Isolation of Cerebellar Granule Cells from Neonatal Rats

Oberdoerster, Jan

doi:10.1002/0471140856.tx1207s09

Cited by 8 publications

(9 citation statements)

References 16 publications

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“…Cerebellar granule cells were acutely isolated in minimum essential medium with Earl's salts (MEM; Life Technologies Inc., Rockville, MD, USA) and 1.5 U ml −1 protease (Sigma, St Louis, MO, USA) as described in Current Protocols in Toxicology (Oberdoerster, ) with a few modifications (Bellum et al ., ; Bawa and Abbott, , ). Isolated cerebellar granule cells were plated onto coverslips coated with poly‐ d ‐lysine (Sigma) or onto chambered coverglasses (Nunc, Rochester, NY, USA).…”

Section: Methodsmentioning

confidence: 99%

The effect of methylmercury exposure on behavior and cerebellar granule cell physiology in aged mice

Bellum

Thuett²,

Bawa

et al. 2012

J of Applied Toxicology

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Epidemiology studies have clearly documented that the central nervous system is highly susceptible to methylmercury toxicity, and exposure to this neurotoxicant in humans primarily results from consumption of contaminated fish. While the effects of methylmercury exposure have been studied in great detail, comparatively little is known about the effects of moderate to low dose methylmercury toxicity in the aging central nervous system. We examined the toxic effects of a moderate dose of methylmercury on the aging mouse cerebellum. Male and female C57BL/6 mice at 16-20 months of age were exposed to methylmercury by feeding a total dose of 5.0 mg kg(-1) body weight and assessed using four behavioral tests. Methylmercury-treated aged mice performed significantly worse in open field, footprint analysis and the vertical pole test compared with age-matched control mice. Isolated cerebellar granule cells from methylmercury-treated aged mice exhibited higher levels of reactive oxygen species and reduced mitochondrial membrane potentials, but no differences in basal intracellular calcium ion levels compared with age-matched control mice. When aged mice were exposed to a moderate dose of methylmercury, they exhibited a similar degree of impairment when compared with young adult mice exposed to the same moderate dose of methylmercury, as reported in earlier studies from this laboratory. Thus, at least in mice, exposure of the aged brain to moderate concentrations methylmercury does not pose greater risk compared with the young adult brain exposed to similar concentrations of methylmercury.

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Section: Methodsmentioning

confidence: 99%

The effect of methylmercury exposure on behavior and cerebellar granule cell physiology in aged mice

Bellum

Thuett²,

Bawa

et al. 2012

J of Applied Toxicology

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“…The isolation of cerebellar granule neurons was performed as previously described by others (Oberdoerster J., 2001) and according to protocols approved by the IAUCC of Thomas Jefferson University. Brifely, rat or mouse pups (wild-type and dpp6 knock-out mice (Zagha et al, 2008)) were sacrificed on postnatal day 7 or 8 and the brains removed.…”

Section: Methodsmentioning

confidence: 99%

The Dipeptidyl-Peptidase-Like Protein DPP6 Determines the Unitary Conductance of Neuronal Kv4.2 Channels

Kaulin¹,

Santiago-Castillo²,

Rocha³

et al. 2009

J. Neurosci.

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The neuronal subthreshold-operating A-type K+ current regulates electrical excitability, spike timing and synaptic integration and plasticity. The Kv4 channels underlying this current have been implicated in epilepsy, regulation of dopamine release, and pain plasticity. However, the unitary conductance (γ) of neuronal somatodendritic A-type K+ channels composed of Kv4 pore-forming subunits is larger (∼7.5 pS) than that of Kv4 channels expressed singly in heterologous cells (∼4 pS). Here, we examined the putative novel contribution of the dipeptidyl-peptidase-like-protein-6 DPP6-S to the γ of native (cerebellar granule neuron, CGN) and reconstituted Kv4.2 channels. Co-expression of Kv4.2 proteins with DPP6-S was sufficient to match the γ of native CGN channels; and CGN Kv4 channels from dpp6 knock-out mice yielded a γ indistinguishable from that of Kv4.2 channels expressed singly. Moreover, suggesting electrostatic interactions, charge neutralization mutations of two N-terminal acidic residues in DPP6-S eliminated the increase in γ. Therefore, DPP6-S, as a membrane protein extrinsic to the pore domain, is necessary and sufficient to explain a fundamental difference between native and recombinant Kv4 channels. These observations may help to understand the molecular basis of neurological disorders correlated with recently identified human mutations in the dpp6 gene.

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“…Cerebellar granule neurons (CGNs) have been used extensively in the study of neurotoxicity (Huang et al, 2010; Giordano et al, 2006; Lee et al, 2009; Giordano and Costa, 2011; Oberdoerster, 2001), and the method described here for isolating these primary cells has been modified from previously published protocols (Giordano and Costa, 2011; Oberdoerster, 2001). Because CGNs are among the most numerous neurons in the brain and are relatively easy to obtain, they yield a large number of cells for experimental use (Giordano and Costa, 2011; Llinás et al, 2004; Banker, 1998), making them well-suited as a general neuronal model system.…”

Section: Basic Protocolmentioning

confidence: 99%

Co‐Culture of Neurons and Microglia

Roqué

Costa

2017

CP Toxicology

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Microglia, the resident immune cells of the brain, have been implicated in numerous neurodegenerative and neurodevelopmental diseases. Activation of microglia by a variety of stimuli has been shown to induce the release of numerous factors that contribute to neuro-inflammation and oxidative stress, two crucial processes linked to disorders of the central nervous system. The in vitro techniques described here will provide a set of protocols for the isolation and plating of primary cerebellar granule neurons, primary cortical microglia from a mixed glia culture, and methods for co-culturing these two cell types, allowing for mechanistic investigations into how the presence of microglia and the factors they release in this shared environment may mediate the effect of toxicants on neuronal function and survival. The protocols presented here allow for flexibility in experimental design, the study of numerous toxicological endpoints, and the opportunity to explore neuroprotective strategies.

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Isolation of Cerebellar Granule Cells from Neonatal Rats

Cited by 8 publications

References 16 publications

The effect of methylmercury exposure on behavior and cerebellar granule cell physiology in aged mice

The effect of methylmercury exposure on behavior and cerebellar granule cell physiology in aged mice

The Dipeptidyl-Peptidase-Like Protein DPP6 Determines the Unitary Conductance of Neuronal Kv4.2 Channels

Co‐Culture of Neurons and Microglia

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