Organotypic Cerebellar Slice Cultures Research Articles

Selectively increased sensitivity of cerebellar granule cells to AMPA receptor-mediated excitotoxicity in a mouse model of Batten disease

Batten disease, a lysosomal storage disorder, is caused by mutations in the CLN3 gene. The Cln3-knockout (Cln3-/-) mouse model of the disease exhibits many characteristic pathological features of the human disorder. Here, we show that Cln3-/- mice, similarly to Batten disease patients, have a deficit in cerebellar motor coordination. To explore the possible cellular cause of this functional impairment, we compared the vulnerability of wild type (WT) and Cln3-/- cerebellar granule cell cultures to different toxic insults. We have found that cultured Cln3-/- cerebellar granule cells are selectively more vulnerable to AMPA-type glutamate receptor-mediated toxicity than their WT counterparts. This selective sensitivity was also observed in organotypic cerebellar slice cultures. Our results suggest that lack of the CLN3 protein has a significant influence on the function of AMPA receptors in cerebellar granule neurons, and that AMPA receptor dysregulation may be a major contributor to the cerebellar dysfunction in Batten disease.

Thiamine deficiency in the pathogenesis of chronic ethanol-associated cerebellar damage in vitro

Nutritional deficiencies associated with long-term ethanol consumption may cause neuronal damage in ethanol-dependent individuals. Thiamine deficiency, in particular, is thought to contribute to ethanol-associated cerebellar degeneration, although damage may occur in adequately nourished alcoholics. Thus, the present study examined the effects of thiamine depletion and ethanol exposure on cytotoxicity in rat cerebellum. Organotypic cerebellar slice cultures were treated starting at 25 days in vitro with 100mM ethanol for 11 days or 10 days followed by a 24-h withdrawal period. This exposure paradigm has previously been shown in hippocampal slice cultures to result in spontaneous cytotoxicity upon ethanol withdrawal. Additional cerebellar cultures were exposed to the thiamine depleting agent pyrithiamine (10–500μM) for 10 or 11 days, some in the presence of ethanol exposure or withdrawal. Other cultures were co-exposed to thiamine (1–100μM), 500μM pyrithiamine, and ethanol for 10 or 11 days. The results demonstrated that neither 11-day ethanol treatment nor withdrawal from 10-day exposure significantly increased cerebellar cytotoxicity, as measured by propidium iodide fluorescence. The 11-day treatment with 100 or 500μM pyrithiamine significantly increased propidium iodide fluorescence ∼21% above levels observed in control tissue. Cultures treated with both ethanol (11 days or 10 days plus withdrawal) and 500μM pyrithiamine displayed a marked increase in cytotoxicity ∼60–90% above levels observed in control cultures. Pyrithiamine and ethanol-induced cytotoxicity was prevented in cultures co-exposed to thiamine (10–100μM) for the duration of pyrithiamine treatment. Findings from this report suggest that the cerebellum may be more sensitive to the toxic effects of thiamine deficiency, as compared with alcohol withdrawal, associated with alcohol dependence.

Corticosterone and dexamethasone potentiate cytotoxicity associated with oxygen-glucose deprivation in organotypic cerebellar slice cultures

Many patients display elevated levels of serum cortisol following acute ischemic stroke. Given that glucocorticoids may potentiate some forms of insult, these studies examined the effects of corticosterone or dexamethasone exposure on cytotoxicity following oxygen-glucose deprivation in the cerebellum, a brain region susceptible to stroke. In organotypic cerebellar slice cultures prepared from neonatal rat pups, 90-min of oxygen-glucose deprivation at 15 days in vitro resulted in significant cytotoxicity at 24-, 48-, and 72-h post-oxygen-glucose deprivation, as measured by uptake of propidium iodide. Exposure of cultures following oxygen-glucose deprivation to the antioxidant trolox (500μM), but not to the glucocorticoid receptor antagonist RU486 (10μM), completely blocked oxygen-glucose deprivation-induced cytotoxicity. Corticosterone (1μM) or dexamethasone (10μM) exposure alone did not significantly increase propidium iodide uptake above levels observed in control cultures. However, corticosterone or dexamethasone exposure after oxygen-glucose deprivation potentiated oxygen-glucose deprivation-mediated propidium iodide uptake at each time point. Trolox, as well as RU486, co-exposure of cultures to corticosterone or dexamethasone after oxygen-glucose deprivation abolished all cytotoxicity. In conclusion, these data demonstrated that glucocorticoid exposure modulated oxygen-glucose deprivation-mediated propidium iodide uptake, which likely involved glucocorticoid receptor activation and pro-oxidant effects.

Developmental expression of matrix metalloproteinases 2 and 9 and their potential role in the histogenesis of the cerebellar cortex

