THE FINE STRUCTURE OF THE LATERAL VESTIBULAR NUCLEUS IN THE RAT : I. Neurons and Neuroglial Cells.

The lateral vestibular nucleus consists of multipolar isodendritic neurons of various sizes The distal segments of some dendrites display broad expansions packed with slender mitochondria and glycogen particles. These distinctive formations are interpreted as being growing tips of dendrites, and the suggestion is advanced that they are manifestations of architectonic plasticity in the mature central nervous system. Unlike large neurons elsewhere, the giant cells (Deiters) contain small Nissl bodies interconnected in a dense mesh-work. The Nissl substance is characterized by randomly arranged cisterns of the endoplasmic reticulum and by a high proportion of free ribosomes. Whether attached or free, ribosomes usually cluster in groups of four to six, and larger polysomal arrays are rare. Free ribosomal clusters also occur in the axon hillock and the initial segment. The neuronal perikarya contain distinctive inclusions consisting of a ball of neurofilaments enveloped by a complex honeycombed membrane. The failure of these fibrillary inclusions to stain with silver suggests that the putative argyrophilia of neurofilaments may reside in an inconstant matrix surrounding them. Giant cells of Deiters are in intimate contact with two kinds of cellular elements-astroglial processes and synaptic terminals. Oligodendroglial cells are only rarely satellites of giant cells; in contrast, they are frequently satellites of small and medium-sized cells.

The axon hillock and the initial segment.

Axon hillocks and initial segments have been recognized and studied in electron micrographs of a wide variety of neurons. In all multipolar neurons the fine structure of the initial segment has the same pattern, whether or not the axon is ensheathed in myelin. The internal structure of the initial segment is characterized by three special features: (a) a dense layer of finely granular material undercoating the plasma membrane, (b) scattered clusters of ribosomes, and (c) fascicles of microtubules. A similar undercoating occurs beneath the plasma membrane of myelinated axons at nodes of Ranvier. The ribosomes are not organized into Nissl bodies and are too sparsely distributed to produce basophilia. They vanish at the end of the initial segment. Fascicles of microtubules occur only in the axon hillock and initial segment and nowhere else in the neuron. Therefore, they are the principal identifying mark. Some speculations are presented on the relation between these special structural features and the special function of the initial segment.

Neuronal perikarya with dispersed, single ribosomes in the visual cortex of Macaca mulatta.

Numerous small and medium-sized neuronal perikarya in layers III and IV of the visual cortex display an unusual pattern of ribosomal distribution. Instead of being aggregated in clusters, spirals, rows, and other regular polysomal configurations, the ribosomes, whether free or attached to the endoplasmic reticulum, are randomly dispersed, with no discernible pattern. The endoplasmic reticulum in such cells is reduced to a few (perhaps only one) meandering, broad cisternae, which delimit broad fields of cytoplasmic matrix occupied almost solely by scattered, single ribosomes. The Golgi apparatus is elaborate. Mitochondria are either small and numerous or large and infrequent. The other organelles, including the nucleus and nucleolus, are not remarkable. Axonal terminals synapse in the normal fashion on the surfaces of these cells and their dendrites. Associated with these cells are more numerous intermediate cells in which a few to many polysomal clusters can be found. It is proposed that the neurons with dispersed, single ribosomes are inactive in protein synthesis and that the suspension of such an important metabolic activity is probably temporary. Thus, these cells are considered to be part of a population undergoing cyclic fluctuations in the intensity of protein synthesis that should be correlated with their specific neural behavior.

Galanin immunoreactivity in hypothalamic neurons: further evidence for multiple chemical messengers in the tuberomammillary nucleus.

