemoafferent activity when nerve terminals are close to type I cells whereas “synaptic” junctions are still few (Zapata et a l . , 1969). It is also possible that microvesicles have nothing to do with nerve depolarization. In this way, type I cell microvesicles can be compared with the apparently ”

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identical vesicles which occur in neurosecretory processes where their function, although unknown, is not synaptic (see Douglas et a l . , 1971). Many substances have been proposed as possible chemical transmitters in the chemosensory process (see Torrance, 1968; Joels and Neil, 1968; Eyzaguirre and Zapata, 1968; Biscoe, 1971; Howe and Neil, 1971). Although this problem is not directly related to this review, it must be said that none of the proposed substances fully accounts for the whole body of experimental results. Another confusing factor is the possible occurrence of species differences: It seems that dopamine inhibits the chemoafferent activity in the cat (Black et a l . , 1972; Sampson, 1972; Zapata, 1975; Nishi, 1977) but may be excitatory in the dog (Black et a l . , 1972) and in the rabbit (Monti-Bloch and Eyzaguirre, 1977). The identification of the transmitterts), therefore, needs further investigations. Nevertheless, type I and/or type I1 cells appear necessary to the generation of chemoafferent impulses since after their destruction, the regenerating nerve fibers do not exhibit any specific chemosensitivity (Verna et a l . , 1975). Furthermore, it has been shown by Zapata et al. (1969) that it is possible to reinnervate the carotid body with mechanoreceptor fibers and to obtain, after several months, chemosensory responses to the usual stimuli. More recently, Zapata et al. (1976, 1977) have shown that after a sinus nerve crush there is a good correlation between the reappearance of chemosensory discharges and the reestablishment of contacts between sensory nerves and type I cells. Therefore, these results suggest that type I and/or type I1 cells are responsible for the receptors’ specificity, although, as underlined by Eyzaguirre et al. (1977), they do not indicate whether the carotid body cells are the primary transducer or if they condition nerve endings to become chemosensitive. However, if we admit that information is conveyed from type I cells to their afferent nerve endings, it is still necessary to account for the occurrence of synaptic-like vesicles in these nerve endings. Hess and Zapata (1972) have suggested that sensory terminals on type I cells could have, in addition to their afferent functions, efferent functional effects. According to these authors, such a dual role may provide the morphological basis for the efferent inhibition described in the carotid body (see Section V). Nishi and Stensaas (1974) and Vazquez-Nin et al. (1977) envisage such inhibitory effects and suggest an axon reflex mechanism for the release of the contents of the vesicles, assuming that impulses generated in an afferent fiber may antidromically invade collaterals of the same axon. Efferent inhibitory effects are also postulated by some authors who consider the nerve terminal itself as a transducer. 2. Nerve Terminal as a Transducer

The hypothesis that carotid body nerve fibers may be chemosensitive by themselves was first evoked by Biscoe and Taylor (1963) and later developed by

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Biscoe (1971) essentially on theoretical grounds. He attributed the transducer function to very small nerve terminals (less than 0.1 p m in diameter) enclosed in type I1 cells, whereas type I cells were thought to be part of an efferent pathway which controls the receptor excitability. However, as we have seen, type I cells receive only minor, if any, efferent innervation, and the very small terminals enclosed in type I1 cells (previously reported by de Kock and Dunn, 1968) do not exist. Nevertheless, the possibility that afferent nerve terminals on type I cells are transducer elements still remains and is particularly supported by McDonald and Mitchell (1975a,b). Their interpretation is principally based on the results of a physiological experiment from Mitchell et al. (1972): These authors have shown that some nerve fibers in a neuroma formed from the cut end of the cat’s sinus nerve show normal chemoreceptive properties, although the neuroma does not contain any type I cell. This possibility is somewhat conflicting with the aforementioned results of Zapata et af. (1969, 1976, 1977) and Verna et al. (1975) which suggest that carotid body cells are necessary for chemoreception. It is possible that the chemoafferent activity recorded by Mitchell et al. (1972) arose from fibers which had reinnervated some type I cells either in the carotid body itself (which was removed in only one of the four studied cats) or in ectopic carotid bodies known to occur along the cat’s common carotid artery (Matsuura, 1973). Mitchell et al. (1972) argue they have “cut all connecting tissue between the carotid bifurcation and the neuroma. However, vascular connections were obviously intact since chemoafferent activities were tested via bloodborne changes; so the possibility that nerve fibers had escaped from the neuroma, along blood vessels toward type I cells elsewhere, cannot be ruled out. Moreover, this possibility was favored by the long delay (12 to 18 months) allowed to nerve regeneration. However, even if we admit with McDonald and Mitchell (1975a,b) that nerve endings on type I cells are chemoreceptive, it is still necessary to envisage some function for type I cells since they do exist. According to McDonald and Mitchell ( 1975a,b) these cells are interneurons modulating the sensitivity of chemoreceptive nerve endings in the following way: When sensory nerve terminals are stimulated, they release an excitatory transmitter; this transmitter induces the cells to release dopamine which, in turn, inhibits the sensory terminals. Thus, type I cells and nerve endings are interconnected by “reciprocal synapses” which form an inhibitory feedback loop. In addition, McDonald and Mitchell (1975a,b) postulate that the few preganglionic sympathetic endings they describe on some type I cells enhance dopamine release from these cells and that synaptic interconnections between type I cells enable them to influence one another. This theory therefore explains most of the ultrastructural and physiological data concerning the carotid body. However, it must be noted that none of the ”

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assumptions which support this theory can be considered as having been conclusively demonstrated. As already mentioned, the evidence that regenerating sinus nerve endings are chemosensitive by themselves (Mitchell et al., 1972) is not entirely convincing. The synaptic nature of the vesicles occurring in afferent nerve terminals is not proved at all: We do not know what they contain; they are presumed to be synaptic only by comparison with similar vesicles in other structures. It may be recalled that synaptic-like vesicles occur in nerve endings of the Pacinian corpuscle, for example, where there is no synapse (Zelena, 1978). McDonald (1977b) argues that conditions stimulating chemoafferent fiber activity (hypoxia, hypercapnia, antidromic electrical stimulation) reduce the number of microvesicles in sensory nerve terminals and also lead these vesicles to become more dispersed. This result is interpreted by the author as reflecting transmitter release but this is a very indirect evidence. The many unsuccessful attempts which have been made to correlate acetylcholine release with synaptic vesicle depletion in motor terminals lead us to be very prudent on this subject. It may be added that microvesicles in nerve endings on type I cells are more frequently sparse than packed at the level of junctions. Moreover, it seems that antidromic stimulation of the sinus nerve does not alter the type I cell membrane potential (Goodman and McCloskey , 1972). Furthermore, the concept of “reciprocal synapse, which has been introduced for dendrodendritic junctions in the olfactory bulb (see Reese and Shepherd, 1972, for references), seems to be questionable and it has been suggested recently that the two elements of these junctions interact by a synaptic mechanism in one direction and a nonsynaptic mechanism in the reverse direction (Ramon-Moliner, 1977). It is possible that afferent nerve endings also exert nonsynaptic effects on type I cells. What can this effect be? McDonald and Mitchell (1975a,b) suggest that the substance released by nerve terminals leads type I cells to liberate dopamine, which, in turn, inhibits the terminal activity. This model does not work for those species in which dopamine seems to be excitatory (dog and rabbit, according to Black et al., 1972, and Monti-Bloch and Eyzaguirre, 1977, respectively). Lastly it has been shown by Hellstrom et al. (1976) and Hellstrom (1977) that hypoxia, by itself, induces dopamine depletion which is quite similar in denervated and intact rat carotid bodies. Thus, things are probably more complicated than expected. ”