The development of the cerebellar cortex depends on intrinsic genetic programs and orchestrated cell-cell/cell-matrix interactions. Matrix metalloproteinases (MMPs) are proteolytic enzymes that play an important role in these interactions. MMP-2 and MMP-9 are involved in diverse neuronal functions including migration, process extension, and synaptic plasticity. We investigated the spatiotemporal pattern of expression/activity of MMP-2/MMP-9 in the developing cerebellum and their role in the histogenesis of the cerebellar cortex. The levels of transcripts of MMP-2/MMP-9 were measured with real-time quantitative polymerase chain reaction. An initial decrease in MMP-2/MMP-9 transcripts was observed between postnatal days 3 (PD3) and PD6, and the mRNA levels remained relatively constant thereafter. Zymographic analysis revealed that the expression/activity of MMP-2/MMP-9 persisted longer than their transcripts; the downregulation occurred around PD9, suggesting a mechanism of translational or post-translational regulation. The gelatinase activity was localized in the external granule layer (EGL) and the internal granule layer during PD3-PD12. The immunoreactivity of MMP-2 was mainly localized in the EGL, the Bergmann glial fibers, and the Purkinje cell layer (PCL), whereas MMP-9 immunoreactivity was detected intensively in the PCL and the extracellular space of the molecular layer. Expression of MMP-9 was relatively weak in the EGL. The immunoreactivity of MMP-2/MMP-9 became undetectable after PD21. A similar expression pattern of MMP-2/MMP-9 was observed in organotypic cerebellar slice cultures. Exposure of organotypic slices to a specific MMP-2/MMP-9 inhibitor significantly increased the thickness of the EGL and concurrently decreased the number of migrating granule neurons in the molecular layer. Thus, MMP-2 and MMP-9 play a role in the postnatal cerebellar morphogenesis.

Lysolecithin induces demyelination in vitro in a cerebellar slice culture system.

Demyelination is a hallmark of several human diseases, including multiple sclerosis. To understand better the process of demyelination and remyelination, we explored the use of an in vitro organotypic cerebellar slice culture system. Parasagittal slices of postnatal Day 10 (P10) rat cerebella cultured in vitro demonstrated significant myelination after 1 week in culture. Treatment of the cultures at 7 days in vitro (DIV) with the bioactive lipid lysolecithin (lysophosphatidylcholine) for 15-17 hr in vitro produced marked demyelination. This demyelination was observed by immunostaining for the myelin components myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). After a transient demyelinating insult with lysolecithin in vitro, the cultures recovered with oligodendrocyte differentiation recapitulating a normal time course; there was initially re-expression of CNPase and MBP during this recovery, and this was followed by MOG. In addition, there seemed to be some limited remyelination during the recovery phase. Lysolecithin thus induces demyelination in an in vitro organotypic cerebellar slice culture system, providing a model system for studying myelination, demyelination, and remyelination in vitro.

Organotypic rat cerebellar slice culture as a model to analyze the molecular pharmacology of GABA A receptors

The preservation of the neuronal circuitry in rat cerebellar slice cultures provides an advantage in monitoring the development and characterizing the pharmacology of GABA A receptor subtypes. Sprague–Dawley rats, 8–11 days of age, were decapitated, their cerebella were cut into 400-μm slices and transferred into culture dishes. Cell viability and organotypic cerebellar organization of the culture remained well preserved up to 3 weeks. Autoradiographic procedures were introduced in these advanced culture technique and employed [ 3H]Ro 15-4513 in the absence and presence of 10 μM diazepam to visualize all benzodiazepine (BZD) and diazepam-insensitive (DIS) binding sites, respectively. Since expression of the α6 subunit variant of the GABA A/BZD receptor is restricted to the cerebellar granule cells and the BZD receptor agonist diazepam has very low affinity for this subunit, changes in DIS [ 3H]Ro 15-4513 binding sites during cultivation time can be attributed to changes in α6 subunit expression. A time-dependent development of total and DIS [ 3H]Ro 15-4513 binding sites were observed in the culture with a trend towards an increase in GABA A receptor α6 subunit levels during the first week. These findings suggest that explant preparations can be used to examine morphological changes in rat cerebellar slices. In addition, these preparations can be utilized to study the pharmacological effects of GABA A/BZD selective drugs on postnatal development of GABA A receptors in rat cerebellum.

N-Cadherin Is Involved in Axon-Oligodendrocyte Contact and Myelination

We have analyzed the influence of the calcium-dependent cell adhesion molecule, N-cadherin, on events leading to CNS myelination. Interactions between axons and oligodendrocyte progenitor (OP) cells and the CG4 OP cell line were examined by video-microscopy. OPs cocultured with dorsal root ganglia explants migrated around the culture and formed numerous contacts with axons. The duration of these contacts depended on the morphology of the OP, with OPs containing four or more processes forming long-lasting contacts and OPs with three or fewer processes forming short-termed contacts. Treatment with N-cadherin function blocking peptides approximately halved the duration of contacts made by cells with four or more processes but contact times for cells with three or less processes were unaffected. The L7 cadherin-blocking antibody and calcium withdrawal had similar effects. Contacts with axons regenerating from explants of adult retina, which do not have N-cadherin on their surface was examined. The contact duration of OPs to adult retina axons was short and similar in length to those formed between OPs and dorsal root ganglion axons in the presence of cadherin blocking reagents. Oligodendrocyte myelination was examined in organotypic rat cerebellar slice cultures, taken before myelination at postnatal day 10 and then allowed to myelinate in vitro over 7 days. When incubated with an N-cadherin function-blocking peptide, myelination of Purkinje cell axons was reduced to about half of control levels, while control peptides were without effect. Cadherin-blockade did not prevent maturation of OPs, since oligodendrocytes showing myelin basic protein immunostaining were still found in these cultures. However, many of the cell processes did not colocalize with calbindin-positive axons. From these data we conclude that N-cadherin is important for the initial contact between a myelinating oligodendrocyte and axons and significantly contributes to the success of myelination.

Organotypic rat cerebellar slice culture as a model to analyze the molecular pharmacology of GABA A receptors

Organotypic rat cerebellar slice culture as a model to analyze the molecular pharmacology of GABA A receptors