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Principles and Techniques of Electron Microscopy

The mucus at the surface of the olfactory mucosa constitutes the milieu in which perireceptor events associated with olfactory transduction occur. In this review, the ultrastructure of olfactory mucus and of the secretory cells that synthesize and secrete olfactory mucus in the vertebrate olfactory mucosa is described. Bowman's glands are present in the olfactory mucosa of all vertebrates except fish. They consist of acini, which may contain mucous or serous cells or both, and ducts that traverse the olfactory epithelium to deliver secretions to the epithelial surface. Sustentacular cells are present in the olfactory epithelium of all vertebrates. In fish, amphibia, reptiles, and birds, they are secretory; in mammals, they generally are considered to be "non-secretory," although they may participate in the regulation of the mucous composition through micropinocytotic secretion and uptake. Goblet cells occur in the olfactory epithelium of fish and secrete a mucous product. Secretion from Bowman's glands and vasomotor activity in the olfactory mucosa are regulated by neural elements extrinsic to the primary olfactory neurons. Nerve fibers described in early anatomical studies and characterized by immunohistochemical studies contain a variety of neuroactive peptides and have several targets within the olfactory mucosa. Ultrastructural studies of nerve terminals in the olfactory mucosa have demonstrated the presence of adrenergic, cholinergic and peptidergic input to glands, blood vessels, and melanocytes in the lamina propria and of peptidergic terminals in the olfactory epithelium. The neural origins of the extrinsic nerve fibers and terminals are the trigeminal, terminal, and autonomic systems.

Fine structural aspects of secretion and extrinsic innervation in the olfactory mucosa.

A comparative study using freeze-fracturing has been made of surface structures of olfactory and nasal respiratory epithelia of frog, ox, rat and dog. Special attention has been paid to cilia and microvilli present at these surfaces, although the observations include various other structures such as small intracellular vacuoles present in the olfactory receptor endings and infrequent brush cells. Within the mucus overlying the olfactory epithelium membranous vesicles, often attached to olfactory cilia, are seen. Some of these show intramembranous particle distributions similar to those of the rest of the cilia, whereas others are devoid of particles. Smooth vesicles are also found in the mucus of other types of epithelium (respiratory epithelium and Bowman's glands). The freeze-fracture morphology of intracellular secretory vacuoles present in olfactory supporting, Bowman's and respiratory glandular cells of the frog is similar in all these epithelia. Quantitative comparisons are made of the different structures of interest. When corrected for cilia which were not observed, mammalian receptor endings bear 17 cilia on average, whereas frog receptor endings have 6 cilia. The relative magnitudes of the diameters of the cilia and microvilli are, except for frog, the same for all species studied. Dimensions of other structures e.g., axons, dendrites and dendritic endings are compared in the various species. Freeze-fracture diameters are usually larger than those seen by techniques using dehydration. Dendritic ending densities range from 4.5 × 106 (frog) to 8.3 × 106 (dog) endings per cm2. Possible sex-dependent differences are only found for these densities and dendritic ending diameters.

Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory structures of frog, ox, rat, and dog. I. A general survey.

A survey of ultra‐rapid cryofixation methods with particular emphasis on applications to freeze‐fracturing, freeze‐etching, and freeze‐substitution

The densities and diameters of intramembranous particles in olfactory and nasal respiratory structures of frog, ox, rat and dog have been compared using the freeze-fracture technique. Dendritic endings and the various segments of the cilia of the olfactory receptor cells of a given species have identical particle densities (700–1,800 particles/μm2 in P-and 100–600 in E-faces). Densities in P-faces of respiratory cilia are about 1/3 of those in the olfactory cilia. E-face particle densities of these respiratory cilia are often higher than P-face densities. Microvillus P-face densities range from 700–2,000 (respiratory cell microvilli) to 1,800–3,400 particles/μm2 (olfactory supporting and Bowman's gland microvilli). Microvillus E-faces show no conspicuous mutual differences. Literature comparisons showed that odour concentrations at threshold are considerably lower (105–1010 times) than the concentrations of olfactory receptor ending intramembranous particles (5 μM–30 μM) expressed in the same units. Relative differences in particle distributions of the various cell structures studied are usually species-independent. Absolute values vary considerably with the species. Relative P-face particle densities of the supporting cell microvilli tend to correlate with those of dendritic ending structures. Particle diameters are usually similar for corresponding structures and fracture faces in the four species. Apical structures of supporting and Bowman's gland cells in rat and dog show rod-shaped particle aggregates in their P-and pits in their E-faces. Neither sex-dependency nor an influence related to physiological treatments on the particle distributions could be demonstrated.

Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory epithelial surfaces of frog, ox, rat, and dog

High-voltage transmission electron microscopy and cryo-ultramicrotomy together with scanning electron microscopy and some conventional transmission electron microscopy of ultrathin sections have been applied to the mucous surfaces of bovine olfactory and respiratory epithelia. Distal segments of olfactory cilia tend to run in parallel and could be followed over distances up to about 30 μm using high-voltage electron microscopy. This technique and scanning electron microscopy showed that on average 12–13 of such cilia could be observed per nerve ending. After correction for obscured cilia this number becomes about 17. High-voltage micrographs and micrographs made from sections prepared with a cryo-ultramicrotome showed the presence of electron-lucent pockets inside the olfactory mucus. The latter technique also showed that the mucus itself is not fibrous, but rather a continuum varying in electron density. The mucus layer contains various granular structures. Ciliary and microvillar membranes appear thicker with cryo-ultramicrotomy than when the sections are prepared with conventional techniques. The cores of the axonemal microtubules in olfactory as well as in respiratory cilia are darkly stained with this technique. Vesicles present inside the nerve endings are also darkly stained. Dimensions and some other numerical values of interest in olfaction are presented.

Bovine Olfactory and Nasal Respiratory Epithelium Surfaces High-Voltage and Scanning Electron Microscopy, and Cryo-Ultramicrotomy

The most frequently occurring cell types in the pars distalis of the pituitary gland of the rainbow trout, (i) the lactotropic, (ii) the gonadotropic, and (iii) the somatotropic cells, were identified in cryosections. Their morphological characteristics were compared with those of Epon-embedded material. Cell location, cell form, position of the nucleus, arrangement of rough endoplasmic reticulum and sizes of secretory granules proved to be useful parameters for identification. The size distribution of secretory granules of corresponding cells in cryosections and Epon sections proved to be similar. Additionally, both the immunoferritin and the unlabeled antibody enzyme method were applied for the immunocytochemical labeling of gonadotropic hormone-producing cells in cryosections. Anti-salmon-GTH as well as anti-carp-GTH serum showed the presence of GTH in both the smaller and the larger granules of the classical GTH cells, but also produced a reaction in TSH cells. Labelling of TSH cells was absent when using anti-beta-carp-GTH. Specificity of the reaction depended upon the degree of dilution of the anti-GTH serum. Results with dilutions of 1:4,000 and 1:8,000 in the unlabeled antibody enzyme method, and of 1:8,000 up to 1:32,000 in the immunoferritin technique were optimal. Acid phosphatase activity in the smaller granules was demonstrated by enzyme cytochemistry in Epon sections. The relationship of the presence of hormone in these granules is discussed. The high sensitivity of the immunocytochemical labeling procedure is discussed with respect to cryo-ultramicrotomy.

Immunocytochemistry of gonadotropic cells and identification of cell types in ultrathin cryosections of the pituitary of the rainbow trout, Salmo gairdneri.

J. L. M. Leunissen

Papers

Bovine Olfactory and Nasal Respiratory Epithelium Surfaces High-Voltage and Scanning Electron Microscopy, and Cryo-Ultramicrotomy

Immunocytochemistry of gonadotropic cells and identification of cell types in ultrathin cryosections of the pituitary of the rainbow trout, Salmo gairdneri.