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Notes: Abstract The inferior olive (IO) appears to be organized functionally in discrete subnuclei that receive transmitter-specific inputs. In particular, the IO receives a GABAergic input that is most densely concentrated in the β -nucleus. In this experiment, we examined the functional specificity of neurons in the β -nucleus of the IO of rabbits by recording their activity during natural vestibular and optokinetic stimulation. Rabbits were anesthetized and positioned in a triaxial servo- controlled rate table with the head fixed at the center of rotation. Contour-rich visual stimuli were rear-projected onto a 70 deg tangent screen and moved at constant velocities. Recording sites in the β -nucleus were verified by subsequent histological analysis of marking microlesions. Neurons in the β -nucleus responded to roll vestibular stimulation about the longitudinal axis. These neurons were excited when the rabbit was rolled onto the side which was contralateral to the recording site, and inhibited when the rabbit was rolled ipsilaterally. Thirty-eight of the 75 β -nucleus neurons that were responsive to roll vestibular stimulation also responded to static tilt, indicating an otolithic as well as a vertical semicircular canal origin of the vestibular input. The modulated activity of none of the neurons could be attributed to stimulation of the horizontal semicircular canals. All the recorded neurons were found in a region of the β -nucleus that was retrogradely labeled following HRP injections into the cerebellar nodulus. Using a “null point” technique, we found that there was a differential projection of information from the anterior and posterior semicircular canals onto to the β -nucleus. Stimulation of the ipsilateral anterior-contralateral posterior semicircular canals modulates activity of the neurons in the caudal 500 μm of the β -nucleus. Stimulation of the ipsilateral posterior-contralateral anterior semicircular canals modulates activity of neurons located more rostrally. β -nucleus neurons and the olivocerebellar circuits in which they participate may constitute an important pathway for the control and adaptive modification of postural reflexes.

Notes: Summary Young and adult cats were operated upon and a number of the vermal cerebellar folia were either transected with a vertical incision or isolated by a horizontal cut. In the proximity of the lesion, Purkinje cell bodies and their dendritic trees became stainable with the Fink-Heimer method. Electron microscopy of the silver stained sections show that the argyrophilic Purkinje neurons undergo an electron dense type of degeneration. Stellate cell dendrites adjacent to the degenerating Purkinje trees are normal, suggesting that the cause of cell death is axotomy close to the perikaryon rather than direct injury. The retrograde Purkinje cell degeneration is fulminant since it is evident 6 hours after the lesion. In Fink-Heimer stained sections the entire dendritic tree is impregnated 1–3 days after the lesion. 4–10 days post-operatively, the flattened dendritic tree becomes fragmented and is partially phagocytized. The silver stained arborizations are approximately 280 μ in width and have an uneven thickness (8–16 μ). In longitudinal and horizontal silver stained sections of lesioned cerebellar folia, uninterrupted fields of degenerating Purkinje cell arborizations can be seen, suggesting that the arborizations overlap. The overlap was demonstrated in electron micrographs of single degenerating arborizations surrounded by normal dendritic trees. The degree of overlap varies with the thickness of the arborization and is in the order of 1–2 μ. This approach indicates that each Purkinje tree occupies an exclusive sheet of molecular layer 8 μ thick and may overlap for as much as 2 μ on each side with neighboring trees. The average thickness of the Purkinje tree is approximately 12 μ. Portions of this work performed by S. Brand are in partial fulfillment of requirements for the degree of Doctor of Philosophy.

