A study of the olfactory pathway

A study of the olfactory bulb in certain mammals by light and electron microscopy of normal and experimental material. The neurons of the glomerular layer of the rat olfactory bulb have been studied using Nissl staining and Golgi-Kopsch impregnation in light microscopy to define the size, shape, and morphological features of individual cell somata, dendrites and axons; these have been correlated with electron microscopic material in which fine structural characteristics were also noted for each cell type, particularly synaptic specialisations. Three neuron types are described: the external tufted and periglomerular cells of classical microscopy, and additional, superficial short-axon cells; a description of the glomerular arborisations of the mitral and deep tufted cells is also included. The tufted and mitral cells snow large, non-spiny glomerular dendritic arborisations, having terminal varicosities, the external tufted cells being more limited in their branching than the deeper cells. External tufted cells have large somata and abundant cytoplasm containing stacks of Nissl material; their main dendrites are characterised by pale cytoplasm and a regular array of neurotubules. Reciprocal dendro-dendritic and somato-dendritic synapses are commonly found, the tufted/mitral cells containing spherical vesicles and contacting by means of asymmetrical membrane thickenings; the other profile involved is a gemmule containing large flattened vesicles and associated with a symmetrical thickening. The periglomerular cells are smaller, with a spiny glomerular arborisation, as well as some other dendrites; all the dendrites of these cells tend to be of irregular outline. They hare a dark nucleus and very little somatic cytoplasm; somatic and dendritic appendages are common and often contain large flattened vesicles. Synapses orientated from the dendritic shaft or gemmule also show such resides, invariably associated with symmetrical thickenings. The superficial short-axon cells are characterised by the entirely periglomerular distribution of their dendrites, which are varicose and rarely branch. Of intermediate soma dimensions, but containing dispersed Nissl material, these cells and their stem dendrites show no synaptic specialisations directed from them. Features of axon initial segments, axo-somatic and axo-dendritic synapses are also described for each call, as well as some unusual glial relationships. Reasons are adduced for relating the superficial short-axon cell to the axon terminal type containing small flattened resides, as well as for considering that the external tufted and periglomerular cells show the same synaptic specialisations at their axon terminals as at their dendritic and somatic synapses. The neuropil of the glomerular layer (consisting of glomeruli and periglomerular regions) of the rat olfactory bulb was studied with the electron microscope with a view to elucidating the type of processes involved andndash; dendrites, appendages and axons andndash; their cellular identity, and the synaptic relationships they establish. The problems encountered in defining these are considered and criteria based on neuron types and from examination of serial sections are put forward. The glomeruli are large structures containing many thousands of processes and are the sole site of termination of the olfactory receptor axons. The terminals of the latter are characteristically electron-dense, allowing identification in normal material; they run through the glomeruli making many synapses by means of spherical vesicles and asymmetrical thickenings on to all types of dendritic profile. The glomerular dendritic arborisations of mitral and tufted cells, which are indistinguishable from each other, start as large, fairly regular, pale profiles but become increasingly varicose as they branch and diminish in size. They regularly show groups of spherical vesicles, often in association with asymmetrical synaptic thickenings directed from the dendrite; these are typically associated with return reciprocal synapses of the symmetrical type from profiles containing large flattened vesicles. These latter profiles are those of the dendrites and gemmules of periglomerular cells; the dendrites are of irregular outline and give rise to many appendages, mostly gemmules making synaptic contact with mitral or tufted cell dendrites. A small number of pale axon terminals containing either small or large flattened vesicles, derived from short-axon and periglomerular cells respectively, synapse with symmetrical thickenings on to the periglomerular cell dendritic processes. Close associations of particular types of axo-dendritic and dendro-dendritic synapses on interconnecting processes, termed synaptic patterns, are described and their significance considered. The nature of the glomerular interactions is discussed and then placed in the context of other, smaller glomeruli in the central nervous system; certain common principles of glomeruli are suggested. The periglomerular region of the olfactory bulb, apart from containing the somata and item dendrites of the cells contributing to the glomeruli, is the sole region of distribution of the periglomerular cell thin dendrites and the short-axon cell dendrites. It is also the major site of termination of all axons to the glomerular layer except the olfactory axons andndash; i.e. tufted cell collaterals, periglomerular cell and short-axon cell axons and centrifugal fibres. Its characteristic neuropil has been studied with the electron microscope to define the cells of origin of the types of neuronal process and their synaptic relationships. Three types of axon terminals have been found: those with spherical, large flattened and small flattened vesicles, which are deduced to derive from tufted cell collaterals or centrifugal fibres, periglomerular cell and short axon-cell axons respectively; those with spherical vesicles are consistently associated with asymmetrical membrane thickenings and those with either type of flattened vesicles with symmetrical thickenings. The thin periglomerular cell dendrites are very irregular and often have a somewhat dense cytoplasm rich in ribosomes; they may become extended into very attenuated glia-like sheets that surround the mitral or tufted cell stem dendrites, from which they may receive synaptic contacts. Such dendrites also receive some synapses from all three types of axon in the periglomerular region. The short-axon cell dendrites are thick and varicose and show no sign of synaptic specialisation orientated from them; they have few spines but receive many asymmetrical type synapses on their shafts. Both axon terminal types synapsing with