Sheep Cerebellum Research Articles

In situ assessment of neuroinflammatory cytokines in different stages of ovine natural prion disease

IntroductionAccording to the neuroinflammatory hypothesis, a cytokine-mediated host innate immune response may be involved in the mechanisms that contribute to the process of neurodegeneration. Specifically, regarding prion diseases, some experimental murine models have evidenced an altered profile of inflammatory intermediaries. However, the local inflammatory response has rarely been assessed, and never in tissues from different natural models throughout the progression of neurodegeneration.MethodsThe aim of this study was to use immunohistochemistry (IHC) to in situ assess the temporal protein expression of several cytokines in the cerebellum of sheep suffering from various clinical stages of scrapie.Results and discussionClear changes in the expression of most of the assessed markers were observed in the affected sheep compared to the healthy control sheep, and from different stages. In summary, this preliminary IHC study focusing in the Purkinje cell layer changes demonstrate that all cytokines or respective receptors studied (IL-1, IL-1R, IL-2R, IL-6, IL-10R, and TNFαR) except for IFNγR are disease-associated signaling proteins showing an increase or decrease in relation to the progression of clinical disease. In the future, this study will be extended to other inflammatory mediators and brain regions, focusing in particular on the release of these inflammatory mediators by astroglial and microglial populations.

Developmental profiles of GFAP-positive astrocytes in sheep cerebellum.

Astroglial account for the largest glial population in the brain and play a variety of vital functions in the development of the central nervous system (CNS). An immunohistochemical study was performed in 19 ovine foetuses ranging from 2 to 5months of gestation, one newborn lamb and three adult sheep. Using the anit-glial fibrillary acidic protein (GFAP) marker, several variations were found in the degree of GFAP positive (GFAP+) astrocyte distribution between the different zones in the cerebellum of sheep during brain development. Our study indicates that the first appearance of astrocytes from restricted zones in the cerebellum occurs around the eighth week of gestation. Bergmann cells were found to be present from around the 15th week of gestation onwards. Our findings suggest that the maturation of astrocytes begins in the caudal parts of the cerebellum, developing from their initial ventral regions to spread first to dorsal regions radially within the white matter, then followed by the more rostral parts of the cerebellum. Astrocytes were also found to proliferate in the vermis before appearing in the cerebellar hemispheres.

Inositol 1,4,5-Trisphosphate Slowly Converts Its Receptor to a State of Higher Affinity in Sheep Cerebellum Membranes

Incubation of cerebellar microsomes with d-myo-inositol 1,4,5-trisphosphate (InsP3) (0.01 1 microM), at 4 or 20 degrees C in a cytosolic-like medium devoid of Ca2+ and Mg2+, followed by InsP3 removal, induced an increase in InsP3 binding determined with 1 nm [3H]InsP3. At 20 degrees C, and pH 7.1, maximal stimulation (1.5 2. 5-fold) was obtained with 1 mum InsP3, and the EC50 was 60 +/- 5 nm. Several lines of evidence suggested that the activating site is identical with the InsP3 binding site: (i) activation and binding exhibited the same inositol phosphate specificity; (ii) addition of decavanadate, a competitive inhibitor of [3H]InsP3 binding, to the preincubation mixture, prevented the activating effect of InsP3; (iii) the concentration of InsP3 giving half-maximal activation was close to that giving half-maximal InsP3 binding. The time course of activation was found to be much slower than that of binding. While a t1/2 less than 0.4 s has been measured recently at neutral pH and 20 degrees C for binding of 0.5 nm [3H]InsP3 (Hannaert-Merah, Z., Coquil, J.-F., Combettes, L., Claret, M., Mauger, J.-P., and Champeil, P. (1994) J. Biol. Chem. 269, 29642-29649), a 20-s preincubation with 1 microM InsP3 was required to half-maximally stimulate binding. Under the present conditions, the InsP3-induced binding increase was only partially reversible. However, this effect was not blocked by antiproteases suggesting that it did not involve proteolysis. Taking advantage of the marked difference in the kinetics of InsP3 binding and InsP3-dependent activation, we performed binding experiments on a short period (3 s) to determine the effect of InsP3 pretreatment on the binding parameters. The data showed that this treatment increased the affinity of the receptor without changing the number of binding sites (control: KD = 107 nm, Bmax = 28 pmol/mg of protein; after preincubation with 1 microM InsP3: KD = 53 nm, Bmax = 32 pmol/mg of protein). The two states of the receptor bound InsP3 with a Hill coefficient close to 1 on a 3-s scale. In agreement with the effect of InsP3 pretreatment, equilibrium binding experiments performed on 10-min incubations revealed an apparent positive cooperative behavior (apparent Hill coefficient = 1.6; apparent KD = 66 nm). These results report a new regulatory process of the InsP3 receptor in cerebellum occurring independently of Ca2+ and on a relatively long time scale.