3. Other Interpretations

Other mechanisms of interaction between nerve fibers and type I/type I1 cells have been proposed by many authors. Some of these will be only briefly reported here, insofar as they have few ultrastructural implications. a. It has been suggested by Paintal (1967, 1968) that the transducer element may be the type I1 cell which is supposed to excite afferent nerve endings by “some physical change. This hypothesis was slightly modified by Jones (1975) according to which stimuli lead type I cells to release acetylcholine which in”

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duces type I1 cells to contract, thereby stimulating the nerve endings they enclose. There are several reports suggesting that the carotid body contains (Eyzaguirre et al., 1965; Jones, 1975) and releases acetylcholine (Metz, 1969). More recent works have shown that this acetylcholine content is not affected by denervation (Fidone et al., 1977) and that the sinus nerve contains no more acetylcholine than noncholinergic nerves (Golberg et al., 1978). These results support the assumption that acetylcholine is located in carotid body cells and not in nerve endings. Furthermore, the ultracytochemical localization of choline acetyltransferase(Ballard and Jones, 1972) and of the high-affinity component of choline uptake (Fidone et al., 1977) suggests that type I cells are capable of acetylcholine synthesis. Unfortunately, the fine structural localization of cholinesterasesin the carotid body is not very enlightening: Cholinesterases seem to be present (in small amounts) all around the type I1 cells and the nerve endings covered by these cells (Ballard and Jones, 1971); the localization of acetylcholinesterase is more controversial (see Jones, 1975). However these observations may be interpreted not only in terms of transmitter hydrolysis but also in terms of the intracellular role of acetylcholine (see Koelle, 1969; Welsch and Pearse, 1969; Satler et al., 1974). Thus, the role of acetylcholine in the carotid body, frequently contested as a chemical transmitter (Heymans and Neil, 1958; Paintal, 1969; Sampson, 1971), is still obscure. Nevertheless, there is no convincing argument which could lead us to think that type I1 cells have a more sophisticated function than Schwann cells. Mills (1972) also has considered type N cells as the possible oxygen sensor, containing cytochrome a3 with a low affinity for oxygen. This author postulates the following mechanism: In hypoxia, type I1 cells release potassium ions which could depolarize type I cells, leading these cells to release an excitatory transmitter acting on the afferent terminals. This interpretation was revised later by Mills (1975) who “no longer excludes the possibility that the respiratory chain with low oxygen affinity is in the type I cell.” b. The originality of the theory proposed by Osborne and Butler (1975) is to suppose that the afferent nerve fibers are endogenously active and therefore spontaneously discharge. The proposed mechanism is the following: First, during normoxia, type I cells release dopamine which inhibits the spontaneous afferent discharge. In hypoxia, this release of dopamine is reduced, allowing the afferent terminals to discharge at a higher rate. This increased activity is accompanied by the release of acetylcholine from the terminals which further reduces the rate of dopamine secretion by type I cells. There is, consequently, a positive feedback loop. This theory, although ingenious, does not fit in with the results of Fidone et al. (1976) who suggest that acetylcholine is not located in nerve endings. Second, in a pharmacological study with a dopamine inhibitor, Docherty and McQueen (1977) showed that, in the cat, inhibition of sensory activity by dopamine is not substantial. Third, hypoxia increases dopamine

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release, at least in the rat (Hellstrom et al., 1976; Hellstrom, 1977) and the rabbit (Gonzalez and Fidone, 1977) carotid body. c. Torrance (1975) proposes that nerve terminals on type I cells are only sensitive to acidity and that hypoxia acts via a pH decrease in the intercellular space between nerve endings and type Utype 11 cells. This hypothesis has the merit of accounting for the convergence of hypoxic and hypercapnic stimuli (see Torrance, 1976, 1977, for full development of his theory). Torrance (1976) suggests that the pH is manipulated by type I1 cells but does not exclude the possibility that nerve endings, alternatively, may be stimulated by a transmitter (possibly polypeptidic) released from type I cells in response to a change in pH at their surface. F. NONINNERVATED TYPEI CELLS Finally we should mention that some type I cells are not innervated. From statistical data, McDonald and Mitchell (1975a,b) concluded that about 50% of type I cells (in the rat carotid body) are not connected to nerve endings but only to other type I cells. However, this estimation has not been corroborated by serial section analysis: Kondo (1976) reconstructed a cell cluster from the same species (rat) but only found one noninnervated cell among the 19 type I cells of the reconstructed cluster. A serial section analysis was also carried out by Nishi (1976) working on the cat carotid body: He only found 3 noninnervated cells out of 22 type I cells. According to this author, type I cells which are not innervated do not differ in appearance and organelle content from innervated cells.

V. Vascular Innervation and Efferent Inhibition It is well known that the carotid body is innervated not only by the sinus nerve but also by autonomic fibers coming from the superior cervical sympathetic ganglion via the ganglioglomerular nerve(s) (see Adams, 1958, for references). Degeneration experiments have shown that carotid body blood vessels are innervated by sympathetic fibers (Biscoe and Stehbens, 1967) and it has been demonstrated that sympathetic stimulation usually increases the chemoafferent activity in the sinus nerve as a consequence of a decrease in the carotid body blood flow (Floyd and Neil, 1952; Eyzaguirre and Lewin, 1961; Biscoe and Purves, 1967). However, it has been recently suggested that sympathetic efferent fibers may have more variable effects, sometimes leading to inhibitory effects, sometimes to different kinds of excitatory effects, or sometimes to no effect at all upon the chemoafferent discharge (O’Regan, 1977). Autonomic fibers are also present in the sinus nerve and there are both morphological and physiological results suggesting the occurrence of sympa-

