Peter J. Snow

Neuropeptides in physiologically identified mammalian sensory neurones

In cats, intracellular dye injection of single sensory neurones of known fibre type and sensory modality has been combined with peptide immunohistochemistry. There was no clear relationship between the sensory function of a neurone and the presence of the neuropeptides substance P, somatostatin, cholecystokinin and vasoactive intestinal polypeptide, in its cytoplasm. In particular, substance P was not detected in many nociceptive sensory neurons even though it could be demonstrated with the same technique in many sensory neurones which did not have cutaneous receptive fields. These results mean that the role, if any, of these neuropeptides in the transmission of pain, must be regarded as complex.

Coexistence of peptide immunoreactivity in sensory neurons of the cat

The coexistence of the neuropeptides substance P, cholecystokinin, somatostatin and vasoactive intestinal polypeptide in cat sensory neurons has been examined using peroxidase-anti-peroxidase immunocytochemistry. Attempts were also made to locate cells containing bombesin, neurotensin, [Met]enkephalin and [Leu]enkephalin but no immunoreactivity was found when antisera to these peptides was used. Cells in the dorsal root ganglia were studied by cutting 5 microns serial wax sections or 15 microns cryostat sections. Coexistence was established by applying the antiserum to each peptide to serially adjacent 5 microns sections and establishing the presence of peptide-like immunoreactivity in each of 4 different sections through a single cell. Results showed that the distribution and combinations of coexistence of these neuropeptides in the cat is extremely complex; three and sometimes all four antisera showing immunoreactivity with a single cell. About 21% of all ganglion cells contained some immunoreactivity but there were certainly some small cells which did not contain any immunoreactivity. The coexistence of these peptides differed markedly from that previously reported in the rat suggesting that interspecific differences in the neuropeptide content of cells might be much greater than they are for classical neurotransmitters. The results are discussed in relation to the possible role of neuropeptides and the regulation of their production by sensory neurons.

The plant lectin Bandeiraea simplicifolia I-B4 identifies a subpopulation of small diameter primary sensory neurones which innervate the skin in the rat

In recent years a variety of glycoconjugates have been found associated with the plasma membrane of mammalian primary sensory neurones. The functional significance of these glycoconjugates remains obscure but their carbohydrate chains have been widely implicated in cell-cell recognition (or adhesion) during development. The plant lectin Bandeiraea simplicifolia I-B4 identifies a characteristic galactose-containing glycoconjugate associated with small diameter primary sensory neurones. In this study we have used a combination of lectin binding and retrograde tracing to examine the distribution of this glycoconjugate on sensory neurones which innervate different target tissues in the rat. The fluorescent tracer diamidino yellow was applied the cut end of peripheral nerves which selectively innervate either the skin, muscle or viscera. Retrogradely labelled neurones were then screened for lectin binding using a lectin-horseradish peroxidase conjugate. The results revealed that Bandeiraea simplicifolia I-B4 binding is associated with over one-third of neurones which innervate the skin, but only a small proportion of neurones innervating muscle or viscera.

Selective labelling of primary sensory afferent terminals in lamina II of the dorsal horn by injection of Bandeiraea simplicifolia isolectin B4 into peripheral nerves

The I-B4 isolectin from Bandeiraea simplicifolia exhibits specific binding to a subpopulation of rat dorsal root ganglion neurons of small diameter which terminate in the substantia gelatinosa of the dorsal horn. Recent double-labelling experiments in the rat have demonstrated that only primary afferents which innervate the skin are recognized by the I-B4 lectin [Plenderleith and Snow (1993) Neurosci. Lett. (in press)]. As the I-B4 lectin appears to bind selectively to a subset of small-diameter primary afferents with cutaneous peripheral projections, we sought to determine whether it could be used as a transganglionic tracer which selectively labels the spinal terminations of cutaneous afferents in superficial dorsal horn. We now report that the I-B4-horseradish peroxidase conjugate labels synaptic terminals in lamina II of the dorsal horn following the injection of the conjugate into the sciatic and saphenous nerves in the rat. Electron-microscopic examination of the dorsal horn revealed many examples of labelled synaptic terminals and unmyelinated axons, but in no cases was label observed in myelinated axons. No label was observed outside of the substantia gelatinosa; thus the I-B4 isolectin is unique among lectins used for transganglionic tracing in that it does not retrogradely label motoneurons. These results, together with previous studies of lectin binding properties of primary sensory afferents, suggest that injection of I-B4 conjugates into peripheral nerves enables the visualization of the central terminations of cutaneous C-fibres. Transganglionic labelling with the I-B4 isolectin from Bandeiraea simplicifolia should facilitate further examination of synaptic relationships of nociceptive cutaneous afferents in the superficial dorsal horn.