Characterization of the co-agonist effects of strontium and calcium on myo-inositol trisphosphate-dependent ion fluxes in cerebellar microsomes

Using sheep cerebellum microsomes previously loaded with 45Ca 2+ or 90Sr 2+, we measured the dependence of inositol 1,4,5-trisphosphate (InsP 3)-induced efflux of these ions on Ca 2+ or Sr 2+ on the cytosolic side. At a low InsP 3 concentration, Ca 2+ in the submicromolar range only poorly activated 45Ca 2+ or 90Sr 2+ efflux, and higher Ca 2+ concentrations were inhibitory. In contrast, Sr 2+ in the micromolar range activated release efficiently, while only very high Sr 2+ concentrations were inhibitory. Experiments were repeated in the presence of a high InsP 3 concentration, which allowed increasing free Ca 2+ to micromolar concentrations without inducing complete inhibition of the InsP 3-dependent efflux. Under these conditions, micromolar Ca 2+ was found to activate efflux to a large extent, similar to that previously found with Sr 2+ Optimal activation by Ca 2+ of the InsP 3-dependent channel occurs at micromolar rather than submicromolar free Ca 2+ concentrations, but at too low an InsP 3 concentration, Ca 2+-induced activation is counteracted by Ca 2+-induced inactivation. Seprate measurements of [ 3H]-InsP 3 binding at a low concentration showed that Sr 2+ and Ca 2+ did not enhance the amount of bound [ 3H]-InsP 3, implying that the activating effect of Sr 2+ and Ca 2+ in cerebellar microsomes is mediated by an increase in the channel opening probability and not by an increase in the receptor's affinity for InsP 3. A similar relationship also holds in the case of the activating effect of nucleotides.

Rapid kinetics of myo-inositol trisphosphate binding and dissociation in cerebellar microsomes.

Using sheep cerebellum microsomes adsorbed on a filter, we measured the kinetics of [3H]inositol 1,4,5-trisphosphate (InsP3) binding and dissociation on the subsecond time scale during rapid perfusion of the filter with [3H]InsP3-containing or InsP3-free media. At 20 degrees C and pH 7.1, in a cytosol-like medium containing MgCl2, the half-time for InsP3 dissociation was as short as 125 ms. The receptor behaved as a simple target for binding of its ligand, with the rate constant for InsP3 binding increasing linearly with InsP3 concentration. Various modulators of InsP3 binding (KCl, NaCl, pH, Mg2+, and Ca2+) were found to affect the receptor's apparent affinity for InsP3 mainly by altering the rate constant for [3H]InsP3 dissociation. ATP (but not InsP3) also accelerated [3H]InsP3 dissociation. In contrast to these modulators, luminal Ca2+ was found to have no effect on the amount of microsome-bound [3H]InsP3.

Rapid filtration studies of the effect of cytosolic Ca2+ on inositol 1,4,5-trisphosphate-induced 45Ca2+ release from cerebellar microsomes

