Lugaro cells target basket and stellate cells in the cerebellar cortex

Lugaro cells are fusiform neurons located just beneath the Purkinje cell layer. Their axon projects profusely in the molecular layer and emits some collaterals in the granular layer. We have analysed the molecular layer postsynaptic targets of a Golgi-impregnated Lugaro cell, using the electronmicroscopic gold-toning procedure and post-embedding anti-GABA immunocytochemistry. All the gold-labeled neuritic and somatic profiles were GABA-positive, thus confirming that the Lugaro cell is an inhibitory interneuron. The axonal varicosities of this cell form multiple symmetrical synaptic junctions with basket and stellate cell somata and proximal dendrites, and, perhaps, with the molecular layer dendrites of Golgi interneurons. Lugaro cells thus seem to exert a feed-back inhibitory control on Purkinje cells.     

Membrane-fusions and cytoplasmic bridges in the cells of the developing cerebellum

In the developing cerebellum of the neonate rats membrane-fusions and cytoplasmic bridges between cells were observed. These membrane-fusions were characterized by the presence of loops of membrane and cytoplasmic bridges between the two limits of the membrane-fusions. They were found between Purkinje cells, Purkinje cells and the migratory cells, mitotically potent cells of the external granular layer, and differentiating granule cells of the internal granular layer. The membrane-fusions were found to be a transient developmental phenomenon. Issues pertaining to the universality of membrane-fusions, their significance in the induction for cell differentiation, and the problem of fixation artifacts are discussed.     

Patterns of inputs to the parietal cortex efferent neurons from the motor cortex and cerebellum in the cat

The focus of the presentation will review the distribution of nitric oxide (NO)-producing sites in the digestive system in mammals and nonmammalian vertebrates and will center on the roles that NO plays in modulating physiological and pathophysiological functions in digestive system.     

NMDA receptor diversity in the cerebellum: identification of subunits contributing to functional receptors

Recent studies of N-methyl-D-aspartate (NMDA) receptors have led to the suggestion that there are two distinct classes of native NMDA receptors, identifiable from their single-channel conductance properties. 'High-conductance' openings arise from NR2A- or NR2B-containing receptors, and 'low-conductance' openings arise from NR2C- or NR2D-containing receptors. In addition, the low-conductance channels show reduced sensitivity to block by Mg2+. The readily identified cell types and simple architecture of the cerebellum make it an ideal model system in which to determine the contribution of specific subunits to functional NMDA receptors. Furthermore, mRNA for all of these four NR2 subunits are represented in this brain region. We have examined NMDA channels in Purkinje cells, deep cerebellar nuclei (DCN) neurons and Golgi cells. First we find that NR2D-containing NMDA receptors give rise to low-conductance openings in cell-attached recordings from Purkinje cells. The characteristic conductance of these events cannot, therefore, be ascribed to patch excision. Second, patches from some DCN neurons exhibit mixed populations of high- and low-conductance openings. Third, Golgi cells also exhibit a mixed population of high- and low-conductance NMDA receptor openings. The features of these low-conductance openings are consistent with the presence of NR2D-containing NMDA receptors, as suggested by in situ hybridization data. On the other hand the existence of high-conductance channels, with properties typical of NR2B-containing receptors, was not expected. Our results provide new evidence about the subunit composition of NMDA receptors in identified cerebellar cells, and suggest that examination of single-channel properties is a potentially powerful approach for determining the possible subunit composition of native NMDA receptors.     

Development and differentiation of glial precursor cells in the rat cerebellum

The development and differentiation of bipotential glial precursor cells has been studied extensively in tissue culture, but little is known about the distribution and fate of these cells within intact animals. To analyze the development of glial progenitor cells in the developing rat cerebellum, we utilized immunofluorescent, immunocytochemical, and autoradiographic techniques. Glial progenitor cells were identified with antibodies against the NG2 chondroitin-sulfate proteoglycan, a cell-surface antigen of 02A progenitor cells in vitro, and the distribution of this marker antigen was compared to that of marker antigens that identify immature astrocytes, mature astrocytes, oligodendrocyte precursors, and mature oligodendrocytes. Cells expressing the NG2 antigen appeared in the cerebellum during the last 3-4 days of embryonic life. Over the first 10 days of postnatal life, the NG2-labeled cells incorporated 3H-thymidine into their nuclei and their total number increased. At all ages examined, the NG2-labeled cells did not contain either vimentin-like or glial fibrillary acidic protein (GFAP)-like immunoreactivity, suggesting that they do not develop along an astrocytic pathway. NG2-labeled cells of embryonic animals expressed GD3 ganglioside antigens, a property of oligodendrocyte precursors, whereas NG2-positive cells of postnatal animals did not express GD3 immunoreactivity. Nevertheless, the NG2-labeled cells of the nascent white matter expressed oligodendrocyte-specific marker antigens. Cells lying outside of the white matter continued to express the NG2 antigen. In adult animals, the NG2-labeled cells incorporated 3H-thymidine. Glial cells isolated from adult animals and grown in tissue culture express the NG2 antigen and display the phenotypic plasticity characteristic of 02A progenitor cells. These findings demonstrate that a population of glial progenitor cells is extensive within both young and adult animals.     

