A time and cost efficient approach to functional and structural assessment of living neuronal tissue.
In this manuscript, we describe a protocol for capturing both physiological and structural properties of living neuronal tissue. An essential aspect of this method is its flexibility and fast turnaround time. It is a streamlined process that includes recording of electrophysiological neuronal activity, calcium imaging, and structural analysis. This is accomplished by placing intact tissue on a modified Millicell Biopore insert. The Biopore membrane suspends the tissue in the perfusion solution, allowing for complete access to nutrients, oxygen, and pharmacological agents. The ring that holds the membrane ensures its structural stability; forceps can be used to grip the ring without contacting the filter or the tissue, for easy transfer between multiple setups. We show that tissue readily adheres to the surface of the membrane, its entire surface is visible in transmitted light and accessible for recording and imaging, and remains responsive to physiological stimuli for extended periods of time.
Department of Ophthalmology, Weill Medical College of Cornell University, Burke Medical Research Institute, White Plains, NY 10605, USA.
Abduqodir H
AH
Toychiev
Bakhodir
B
Sagdullaev
Christopher W
CW
Yee
Elena
E
Ivanova
Botir T
BT
Sagdullaev
D000814:Aniline Compounds / Q000032:analysis; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001921:Brain / Q000502:physiology / Q000648:ultrastructure; D002118:Calcium / Q000032:analysis; D003365:Costs and Cost Analysis; D003470:Culture Media; D004594:Electrophysiology / Q000295:instrumentation* / Q000379:methods; D005452:Fluoresceins / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000235:genetics; D008567:Membranes, Artificial; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques; D020419:Photoreceptor Cells, Vertebrate / Q000502:physiology / Q000528:radiation effects; D011138:Polytetrafluoroethylene; D012160:Retina / Q000502:physiology* / Q000528:radiation effects; D051245:Retinal Bipolar Cells / Q000502:physiology / Q000528:radiation effects; D012165:Retinal Ganglion Cells / Q000502:physiology / Q000528:radiation effects; D012171:Retinal Vessels / Q000648:ultrastructure; D012235:Rhodamines; D013997:Time Factors; D046509:Tissue Culture Techniques / Q000191:economics / Q000295:instrumentation*
D000814:Aniline Compounds; D001426:Bacterial Proteins; D003470:Culture Media; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D008567:Membranes, Artificial; C503942:Oregon green 488 BAPTA-1; D012235:Rhodamines; C054562:yellow fluorescent protein, Bacteria; D011138:Polytetrafluoroethylene; C050829:sulforhodamine 101; D002118:Calcium