I'm trying to ricreate in vitro the developmental shift of [Cl]i which take place in neuron during the first two postnatal weeks. Since I haven't observed any change in primary neuron cultures from P2 mice I'm considering to coat dishes whith a substrate which mimics CNS's ECM (I suppose that interaction between ecm proteins and cell molecules can play a role in the process and in the maturation of a sinchronized cortical network which is subject of study in my lab). I found that MAXGEL ECM from Sigma-Aldrich (http://www.sigmaaldrich.com/catalog/product/sigma/e0282?lang=it®ion=IT) contains the most relevant ECM so I'm going to give it a try. I don't exactly know if the matrix interpheres with patch-clamp experiments (bad gigaseal formation, etc), does anybody has any experience with it?
Dear Simone,
I personally don't have experience with patchclamp recording on this matrix but have a look at those papers, it it should be feasible...
Coates, P. W., and R. D. Nathan, “Feasibility of Electrical Recordings from Unconnected Vertebrate CNS Neurons Cultured in a Three-Dimensional Extracellular Matrix,” J. Neurosci. Methods, Vol. 20, No. 3, 1987, pp. 203–210.
Ma, W., et al., “CNS Stem and Progenitor Cell Differentiation into Functional Neuronal Circuits in Three-Dimensional Collagen Gels,” Exp. Neurol., Vol. 190, No. 2, 2004, pp. 276–288.
Irons, H. R., et al., “Three-Dimensional Neural Constructs: A Novel Platform for Neurophysiological Investigation,”J. Neural Eng., Vol. 5, No. 3, 2008, pp. 333–341.
Good luck!
Dear Simone,
I have used this matrix on collagen coated glass cover slips with excellent results. I have used hippocampal neurons, DRGs and cortical neurons for patch clamping using these cover slips with no problem. You may also like to add NGF in the culture system for encouraging survival of the neurons. What medium are using by the way?
I'm using neurobasal+Glutamine+B27; in the past I used AraC from 10 div to avoid excessive glial growth (cell visualization and seal formation became difficult in these conditions), I'll try to avoid it to see if the switch is triggered by something secreted from glial cells
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