Identification of Select Autonomic and Motor Neurons in the Rat Spinal Cord Using Retrograde Labeling Techniques and Post-embedding Immunogold Detection of Fluorogold in the Electron Microscope

Abstract

It is of interest to combine intracellular neuronal labeling techniques with immunohistochemistry for ultrastructural studies, but experimental options for investigators have been limited. Recently, protocols have been developed to allow for electron microscopic detection of retrogradely labeled spinal cord neurons in combination with post-embedding immunogold studies. Retrogradely labeled preganglionic sympathetic and parasympathetic neurons are identified in the rat spinal cord after injection of fluorogold (FG) into the major pelvic ganglia. Similarly, injection of FG into the rat external urethral sphincter muscle retrogradely labels motoneurons in the dorsolateral (DL) nucleus of the L6 spinal cord segment. Tissues are next preserved by intravascular perfusion with a fixative solution containing 2 % paraformaldehyde and 1–2 % glutaraldehyde. Vibratome sectioning of the spinal cord tissue is followed by cryo-protection, snap freezing, and dehydration in methanol containing 1–2 % uranyl acetate using a freeze substitution protocol. Finally, the sections are embedded in the Lowicryl HM20 resin at low temperatures. Post-embedding immunogold detection of FG localizes the tracer in the lysosomes of labeled neurons. The protocol is versatile and can be combined with immunogold detection of, e.g., neurotransmitters and membrane transporters and therefore may have broad applicability to ultrastructural studies on the central nervous system.