Optimized protocols for the simultaneous preparation of primary neuronal cultures of the neocortex, hippocampus and cerebellum from individual newborn (P0.5) C57Bl/6J mice

Ahlemeyer, Barbara; Baumgart-Vogt, Eveline

doi:10.1016/j.jneumeth.2005.05.022

Cited by 66 publications

(52 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Primary microglia were isolated from mixed glial cultures prepared from cerebral cortices of C57BL/6 neonates of either sex, as described previously (27,28). Cultures of mouse primary neurons were prepared from C57BL/6 day 17 embryos or neonates as described previously with modifications (29,30,31). Briefly, the dissected hippocampi were digested, dissociated by trituration, and then seeded at a density of 1 ϫ 10 5 cells per well on poly-L-lysine-coated coverslips in 24-well plates or glass chamber slides.…”

Section: Methodsmentioning

confidence: 99%

Neurons Are Host Cells for Mycobacterium tuberculosis

et al. 2014

View full text Add to dashboard Cite

Section: Methodsmentioning

confidence: 99%

Neurons Are Host Cells for Mycobacterium tuberculosis

et al. 2014

View full text Add to dashboard Cite

“…The protocols are described in SI Experimental Procedures and earlier reports (17,18,(47)(48)(49)(50)(51). All animal procedures were performed in accordance with the National Institutes of Heath Guide for the Care and Use of Laboratory Animals, under protocols approved and strictly followed by the Johns Hopkins Animal Care and Use Committees.…”

Section: Methodsmentioning

confidence: 99%

Phosphatidylinositol 4,5-bisphosphate alters pharmacological selectivity for epilepsy-causing KCNQ potassium channels

Zhou

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Pharmacological augmentation of neuronal KCNQ muscarinic (M) currents by drugs such as retigabine (RTG) represents a first-in-class therapeutic to treat certain hyperexcitatory diseases by dampening neuronal firing. Whereas all five potassium channel subtypes (KCNQ1-KCNQ5) are found in the nervous system, KCNQ2 and KCNQ3 are the primary players that mediate M currents. We investigated the plasticity of subtype selectivity by two M current effective drugs, retigabine and zinc pyrithione (ZnPy). Retigabine is more effective on KCNQ3 than KCNQ2, whereas ZnPy is more effective on KCNQ2 with no detectable effect on KCNQ3. In neurons, activation of muscarinic receptor signaling desensitizes effects by retigabine but not ZnPy. Importantly, reduction of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) causes KCNQ3 to become sensitive to ZnPy but lose sensitivity to retigabine. The dynamic shift of pharmacological selectivity caused by PIP 2 may be induced orthogonally by voltagesensitive phosphatase, or conversely, abolished by mutating a PIP 2 site within the S4-S5 linker of KCNQ3. Therefore, whereas drugchannel binding is a prerequisite, the drug selectivity on M current is dynamic and may be regulated by receptor signaling pathways via PIP 2 . . They open at subthreshold voltages and the channel activity is functionally down-regulated by G protein coupled receptor (GPCR) signaling pathways through intracellular secondary messengers, primarily phosphatidylinositol 4,5-bisphosphate (PIP 2 ) (1, 2). In the nervous system, KCNQ2 and KCNQ3 subtypes are major determinants for potassium current sensitive to muscarinic (M) receptor signaling, hence also known as M current (3). The combination of sensitivity to subthreshold voltages and down-regulation by receptor signaling makes M current a critical component for controlling membrane potential in the nervous system, and consequently neuronal firing (4-6). KCNQ2-5 channels, with few exceptions (7), are primarily expressed in the brain and peripheral nervous system. KCNQ1, although it is found in brain (8), is more abundantly expressed in the heart to mediate slow delayed rectifier K + current (also known as I Ks ) that contributes to the termination of action potential, hence regulating QT duration (9).Retigabine (RTG) or ethyl N-[2-amino-4-[(4-fluorophenyl) methylamino] phenyl]carbamate, a positive allosteric modulator (or an opener) for neuronal KCNQ channel, is a first-in-class therapeutic for antiepileptic treatment (10-12). Its clinical use to dampen the hyperexcitatory activity has greatly intensified the interest in a more detailed understanding of chemical augmentation of voltage-sensitive channel activity. Retigabine has selectivity for KCNQ2, -3, -4, and -5 potassium channels but not KCNQ1 potassium channels (13). Tryptophan (W) 236 in KCNQ2 is conserved among the sensitive channels. Interestingly, KCNQ2 channels with W236L mutation (or KCNQ2 W236L ) are functional but no longer sensitive to retigabine (14,15). This and other evidence supports the idea that W236 is a ...