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thetic and parasympathetic fibers in this nerve. Both kinds of fibers seem to have opposite vasomotor effects. Thus, parasympathetic fibers appear to be vasodilatory, leading to a decrease in chemoafferent activity. However, a new controversy arose a few years ago concerning the possible occurrences of nonvasomotor inhibitory fibers in the sinus nerve. Some authors think that such fibers do exist, but others disagree with this and ascribe all inhibitory effects to vasomotor fibers. Besides, both light and electron microscope studies mention a barosensory nerve supply to carotid body blood vessels. From a morphological point of view, we must consider therefore the courses and destinations of vasomotor and barosensory fibers and look for possible nonvasomotor inhibitory fibers. A. VASOMOTOR INNERVATION

The first ultrastructural demonstration of nerve fibers related to carotid body blood vessels was carried out by Biscoe and Stehbens (1966). They described unmyelinated nerve fibers, sometimes as small as 0.1 pm in diameter, more or less invested by Schwann cells and situated close to blood vessels. Among these fibers, nerve endings were identified by their mitochondria and microvesicles (about 50 nm) and large dense-cored vesicle (65 to 100 nm) contents. However, in this study, the origins of nerve fibers and endings were not determined. Now it appears that carotid body blood vessels receive sympathetic and possibly parasympathetic postganglionic nerve fibers. 1 . Sympathetic Nerve Fibers a. Identification. The above-mentioned results of Biscoe and Stehbens (1966) were enlarged upon in 1967 when they described degenerative changes in the perivascular nerve fibers after cutting the ganglioglomerular nerve(s), i.e., after postganglionic sympathectomy. However, no changes were seen after cutting the preganglionic cervical sympathetic, This observation therefore established, ultrastructurally, the existence of a vascular postganglionic sympathetic innervation in the carotid body. This conclusion has been largely confirmed afterward, using different methods. It is, indeed, very easy to demonstrate postganglionic sympathetic fibers thanks to their ultrastructural and cytochemical feature-. In conventional electron microscopy these fibers are characterized by varicosities containing some large dense-cored vesicles and numerous small vesicles (about 60 nm in diameter) which are either “empty” or contain one dense granule. These different kinds of vesicles are known to contain noradrenaline (Bisby and Fillenz, 1971) and the occurrence of even a limited number of small granulated vesicles is a good criterion to identify noradrenergic neurons (see Taxi, 1969). It is also possible to demonstrate noradrenergic terminals by highresolution autoradiography after administration of tritiated noradrenaline. Finally, aminergic nerve endings can be located with the light microscope using the

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formol-induced fluorescence method (Falck et al., 1962). All these methods have been used in carotid body studies and converge to prove the existence of vascular postganglionic sympathetic innervation: Perivascular nerve profiles in the carotid body appear to contain small granulated vesicles (Fig. 10) (Rees, 1967b; Kondo, 1971; McDonald and Mitchell, 1975a,b); a dense network of aminergic varicose nerve fibers has been demonstrated by Falck’s method (Rees, 1967b; Korkala et al., 1974; Verna, 1975), but it must be pointed out that fluorescent fibers appear related not only to blood vessels but also sometimes to type I cells (see Section V,A,c below). Finally, labeled nerve fibers may be seen in the vicinity of blood vessels after tritiated noradrenaline administration (Verna, 1975). Thus, the existence of noradrenergic sympathetic fibers related to carotid body blood vessels is well proved. There are, usually, several perivascular nerve fibers enclosed in a given Schwann cell but these fibers may be of different origin (sympathetic and parasympathetic according to McDonald and Mitchell, 1975b). Noradrenergic nerve endings (or, more exactly, varicosities) are often more or less devoid of Schwann cell sheaths but the distance between noradrenergic endings and smooth muscle cells or pericytes is rarely less than 0.1 pm (“at distance” junction). Although there is, unfortunately, no report about the distribution of nerve fibers along blood vessels, it seems that noradrenergic

FIG. 10. Sympathetic nerve profile (N) next to a capillary (Ca) (rabbit carotid body). Sympathetic endings contain both large dense-cored vesicles (arrowheads) and small vesicles some of which have a dense core (arrows). E, Endothelial cell; F, cytoplasmic processes of fibrocytes; M, mitochondria; Py,cytoplasmic processes of pericytes; S , Schwann cell. Bar = 0.5 pm.

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fibers are abundant around carotid body arteries, arterioles, and capillaries, but absent at the venous level. It must be emphasized that there are no smooth muscle cells but only pericytes around capillaries. b. Origin and Courses of Sympathetic Nerve Fibers. i. The ganglioglomerularnerve(s) pathway. As a rule, it can be said that carotid body aminergic fibers are unmyelinated axons of ganglion cells located in the superior cervical ganglion which reach the carotid body by the ganglioglomerular nerve(s). This has been shown by degeneration studies with the electron microscope (Biscoe and Stehbens, 1967; Rees, 1967b; Nishi and Stensaas, 1974; McDonald and Mitchell, 1975b) and with Falck’s method (Rees, 1967b; Verna, 1975). However, there are two possible variants: postganglionic fibers leaving the superior cervical ganglion to reach the carotid body via the sinus nerve and postganglionic fibers arising from sympathetic ganglion cells located in the carotid body itself. ii. The sinus nerve pathway. The occurrence of sympathetic nerve fibers in the cat sinus nerve has been suggested on physiological grounds (Biscoe and Sampson, 1968; Neil and O’Regan, 1971a). According to Biscoe and Sampson (1968) these fibers arise from postganglionic branches of the superior cervical ganglion joining the glossopharyngeal nerve and, then, course down the sinus nerve toward the carotid body. However, there is no histological study as to the destination of these fibers. Biscoe and Sampson (1968) have minimized their physiological importance, for the additional reason that such fibers are not always present. Furthermore, electrophysiological recordings have failed to demonstrate sympathetic fibers in the rabbit sinus nerve (Laurent and Jager-Barres, 1969) and, histologically, section of the rabbit sinus nerve does not seem to significantly affect the abundance of the carotid body noradrenergic innervation (A. Verna, unpublished observations). iii. Intrinsic sympathetic ganglion cells. McDonald and Mitchell (1975b) have observed few postganglionic sympathetic endings which remain intact after sinus nerve section and superior cervical ganglion removal (only three examples in sections from two rat carotid bodies). These nerve endings were indentified by their small granulated vesicles and were supposed to arise from sympathetic ganglion cells in the carotid body. Sympatheticganglion cells are usually located outside innervated organs (contrary to parasympathetic neurons), but their presence within the carotid body may be explained by the intimate connections which occur between the carotid body and the superior cervical ganglion anlagen during embryonic development (see Section VII). It is possible that the few myelinated nerve fibers present in the ganglioglomerularnerve(s) (Eyzaguirre and Uchizono, 1961) are sympathetic preganglionic fibers reaching intrinsic sympathetic neurons in the carotid body. However, we must underline that these neurons are few in number and, consequently, most sympathetic postganglionic nerve fibers in the carotid body arise from ganglion cells in the superior cervical ganglion.