Calbindin immunoreactivity in Purkinje cells of the bullfrog cerebellum during thyroxine-induced metamorphosis

Calbindin-immunoreactive Purkinje cells were identified in the cerebella of frog tadpoles that had been treated with thyroxine to accelerate metamorphosis. The dorsal part of the cerebellar plate contained the full complement of Purkinje cells which were all CaBP-immunoreactive, while in the ventral part of the cerebellum Purkinje cells acquired CaBP-immunoreactivity only after several days of thyroxine treatment. The ventral group of Purkinje cells was separated from the dorsal group by a distinct gap, which is the site of a shallow sulcus in adult frogs. Additionally, following thyroxine treatment, the numbers of CaBP-immunoreactive Purkinje cells in the ventral group were only half the numbers seen in frogs that metamorphosed spontaneously. We suggest that the variation in the CaBP-immunoreactivity of the dorsal and ventral groups of Purkinje cells, along with the gap in the Purkinje cell layer between the two groups, may be indicative of two distinct populations of Purkinje cells, with distinct patterns of generation, maturation, and perhaps, origin and connectivity, in the cerebellum of frogs.     

ON-OFF amacrine cells in cat retina

We studied the morphology, photic responses, and synaptic connections of ON-OFF amacrine cells in the cat retina by penetrating them with intracellular electrodes, staining them with horseradish peroxidase, and examining them with the electron microscope. In a sample of seven cells, we found two different morphological types: the A19, which ramifies narrowly in stratum 2 (sublamina a) of the inner plexiform layer, and the A22, which ramifies mostly in stratum 4 (sublamina b) but extends some dendrites to sublamina a. Both of these cell types have axon-like processes that extend > 800 microns from the conventional dendritic arbor. ON-OFF amacrine cells in our sample had receptive fields (1.7 +/- 0.3 mm diameter) that were broader than their dendritic arbors (425 +/- 35 microns diameter) and that extended over the region of axon-like processes. In addition, we found many features in common with ON-OFF amacrine cells in poikilotherm vertebrates: a broad receptive field without surround antagonism, two sizes of spike-like events, narrow dynamic range (1 log unit intensity), and excitatory postsynaptic potentials at light on and light off. Two A19 amacrine cells were examined in the electron microscope: most synaptic inputs (93 and 76%, respectively) to either cell were from amacrine cells, with minor inputs from cone bipolar cells. Synaptic outputs were to bipolar, amacrine, and ganglion cells, including the OFF-alpha cell.     

The growth of the dendritic trees of Purkinje cells in the cerebellum of the rat

The growth of Purkinje cell dendritic trees in the cerebellum of the rat was studied over the first 50 days of life, using the technique of network analysis and the Golgi-Cox impregnation method. Our findings showed that a growth spurt occurred from the 10th to 30th day post partum (pp) and involved the production of a massive number of branches of fairly constant length. Growth of the tree occurred firstly in the lateral domain, so that by 15 days pp most trees were of adult width. Thereafter, increases in height occurred until 30 days pp. Associated with this change in direction of growth, from the mainly transverse to the vertical plane, was a deviation from the normal random pattern of branching of the tree, but this was reestablished when reorientation was complete, and growth in the vertical plane underway. The lengths of proximal segments increased once they had become established, but distal branches probably maintained a constant length. The above results, together with changes in segment length, trichotomy, branching probability, and growth cone morphology during development have been discussed in relation to current concepts of dendritic growth.     

DNA fragmentation in granular cells of human cerebellum following global ischemia

Vulnerability of human cerebellum in two autopsy cases following global brain ischemia was examined histologically by using a specific in situ nick-end labeling method for DNA breaks. In both cases, DNA fragmentation was observed in approximately one-third of the granular cells in cerebellar cortex, whereas Purkinje cells were still alive and no DNA fragmentation was recognized in the nuclei. The present study suggests that some granular cells of cerebellar cortex are more vulnerable to transient ischemia than Purkinje cells and death of granular cells is induced by an apoptotic DNA fragmentation following global brain ischemia.     