symmetrical thickenings are also found on the shafts. The evidence obtained from the study of normal material is summarised and the various cellular roles considered. In the light of observations on the olfactory bulb, it is suggested that dendrites may be divided into two major classes: those that only receive synapses (Class A) and those that make synaptic contacts as well as receiving them (Class B), After a summary of the findings from normal material and a brief description of an unusual feature of the olfactory bulb in the monkey, a group of experimental studies on this site in the rat and rabbit are described. The degeneration of axon terminals in the glomerular layer of the rat olfactory bulb has been studied, concentrating particularly on the sequence of degeneration in the olfactory nerve terminals and the long-term events in the degeneration process in several terminal types. Olfactory nerve terminal degeneration is divided into five stages, representing parts of the sequential changes taking place in the terminal after fibre section. The main features in the sequence are as follows: I. Swelling of the terminal and its vesicles. II. Initial shrinkage of the terminal, while vesicles remain swollen and some are distorted. III. Further shrinkage and darkening of the terminal, distortion of many of the vesicles and some mitochondrial swelling. IV. Extreme shrinkage of the terminal and loss of cytoplasmic detail; synaptic contact still intact or partially separated. V. Disappearance of the presynaptic terminal and persistence of the post-synaptic thickening. The validity of the observation of the persistence of post-synaptic membrane thickenings is considered and evidence adduced in its favour, both qualitative and quantitative. Characteristics of the newly apposed profiles are described, including cisternae and alveolate vesicles; the extra-cellular material of the synaptic cleft is considered in the light of its association with persisting thickenings. The relevance of these associated structures is discussed in terms of function and development. Spontaneous degeneration of olfactory nerves and the degeneration of the vomeronasal nerves in the accessory olfactory bulb are described, as well as Stage V in the degeneration of other terminal types in the glomeruli, following various lesions; persistence of post-synaptic thickenings after the degeneration of terminals showing symmetrical membrane thickenings is included. Observations on the persistence of post-synaptic thickenings in various sites in the central and peripheral nervous systems are reviewed in the context of the present study; the problems arising out of the morphological identification of apposition or reinnervation are discussed. A study of the transneuronal degenerative changes in the cells of the olfactory bulb of the rabbit and rat following removal of the olfactory mucosa has also been made. The principal features of this type of change are cell shrinkage, a concentration of cytoplasmic and nuclear constituents, an alteration in the organisation of nucleic acid material, and a swelling of membrane-bound systems; some elements of cellular organisation appear unchanged, including synaptic specialisations. The changes are described for each part of the neuron andndash; soma, dendrite, axon and terminal andndash; as well as the variants found in different cells. The degree of morphological alteration appears to be related to the extent of denervation for any individual cell and does not seem to be sentential. These changes are related to findings on altered impregnation properties of the sane cells with the Golgi technique and are discussed in the context of the functional alteration produced by deafferentation. A new interpretation of some 'dark neurons' is also suggested on the basis of those findings. The termination of the centrifugal fibres running in the lateral olfactory tract to the glomerular layer of the rat olfactory bulb has been determined with the electron microscope; this has been done with material perfused at various times after section of the lateral olfactory tract, as well as after a combination of this lesion with the long-term degeneration of olfactory nerves. The axon terminals are sparse at the glomerular level, but undergo typical degenerative changes; they are distributed solely in the periglomerular region and intermediate zone. The most common post-synaptic profiles are the processes of periglomerular cellS, but a few centrifugal fibres terminate on short-axon, tufted and mitral cell dendrites. Evidence is produced to suggest that the anterior olfactory nucleus does net project as far as the glomerular layer. The findings are discussed in relation to previous studies with normal material and silver degeneration methods on similar experimental material; the functional implications of the Centrifugal pathways in the bulb are briefly discussed. Lesions have been made in the superficial layers of the dorsal surface of the rat olfactory bulb; the resulting axonal degeneration haft been studied with light and electron microscopy, in order to determine the mode and extent of interconnection of the intrinsic interneurons of the glomerular layer. Although the lesions necessarily involve extrinsic pathways, the axonal distribution of each of the intrinsic neurons may be deduced from this evidence and a knowledge of normal and experimental material. Tufted cell collaterals extend for 6andndash;12 glomeruli, periglomerular cell axons for 4andndash;5 glomeruli and superficial short-axon cell axons for 2andndash;3 glomeruli; these figures correspond well with observations of Golgi-impregnated material. The experimental approach to the study of intrinsic connections and its applicability to other sites is considered, and the results are discussed from the point of view of neural coding and odour specificity. In the last chapter concerned with results from this study, mention is made of the technique of combining Golgi-impregnation for light microscopy and electron microscopy of the same cell; its practical application is considered and examples are given from the study of a middle tufted cell by this technique. Finally, the general discussion brings together the results on the organisation of the olfactory bulb, particularly the glomerular layer. The discussion centres around two principal issues arising from these studies: the structure and function of individual neurons and their specialisations on the one hand, and the principles of neuronal organisation on the other; where possible, these are related to findings and function in other parts of the central nervous system, as well as to the function of the olfactory bulb in the olfactory pathway.