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C. Functional Significance of Postganglionic Sympathetic Innervation. Sympathetic innervation is classically thought to be vasoconstrictory in the carotid body and therefore to mediate a decrease in blood flow (Daly et a l . , 1954; Purves, 1970). However, the effects of sympathetic excitation are far from clear: It has been suggested that sympathetic nerve fiber activity also tends to diminish the rate of carotid body oxygen consumption (Purves, 1970) but the mechanism of this effect is unknown (direct effect on carotid body cells? redistribution of blood?). More recently, O’Regan (1977) has studied the effects of sympathetic stimulation on chemoafferent activity. This author describes occasional inhibitory effects and two kinds of excitatory effects: The first kind is an early, transient increase whereas the second one is a slowly developing increase. The latter is presumed to be a vasoconstrictor a-adrenergic effect but the first excitatory effect is resistant to a-blockade and, thus, presumed to be nonvasomotor in nature. In this context, it may be worthwhile to recall that aminergic nerve endings have been observed close to type Vtype I1 cells by several authors. Kondo (1971) has shown a nerve terminal containing small granulated vesicles separated from a type I cell by only a slender type I1 cell process (guinea pig carotid body). Noradrenergic nerve profiles invested by thin processes of type I1 cells have been observed, autoradiographically, in the rabbit carotid body (Verna, 1975). McDonald and Mitchell (1975b) also report (but without giving any illustration) they have observed, in the rat carotid body, a few sympathetic nerve endings in contact with type I cells. This assertion has been repeated (but again without illustration) by Knoche and Kienecker (1977) concerning the rabbit carotid body. It seems therefore that carotid body-specific cells are surrounded by noradrenergic nerve endings (as clearly indicated by Falck’s method) which can even be in contact with them. Consequently, it may be legitimate to envisage sympathetic actions not only on carotid body blood vessels but also on the type I cell/type I1 cell/afferent ending complex. Nonvasomotor sympathetic actions have also been envisaged concerning the cochlea (Densert, 1974; Densert and Flock, 1974). However, it must be added that, after some controversies, the effects of noradrenaline upon chemosensory discharges appear to be weak and of short duration, at least in the cat carotid body (Llados and Zapata, 1978). Nevertheless, sympathetic effects exerted at the level of cell clusters, in addition to those on blood vessels, could explain the variable effects observed after sympathetic stimulation (O’Regan, 1977) or noradrenaline administration (Llados and Zapata, 1978).

2 . Parasympathetic Nerve Fibers The existence of a parasympathetic vascular innervation has been suggested by de Castro (1926, 1928, 1951) and de Castro and Rubio (1968). According to these authors there are parasympathetic ganglion cells in the carotid body which

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are innervated by preganglionic fibers coursing in the sinus nerve. The postganglionic fibers were supposed to innervate smooth muscle cells of carotid body arteries or arterioles. However, this problem has received little attention and it was only in 1975 that ultrastructural evidence was presented on this subject by McDonald and Mitchell (1975b). These authors have described, in the rat carotid body, perivascular nerve endings containing small vesicles without dense cores and therefore, of nonsympathetic nature. These endings did not degenerate after severance of all nervous connection to the carotid body and, consequently, were presumed to arise from parasympathetic ganglion cells in the carotid body. Furthermore, McDonald and Mitchell (1975b) demonstrated that most of carotid body ganglion cells were denervated by cutting the glossopharyngeal nerve. Thus, these results are concordant not only with the light microscope studies of de Castro but also with physiological observations showing the occurrence in the cat sinus nerve of fibers whose activity increases the carotid body blood flow, an effect which is blocked by atropine (Neil and O’Regan, 1969a, 1971a). However, it is surprising that McDonald and Mitchell (1975b) describe parasympathetic vasomotor endings as being abundant since they only found 25 ganglion cells in one carotid body. Moreover, this number may be exceptional since, in the same species (rat), Hess (1977) found a mean number of about 5 ganglion cells per carotid body (actually from 1 to 8 neurons in 12 carotid bodies). Since type I cell processes also contain synaptic-like vesicles without dense cores and, of course, do not degenerate after denervation, there is an obvious possibility of confusion. Alternatively, it is possible that parasympathetic terminals innervate only certain portions of the vascular apparatus and it is regrettable that McDonald and Mitchell (1975b) describe the vasomotor innervation only in terms of endings on “blood vessels.” According to de Castro and Rubio (1968) postganglionic parasympathetic fibers innervate carotid body arteries and arterioles and are the efferent pathway of a reflex which tends to maintain a constant flow in spite of arterial pressure variations. The afferent part of this reflex has been attributed by these authors to baroreceptor endings in the wall of carotid body arteries.

B. BAROSENSORY INNERVATION De Castro (1940, 1951) has described, in silver-impregnated material, baroreceptor nerve endings in the adventitia of carotid body arterioles and in the arterial segment of arteriovenous shunts. Presumed baroreceptor endings were also described, in light microscopy, by Abraham (1968) around carotid body arteries of some species (dog), in the connective tissue of others (horse and bovides), and in vein walls of human carotid body. This question has been recently investigated with the electron microscope by Gorgas and Bock (1977). They describe mitochondria-rich nerve endings located in the tunica adventitia of

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small arteries (one to two layers of smooth muscle cells). These endings are branched lanceolate terminals which adhere helically or circularly to the arterial wall. Furthermore, Gorgas and Bock (1977) describe junctions between these presumed baroreceptor endings and slender processes of type I cells. Unfortunately, although their interpretation may be correct, it is absolutely impossible to affirm that a given nerve ending is barosensory or not on morphological criteria alone. This is more especially the case in the carotid body insofar as the ultrastructure of the putative barosensory terminals described by Gorgas and Bock (1977) is identical to the ultrastructure of the afferent nerve endings on type I cells. This criticism may also be applied to the description of axon swellings in the carotid body connective tissue as possible baroreceptor endings (Kobayashi, 1971b). Although such axon swellings which do not contact type Utype I1 cells actually exist (Nishi and Stensaas, 1974) their origin and significance are open to question. C. THEPROBLEM OF THE EFFERENT INHIBITION The problem of so-called efferent inhibition is, primarily, a physiological one. Its origin lies in the electrophysiological observations of Biscoe and Sampson (1968) who recorded spontaneous nervous activities from the central cut end of the sinus nerve (in the cat). This efferent activity was also demonstrated by Laurent and Jager-Barres (1969) in the rabbit and again in the cat by Neil and 0 ’Regan ( 1969b, 1971b) , Possible physiological effects of sinus nerve efferent activity were investigated by Neil and O’Regan (1969a, 1971a) and Fidone and Sat0 (1970). It was shown by these authors that electrical stimulation of the sinus nerve decreases the chemoafferent activity in that nerve. However it is necessary, in this kind of experiment, to distinguish true inhibitory effects from antidromic depression of afferent fibers due to stimulus spread along the nerve. This was done by Fidone and Sat0 (1970) who concluded that true efferent inhibition, mediated by unmyelinated fibers, may be elicited in the sinus nerve but appears to be less potent than antidromic depression. Furthermore, Neil and O’Regan (1969b, 197lb) demonstrated that the efferent activity recorded from slips of otherwise intact sinus nerves markedly increases when the chemoafferent activity is raised by systemic hypoxia or asphyxia. These results suggest a feedback inhibitory mechanism similar to the efferent control of other receptors such as the auditory system (Fex, 1962) or the lateralline organ of fishes (Russell, 1968). However, the results of Neil and O’Regan were severely criticized by Goodman (1973) who attributed their results to antidromic depression and vasomotor effects. There are, indeed, vasomotor nerve fibers present in the cat sinus nerve: Neil and O’Regan themselves (1969a, 197la) have demonstrated that stimulation