Identification of acutely isolated cells from developing rat cerebellum

Immunocytochemical staining was used to identify nerve and glial cells from postnatal rat cerebelli in situ and following tissue dissociation. Purkinje cells were identified using antibodies for the calcium-binding proteins calbindin and PEP19. Purkinje cells isolated during the second postnatal week were 15-20 microns in diameter and relatively abundant and displayed thin perisomatic processes. These features were used to identify Purkinje cells with scanning electron microscopy, which revealed extensive membrane infoldings. Golgi and nuclear cells were identified using antibodies against rat-303 antigen. Pale, nuclear, and Purkinje cells were identified using antibodies for rat-302 antigen. Although staining for rat-302 and rat-303 was weak during the second postnatal week, we were able to identify Golgi and pale cells even after tissue dissociation. Isolated Golgi cells were 8-10 microns in diameter and fewer in number than Purkinje cells and did not counterstain with calbindin antibodies. Isolated pale cells were 8-10 microns in diameter, rare, and resistant to calbindin antibodies. Isolated neurons from cerebellar nuclei were not located with either 302 or 303 staining, suggesting that they remained in the tissue. Golgi-Bergmann cells and astrocytes were identified using antibodies for glial fibrillary acidic protein. Isolated glial cells were 12-15 microns in diameter, more numerous than Purkinje cells, and unstained with calbindin antibodies. With phase-contrast optics, glial cells appeared flatter than neuronal cell types and had acentric nuclei. These results demonstrate that specific cell types in developing rat cerebellum can be identified after acute isolation, which should facilitate analysis of their endogenous properties.     

Visuo-oculomotor properties of cells in the superior colliculus of the alert cat

Visual responses and eye movement (EM) -related activities were studied in single units of the superior colliculus (SC) of alert cats. Spontaneous EMs were encouraged by training. Throughout the SC (i.e., in intermediate and deep layers as well as in superficial layers), units were found to respond well to visual stimuli. Strong and consistent responses could be elicited by very dim, low-contrast stationary stimuli. Visual responses varied from phasic to tonic; some units responded tonically to stationary stimuli in the center of the receptive field, and phasically to peripheral stimuli. Many cells responded more vigorously to moving than to stationary stimuli, but very few responded exclusively to stimulus movement. The vast majority of cells were directionally selective. A small number of units were sensitive to the absolute, as well as the retinal, position of visual stimuli. These cells were activated by visual stimuli which fell in the receptive field only if the cat's gaze was fixated on one half of the screen. It seems that these cells must receive information about both eye position and the retinal (receptive field) position of the stimulus. It is possible that they reflect coding of target location within a head (or body) frame of reference. EM-related units were of two types: (1) about 20% of the sample responded prior to spontaneous or visually-triggered EMs, and (2) another 10% (or more) responded with, but not before, EMs. Some cells in the second group discharge almost synchronously with EMs and, thus, cannot plausibly be said to respond to the movement of images across the retina. All cells in the first group were directionally selective. The percentage of EM-related cells in the deep layers of SC is lower in cat than in monkey. Possible reasons for such differences are discussed.     

Filopodia and growth cones in the vertically migrating granule cells of the postnatal mouse cerebellum

The significance of cholinergic modulation for associative memory performance in the piriform cortex was examined in a study combining cellular neurophysiology in brain slices with realistic biophysical network simulations. Three different physiological effects of acetylcholine were identified at the single-cell level: suppression of neuronal adaptation, suppression of synaptic transmission in the intrinsic fibers layer, and activity-dependent increase in synaptic strength. Biophysical simulations show how these three effects are joined together to enhance learning and recall performance of the cortical network. Furthermore, our data suggest that activity-dependent synaptic decay during learning is a crucial factor in determining learning capability of the cortical network. Accordingly, it is predicted that acetylcholine should also enhance long-term depression in the piriform cortex.     