By using a specific antibody against the 29 amino-acid peptide galanin (Gal) with light and electron microscopic immunocytochemistry, we have studied the distribution of Gal immunoreactivity in the posterior hypothalamic magnocellular neurons of the rat. In colchicine-treated rats, a large number of Gal-immunoreactive cells were identified within all subdivisions of the tuberomammillary nucleus. The majority of these cells are large multipolar or fusiform neurons, with long, sparsely branching dendrites. A small number project to the ventral hippocampus, as shown by experiments with the retrograde tracing of Fast Blue. Ultrastructural examination of the Gal-immunoreactive cells confirms their indentity as magnocellular neurons, with dense deposits of immunoreaction product, particularly in small ribosomal arrays and in large, dense-cored vesicles. Axosomatic synapses occur on these neurons. The axonal boutons synapse with asymmetric and symmetric junctions and contain small synaptic vesicles as well as numerous large, dense-cored vesicles, which display Gal immunoreactivity. Sequential staining of thin, alternate sections with antibodies against Gal and L-histidine decarboxylase (HDCase; EC 4.1.1.22) showed colocalization of Galand HDCase-immunoreactivities in a majority of tuberomammilary neurons. The finding of Gal immunoreactivity within histamine-producing neurons of the tuberomammillary nucleus adds to the multiplicity of potential neuronal messengers utilized by these cells.

Distribution of tyrosine-hydroxylase-immunoreactive neurons in the hypothalamus of rats.

The distribution and morphology of cells containing tyrosine hydroxylase (TH) immunoreactivity in the hypothalamus of rats were studied by using a modified immunoperoxidase technique. The TH cell system is more complexly organized than was previously thought. On the basis of their clustering patterns, hypothalamic TH neurons could be subdivided into two groups: dorsal and ventral. The ventral group consists of a prominent aggregate of cells located in the caudal part of the arcuate nucleus. From here, cells extend around the caudal part of the ventromedial and dorsomedial nuclei and the base of the diencephalon. Tyrosine hydroxylase-positive cells are present throughout the arcuate nucleus, except in its ventromedial part. Anteriorly, immunoreactive cells appear in the suprachiasmatic and supraoptic nuclei, in the retrochiasmatic area, and in the ventral part of the anterior hypothalamic nucleus. The dorsal group has its main concentration of cells in the medial part of the zona incerta, from which two clusters of cells, one medial and one lateral, extend rostralward. The medial group comprises cells in the medial part of the dorsomedial, paraventricular, and anterior hypothalamic nuclei. These cells adjoin the periventricular cells. The lateral group of cells emanating from the zona incerta occupies the lateral part of the dorsomedial and anterior hypothalamic nuclei and the dorsal hypothalamic area. The dorsal and ventral TH cell groups are in continuity medially in the periventricular layer, and laterally through the cells that surround the ventromedial nucleus. Although the cells vary widely in size, shape, and dendritic arborization pattern, there are two main cell types. Small (21 X 11 microns), round to fusiform cells, with two or three dendrites arborizing simply, were frequently seen in the arcuate, suprachiasmatic, periventricular, supramammillary nuclei and at the borders of the ventromedial nucleus. The other cell type is larger (40 X 15 microns) and multipolar, with three to five frequently branching dendrites. The dendritic field is large and the cells are intensely TH-immunoreactive. Although the larger cells occur occasionally in every hypothalamic nucleus, their principal locations are in the dorsal parts of the dorsomedial, posterior hypothalamic nuclei and the dorsal and lateral parts of the zona incerta, and in the areas dorsal and medial to the mammillothalamic tract at caudal hypothalamic levels. In this paper we give a detailed description of TH-immunoreactive fibers and terminals in the hypothalamus and a comparison with previous studies of catecholamine cells in the hypothalamus.

Autoradiographic experiments to examine uptake, anterograde and retrograde transport of tritiated serotonin in the mammalian brain.

In an attempt to define the potential application of neurotransmitter-specific transport as a method of tracing fiber connections, we have examined the uptake and subsequent ortho- and retrograde transport of tritium-labeled serotonin (3H-5HT) in the cerebellum-raphe pallidus system. Injection of various concentrations of 3H-5HT followed by different post-injection survival times revealed different labeling patterns in the injected sites and different patterns of transport. The most striking feature is that nonseroitonin neurons as well as serotonin cells were able to take up and transport the tritium label in both otho- and retrograde fashion. The non-sertonin-specific nature of this uptake and transport is more obvious at higher concentrations of 3H-5HT (more than 9 X 10(-5) M), with longer survival times and following pretreatment with monoamine oxidase inhibitors. At a concentration of 9 X 10(-6) M 3H-5HT, only specific uptake seems to take place as evidenced by label in known serotinin cells and fiber systems; however, it was impossible to detect by autoradiography any ortho- or retrograde transport at this low concentration. Non-specific uptake and transport were observed following injection into the vestibular nuclei and oculomotor complex. This suggests that non-specific uptake and the transport of 3H-5HT or metabolites may also occur in other regions of the central nervous system.