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of the sinus nerve efferents usually increases the carotid body flow, an effect which can be blocked by atropine. It is well known that chemoafferent activity is inversely related to carotid body blood flow (Daly et al., 1954). Thus, there are three possible mechanisms which can decrease the chemoafferent discharge: (1) antidromic depression, either artifactual in the case of electrical stimulation or physiological if primary afferent depolarization or axon reflexes occur; (2) increase in the carotid body blood flow; (3) efferent inhibition directly exerted on chemoreceptors. According to Goodman (1973) the latter mechanism does not exist and the so-called “efferent inhibition” is only of vasomotor origin. But O’Regan (1975) showed that inhibition can still be observed in the ischemic carotid body, i.e., in the absence of blood flow and, therefore, of vasomotor effects. Furthermore, Willshaw (1975) was able to measure the carotid body blood flow and found that the inhibition is not accompanied by any significant change in blood flow. On the other hand, Belmonte and Eyzaguirre (1974) and McCloskey (1975) did not observe efferent inhibition in the absence of carotid body perfusion. Thus, the influence of vasomotor effects on chemoafferent discharge is not contested but the question remains: Are there other, more direct, inhibitory mechanisms? 1. Efferent Influences on Type I Cells The so-called efferent inhibition being mediated by sinus nerve fibers, we shall not discuss here the possible effects exerted by preganglionic sympathetic nerve endings on type I cells described by some authors and contested by others (see Section IV). This being said, the question now is: Do sinus nerve efferents affect type I cells? As we have seen, such fibers (of parasympathetic nature) may end on ganglion cells but there is no evidence of sinus nerve efferent fibers terminating on type I cells. Nevertheless, it has been shown that centrifugal sinus nerve activity may affect the type I cell content (Yates et al., 1970) and synthesis and release (Mills and Slotkin, 1975; Sampson et al., 1975) of catecholamines. Thus, we have to explain efferent effects without efferent terminals. One possibility is to admit, as we have already mentioned (see Section IV,E,l), that in addition to their afferent activity, sensory nerve terminals may exert efferent effects on type I cells. This phenomenon may occur under artificial circumstances, such as electrical stimulation of the sinus nerve, or under natural circumstances, either by an axon reflex mechanism as suggested by Nishi and Stensaas (1974) and VBzquez-Nin et al. (1977) or by the mechanism proposed by McDonald and Mitchell (1975a,b), i.e., a transmitter release (at the level of so-called reciprocal junctions) concomitant to nerve ending excitation. However, these mechanisms do not involve efferent fibers, which are known to be present in the sinus nerve, so we must consider yet another possibility of nonvasomotor inhibitory effects, namely, efferent control of the afferent nerve endings themselves.

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2. Efferent Influences on Afferent Nerve Fibers It has been suggested by some authors (Verna, 1973; King et al., 1975; Morgan et al., 1975) that the activity of the type I cell afferent innervation may be controlled by efferent fibers terminating on the afferent endings themselves. This interpretation was based on electron micrographs showing presumed nerve profiles (containing synaptic-like vesicles) which were presynaptic to nerve endings in contact with type I cells. Unfortunately, none of the authors who have described these junctions between nerve endings has been able to prove the neural nature of the presumed efferent terminal. So, it cannot be excluded, as pointed out by McDonald and Mitchell (1975a,b), that the presumed efferent “fibers” actually were type I cell processes since we know that such processes may contain numerous synaptic-like vesicles (see Section IV,C,2). Another possibility of efferent influences on the chemoafferent activity has been suggested which does not involve efferent fibers but involves interactions between afferent fibers running close together (Willshaw, 1977, discussion of his paper). However, this hypothesis has so far received no experimental support. Thus, there are both physiological and morphological data which explain “efferent inhibition” by activity of vasomotor fibers, whereas there are no conclusive morphological results, only physiological ones, supporting the existence of nonvasomotor “efferent inhibition” mediated by sinus nerve efferent fibers.

VI. Ultrastructural Changes after Stimulation of Chemoreceptor and Pathology Since it has been postulated (de Castro, 1928) and then demonstrated (Heymans and Bouckaert, 1930) that the carotid body is an arterial chemoreceptor, many attempts have been made to demonstrate morphological changes consecutive with stimulation by hypoxia, hypercapnia, asphyxia, and so on. So, Hollinshead ( 1945) has described cytological modifications of carotid body cells after severe hypoxia and similar investigations were also made with the electron microscope as early as 1958 by Hoffman and Birrel. The first studies were principally focused on type I cells but more recent reports also consider changes in nerve ending ultrastructure. A. CHANGES IN TYPEI CELLS The first report of changes in carotid body ultrastructure following anoxia was from Hoffman and Birrel (1958) who described the disappearance of type I cell dense-cored vesicles as a consequence of severe anoxia. However, these results are unreliable because of the poor fixation methods available at that time. Bliimcke et af. (1967a) also have studied the effects of oxygen deficiency on the rat carotid body ultrastructure. They submitted rats to gas mixtures containing

