Transmembrane dye labeling and immunohistochemical staining of electrophysiologically characterized single neurons.
Numerous studies have used whole-cell patch recording to characterize the electrophysiology of neurons and, via intracellular dye filling, the detailed morphology of the same cells. However, it has been difficult to demonstrate the presence of small soluble molecules within such cells, because washout of the soluble contents of the cell into the patch pipette precludes their later detection by immunohistochemistry. This leaves a major gap in our understanding of circuits made up of neurochemically heterogeneous neurons. To bridge this gap we have developed a transmembrane labeling approach, employing membrane-permeant dye in conjunction with perforated patch electrophysiology. Using this method we have successfully recorded from juxtaglomerular cells in the olfactory bulb, reconstructed the morphology of the cells, and demonstrated expression of soluble neurochemical markers within the same cells. This new technique provides a reliable means to link the physiology, morphology, and neurochemistry of single identified neurons studied using patch-clamp recording.
Puche
Adam C
AC
Department of Anatomy and Neurobiology, Program in Neuroscience, School of Medicine, The University of Maryland, Rm. 222, 685 West Baltimore St., Baltimore, MD 21201, USA.
Heyward
Philip
P
Shipley
Michael T
MT
D000818:Animals; D002766:Cholecystokinin / Q000378:metabolism; D004594:Electrophysiology / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D009474:Neurons / Q000502:physiology*; D009830:Olfactory Bulb / Q000166:cytology; D018408:Patch-Clamp Techniques / Q000379:methods*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D005452:Fluoresceins; C069306:5-chloromethylfluorescein; D002766:Cholecystokinin; D014446:Tyrosine 3-Monooxygenase