Cytoplasmic inclusions of neurons in the monkey visual cortex (area 19).

Several unusual neuronal inclusions were found in certain cells of the rhesus monkey visual cortex (Area 19): 1. Filamentous bodies, present in the small stellate cells of layer IV, globoid, 0.3-0.6 mum in diameter, consisting of fine 50 A filaments in a hexagonal meshwork. These are often associated with the labyrinthine bodies. 2. Labyrinthine bodies found exclusively in the small stellate cells of layer IV, including certain neurons with dispersed ribosomes. These are 0.4-0.7 mum in diameter and consist of 900 A wide tubes which interconnect with one another. The walls of these tubes are continuous and made up of a sheet or honeycomb lacework of small hexagonal 150 A subunits. 3. This inclusion, an aggregate 0.3-0.7 mum in size, consists of small membrane-bounded vesicles with a single dense granule associated with other non-membrane bound small dense droplets. The inclusions are always associated with the maturing face of the Golgi complex of certain layer IV pyramidal cells; as such, they may be an unusual product of the Golgi apparatus. These observations were confirmed by examination of stereo pairs of electron micrographs. Speculations are made with regard to possible functions for these inclusions.

Gamma-aminobutyric acid pathways in the cerebellum studied by retrograde and anterograde transport of glutamic acid decarboxylase antibody after in vivo injections.

Injections of characterized antibody against glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of gamma-aminobutyric acid (GABA), were made into the cerebellum. Small cortical injections of anti-GAD antibody produced labeled stellate, basket, Purkinje, and Golgi cells and their processes at the injection site. Anterograde transport of GAD antigen-antibody complexes in Purkinje cell axons caused intense labelling of terminals in deep cerebellar and several vestibular nuclei. Small groups of mossy fiber rosettes labeled and produced retrograde labeling and GAD immunoreactivity in a small number of pleomorphic neurons in the deep cerebellar nuclei. Injections into the dentate nucleus produced retrograde labeling in Purkinje cell bodies and anterograde label in a small number of mossy fiber rosettes. All projections conformed to previously reported topographic distributions of corticonuclear and nucleocortical cerebellar pathways. These findings confirm the GABA content of most Purkinje cell-deep nuclei connections and provide new evidence for a GABA component in part of the nucleocortical pathway in the cerebellum. Immunocytochemical controls for specificity were conducted by injections of preimmune rabbit serum as a substitute for GAD antibody. Only nonspecific labeling was obtained in these cases. Colchicine caused a cumulative enhancement of GAD immunoreactivity in all cases. The present studies indicate that the method of in vivo antibody injections can be utilized to study chemically specific connections in nervous tissue.

The nucleus paragigantocellularis lateralis in the rat. Conformation and cytology.

The nucleus paragigantocellularis lateralis (PGCL) is located in the ventral portion of the rostral medulla. Serial sections of the rat brainstem were examined in the three cardinal planes and the boundaries of the PGCL were determined. In order to visualize the shape and extent of the nucleus, a three-dimensional reconstruction of the PGCL was made from a series of coronal sections. Measurements of neuronal areas, lengths, and widths indicate that a number of neuronal types are present. Small neurons measure less than 150 micron2 and large neurons greater than 250 micron2. Some neuronal types are distributed preferentially throughout the PGCL, and on this basis the nucleus may be divided into caudal and rostral subgroups. Most large neurons (greater than 250 micron2) are found in the caudal portion. Certain neurons contain intranuclear rods, and these neurons are often disposed in small groups, especially common the caudal PGCL. Two morphologically distinct neuronal types incorporate 3H-serotonin when this marker is infused into the ventricular system; the other neurons not marked by this method probably contain other, different transmitters. On the basis of neuronal measurements and staining qualities, it is ascertained that the PGCL is a parvocellular reticular nucleus characterized by many neuronal types.

The nucleus paragigantocellularis lateralis in the rat. Demonstration of afferents by the retrograde transport of horseradish peroxidase.