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10,5, and 2.5% oxygen for 5 to 20 minutes but only found changes in type I cells after extreme hypoxia (10 minutes in 2.5% 0 2 ) . In these conditions, Blumcke et al. (1967a) observed an increased frequency of exocytosis pictures, suggesting the discharge of type I cell dense-cored vesicles. They also describe other changes in these cells such as swelling of the mitochondria, disintegration of the chromatin in the nucleus, and distension of the perinuclear cleft. Moreover, they show by formol-induced fluorescence microscopy that, after extreme oxygen deficiency (20 minutes in 2.5% 0 2 )the , catecholamine content of type I cells disappears. Blumcke et al. (1967a) conclude from their observations that type I cells release their catecholamines (by exocytosis) in response to hypoxia. Although this conclusion may be right (see Mills and Slotkin, 1975; Hellstrom et af., 1976; Hellstrom, 1977) the results of such extreme stimulations (the authors confess that rats lost consciousness after 1 minute in 2.5% 0,) seem to be doubtful. The swelling of the mitochondria was certainly unspecific and the exocytosis pictures were not convincing due to very marked osmotic artifacts enlarging the intercellular spaces up to 100 nm. In another paper, Blumcke et al. (1967b) considered the effects of hypercapnia on type I cells. Here again they used unphysiological stimuli (up to 75% C02) and, hence, it was not surprising that type I cell cytoplasm showed a “high degree of oedema. The authors also described a disintegration of type I cell dense-cored vesicles leading their dense cores to disappear in the cytoplasm. These aspects were probably due, in fact, to the use of collidine buffer in the fixative (Chen et al., 1969). Using somewhat better fixation methods, Al-Lami and Murray (1968a) reported that hypoxia (9% O2 for 45 minutes) leads to a slight increase in the relative number of type I cell dense-cored vesicles (in the cat carotid body). They concluded from this observation that type I cells do not release the content of their dense-cored vesicles after hypoxia. Such a conclusion is purely speculative since the observation could be also explained by an increased rate of synthesis which compensates an increased rate of discharge. The same remark may be made for the conclusion of Chen et al. (1969), who demonstrated, with a cytochemical method, that reserpine depletes the amine content of type I cell dense-cored vesicles whereas hypoxia does not. However this result does not validate their suggestion that type I cells do not release catecholamines in response to hypoxia. At the same time, Zapata et al. (1969) observed no detectable changes in the carotid body cytology induced by hypoxia (5% O2 in N, for 3 hours in cats) and, more recently, McDonald and Mitchell (1975a) found no striking changes in type I cells after hypoxia, except a slight enlargement of mitochondria and a decrease in the electron density of their matrix. On the other hand, Hellstrom (1977) reported similar changes about type I cell mitochondria but suggested that there is a small decrease in the number of dense-cored vesicles after hypoxia. ”

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In the above-reported studies only temporary periods of hypoxia were used (no more than a few hours) but other investigators have considered the consequences of chronically hypoxic conditions on the carotid body. It has been shown that subjects (man and animals) living at high altitudes have enlarged carotid bodies (Arias-Stella, 1969; Edwards et al., 1971b; Barer et al., 1972; Blessing and Wolff, 1973; Laidler and Kay, 1975). According to Blessing and Wolff (1973) and Laidler and Kay (1975) the enlargement of the carotid body in animals submitted to high altitude is due to an increased volume of cellular structures and capillaries. From an ultrastructural point of view, Edwards et al. (1972) reported only some minor changes between sea-level and high-altitude guinea pig carotid bodies. On the other hand, Moller et al. (1974) claims there is a marked increase in the number of dense-cored vesicles and mitochondria in type I cells of hypoxic rabbits. This assertion is only based on subjective grounds since the authors write “it is our impression that the number of dense-cored vesicles and mitochondria in the type I cells is at least doubled in the hypoxic rabbits. Such an “impression” is not conclusive because the amount of dense-cored vesicles in rabbit type I cells varies greatly from cell to cell (in a 1 to 10 range; A. Verna, unpublished observations). There is consequently an obvious problem of sampling. Contrary to Moller et al. (1974), Blessing and Kaldeweide (1975) found a noticeable decrease in the number of dense-cored vesicles following adaptation to a simulated altitude of 7000 m (in rats). Furthermore they observed dilated capillaries and thrombosis leading to type I cell degeneration. However, Laidler and Kay (1978) have made a more precise study: They have used stereological methods to investigate carotid bodies from rats living at a simulated altitude of 4300 m for 4 to 5 weeks. They found a 3-fold increase in the volume of type I cells (due to an increased volume of cytoplasm) in hypoxic rats. On the other hand, no change in the volume proportion of type I cells occupied by mitochondria was reported. The authors deduced from their data that the number of mitochondria in each type I cell was increased, but it must be noted that possible changes in length of mitochondria have not been considered. Dense-cored vesicles were reported to increase in diameter but without significant change in volume proportion of cytoplasm due to a reduction in their mean number per volume unit of cytoplasm. Thus, chronically hypoxic conditions really seem to affect type I cell ultrastructure. However, it is difficult to deduce from these results any conclusion whatsoever. Laidler and Kay (1978) suggest that type I cells react to hypoxia by an increased metabolic activity accompanied by an increased rate of dense-cored vesicle release. However, other interpretations may be proposed as well, as long as no changes are reported in the number of ribosomes. Finally, the effects of acute hemorrhagia were also studied by Korkala and Waris (1975) who have observed (and confirmed with a statistical method) that type I cell dense-cored vesicles move toward nerve endings in response to severe hemorrhagia. The authors concluded that type I cells probably have an inhibitory ”

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modulating function, a conclusion which is not, actually, founded on their morphological observations.

B. CHANGES IN NERVE ENDINGS Modifications of nerve ending ultrastructure after chemoreceptor excitation have been reported only by McDonald and Mitchell (1975a, 1976) and McDonald (1977a,b). These authors describe conspicuous changes in afferent nerve terminals on type I cells following increased activity of these terminals (produced by hypoxia, by hypercapnia, or by antidromic electrical stimulation of the sinus nerve). These changes concern mitochondria and synaptic-like vesicles. After 1 minute of severe hypoxia (100% N2) the mitochondria become swollen and exhibit a more electron-lucent matrix than the mitochondria in hyperoxic animals. However, efferent nerve terminals do not show mitochondria1 swelling in response to hypoxia. In addition, the abundance and packing density of the synaptic-like vesicles in sensory endings on type I cells are decreased by stimulation of the chemoafferent activity: After hypoxia produced by ventilating rats for 10 minutes with 10 or 5% O2 in N2, the mean concentration of synaptic-like vesicles is reduced by about 27%. This observation is interpreted by the authors as reflecting a rate of exocytosis which exceeds the rate of vesicle formation, these vesicles being thought to contain a synaptic transmitter (McDonald and Mitchell, 1975a, 1976; McDonald, 1977a,b) (see Section IV,E,2).

