Ian A. Hendry

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 response of adrenergic neurones to axotomy and nerve growth factor.

Division of the axons of adrenergic neurones by crushing the postganglionic nerve trunks of rat superior cervical ganglia (SCG) at 6 days of age resulted in a permanent atrophy of the SCG reflected by a persistent decrease in the total protein content and in the activities of the enzymes tyrosine hydroxylase and DOPA decarboxylase. Administration of nerve growth factor (NGF) to rats with unilateral axotomy at a dose of 10 mug/g/day for the period 7-21 days of age resulted in hypertrophy of both normal and axotomised SCG. There was a progressive rise in the total protein content and in the activities of the two enzymes till the end of the treatment period in both SCG. After treatment ceased there was a progressive fall in the total protein content and activities of the two enzymes reaching a stable level after 4 weeks. The level reached for treated unoperated SCG remained elevated when compared to untreated control SCG. Axotomised treated SCG had approximately the same biochemical parameters as untreated control SCG and very much elevated over untreated axotomised SCG. These final levels persisted for at least 56 days after treatment had ceased. Animals showed a persistent ptosis after axotomy at 6 days of age but treatment with NGF resulted in a functional recovery by 11 weeks of age. It is suggested that there is normally a retrograde transfer of a factor durind development from the target cell to the perikarya of the neurone permitting survival if the appropriate connections are made. Failure to make such a contact results in cedd death. The cell death occurring normally, and the cell death resulting from axotomy, can both be prevented by NGF treatment leading to an hypertrophy of both SCG. This consistent with the hypothesis than NGF is the retrograde trophic agent for the sympathetic nervous system in the developing animal.

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.

Retrograde axonal transport of signal transduction proteins in rat sciatic nerve

Neurons require a mechanism to transmit stable signals over the large distance from the nerve growth cone or terminal to the cell body, in order that information from the target tissue can be relayed to the cell body where it is required. Nerve growth factor (NGF), a target-derived neurotrophic factor, is thought to signal over this distance by receptor mediated internalization of NGF, followed by retrograde axonal transport of the NGF-receptor complex. In this paper we show, by immunohistochemistry of rat sciatic nerve, accumulation of phosphotyrosine immunoreactivity only on the distal side of a nerve crush, suggesting axonal transport of tyrosine kinases and/or tyrosine phosphorylated proteins primarily in a retrograde direction. Furthermore, we also show retrograde axonal transport of phosphoinositide 3-kinase, ERK, MEK and MEK kinase, of which all but MEK kinase are known to be activated downstream of tyrosine receptor kinase activation. The retrograde transport of these proteins suggests that they may be involved in transmission of signals along the axon, relaying neurotrophic factor receptor activation at the nerve terminal to the nerve cell body.

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.

Molecular mechanisms regulating the retrograde axonal transport of neurotrophins.

Neurotrophins are released from target tissues following neural innervation and bind to specific receptors situated on the nerve terminal plasma membrane. The neurotrophin-receptor complex undergoes retrograde axonal transport towards the cell soma, where it signals to the nucleus. This process allows neurotrophins to perform their numerous functions, which include the promotion of neuronal survival and the outgrowth of axons towards certain target tissues. The molecular events controlling each of the components of retrograde axonal transport are beginning to become defined. There is good evidence for the participation of phosphatidylinositol 3-kinase, phosphatidylinositol 4-kinase and the actin cytoskeleton in neurotrophin retrograde axonal transport in vivo. It also appears that the retrograde motor protein dynein mediates the retrograde axonal transport in vivo of neurotrophins such as nerve growth factor. This review discusses the role of the neurotrophin receptors in binding and axonal transport, the endocytic processes required for neurotrophin internalization, the targeting and trafficking of neurotrophins, and the propagation of neurotrophin-induced signals along the axon.

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.

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.

The effects of axotomy on the development of the rat superior cervical ganglion.

The effects of division of the postganglionic axons of the adrenergic neurones in the superior cervical ganglion of the rat were examined with regard to the total ganglionic protein content and tyrosine hydroxylase and DOPA decarboxylase activities. Axotomy before the twelfth postnatal day results in a marked atrophy of the ganglion and a reduction in the total enzyme content of the ganglion. Axotomy after postnatal day 21 results in the normal adult ganglion response with a large increase in the total protein content of the ganglion and only minor changes in the total enzyme content of the ganglion. Axotomy between the postnatal days 12 and 21 results in an intermediate response. Thus it can be concluded there is a critical period during the development of the ganglion during which the adrenergic neurones undergo a maturation governed by their contact with the peripheral target cell via axons. These results suggest a trophic role of the end organ on the adrenergic neurone.