A.W. Duggan

Cutaneous stimuli releasing immunoreactive substance P in the dorsal horn of the cat.

In barbiturate-anaesthetized spinal cats, antibody microprobes were used to examine immunoreactive substance P (irSP) release at sites within the spinal cord following cutaneous stimuli. A basal level of irSP release was detected in the region of the substantia gelatinosa of the lumbar spinal cord. No increase in this irSP release was produced by non-noxious thermal or mechanical cutaneous stimulation. Noxious thermal, mechanical or chemical cutaneous stimuli all increased release of irSP in the region of the substantia gelatinosa and in the overlying pia mater. The results support a role for SP in the transmission of information from nociceptors to spinal neurones.

Noxious heating of the skin releases immunoreactive substance P in the substantia gelatinosa of the cat: A study with antibody microprobes

Using a new method, the antibody microprobe technique, the release of immunoreactive substance P (SPiR) in the dorsal horn in response to noxious heating of the skin, was studied in barbiturate anaesthetized spinal cats. Release of SPiR was not produced by immersing the ipsilateral hind paw in water at 37 degrees C. With water at 50 and 52 degrees C, however release was consistently detected in the region of the substantia gelatinosa. These results directly show a central release of SPiR with excitation of nociceptors by heat.

Inhibition of the spinal transmission of nociceptive information by supraspinal stimulation in the cat.

&NA; In cats anaesthetized with &agr;‐chloralose or sodium pentobarbitone a study was made of the effects of supraspinal electrical stimulation on the excitation of dorsal horn neurones by noxious and non‐noxious cutaneous stimuli. Stimulation near the dorsal raphe of the midbrain non‐selectively reduced the responses of neurones to both noxious and non‐noxious stimuli. Intravenous naloxone (0.3–0.6 mg/kg) had no effect on this inhibition. Electrical stimulation near the medullary raphe selectively reduced the excitation of dorsal horn neurones by noxious cutaneous stimuli. Excitation of neurones by deflection of hairs was unaffected. The inhibition of nociceptive responses outlasted the period of raphe stimulation by up to 6 min. Intravenous naloxone (0.6–1.0 mg/kg) also failed to affect this selective inhibition.

Laminar localization of the sites of release of immunoreactive substance P in the dorsal horn with antibody-coated microelectrodes

The localization of the sites of release of immunoreactive substance P (SP) in the spinal cord after peripheral nerve stimulation has been examined in the anaesthetized cat. A new technique using antibodies bound to the outside of glass microelectrodes has allowed the identification of these sites with a spatial precision previously unobtainable. A basal release of SP was detected and this was not increased by electrical stimulation of large myelinated primary afferent fibres. Excitation of unmyelinated primary afferents resulted in the release of high concentrations of SP confined to the region of the substantia gelatinosa and lamina V-VI.

The location of brainstem neurones tonically inhibiting dorsal horn neurones of the cat.

In an investigation of the origin of tonic descending inhibition of dorsal horn neurones by impulses in unmyelinated primary afferents, brainstem regions were electrolytically lesioned. With each neurone studied, tonic descending inhibition was measured before and after brainstem lesions by cooling a segment of spinal cord cephalic to the recording site. Such inhibition was not reduced by lesions of areas which, when stimulated, produce analgesia. These included the periaqueductal grey and the raphé areas of the midbrain and pons-medulla. Tonic descending inhibition was reduced by bilateral lesions of the ventrolateral caudal medulla in the region of the lateral reticular nuclei. Lateral reticular areas may have a functional role in the control of pain.

The preparation and use of antibody microprobes

A new method of detecting release of neuropeptides in the central nervous system is described. Glass micropipettes are treated with gamma-aminopropyltriethoxysilane resulting in a fine outer coating of a siloxane polymer containing free amino groups. Glutaraldehyde is then used to covalently couple protein A which in turn binds antibodies to a particular peptide. Following use in the central nervous system, microprobes are incubated in a radiolabelled form of the peptide being studied and release is detected on autoradiographs as localized zones of inhibition of binding of the labelled peptide. The spatial resolution of the method is at least 100 micron. Necessary tests of the validity of the technique are also described.

Somatostatin: evidence for a role in thermal nociception

In barbiturate-anaesthetized spinalized cats, antibody microprobes were used to investigate the release of immunoreactive somatostatin (irSS) in the lumbar dorsal horn in response to cutaneous stimuli. In the absence of applied stimulation, a significant basal release of irSS was present in the region of the substantia gelatinosa. Such release was not increased by innocuous or noxious cutaneous mechanical stimuli nor by innocuous thermal stimuli, but was increased by noxious thermal stimulation. The magnitude of this noxious heat-evoked release was estimated by comparing in vivo microprobes with those used to detect known concentrations of somatostatin in vitro. Pairs of microprobes were used to detect simultaneous release of both irSS and immunoreactive substance P in the substantia gelatinosa. The results support the putative role of somatostatin in the spinal transmission of thermal nociceptive information.

Periaqueductal grey stimulation: an association between selective inhibition of dorsal horn neurones and changes in peripheral circulation

Abstract In barbiturate‐anaesthetized and paralysed cats, dorsal horn neurones were studied during electrical stimulation of the periaqueductal grey matter (PAG) and the midbrain ventral tegmentum (VT). Responses to impulses in unmyelinated primary afferents were selectively inhibited by stimulation in the PAG, whereas stimulation in the VT non‐selectively reduced both these responses and those to innocuous cutaneous stimuli. Stimulation in the PAG but not the VT produced changes in peripheral circulation. This was observed as a rise in the levels of carbon dioxide in expired air, a rise in muscle temperature in the hind limb and a fall in skin temperature of the pinna or glabrous skin. The combination of suppression of spinal transmission of impulses related to pain and an increase in perfusion of muscles may be a mechanism appropriate to coping with a potentially injurious environment.

Bicuculline and spinal inhibition produced by dorsal column stimulation in the cat

&NA; In barbiturate anaesthetized cats, dorsal column stimulation inhibited ascending volleys recorded in the antero‐lateral spinal fasciculus from electrical stimulation of the contralateral tibial nerve and the excitation of neurones of the dorsal horn by noxious heating of the skin. The inhibition was non‐selective. Intravenous bicuculline (0.2–0.6 mg/kg) reduced dorsal column induced inhibition of ascending volleys. Bicuculline but not strychnine, administered electrophoretically from micropipettes, reduced dorsal column induced inhibition of the excitation of dorsal horn neurones by noxious heat. These findings suggest that the inhibition studied was produced by release of &ggr;‐aminobutyric acid. This amino acid may play a role in the clinical suppression of pain produced by dorsal column stimulation.

Analysis of antibody microprobe autoradiographs by computerized image processing

A computerized digitizing method to analyze the autoradiographs derived from the antibody microprobe technique is described. The method enables the averaging of groups of microprobes that have been placed in similar locations in the central nervous system during the same physiological stimulation. Control and experimental groups of microprobes can be compared, and a level of significance for the release of a neuropeptide obtained. In addition estimates can be made of the concentration of peptide present in regions of release.