A sequential fluorescence method for neurotransmitter-specific retrograde tracing in the central nervous system of the rat; utilizing True Blue and immunohistochemistry in combination with computer-assisted photography.
Aiming to map the distribution of spinally projecting, hypothalamic neurons containing neuronal nitric oxide synthase (nNOS), True Blue (TB) is injected into the rat spinal cord. After survival times of 7-14 days the animals are anaesthetized and perfused transcardially with a solution containing paraformaldehyde and sucrose. After dissection, the injection site is further fixed for 4-8 h, cut in a cryostat, and documented by computer-assisted digital photography. The brain region of interest is fixed for 4 h, rinsed in phosphate buffer for 48 h, sectioned, and photographically documented utilizing filter settings for visualization of TB. The brain sections are then immunohistochemically processed using a primary antibody against nNOS and a Texas Red (TR)-labelled secondary antibody and once again photographically documented, now using filter settings for visualization of TB and TR, respectively. Utilizing the Photoshop program, the TB containing cells can then be exactly aligned and the presence of TB and/or TR fluorescence in the same cell bodies are evaluated. This method for neurotransmitter-specific retrograde tracing derives its high sensitivity from the optimization of fixation/rinsing parameters, the use of appropriate fluorophores, and sequential digital microphotography.
Nylén
Anders
A
Department of Clinical and Experimental Pharmacology, Institute of Laboratory Medicine, Lund University Hospital, Lund University, S-221 85 Lund, Sweden. anders.nylen@klinfarm.lu.se
Larsson
Bengt
B
Skagerberg
Gunnar
G
D000818:Animals; D000906:Antibodies; D001572:Benzofurans; D002490:Central Nervous System / Q000378:metabolism*; D005260:Female; D005453:Fluorescence; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007150:Immunohistochemistry / Q000379:methods*; D009434:Neural Pathways / Q000033:anatomy & histology / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000737:chemistry* / Q000378:metabolism; D010784:Photomicrography / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012996:Solutions; D016707:Tissue Fixation; D014966:Xanthenes
D000906:Antibodies; D001572:Benzofurans; D005456:Fluorescent Dyes; D018377:Neurotransmitter Agents; D012996:Solutions; D014966:Xanthenes; C038675:true blue; C034657:Texas red