Primary Cultures Of Rat Cerebellum Research Articles

Chronic interleukin‐6 exposure alters metabotropic glutamate receptor‐activated calcium signalling in cerebellar Purkinje neurons

Chronic central nervous system expression of the cytokine interleukin-6 (IL-6) is thought to contribute to the histopathological, pathophysiological, and cognitive deficits associated with various neurological disorders. However, the effects of chronic IL-6 expression on neuronal function are largely unknown. Previous studies have shown that chronic IL-6 exposure alters intrinsic electrophysiological properties and intracellular Ca2+ signalling evoked by ionotropic glutamate receptor activation in cerebellar Purkinje neurons. In the current study, using primary cultures of rat cerebellum, we investigated the effects of chronic IL-6 exposure on metabotropic glutamate receptor (mGluR)-activated Ca2+ signalling and release from intracellular Ca2+ stores. Chronic exposure (6-10 days) of Purkinje neurons to 500 units/mL IL-6 resulted in elevated resting Ca2+ levels and increased intracellular Ca2+ signals evoked by the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) compared to untreated control neurons. Chronic IL-6 treatment also augmented Ca2+ signals evoked by brief 100 mm K+ depolarization, although to a lesser degree than responses evoked by DHPG. Depleting intracellular Ca2+ stores with sarcoplasmic-endoplasmic reticulum ATPase inhibitors (thapsigargin or cyclopiazonic acid) or blocking ryanodine receptor-dependent release from intracellular stores (using ryanodine) resulted in a greater reduction of DHPG- and K+-evoked Ca2+ signals in chronic IL-6-treated neurons than in control neurons. The present data show that chronic exposure to elevated levels of IL-6, such as occurs in various neurological diseases, alters Ca2+ signalling involving release from intracellular stores. The results support the hypothesis that chronic IL-6 exposure disrupts neuronal function and thereby may contribute to the pathophysiology associated with many neurological diseases.

Expression of gabaa receptors by reactive astrocytes in explant and primary cultures of rat cns

The presence of GABAA-receptors on astrocytes was studied in explant and primary cultures of rat cerebellum, hippocampus and spinal cord by means of immunohistochemistry. For these studies we have used the monoclonal antibody bd 17 against the β2- and β3-subunits of GABAA-receptor. In explant cultures many neurones were intensely stained with the GABAA-receptor antibody whereas adjacent astrocytes revealed little or no immunoreactivity. In the far outgrowth zone of explant culture, however, many immunostained astrocytes were observed. In primary astrocyte cultures, only a few cells were stained by the antibody. Astrocytes which became reactive after producing an artificial scar or after addition of certain compounds such as dibutyryl cyclic AMP, interleukin-6, basic fibroblast growth factor and kainic acid, also revealed GABAA-receptor immunoreactivity. Furthermore, these astrocytes were intensely stained for glial fibrillary acidic protein and vimentin. From our studies we conclude that only a subpopulation of normal astrocytes are immunopositive for the GABAA-receptor antibody whereas astrocytes which become reactive following injury of the tissue or after addition of dibutyryl cyclic AMP, the cytokine interleukin-6, fibroblast growth factor or the neurotoxin kainic acid express GABAA-sites.

Immunocytochemical analysis of gangliosides in rat primary cerebellar cultures using specific monoclonal antibodies

We studied the expression of ganglioside antigens in primary cultures of rat cerebellum using an immunocytochemical technique with mouse monoclonal antibodies (MAbs) specific for various gangliosides. Twelve MAbs that specifically recognize each ganglioside were used. Our study revealed that there is a cell type-specific expression of ganglioside antigens in the primary cultures. A number of b-series gangliosides were detected in the granule cells, whereas a-series gangliosides were not intensely expressed. GD1b was detected in the granule cells. GD2 appeared to be present in a subset of the granule cells or a type of small neurons. GD3 was associated not only with the granule cells, but also with both astrocytes and oligodendrocytes. An O-Ac-disialoganglioside, which was suggested to be O-Ac-LD1, was restrictedly detected in Purkinje cells. The other gangliosides were not detected clearly in these cells. These results suggest that several gangliosides may be useful markers for identifying cells in primary cultures of the rat cerebellum; particularly b-series gangliosides such as GD2 and GD1b for the granule cells and O-Ac-LD1 for Purkinje cells.

Developmental characterization of thymosin beta 4 and beta 10 expression in enriched neuronal cultures from rat cerebella.