Injections of horseradish peroxidase (HRP) were placed in the middle or caudal portion of the nucleus paragigantocellularis lateralis (PGCL) and 24 h later the entire spinal cord and brain were processed and examined for labeled neurons. Spinal afferents arise from all levels of the cord. Rexed's lamination scheme was adapted to the spinal cord of the rat and labelled neurons were localized to laminae IV, V, VII, VIII and X mainly on the side contralateral to the injection. At cervical levels, labeled neurons were consistently found bilaterally. The medial reticular nuclei of the medulla and pons contained HRP-labelled perikarya, which were concentrated most heavily in the nuclei reticularis medullae oblongatae ventralis, gigantocellularis, and pontis caudalis predominantly ipsilateral to the injection. The medial vestibular nucleus was consistently labeled. HRP-labeled perikarya were found bilaterally within the commissural portion and in the medial part of the nucleus of the solitary tract on the side of the injection. The rostral portion of the PGCL receives afferents from some secondary auditory nuclei: the ipsilateral inferior colliculus and the posterior ventral cochlear nucleus bilaterally. Thus, the rostral PGCL may be involved in auditory feedback loops. The caudal raphe nuclei are a major source of afferents to the caudal PGCL. The lateral hypothalamic area, paraventricular nucleus, and zona incerta also contain labeled neurons when injections are centered in the caudal portion of the nucleus.

Neurofilaments and microtubules in anterior horn cells of the rat.

Dendrites arising from the larger nerve cells in the anterior horn contain fascicles of neurofilaments in addition to the usual dendritic components From a comparison of neurofilaments and microtubules, and their respective subunits it is concluded that direct interconversion between them is improbable In transverse section the wall of the neurofilament is composed of 4-6 circular densities about 30 A in diameter Short spokelike side arms project from the circular densities into the surrounding cottony matrix in which the filaments are embedded.

The general architecture of sensory neuroepithelia.

All neuroepithelia are sheets of cells lining an internal or external surface of the body and resting on a basement membrane. They consist of at least two kinds of cell, receptor cells and sustentacular (supporting) cells. Some contain undifferentiated precursor cells and senescent or degenerating cells. The potential for plasticity and regeneration in different sensory neuroepithelia varies widely according to their origins and structure in any individual animal and according to the species in which they occur. Four sensory neuroepithelia are described as examples of the range of construction, complexity, and life history.

Immunocytochemical localization of cyclic GMP: light and electron microscope evidence for involvement of neuroglia.

Guanosine 3',5'-cyclic monophosphate (cGMP) immunoreactivity in the rat's cerebellum was studied with light and electron microscopy by the indirect fluorescence method and the peroxidase-antiperoxidase method. Labeled cells included neuroglial cells in the cerebellar cortex, white matter, and deep nuclei; some stellate and basket cells in the cortex; and some large neurons in the deep nuclei. No evidence was found for sagittal microzonation in the cGMP distribution. In the labeled cells, cGMP immunoreactive sites were localized to surface membranes, organelles, and the cytoplasmic matrix. Specificity was indicated by the same pattern of labeling after treatment with cGMP immunoglobulin that had been adsorbed with adenosine 3',5'-cyclic monophosphate (cAMP) and by the failure to label after treatment with normal rabbit sera or with cGMP immunoglobulin that had been adsorbed with 1 mM cGMP. Cerebella treated with cAMP antisera, however, showed immunoreactivity in Purkinje cells, granule cells, and Golgi cells in addition to neuroglia in cortex and deep nuclei. Sequential norepinephrine and glutamate superfusions generally intensified cGMP immunoreactivity, not only in neuroglial cells but also in the background. Under these conditions some Purkinje cells and some granule cells were also labeled. Increased cGMP immunoreactivity was also obtained by treatment with harmaline, gamma-aminobutyric acid and aminooxyacetic acid, muscimol, gamma-aminobutyric acid, or apomorphine in order of decreasing effectiveness. Serotonin and colchicine produced no detectable increase of cGMP immunoreactivity above normal, and diazepam and sodium pentobarbital decreased it. In these experiments, diethyl ether was preferable to sodium pentobarbital for anesthesia on account of the depressive action of the latter on cGMP immunoreactivity. Thus, drugs that increase cerebellar activity enhance cGMP levels, whereas those that decrease cerebellar activity decrease cGMP levels. However, it is not clear whether these fluctuations in cGMP levels are a direct consequence of neurotransmitter function or are sequelae to other related events. The present study suggests that some neurons and many neuroglial cells are the major sites of cGMP in the cerebellum.