Using microsomal membrane vesicles derived from sheep cerebellum, we measured the rate of inositol 1,4,5-trisphosphate (InsP3)-dependent 45Ca2+ efflux from 45Ca(2+)-loaded compartments during rapid perfusion with a medium containing InsP3 and various concentrations of free 40Ca2+ on the cytosolic side (pH 7.1, 5 mM Mg2+, in the absence of ATP at 20 degrees C). At 0.15 microM InsP3 and pCa 6.5, half-45Ca2+ release was attained within less than 200 ms. At low Ca2+ concentrations, the initial rate of 45Ca2+ release depended smoothly on InsP3 concentration, and InsP3 activated release with moderate positive cooperativity. Preliminary experiments performed at various free 40Ca2+ concentrations were consistent with a bell-shaped 40Ca2+ dependence of 45Ca2+ release. In the range of micromolar or higher free 40Ca2+ concentrations, the apparent inhibition of 45Ca2+ release was dependent on InsP3 concentration, and 45Ca2+ release for intermediate InsP3 concentrations was transient; under selected conditions, a second perfusion period, identical to the first one but separated from it by a short recovery period, was found to allow renewed 45Ca2+ efflux. At high Ca2+ concentration, fast reversible Ca(2+)-dependent desensitization of the channel, and not heterogeneity, was therefore responsible for the termination of InsP3-triggered 45Ca2+ efflux at submaximal concentrations of InsP3. At lower Ca2+ concentrations, a large fraction of the apparent activating effect of submicromolar 40Ca2+ concentrations on 45Ca2+ efflux that we had observed in the preliminary experiments proved to be the artifactual consequence of an inhibitory effect exerted by metal-free Ca2+ chelators on InsP3-dependent efflux at nanomolar 40Ca2+ concentrations. 1,2-Bis(2-aminophenoxy)ethane-N,N,N'-N'-tetraacetic acid, EGTA, and fluo-3 were all effective inhibitors. When this inhibition was taken into account, a rise in free 40Ca2+ concentration from 30 to 300 nM only weakly enhanced 45Ca2+ fluxes in the presence of a low concentration of InsP3. As a result, submicromolar free 40Ca2+ appears to be only a poor activator of InsP3-induced Ca2+ release under these experimental conditions.

Corticotropin-releasing factor in cerebellar afferent systems: a combined immunohistochemistry and retrograde transport study

The flocculus and paraflocculus of cat and sheep cerebellum were studied with immunohistochemical methods, using antisera to corticotropin-releasing factor (CRF). CRF immunoreactivity was present within 3 populations of varicose nerve fibers. One population of CRF-immunoreactive (CRF-IR) fibers appeared to appose Purkinje cell somata and to follow their dendrites into the molecular layer. This arrangement suggested they were climbing fibers. A second group of CRF-IR profiles reminiscent of mossy fibers was widely distributed throughout the granule cell layer. A third population of CRF-IR fibers was present as a beaded plexus lying parallel to the pial surface, above and subadjacent to the Purkinje cell layer. The fibers of this plexus extended into the Purkinje cell layer and surrounded these somata. The source of some of the CRF-IR fibers within the flocculus and paraflocculus was determined by a retrograde axonal transport study utilizing the fluorescent tracer Fast blue (FB) in combination with the immunohistochemical localization of CRF. It was determined that CRF-IR perikarya within the inferior olivary nucleus gave rise to a population of climbing fibers within those lobules. Furthermore, all divisions of the inferior olive were found to contain CRF-IR somata. This latter finding suggests the potential for CRF-IR climbing fiber projections from the inferior olive to other regions of the cerebellar cortex. The existence of CRF-IR mossy fibers and fibers within the ganglionic plexus suggests the possibility of CRF-IR afferent projections from other regions of the brain stem to the flocculus and paraflocculus.

The effects of intrauterine growth retardation on the development of the Purkinje cell dendritic tree in the cerebellar cortex of fetal sheep: A note on the ontogeny of the Purkinje cell

The development of the fetal sheep cerebellum at 80, 100, 120 and 140 days gestation (term = 146 days) and 3 months postnatally was studied using Nissl stained sections and rapid Golgi preparations. The most rapid expansion of the Purkinje cell dendritic tree occurred between 100 and 120 days of gestation (5–6 fold increase in area). By 140 days it had acquired its adult form after which time growth continued mainly in the vertical direction. The effects of intrauterine growth retardation on the growth of granule and Purkinje cell dendrites in the cerebellar cortex of fetal sheep (140 days) were investigated in Golgi preparations. Compared with control cerebella the length (but not the number) of granule cell dendrites was reduced by 14% (P<0.01); the area of the Purkinje cell dendritic field was reduced by 20% (P<0.01); the branching density was reduced by 8% (P<0.01); the total branch length was reduced by 27% (P<0.002); the density of dendritic spines per row was not affected. These factors resulted in a decrease of 26% (P<0.002) in the total number of dendritic spines per row per Purkinje cell.These findings show that the growth of granule cell dendrites and the Purkinje cell dendritic tree have been significantly affected by chronic intrauterine deprivation. Such structural abnormalities could affect the pattern of neuronal connectivity and could be associated with functional deficits.

