C.R. Morton

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.

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.

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.

Lateral reticular regions and the descending control of dorsal horn neurones of the cat: Selective inhibition by electrical stimulation

In barbiturate-anaesthetized cats, brainstem sites were electrically stimulated while studying the synaptic responses of lumbar dorsal horn neurones. The excitation of these neurones by impulses in unmyelinated primary afferents was selectively inhibited by stimulation of the ventrolateral medulla in the region of the caudal lateral reticular nucleus. The significance of this inhibition was heightened by the finding that stimulus currents producing inhibition from this area were less effective in the raphé region and not effective at intervening or dorsal sites. Bilateral lesions of the inhibition-producing ventrolateral sites reduced tonic descending inhibition of the responses of dorsal horn neurones to impulses in C fibres. The lateral reticular regions of the medulla may thus exert a considerable control over the transmission of nociceptive information in the cat spinal cord.

The effects of lesions of medullary midline and lateral reticular areas on inhibition in the dorsal horn produced by periaqueductal grey stimulation in the cat

In barbiturate-anaesthetized cats, the excitation of lumbar dorsal horn neurones by impulses in unmyelinated primary afferent fibres was inhibited by electrical stimulation in the periaqueductal grey matter. This inhibition was slightly reduced by extensive electrocoagulation of the medullary midline and para-medial areas including the raphé, but significantly reduced by small bilateral lesions in the region of the caudal lateral reticular nuclei. When the lateral lesions were made subsequent to midline coagulation, the inhibition from periaqueductal grey stimulation was abolished. An important component of spinal inhibition from periaqueductal grey stimulation appears to relay in lateral reticular areas of the medulla.

Dynorphin A: in vivo release in the spinal cord of the cat

The antibody microprobe technique was used to study the release of immunoreactive dynorphin A within the lower lumbar spinal cord of anaesthetised cats. A basal release was observed in the dorsal horn, centered in the region of lamina I, but was abolished by spinal cord transection at the thoracolumbar junction. Release of dynorphin A in the lamina I region was evoked by high-frequency electrical stimulation of unmyelinated primary afferent fibres, whereas stimulation of myelinated (including A delta) afferents was ineffective. Evidence was also obtained for release in laminae V-VI and at the spinal cord surface. These results suggest that in the lumbar spinal cord of the cat, dynorphin A is released in the superficial dorsal horn by impulses in descending pathways and in somatic unmyelinated primary afferent fibres.