C. PATHOLOGY 1. The Carotid Body in Respiratory Diseases and Anemia The structure of the carotid body has been investigated in patients suffering diseases which may affect the chemoreceptors by a correlative hypoxia. For example, Simhrszky and Lapis (1970) have studied the ultrastructure of carotid bodies in individuals suffering bronchial asthma. These carotid bodies were removed for therapeutic purposes (a somewhat surprising practice) but their ultrastructure appeared to be normal. On the other hand, carotid body enlargement has been described as a response to chronic hypoxia due to chronic bronchitis (Heath et al., 1970), emphysema (Edwards et al., 1971a), or cyanotic congenital heart disease (Lack, 1977). The effects of chronic anemia upon carotid body histology have also been investigated. Tramezzani et al. (1971) reported that carotid bodies enlarge in cats made chronically anemic by daily bleeding. The authors suggested, together on experimental grounds, that the carotid body controls erythropoiesis. This asser-

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tion led Winson and Heath (1973) to correlate carotid body weight to hemoglobin level but carotid bodies were found not to be heavier in anemia. Furthermore, the experimental data reported by Tramezzani et al. (1971) to support their statement were confirmed neither by Lugliani et al. (1971) nor by Hansen et al. (1973) who rejected the suggestion that the carotid body produces erythropoietin. Finally, there has also been an attempt to relate the carotid body size to the sudden infant death syndrome, but with inconclusive results (Naeye et al., 1976). 2. Carotid Body Tumors Carotid body tumors, although occurring infrequently, have been known for almost a century (Shamblin et al., 1971) and there is a considerable volume of literature on this subject. However, it seems that some confusion exists regarding the terminology of this disease. Carotid body tumors are sometimes referred to as “glomus tumors” but this term more frequently designates glomera of the skin. Lattes (1950) first used the name “nonchromaffin paraganglioma” for tumors of the carotid body and aortic body but also for those of the ganglion nodosum and the jugular body. This term was opposed to “chromaffin paraganglioma” or ‘‘pheochromocytoma” (which designates tumors of the adrenal medulla for example), but, as pointed out by Karnauchow (1963, chromaffinity is not a good criterion to subclassify these tumors. As in the normal carotid body, the chromaffinity of carotid body tumors is negative or doubtful (LeCompte, 1951), or positive only at the level of a few cells (Pryse-Davies et al., 1964). For this reason, Totten (1973) recommends the use of only “paraganglioma” accompanied by the anatomical site of the tumor. But the most usual term is perhaps “chemodectoma,” an appellation introduced by Mulligan (1950). Unfortunately, this term is used not only for carotid and aortic body tumors but also for tumors of other structures whose chemoreceptor function is not established. Only carotid and aortic body tumors are considered here. According to Shamblin et al. (1971) about 500 cases of human carotid body tumors have been reported up to 1970. These tumors are consequently infrequent. Among these cases only a few percent were malignant. The incidence of metastases is very low but some cases have been reported (Fanning et al., 1963; Whimster and Masson, 1970; Hortnagl et al., 1973; Villiaumey et al., 1974). The tumors usually grow slowly but may be bilateral or even multicentric. Familial cases have been described. One report suggests that carotid body tumors are more frequent in people living at high altitude (Saldana et al., 1973). Symptoms and clinical signs are ordinarily minimal and often due to compression of neighboring structures. However, a few cases of catecholamine secretion have been reported (Glenner et al., 1962; Hamberger et al., 1967; Hortnagl et al., 1973).

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Carotid and aortic body tumors have also been described in other mammals and more especially in the dog (Parodi and Lekkas, 1970; Cheville, 1972). Whereas carotid body tumors are preponderant over aortic ones in man, the reverse is true for the dog (Parodi and Lekkas, 1970). Histologically, carotid body tumors are characterized by a lobulated and richly vasularized structure. Because of this vascular nature, biopsy is a hazardous procedure. The tumor cells are grouped in clusters and frequently show enlarged nuclei, Nuclear pleomorphism is sometimes considered as indicating a malignant character (Hortnagl ef al., 1973) but difficulty in judging carotid body tumors benign or malignant on histological grounds has been underlined by many authors (LeCompte, 1951; Pryse-Davies et al., 1964). Mitoses are always rare. Despite the inconstant results of the chromaffin reaction, catecholamines have been characterized in carotid body tumors with fluorescence microscopy (Grimley and Glenner, 1967). Nerve endings have been described by some authors (Costero and Barroso-Moguel, 1961; Pryse-Davies et al., 1964) but have not been observed by others (Grimley and Glenner, 1967; Macadam, 1969; Alpert and Bochetto, 1974). Type I and type I1 cells have been characterized in carotid body tumors. However, type I1 cells are usually few or even absent. Type I cells are polygonal and have a “light, ” “dark, or intermediate appearance (Alpert and Bochetto, 1974). Dense-cored vesicles have been described in these cells by many authors (Grimley and Glenner, 1967; Toker, 1967; Macadam, 1969; Capella and Solcia, 1971; Hortnagl ef al., 1973; Alpert and Bochetto, 1974). It has been shown by Lishajko (1970) that isolated dense-cored vesicles from a human carotid body tumor release and take up dopamine. On the other hand, Capella and Solcia (197 1) suggest that some tumoral cells may contain serotonin (the others containing some polypeptidic substance), whereas Hortnagl et al. (1973) characterize noradrenaline and dopamine P-hydroxylase in a liver metastase of a carotid body tumor. Type I cell mitochondria are more or less numerous but frequently swollen due to poor fixation conditions. In conclusion, the study of carotid body tumors seems to add little to our knowledge of the normal carotid body. ”

VII. Embryology and Development The embryology of the carotid body has been very controversial until recent years, particularly with respect to the origin of type I cells (see Adams, 1958, for historical review). According to Rogers (1965) the carotid body appears as a primary condensation of cells on the third aortic arch, but this author concluded that it was impossible to know whether these cells derive from local mesenchyme or migrate from the neural crest. However, this problem has been solved recently, at least for birds, by Le Douarin et al. (1972) and Pearse ef al. (1973).