Notes: Summary Experimental light and electron microscopic studies were carried out to determine the length of parallel fibers in the cat cerebellar cortex. Using a fine surgical knife, vermal and hemispheral folia were cut perpendicular to their long axis. The animals were sacrificed 1–10 days after the operation. Sections of the transected folia were then stained with a Fink-Heimer procedure. The resulting degeneration appeared as fine dots that extended lateral to the lesion, as predictable from the course of the parallel fibers. Densitometer readings indicate that the density of degeneration declines gradually lateral to the lesion. The specificity of the silver impregnation was checked by processing silver stained sections for electron microscopy. This confirmed the location of the silver precipitate on degenerating parallel fibers. The pattern of parallel fiber degeneration in the molecular layer has a trapezoidal configuration centered on the lesion. The shorter parallel fibers are located at the base of the molecular layer and extend for 5 mm. The parallel fibers become progressively longer as they approach the pial surface where they attain a maximum length of 7 mm. Our studies suggest that in folia longer than 7 mm parallel fibers are 6 mm long on the average. In addition, it was determined on Golgi sections that the average center-to-center distance between en passant boutons of individual parallel fibers is 5.2 μm. The data indicate that an average parallel fiber, 6 mm long, forming approximately 1100 boutons, may synapse with each Purkinje dendritic tree it traverses.

Notes: Summary The cerebellum develops from a germinal zone at the rhombic lip of the metencephalon. This region, like the telencephalic vesicle which gives rise to the cerebral cortex, presumably consists of germinative units showing a rather repetitive neurogenetic pattern. In all cerebellar folia, cortical neurons, and especially the Purkinje cells, express highly stereotyped phenotypes, although some variations in their chemical make-up have been uncovered with monoclonal antibodies. Here, we demonstrate for the first time that three independent murine mutations,Lurcher, Purkinje cell degeneration andstaggerer, which result in the postnatal degeneration of Purkinje cells, also cause the elimination of cartwheel cells of the dorsal cochlear nucleus. The cerebellar granule cell mutation,weaver, which spares most Purkinje cells in the lateral cerebellum, also spares cartwheel cells. These data support the notion that the cerebellar germinative zone extends to the caudal portion of the rhombic lip, which gives rise to the dorsal cochlear nucleus.

Notes: Summary Ciliary and choroid neurons of the avian ciliary ganglion innervate different targets in the eye bulb. By light microscopic immunocytochemistry, somatostatin (SOM) has been localized to a subset of ganglionic neurons believed to be, for the most part, choroid neurons. Although several studies have been published on the physiology, afferent and efferent innervation, and response to experimental injury of this population of cells, their morphological features are still unclear. This has led us to perform a fine structural and immunocytochemical study on the ciliary ganglia of adult chickens and quails to provide the first thorough characterization of the choroid neurons and to analyze whether or not they can be unequivocally identified by expression of SOM. Here, we show that standard and immuno-electron microscopy provide firm criteria for the distinction of ciliary and choroid neurons, whose populations overlap in cell size and territory of distribution. The satellite cell sheaths form compact myelin lamellae around ciliary neurons and flattened processes around choroid neurons. Moreover, ciliary neurons are innervated by a larger number of boutons than choroid neurons. Chicken ciliary neurons are invested by boutons only over one pole of the cell body, while their quail counterparts have an almost complete shell of presynaptic boutons over the entire cell body. Ciliary neurons form mixed synaptic junctions (chemical and electrical), while choroid neurons form only chemical synapses. Crest synapses are present in ciliary neurons of both species. Nematosomes occur in both ciliary and choroid neurons. Choroid neurons contain a larger complement of large dense core vesicles than ciliary neurons and their Golgi apparatuses are more prominent. In the light microscope, somatostatin-immunostaining appears noticeably different in the two species: mostly granular in the chicken and skein-shaped in the quail. Immuno-electron microscopy reveals that somatostatin-like immunoreactivity is localized to Golgi apparatus and large dense core vesicles. Somatostatin is expressed by all the choroid neurons, but not by the ciliary neurons. This neuropeptide is, therefore, a true cell population marker.