The beta 4 and beta 10 thymosins are G-actin binding proteins that exhibit complex patterns of expression during rat cerebellar development. Their expression in vivo is initially high in immature granule cells and diminishes as they migrate and differentiate, ceasing altogether by postnatal day 21. Thymosin beta 4 is present in a subset of glia throughout postnatal development, and its synthesis is also induced in maturing Bergmann glia. In contrast, thymosin beta 10 is only present at very low levels in a very small subpopulation of glia in the adult cerebellum. To study the factors differentially regulating expression of the beta-thymosins, we characterized their patterns of expression in primary cultures of rat cerebellum. Both beta-thymosins were initially expressed in granule cells, although expression, especially of thymosin beta 4, was truncated compared with the in vivo time course. As in vivo, thymosin beta 4 was synthesized at much higher levels in astrocytes and microglia in cultures from postnatal cerebellum than was thymosin beta 10. Unlike in vivo, the latter was expressed in glia cultured from fetal cerebellum. The similarities between the in vivo and in vitro expression of the beta-thymosins show that modulation of tissue culture conditions could be used to identify factors regulating beta-thymosin expression in vivo. The differences would identify regulatory mechanisms that are not evident from the in vivo studies alone.

Immunocytochemical analysis of gangliosides in primary cultures of rat cerebellum with monoclonal antibodies for specific gangliosides

Immunocytochemical analysis of gangliosides in primary cultures of rat cerebellum with monoclonal antibodies for specific gangliosides

Cerebellar neurons and glia respond differentially to endothelins and sarafotoxin S6b

Endothelins (ETs) and sarafotoxin-S6b belong to a family of extremely potent vasoconstrictors which may also have a role as neuropeptides or neuromodulators in the central nervous system (CNS). We show, using single cell dynamic video imaging of intracellular free calcium ions ([Ca 2+] i), that binding of ET to its receptors modulates [Ca 2+] i of neurons as well as glial cells in primary cultures of rat cerebellum. At least two receptor subtypes, differing in both their ligand specificity and distribution, appear to be involved in the action of ETs and sarafotoxin S6b on these cells. One of these receptors may be a previously undescribed neuronal form of ET receptor. This is the first demonstration of a direct effect of ETs on neurons as well as glia in the CNS. These data support a possible role for ET as a neurotransmitter or neuromodulator in the CNS.

In Vitro Development of Rat Cerebellar Neurons of Early Embryonic Origin. An Anatomical and Electrophysiological Study.

The development of the major morphological and electrophysiological properties of presumptive Purkinje cells (PCs) was studied in primary cultures of rat cerebellum dissociated on the 14th embryonic day, when PCs are minimally differentiated and migrate in vivo. PCs were identified with a specific antibody to calbindin D-28K (CaBP), which allowed visualization of the different morphological types of PCs between 3 and 29 days in vitro (DIV). CaBP-immunopositive cells were first detected at 3 DIV. Thereafter, the shape of these cells resembled some of those described in vivo. After 20 DIV, 95% of the CaBP-immunopositive cells had characteristic PC dendritic trees, although they were very atrophic. Glial cells immunopositive for the glial fibrillary acidic protein (GFAP) were first seen at 3 DIV. Thereafter GFAP-immunopositive cells resembled Bergmann cells or velate astrocytes. Neurons regarded as PCs were studied electrophysiologically using the patch-clamp whole-cell configuration. Voltage-dependent, tetrodotoxin-sensitive fast inward currents were virtually absent at 2 - 4 DIV, but increased between 7 and 14 DIV to reach two-thirds of the amplitude obtained after 15 DIV. These currents were large enough to give rise to overshooting spikes as early as 7 DIV in the current-clamp mode. This time schedule is in keeping with that of PCs developed in situ. The tetraethylammonium-sensitive, slowly inactivating outward currents had reached two-thirds of the amplitude obtained after 15 DIV by 3 - 4 DIV. Their amplitude remained stable between 4 and 7 DIV, and increased to their maximal value during 7 - 14 DIV, with a marked shortening of action potentials. 4-Aminopyridine-sensitive, fast-inactivating outward currents might also be associated with development, since they were present in 66% of the cells between 7 and 14 DIV but in only 39% from 15 to 29 DIV; however, their amplitude did not vary with time. Presumptive PCs bore l-glutamate-activated receptors, which preceded the emergence of kynurenate-sensitive, spontaneous synaptic currents at 7 DIV. These currents were sometimes intermingled with inhibitory currents, although presumptive PCs were sensitive to gamma-aminobutyrate at 7 DIV. The present model represents some unequivocal features of PC development, although the PCs used had undergone minimal differentiation in vivo and were cultured in a very disturbed cellular environment.