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These authors have demonstrated that carotid body type I (and possibly type 11) cells are of neural crest origin. This result was obtained from heterospecific grafting experiments together with histological and histochemical studies making it possible to identify the cells of the graft among the cells of the receiver. Although these studies were made on avian species, there is no reason to postulate a different origin for the mammalian carotid body type I cells. The development of the carotid body anlage has been investigated recently with the light, fluorescence, and electron microscopes. It has been shown with the formol-induced fluorescence method that type I cells of the midterm human fetus contain catecholamines (Hervonen and Korkala, 1972; Korkala and Hervonen, 1973). Furthermore, it was shown by Korkala and Hervonen (1973) that a connecting cell cord links the carotid body to the sympathetic anlage in the 7-week-old human fetus. This process, which is made of small fluorescent cells, disappears after the tenth week of development. The authors suggest a migration of cells from the sympathetic trunk to the carotid body. In the newborn and 2-week-old rat, Korkala et al. (1974) observed a bundle of fluorescent nerve fibers which connect the sympathetic superior cervical ganglion to the carotid body. Occasionally, fluorescent cells, identical to carotid body type I cells, were seen inside or beside the fluorescent nerves. In the mouse embryo, catecholamine-containing cells were identified in the carotid body anlage at 2 weeks of gestation. These cells were able to take up L-dopa which increases their fluorescence but does not modify the number of fluorescent cells (Fontaine, 1974). There are two recent electron microscope studies on the embryonic development of the carotid body. The first one is devoted to the midterm human fetus (Hervonen and Korkala, 1972). The histochemically demonstrated occurrence of catecholamines in cells of this material has been correlated by Hervonen and Korkala (1972) with the occurrence of dense-cored vesicles in many cells, as shown by electron microscopy. These dense-cored-vesicle-containing cells (that is, type I cells) occur in small groups surrounded by processes of other cells which do not contain dense-cored vesicles (type I1 cells). However, type I cells are not always completely enveloped by type I1 cells (as in the adult organ). The dense-cored vesicle size does not differ noticeably from cell to cell (but the authors give no quantitative data). Type I1 cells surround not only type I cell groups but also capillaries and nerve fibers. Some type I1 cells were occasionally found by Hervonen and Korkala (1972) in contact with both endothelial and type I cells. Capillaries are abundant but do not show fenestrations. Type I cells are already richly innervated: Many type I cells are in contact with nerve endings which contain agranular vesicles (40 to 50 nm) and a few larger dense-cored vesicles. Several sites of increased electron density were observed by Hervonen and Korkala (1972) along the course of the longer nerve endings. These authors, in conclusion to their study, underlined that the fetal carotid body looks surpris-

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ingly mature and suggest there may be some sort of chemoreceptor activity during pregnancy, as previously described by Biscoe et al. (1969). The second electron microscope study of the embryonic development of the carotid body is from Kondo (1975) and concerns the rat. In the 11-mm embryo (13 days of gestation) this author describes the carotid body anlage as a thickening of the wall of the third branchial artery. This thickening is made of undifferentiated cells and small blood vessels. There are also unmyelinated nerve fibers which contain clear vesicles about 40 to 70 nm in diameter and dense-cored vesicles about 100 nm in diameter. Although some nerve fibers were enveloped by processes of undifferentiated cells, membrane specializations were absent. In the 12-mm embryo (14 days of gestation) dense-cored vesicles appear in some of the undifferentiated cells, particularly those located at the boundaries of the anlage. At this stage, bundles of small unmyelinated nerve fibers are common and sometimes touch undifferentiated cells or cells containing dense-cored vesicles. However there are no junctional specializations. The latter appear in the 17-mm embryo (16.5 days of gestation). Kondo (1975) describes two kinds of junctions with embryonic type I cells, one with membrane densification and vesicles clustered inside the nerve ending, the other with dense material and vesicles inside the type I cell. At this stage, fenestrations of the blood vessels occur. Some type I cells exhibit slender processes as long as 25 pm. Finally, in the 20-mm embryo (17 to 17.5 days of gestation) the carotid body anlage is completely separated from the wall of the internal carotid artery. Undifferentiated cells show a tendency to envelop adjacent type I cells. The number of junctions with nerve endings remains small but the length of the terminals increases and more vesicles are associated with the membrane specializations. These studies are therefore concordant as to the mature appearance of the carotid body before birth. However, the problem of the factors controlling type I cell differentiation is still obscure. Korkala and Hervonen (1973) suggest humoral influences coming from the blood vessels but Kondo (1975) points out that type I cells can be first identified at the anlage periphery and suggests that these cells migrate from a distant site, already differentiated, at least partially. It is obvious that this kind of study comes up against the difficulty involved in identifying undifferentiated cells.

VIII. Concluding Remarks Despite some remaining problems concerning type I cell subdivision or autonomic innervation, for example, it can be said that the carotid body ultrastructure is well known. Conclusive results, recently obtained, have put an end to the confusion about the nature of nerve endings on type I cells. It is clear now that the elementary structure of the carotid body is a dense-cored vesicle-containing cell situated close to a capillary and innervated by a sensory fiber, the cell and the

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nerve ending being more or less invested by a satellite cell. However, further data about the three-dimensional structure of the carotid body would be desirable. Such investigations have been undertaken by means of reconstruction from serial sections, a method recently improved by use of computers (see Seidl et al., 1977; Lubbers et al., 1977). Unfortunately, morphological methods do not make it possible to identify the transducer element of the chemosensory process. Morphologists can stimulate chemoreceptors and look for specific changes by comparing the structure of stimulated and unstimulated carotid bodies. However, as we have seen, these changes are subtle and difficult to interpret. On this point, it could even appear somewhat paradoxical to study the structure of unstimulated chemoreceptors after a chemical fixation! Morphologists can also compare the structure of type I cells to the structure of other cells. This procedure leads to conclusions such as: Type I cells are sensory cells, type I cells are secretory cells, type I cells are interneurons, type I cells are paraneurons . . . ! These varied interpretations show with some obviousness that type I cells, actually, have no real specific cytological character. They even look like relatively undifferentiated cells with a high nucleus/cytoplasm ratio. Nevertheless, type I cells (andor type I1 cells) appear necessary to the chemosensory process (Verna et al., 1975; Zapata et al., 1976, 1977) but their exact role, if any, in the mechanism of chemoreceptor excitation has not been elucidated (see Acker and Pietruschka, 1977; Roumy and Leitner, 1977; Torrance, 1977, for current hypotheses). On this subject, intracellular recordings from carotid body slices (Eyzaguirre et al., 1977) or on carotid body cells in culture (Acker and Pietruschka, 1977) look promising. It has been shown by these methods that type I cells have a membrane potential of relatively low value, that they do not generate spikes, and that their membrane potential and input resistance are affected by various chemical and physical stimuli. But, once again, results are difficult to interpret: Among stimuli increasing the chemoafferent discharge, some hyperpolarize (temperature increase), others depolarize (acidity, interuption of superfusion, hyperosmolarity), and others do not seem to affect type I cell membrane potential and input resistance (COz, hypoxia, cyanide, acetylcholine) (Eyzaguirre et al., 1977). These approaches are, nevertheless, at the root of the problem which is, finally, the function(s) of these mysterious cells called type I cells.

REFERENCES Abbott, C. P., and Howe, A . (1972). Acra Anat. 81, 609. Abbott, C. P., Daly, M. de B . , and Howe, A. (1972). Acfu Anar. 83, 161. Abrahbm, A. (1968). In “Arterial Chemoreceptors” (R. W. Torrance, ed.), p. 57. Blackwell, Oxford.

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