Notes: Summary A subset of cerebellar mossy fibres is rich in choline acetyltransferase, the rate-limiting enzyme for the synthesis of acetylcholine. These choline acetyltransferase-positive mossy fibres are concentrated in the vestibulocerebellum and originate predominantly from the medial vestibular nucleus. The granular layer of the vestibulocerebellum is also enriched in unipolar brush cells, an unusual type of small neuron that form giant synapses with mossy fibres. In this immunocytochemical light and electron microscopic study, we explored whether choline acetyltransferase-positive mossy fibres innervate unipolar brush cells of the rat cerebellum. We utilized monoclonal antibodies to rat choline acetyltransferase of proven specificity, and immunoperoxidase procedures with 3,3′-diaminobenzidine tetrahydrochloride as the chromogen. A high density of choline acetyltransferase-positive fibres occurred in the nodulus and ventral uvula, where they showed an uneven, zonal distribution. Immunostained mossy fibre rosettes contained high densities of round synaptic vesicles and mitochondria. They formed asymmetric synaptic junctions with dendritic profiles of both granule cells and unipolar brush cells. The synaptic contacts between choline acetyltransferase-immunoreactive mossy fibres and unipolar brush cells were very extensive, and did not differ from synapses of choline acetyltransferase-negative mossy fibres with unipolar brush cells. Analysis of a total area of 1.25 mm2 of the nodulus from three rats revealed that 14.2% of choline acetyltransferase-immunoreactive mossy fibre rosettes formed synapses with unipolar brush cells profiles. Choline acetyltransferase-positive rosettes accounted for 21.7% of the rosettes forming synapses with unipolar brush cells. Thus, the present data demonstrate that unipolar brush cells are innervated by a heterogeneous population of mossy fibres, and that some unipolar brush cells receive cholinergic synaptic input from the medial vestibular nucleus. The ultrastructure of these synapses is compatible with the possibility that choline acetyltransferase-positive mossy fibres co-release acetylcholine and glutamate. As the granular layer of the vestibulocer-ebellum contains nicotinic binding sites, the choline acetyltransferase-positive mossy fibres may be a model for studying nicotinic neurotransmission in the CNS.

Notes: Summary Stellate neurons in the outer two layers of the rat dorsal cochlear nucleus (DCN) were studied by the Golgi-EM method. Stellate cell bodies are usually spherical or ovoidal and range from 9 μm to 14 μm in mean diameter. The smallest cells are situated underneath the ependymal layer and the largest cells in layer 2. Primary dendrites are short, thin and smooth and arise abruptly from the perikaryon, without a tapering main stem. Meandering secondary and tertiary dendrites extend in all directions, carry few pleomorphic spines lacking a spine apparatus and often show artifactual beading. The axons are impregnated only for a short distance (10–45 μm). The nucleus is indented, the nucleolus varies in position, and the chromatin, evenly dispersed in the centre, forms small clumps along the nuclear envelope. The cytoplasm is rich in free polyribosomes and contains scattered cisterns of granular endoplasmic reticulum. Varicosities of thin fibres, containing round synaptic vesicles, form asymmetric synapses on perikarya, dendritic shafts and spines of stellate cells. Such fibres run parallel to the long axis of the DCN or are oriented radially and are interpreted as axons of cochlear granule cells. Two kinds of bouton containing pleomorphic vesicles, one kind electron lucent and the other electron dense, form symmetric synapses on perikarya and dendritic shafts of stellate cells. The lucent boutons occur more frequently than the dense boutons, especially on the distal dendritic branches. The boutons with pleomorphic vesicles presumably represent terminals of local circuit neurons, probably the stellate and cartwheel cells. In addition, stellate cells show numerous dendro-somatic and dendro-dendritic appositions characterized by gap junctions and puncta adhaerentia. Most of the dendrites involved in these appositions resemble stellate cell dendrites and it is concluded that DCN stellate cells are coupled electrotonically with one another. The axons of stellate cells acquire a thin myelin sheath. Since the Golgi impregnation did not stain axons of stellate cells past this point, we were unable to demonstrate the synaptic targets of stellate cells.