show abstract

“…P19 cells were cultured and differentiated according to previously published protocols (Yao et al 1995). Cortical neural precursors were cultured according to Feng et al (2005) with minor modifications (Ahlemeyer and Baumgart-Vogt 2005;Feng et al 2005). Briefly, cortices from E10.5 or E14 mouse embryos were dissected in Hank's balanced salt solution (HBSS).…”

Section: Tissue Culturementioning

confidence: 99%

“…Cultures were fed with 10 ng/mL basic fibroblast growth factor (bFGF) (Peprotech) at the time of plating. Cerebellar culture was carried out according to Ahlemeyer and Baumgart-Vogt (2005). Cerebelli were dissected out from P3-P5 mice in Neurobasal medium (Invitrogen) containing 0.02% BSA and were digested at 37°C in 0.25% trypsin in HBSS.…”

Section: Tissue Culturementioning

confidence: 99%

A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons

Boutz¹,

Stoilov²,

Li³

et al. 2007

Genes Dev.

495

608

View full text Add to dashboard Cite

Many metazoan gene transcripts exhibit neuron-specific splicing patterns, but the developmental control of these splicing events is poorly understood. We show that the splicing of a large group of exons is reprogrammed during neuronal development by a switch in expression between two highly similar polypyrimidine tract-binding proteins, PTB and nPTB (neural PTB). PTB is a well-studied regulator of alternative splicing, but nPTB is a closely related paralog whose functional relationship to PTB is unknown. In the brain, nPTB protein is specifically expressed in post-mitotic neurons, whereas PTB is restricted to neuronal precursor cells (NPC), glia, and other nonneuronal cells. Interestingly, nPTB mRNA transcripts are found in NPCs and other nonneuronal cells, but in these cells nPTB protein expression is repressed. This repression is due in part to PTB-induced alternative splicing of nPTB mRNA, leading to nonsense-mediated decay (NMD). However, we find that even properly spliced mRNA fails to express nPTB protein when PTB is present, indicating contributions from additional post-transcriptional mechanisms. The PTB-controlled repression of nPTB results in a mutually exclusive pattern of expression in the brain, where the loss of PTB in maturing neurons allows the synthesis of nPTB in these cells. To examine the consequences of this switch, we used splicing-sensitive microarrays to identify different sets of exons regulated by PTB, nPTB, or both proteins. During neuronal differentiation, the splicing of these exon sets is altered as predicted from the observed changes in PTB and nPTB expression. These data show that the post-transcriptional switch from PTB to nPTB controls a widespread alternative splicing program during neuronal development.[Keywords: Alternative splicing; neuronal development; nonsense-mediated decay; polypyrimidine tract-binding proteins; splicing microarray; ultraconserved element] Supplemental material is available at http://www.genesdev.org. Alternative pre-mRNA splicing is a common mechanism for diversifying genetic output in metazoan organisms (Black 2003;Matlin et al. 2005). Alternative choices in exons and splice sites can create substantial changes in the encoded protein and its activity. Changes in splicing can also affect downstream regulatory processes such as nonsense-mediated decay (NMD), and thus direct additional levels of post-transcriptional gene regulation (Lewis et al. 2003;Lejeune and Maquat 2005;Hughes 2006). Transcripts exhibiting multiple splicing patterns are especially prevalent in the mammalian nervous system, where alternative splicing affects important processes such as axon guidance, synaptogenesis, and the regulation of membrane physiology (Black and Grabowski 2003;Lipscombe 2005;. The choice of splicing pattern within a transcript is generally controlled by RNA-binding proteins that bind to the pre-mRNA to enhance or silence particular splicing events (Black 2003;Matlin et al. 2005). Some splicing regulators are expressed in a tissue-specific manner and have been sh...

show abstract

Optimized protocols for the simultaneous preparation of primary neuronal cultures of the neocortex, hippocampus and cerebellum from individual newborn (P0.5) C57Bl/6J mice

Cited by 66 publications

References 39 publications

Neurons Are Host Cells for Mycobacterium tuberculosis

Neurons Are Host Cells for Mycobacterium tuberculosis

Phosphatidylinositol 4,5-bisphosphate alters pharmacological selectivity for epilepsy-causing KCNQ potassium channels

A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons

Contact Info

Product

Resources

About