HyQ Trypsin from H圜lone SH30042.01) incubate in 37 ☌ water bath for ~15 min remove trypsin/EDTA as much as possible with a disposable plastic pipette,ĭissociate the digested tissues mechanically by gentle pipetting with a 5 ml pipette (~10 strokes, avoid creating air bubbles), add 2 ml medium, let settle for 30 sec, then transfer 2 ml supernatant into a 15 ml tube. Pool dissected cortical tissues, mince briefly and transfer to a 15 ml tube containing 4 ml pre-warmed trypsin/EDTA (0.25% trypsin, 0.53 mM EDTA Hold brain stem with one forceps, separate hemispheres with another forceps, remove hippocampus and striatum, separate cortex and carefully peel the cortical tissue off from meninges. Remove the whole brain with a curved forceps (in a scoop motion) and place in a 100 mm petri dish containing 10 ml ice-cold Ca2 +- and Mg 2+-free HBSS. Prepare high-density cortical neuron cultures from Sprague-Dawley rat E18 embryos and culture in glia-conditioned serum-free medium 1-2.Įuthanize one pregnant rat (E18) according to ACUC procedures quickly remove uterus (with fetuses in it) and place in a 100 mm Petri dish on ice.Ĭut open uterus and amniotic membrane, hold fetus by the neck (umbilical cord intact) with one forceps, use another forceps to peel scalp from back to front, and slit the skull open with a sharp forceps tip along the midline from back to front.
Preparation of primary cortical neuron cultures These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of theseġ. Vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological This system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these Neuropathological studies have demonstrated thatĪ number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. Signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Within dendritic spines, a complex network of proteins link extracellular In response to classical Hebbian plasticityĪs well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neuralĬircuits and the processing and storage of information within the brain. These structures are rich in actin and have been shown to be highly dynamic. Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synapticĬompartment of synapses.
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