Notes: Summary During antibody screening on sections of rat cerebellum, we noticed a group of small neurons which exhibited unusual staining properties. They were robustly immunopositive for the high molecular weight neurofilament protein, moderately immunostained with antibodies to the low molecular weight neurofilament protein and α-internexin, but only faintly immunoreactive (in PAP sections) or essentially immunonegative (in immunofluorescent sections) with all members of a panel of antibodies directed against the middle molecular weight neurofilament protein. Since neurons generally react equally well with phosphate-independent, (antibodies to) low, middle and high molecular weight neurofilament protein, we conclude that middle molecular weight neurofilament protein is present in these cells in an unusually low relative amount. These cells are found in the granular layer and appear concentrated in the flocculus, ventral paraflocculus, and vermis, particularly in the ventral uvula and nodulus (lobules IXd and X). Previous studies performed by Hockfield defined a population of neurons of similar appearance and distribution using the monoclonal antibody Rat-302, which recognized an uncharacterized 160 kDa protein. We show here that the cells described by Hockfield are identical to those we have found and furthermore that the Rat-302 antibody specifically recognizes the dephosphorylated form of the lysine-serine-proline repeated sequences of high molecular weight neurofilament protein. These cells were studied by pre-embedding immunoelectron microscopy. The nucleus is deeply indented and shows little condensed chromatin. The cytoplasm contains scattered microtubules and a larger number of neurofilaments than expected in a small cell. There are numerous large dense core vesicles, an unusual organelle consisting of ringlet subunits, and relatively little granular endoplasmic reticulum. A thin axon and a single stout dendritic trunk emanate from the perikaryon. Although the cell body and the dendritic shaft may form either complex contacts with mossy fibres (resembling those previously termed en marron synapses) or simple symmetric synapses with small boutons containing pleomorphic vesicles, most of the synaptic relations are established on the shafts of brush-like branchlets that form at the tip of the dendrite and enter one or two glomeruli. Each branchlet forms an extraordinarily extensive asymmetric synapse with the mossy fibre rosette and the subsynaptic region shows a microfibrillar web connected to the postsynaptic density. In addition to other organelles, the branchlets contain numerous mitochondria and large dense core vesicles. Short, non-synaptic appendages with few cytoplasmic organelles emanated from the cell body, dendritic shaft and branchlets. The immunoreaction products of all neurofilament antibodies were similarly distributed within the small cells, and were absent from the granular reticulum, the Golgi apparatus, the appendages and the subsynaptic region. These high molecular weight neurofilament protein rich small cells correspond to the pale cells, the calretinin and secretogranin positive small cells and the unipolar brush neurons newly described with the Golgi method. Unlike the multipolar Golgi neurons, unipolar brush cells are not immunopositive for the inhibitory neurotransmitters GABA or glycine. The unusual concentration of secretogratin and two different types of calcium-binding protein (calretinin and high molecular weight neurofilament protein) along with the high content of mitochondria suggest that these cells subserve a function that requires an unusual degree of metabolic activity, perhaps as a result of their unusually rich synaptic connections.

Notes: Abstract Immunocytochemical staining with antibodies to the class III intermediate filament protein peripherin reveals discrete subpopulations of neurons and nerve fibres throughout the rat central nervous system. Some of these fibres enter the cerebellar granular and molecular layers. Here we use light and electron microscopic immunocytochemistry and confocal fluorescence microscopy to identify the peripherin positive fibres in the molecular layer of the cerebella of various mammals. 1) The peripherin positive fibres in the molecular layer have morphological attributes of climbing fibres, and peripherin positive fibres are also detected in the olivo-cerebellar tract. Furthermore peripherin positive neurons can be seen in the inferior olive, from which climbing fibres originate. (2 ) The peripherin positive molecular layer fibres rapidly degenerate in rats treated with 3-acetylpyridine (3-AP), a reagent which destroys neurons in the inferior olive, and the time course of degeneration of these mirrors that previously described for 3-AP induced destruction of climbing fibres. (3) Cerebella of other mammal species tested (mouse, rabbit, pig, cow and human) revealed a similar peripherin staining pattern in the cerebellum, including fibres in the molecular layer with the morphology of climbing fibres. (4) We also noted peripherin positive spinocerebellar and vestibulocerebellar mossy fibres in the cerebellar granular layer of folia known to receive these inputs. (5) A subset of perivascular nerve fibres are also peripherin positive. These results show that peripherin is a useful marker for mammalian cerebellar climbing fibres, and that a subset of morphologically distinct cerebellar mossy fibres are also peripherin positive.