Strategies for cellular identification in nucleus tractus solitarius slices.
The indistinct regional anatomy and intermixing of second order neurons with projection and interneurons make cellular studies more difficult within the nucleus tractus solitarius (NTS). Here, we outline experimental strategies to join in vitro electrophysiological with neuroanatomical protocols to discriminate specific subpopulations of NTS neurons. Horizontally cutting the brain stem produces slices in which electrical activation of the solitary tract (ST) is free of local interneuron contamination. Such ST excitatory synaptic currents (EPSCs) functionally identify second order NTS neurons by their minimal variation of latency (jitter). Sapphire blades, cold cutting temperatures and a mechanically stable microtome were critical to consistently obtain viable slices that were optimized for infrared and fluorescence microscopy. Anterogradely transported carbocyanine dye implanted on the aortic depressor nerve anatomically identified second order NTS neurons and their ST synaptic performance conformed to the minimal jitter signature of second order neurons. Retrograde tracers and green fluorescent protein labeled neurons afford two additional promising approaches for discriminating NTS neuron phenotypes in broader system contexts. Detailed methods and troubleshooting are described. Coupling tracing techniques with electrophysiology adds important new dimensions to NTS studies and such strategies provide bridging information between cellular mechanisms, neuroanatomy and systems integration.
Doyle
Mark W
MW
Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR 97239-3098, USA.
Bailey
Timothy W
TW
Jin
Young-Ho
YH
Appleyard
Suzanne M
SM
Low
Malcolm J
MJ
Andresen
Michael C
MC
D000818:Animals; D001370:Axonal Transport / Q000502:physiology; D004210:Dissection / Q000295:instrumentation / Q000379:methods*; D004594:Electrophysiology / Q000295:instrumentation / Q000379:methods*; D019706:Excitatory Postsynaptic Potentials / Q000502:physiology; D005456:Fluorescent Dyes; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008867:Microtomy / Q000295:instrumentation / Q000379:methods; D009434:Neural Pathways / Q000166:cytology / Q000502:physiology; D009474:Neurons / Q000145:classification / Q000166:cytology / Q000502:physiology; D009482:Neurophysiology / Q000295:instrumentation / Q000379:methods*; D009924:Organ Culture Techniques / Q000295:instrumentation / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011930:Reaction Time / Q000187:drug effects / Q000502:physiology; D017552:Solitary Nucleus / Q000166:cytology / Q000502:physiology*; D013055:Spectrophotometry, Infrared / Q000295:instrumentation / Q000379:methods; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods*; D009435:Synaptic Transmission / Q000502:physiology
D005456:Fluorescent Dyes