Cerebellar cortical afferents from the dorsal column nuclei in sheep, demonstrated with retrograde transport of horseradish peroxidase

Following horseradish peroxidase injections in the cortex of the sheep cerebellum (except the ventral part of the anterior lobe, the flocculus and ventral paraflocculus), labeled cells were found in nucleus gracilis, medial cuneate and lateral cuneate. The present findings provide evidence that the projection of the dorsal column nuclei to the cerebellum in the sheep is more extensive than has been reported for the common laboratory animals. Projections to the vermis are bilateral, projections to the hemisphere are mainly ipsilateral.

Neurophysin in the brain and pituitary gland of normal and scrapie-affected sheep—II: Its occurrence in the cerebellum in dystrophic axon terminals with lysosome-lipofuscin accumulation: A possible anomaly of neuronal sulphur-protein metabolism

Neurophysin (the protein present in neurosecretory granules of the posterior pituitary) has been demonstrated by immunofluorescence using a cross-species-reactive porcine neurophysin-II antiserum of high specificity in the cerebellum of sheep affected with the heredo-familial neurological disorder known as natural scrapie, but not in that of normal sheep. Neurophysin occurred in the granular and Purkinje cell layers, probably in axon terminals in the cerebellar glomeruli and on Purkinje cell bodies, in foci which on subsequent staining for neurosecretory material gave a ‘Gomori-positive’ reaction. Many foci of ‘Gomori-positive’ material did not show the presence of neurophysin. The cerebellum of a scrapie-affected sheep absorbed the specific neurophysin antibody from the antiserum almost as completely as did porcine neurophysin-II. Yellow autofluorescent bodies corresponding to lysosomes and lipofuscin, including giant forms, were greatly increased in the scrapie sheep. The data suggest that in scrapie there is an anomaly of neuronal protein metabolism affecting primarily the terminal axons of certain neurones with a prominent sulphur-protein metabolism. A slowly progressive and sequential terminal axon dystrophy ensues, during which neurophysin or a related compound appears in ectopic sites; the necessary basic metabolic pathways involved are probably widespread in cells outside the hypothalamo-neurohypophysial systems. The anomaly may represent acceleration of the normal process of ageing and be under single gene control. The applicability of the protein transcription error theory of senescence is discussed with particular reference to a possible anomaly of neuronal methionine metabolism with or without aberrant lysosome function.

Neurosecretion-like material in the hindbrain of ageing sheep and sheep affected with natural scrapie.

THE earliest lesion in the cerebellum of sheep affected with natural scrapie is a degeneration of the mossy terminals within the granular layer of the cerebellar cortex1,2. With this in mind we have made a combined electron and light microscope study of these early lesions. We used sheep from a supervised breeding population, the normal controls being genetically non-susceptible to natural scrapie3.

The oxidation of glycine by D-amino acid oxidase in extracts of mammalian central nervous tissue.

Abstract— Glycine was a substrate for d‐amino acid oxidase purified from extracts of cat spinal cord and sheep cerebellum. d‐Aspartate and N‐methyl‐d‐aspartate were oxidized at a rate similar to that of glycine by the purified sheep cerebellum extract; d‐α‐alanine and d‐serine were oxidized appreciably faster than glycine, while GABA and d‐glutamate were not oxidized at a measurable rate. p‐Mercuribenzoate and kojate inhibited the oxidation of glycine by the purified sheep cerebellum extract. d‐Amino acid oxidase activity was higher in the grey than in the white matter of cat spinal cord, while the reverse was true for the cerebral cortex; the activity in the cord and cerebral cortex was much lower than that in the cerebellum.