Long-term outcome after frontal sinus obliteration with an ionomer bone substitute in the cat. I: Histological results

BACKGROUND: Osteoplastic frontal sinus surgery in combination with sinus obliteration may occasionally be performed for indications such as severe sinus inflammations, extensive wall fractures involving the nasofrontal duct, or osteomas. The material most frequently used for this obliteration is autogenous adipose tissue. Autografts such as muscle, cartilage, and bone chips have been also recommended for this purpose. In order to avoid the surgical procedure required for harvesting an autograft from other body sites, alloplastic materials have been suggested for frontal sinus obliteration. METHODS: In an animal study using cats, the mucous lining of the frontal sinus was removed, the nasofrontal duct sealed with semifluid ionomer cement, and the cavity filled up with a solid and porous ionomer-based microimplant. RESULTS: Histological investigations were performed up to two years after surgery. Increasing obliteration of the sinus cavity by bone regeneration, starting from the sinus wall, as well as formation of connective tissue between the cement grains was detected as early as one month after implantation. Osteoblasts lining the osteoid layer were considered to be an indication of active bone regeneration. Osteogenesis inside the sinus cavity continuously progressed during the following months. Two years after implantation, osteoid and newly mineralized bone encircled the microimplants leading to an almost complete obliteration. There was no evidence for mucosal regeneration or foreign body reaction. CONCLUSIONS: Because of its osteoconductive effect, biocompatibility, and biostability the ionomer-based microimplant is a suitable alloplastic material for frontal sinus obliteration.     

Threshold setting by the surround of cat retinal ganglion cells

1. The slope of curves relating the log increment threshold to log background luminance in cat retinal ganglion cells is affected by the area and duration of the test stimulus, as it is in human pyschophysical experiments. 2. Using large area, long duration stimuli the slopes average 0-82 and approach close to 1 (Weber's Law) in the steepest cases. Small stimuli gave an average of 0-53 for on-centre units using brief stimuli, and 0-56 for off-centre units, using long stimuli. Slopes under 0-5 (square root law) were not found over an extended range of luminances. 3. On individual units the slope was generally greater for larger and longer test stimulus, but no unit showed the full extent of change from slope of 0-5 to slope of 1. 4. The above differences hold for objective measures of quantum/spike ratio, as well as for thresholds either judged by ear or assessed by calculation. 5. The steeper slope of the curves for large area, long duration test stimuli compared with small, long duration stimuli, is associated with the increased effectiveness of antagonism from the surround at high backgrounds. This change may be less pronounced in off-centre units, one of which (probably transient Y-type) showed no difference of slope, and gave parallel area-threshold curves at widely separated background luminances, confirming the importance of differential surround effectiveness in changing the slope of the curves. 6. In on-centre units, the increased relative effectiveness of the surround is associated with the part of the raised background light that falls on the receptive field centre. 7. It is suggested that the variable surround functions as a zero-offset control that sets the threshold excitation required for generating impulses, and that this is separate from gain-setting adaptive mechanisms. This may be how ganglion cells maintain high incremental sensitivity in spite of a strong maintained excitatory drive that would otherwise cause compressive response non-linearities.     

Numerical aspects of pontine, lateral reticular, and inferior olivary projections to two paravermal cortical zones of the cat cerebellum

Two different olivo-cortico-nuclear zones in the cat cerebellum have been compared quantitatively as regards the numbers of cells projecting to them from within several sources of mossy fibres (MFs), namely the basal pontine nuclei (BPN), nucleus reticularis tegmenti pontis (NRTP), and the ipsilateral lateral reticular nucleus (LRN). The zones chosen were the C3 zone in lobule V of the anterior lobe and the C1 zone in pars copularis of the paramedian lobule (PMLpc), localised by recording climbing fibre-mediated potentials evoked on their surface as a result of volleys set up in their spino-olivocerebellar paths. The zones were injected with fluorescent-labelled latex microspheres and cell bodies, retrogradely labelled in the MF source nuclei and in the contralateral inferior olive, were counted and mapped. Evidence was obtained that tracer efficiency was very high in both the MF projections and the olivo-cerebellar projection and that each olivocerebellar axon may provide only one climbing fibre to the upper part of a lobule V folium but an average of nearly two to the same part of a PML folium. When the numbers of labelled cells in each MF source nucleus were expressed as a percentage of the total number of labelled pontine cells, the biggest source for lobule V was the contralateral BPN, followed by LRN, contralateral NRTP, ipsilateral BPN, and ipsilateral NRTP. For PMLpc, the order was similar except that ipsilateral BPNs exceeded contralateral NRTPs, but the dominance of contralateral BPN as a source was much greater. Cell totals were converted into projection densities (i.e., numbers of cells labelled per square millimetre of cortical sheet involved in the injection site); densities for PMLpc were found to be almost three times greater than those for lobule V for contralateral BPN but the two densities were not significantly different for ipsilateral BPN. The three other MF sources projected at higher densities to lobule V than to PML. These findings indicate that two cortical zones, both of which receive climbing fibres from the rostral part of the dorsal accessory olive and project to nucleus interpositus anterior, nevertheless differ markedly in regard to both the relative and the absolute sizes of the projections they receive from several of their most important sources of MFs.