Effects of β-xylosides on proteoglycan biosynthesis and morphology of PC12 pheochromocytoma cells and primary cultures of rat cerebellum

We have examined the effects of beta-xylosides, which act as exogenous acceptors for glycosaminoglycan chain initiation, on the morphology and proteoglycan biosynthesis of PC12 pheochromocytoma cells, and on monolayer, aggregate and explant cultures of early postnatal rat cerebellum. PC12 cells cultured for 13 days in the presence of nerve growth factor (NGF) and beta-xyloside, and labeled during days 11-13 with sodium [35S]sulfate, showed an 8- to 11-fold increase in [35S]sulfate-labeled macromolecules released into the culture medium. Most of the increase was accounted for by chondroitin sulfate, which was in the form of free glycosaminoglycan chains, which were not acid-precipitable. The presence of beta-xyloside also led to a 65-115% increase in [35S]sulfate incorporation into cell-associated glycosaminoglycans and glycoproteins of untreated and NGF-treated PC12 cells, respectively. beta-Xyloside treatment reduced the size of the chondroitin sulfate chains in both the cells and medium from approximately 34,000 to 10,000 Mr, but had much less effect on heparan sulfate, which decreased in size from 16,000 to 13,000-14,500 Mr (in the medium and cells, respectively). beta-Xyloside inhibition of proteoglycan biosynthesis was accompanied by significant morphological effects in NGF-treated PC12 cells, consisting of an increase in length and decrease in the branching, diameter and adhesion to the collagen substratum of the PC12 cell processes. p-Nitrophenyl- and 4-methylumbelliferyl-beta-D-xylosides produced similar effects, which were not seen with p-nitrophenyl-beta-D-galactoside. beta-Xylosides also produced distinct alterations in the adhesion and morphology of monolayer, aggregate, and explant cultures of early postnatal rat cerebellum, which occurred together with inhibition of chondroitin sulfate proteoglycan biosynthesis and a decrease in glycosaminoglycan chain size. These studies indicate that chondroitin sulfate (and probably also heparan sulfate) proteoglycans play a significant role in modulating cell-cell and cell-matrix interactions in nervous tissue development and differentiation.

Free calcium rise and mitogenesis in glial cells caused by endothelin

The peptide endothelin causes a biphasic rise in intracellular free calcium levels in cultured Type-1 astrocytes and C 6 glioma cells, suggesting that glial cells may be the physiological target of endothelin in the brain. Endothelin also causes a calcium-dependent increase [ 3H]thymidine incorporation in primary cultures of rat cerebellum, indicating that, among other possible roles, this peptide may mediate mitogenesis in the brain.

Expression of neurofilament immunoreactivity in developing rat cerebellum in vitro and in vivo.

The developmental expression of neurofilaments immunoreactivity was examined in frozen sections and in primary cultures of rat cerebellum by immunocytochemistry with a series of monoclonal antibodies and with a polyclonal antibody. In tissue sections immunocytochemical staining with all the antibodies used was observed in basket cells where adult-like appearance could be detected by 14 days of age and adult-level intensity was achieved by about 25 days. Granule cells remained unstained. Intense staining appeared in cerebellar white matter as early as 7 days after birth. In contrast, neurofilaments immunoreactivity was detected in cultured granule cells from 7-day-old cerebellum. Only polyclonal antibodies reacting with the highly conserved middle alpha-helical domain of the neurofilament subunits were reactive in culture. Staining could be detected in the nerve cell bodies from the first day after plating; thereafter staining intensity increased and was also distributed in neurite extensions. We conclude that unlike their counterparts in vivo cultured embryonic granule cells can express certain neurofilaments immunoreactivity.

Triiodothyronine increases glutamine synthetase activity in primary cultures of rat cerebellum.

Dissociated cells from 2-3 day-old rat cerebella were cultured in absence of thyroid hormones using conditions yielding mainly glial cells. After 7, 14 and 21 days in vitro, triiodothyronine (60 nM) was added to a set of dishes and glutamine synthetase activity was measured after 24, 48, and 72 h in both control and triiodothyronine-treated cultures. Basal glutamine synthetase activity increased more than 6 X between 7 and 21 days of culture. Triiodothyronine produced significant increases of glutamine synthetase activity after 72 h in 7-day-old cultures (+ 16%), after 48 h in 14-day-old cultures (+ 45%) and after 24 h in 21-day-old cultures (+ 27%). This effect depends on the initial plating density and is not observed if cells are plated at less than 1 cerebellum equivalent per 60 mm dish. Dose-response experiments indicated that 10(-8) M of triiodothyronine induces maximal response whereas half-maximal response is achieved around 10(-10) M. These results show that physiological amounts of thyroid hormone can influence the maturation of astrocytes in culture.

Muscarinic acetylcholine receptor regulation and protein phosphorylation in primary cultures of rat cerebellum

Exposure of primary cell cultures of rat cerebellum to the muscarinic agonist carbachol led to a 50% loss in apparent muscarinic receptor number detectable in a ligand binding assay using [ 3H]quinuclidinyl benzilate ([ 3H]QNB). The loss in [ 3H]QNB binding was time-dependent with a half-life of 2.9 h. Examination of membrane protein phosphorylation indicated that exposure to carabachol resulted in an increase in the level of phosphorylation of 6 polypeptides. The increased phosphorylation of 3 of these polypeptides, of molecular weights, 75,000, 67,000 and 62,000, followed a similar time-course to that of carbachol-induced loss of receptor binding. These results are consistent with the idea that the muscarinic receptor regulation involves a protein phosphorylation mechanism.