534
Brain Research, 122 11977) 534-.541.)
Elsevier/North-Holland Biomedical Press, Amsterdam
Printed in The Netherlands
Short Communications
Ultrastructural localization of substance P in neurons of rat spinal cord
VIRGINIA M. PICKEL, D O N A L D J. REIS and SUSAN E. LEEMAN Laboratoo" o["Neurobiology, Department O! Neuroh~,,l', Cornell University Medical Cotlegc, ;~ew )or],, N.Y. 10021 and (S.E.L.) Department of Physiology, Harvard Medical School, Boston, Mass. 02115 (U.S.A.~
(Accepted November 19th, 1976)
Substance P is a polypeptide originally detected in the equine brain and gut by yon Euler and Gaddum 4 and first independently isolated from rat brain as a peptide with sialogogic properties by Chang and Leeman e,ia. The peptide is widely, but unequally, distributed throughout the central nervous system la,el. Its high concentrations, as determined by bioassay or more recently by radioimmunoassay, m areas receiving sensory afferent fibers has led to the speculation that it may be a neurotransmitter in primary sensory systems v',l:~.~,-'~ That substance P is contained within sensory neurons has been recently demonstrated by immunofluorescence in which, by light microscopy, the peptide was localized to neuronal perikarya in dorsal root ganglia and to neuronal processes within the dorsal horn of the spinal cord ~,9. However, light microscopy cannot establish whether substance P is contained within axon terminals and, if so, whether it is associated with any specific subcellular organelles. The present study therefore sought to determine the ultrastructural localization of substance P within axon~ of sensory neurons which terminate in the dorsal horn of the rat spinal cord. This was achieved by localization of antibody to substance P by the peroxidase-antiperoxidase (PAPJ method 19, which offers the high resolution and specificity necessary I'or electron microscopy. Synthetic substance P (Beckman Instruments Co.), coupled to succinylated thyroglobulin, was suspended in saline and emulsified in Freund's complete adjuwlnt supplemented with killed tubercle bacilli. Antiserum was raised in rabbits following multiple intradermal injection of the conjugate according to the method of Vaitukaitis et al. ~>. The titer of the antiserum used in these experiments was such that 50
Brain Research, 122 11977) 534-.541.)
Elsevier/North-Holland Biomedical Press, Amsterdam
Printed in The Netherlands
Short Communications
Ultrastructural localization of substance P in neurons of rat spinal cord
VIRGINIA M. PICKEL, D O N A L D J. REIS and SUSAN E. LEEMAN Laboratoo" o["Neurobiology, Department O! Neuroh~,,l', Cornell University Medical Cotlegc, ;~ew )or],, N.Y. 10021 and (S.E.L.) Department of Physiology, Harvard Medical School, Boston, Mass. 02115 (U.S.A.~
(Accepted November 19th, 1976)
Substance P is a polypeptide originally detected in the equine brain and gut by yon Euler and Gaddum 4 and first independently isolated from rat brain as a peptide with sialogogic properties by Chang and Leeman e,ia. The peptide is widely, but unequally, distributed throughout the central nervous system la,el. Its high concentrations, as determined by bioassay or more recently by radioimmunoassay, m areas receiving sensory afferent fibers has led to the speculation that it may be a neurotransmitter in primary sensory systems v',l:~.~,-'~ That substance P is contained within sensory neurons has been recently demonstrated by immunofluorescence in which, by light microscopy, the peptide was localized to neuronal perikarya in dorsal root ganglia and to neuronal processes within the dorsal horn of the spinal cord ~,9. However, light microscopy cannot establish whether substance P is contained within axon terminals and, if so, whether it is associated with any specific subcellular organelles. The present study therefore sought to determine the ultrastructural localization of substance P within axon~ of sensory neurons which terminate in the dorsal horn of the rat spinal cord. This was achieved by localization of antibody to substance P by the peroxidase-antiperoxidase (PAPJ method 19, which offers the high resolution and specificity necessary I'or electron microscopy. Synthetic substance P (Beckman Instruments Co.), coupled to succinylated thyroglobulin, was suspended in saline and emulsified in Freund's complete adjuwlnt supplemented with killed tubercle bacilli. Antiserum was raised in rabbits following multiple intradermal injection of the conjugate according to the method of Vaitukaitis et al. ~>. The titer of the antiserum used in these experiments was such that 50