The electrophysiological and morphological characteristics of feline dorsal root ganglion cells

The electrophysiological characteristics of physiologically typed L7 and S1 dorsal root ganglion (DRG) cells have been studied in the cat by intracellular recording. The injection of the fluorescent dye Lucifer Yellow has enabled us to study the morphology of these neurons. Our results show that over the entire range of primary sensory afferents there is a linear relationship between the peak rate of rise of somatic action potentials (dv/dt) and axonal conduction velocity. There is a prominent inflexion on the repolarizing phase of the somatic action potentials of group III and group IV afferents. This is not seen in the action potentials of group II or group I afferents. These results correlate with the observation that the total action potential duration (APD) is inversely related to conduction velocity. Primary afferent somata were observed to have an ellipsoidal shape with the long axis in the rostrocaudal dimension. It was observed that for all afferents studied the volume of a dorsal root ganglion cell was linearly related to its peripheral axonal conduction velocity. We were able to show further that group IV somata, some of whose axons supplied nociceptors, were among the smallest in the ganglion.

The coexistence of neuropeptides in feline sensory neurons.

The coexistence of immunoreactivity to the peptides substance P, bombesin, calcitonin gene-related peptide and somatostatin has been determined in single, lumbar and sacral dorsal root ganglion cells in the cat. Colchicine pretreated L7 and S1 dorsal root ganglia were embedded in wax and cut into 5 microns sections. Groups of four, serially adjacent sections were reacted with antisera to one of four peptides using avidin-biotin immunocytochemistry. It was thus possible to determine the coincidence of the four peptides in single cell bodies by examining the immunoreactivity in a ganglion cell in one section and then locating the same cell in three adjacent sections. As a comparison, this procedure was repeated on a different population of ganglion cells using antiserum to substance P, bombesin and calcitonin gene-related peptide only. The results indicate that different combinations of three or four peptides may occur in single, small diameter sensory neurons in the cat. It would appear that immunoreactivity to bombesin and/or calcitonin gene-related peptide coexists with immunoreactivity to substance P in some dorsal root ganglion cells. However, immunoreactivity to each of these peptides was also found to occur alone in single cells. Immunoreactivity to calcitonin gene-related peptide but not to the other three peptides was found to occur in some medium-sized cell bodies (up to 70 microns). Somatostatin-like immunoreactivity was found to have a high level of coexistence with substance P-like immunoreactivity in cells which contained immunoreactivity to these two peptides only. Immunoreactivity to all the four peptides tested was found to occur in 18-26% of ganglion cells which contained at least one peptide.

Ascending projections to nucleus parafascicularis of the cat

Experiments utilizing the retrograde transport of horseradish peroxidase (HRP) were performed in order to locate the cells of origin of ascending projections to the parafascicular nucleus (Pf) of the cat. HRP-labelled cells were identified in several regions of the brain stem including: trigeminal nuclei, vestibular nuclei, nucleus coeruleus, tegmental field, deep layers of the superior colliculus, substantia nigra, dorsal median nucleus of Raphe and periaqueductal grey substance. Of these, the periaqueductal grey contained approximately 40% of labelled neurones. A weak spinal cord projection to Pf originated bilaterally from laminae VI and VII-VIII at C1 and C2 and some neurones were also found contralaterally at C7 and L7.

The plant lectin soybean agglutinin binds to the soma, axon and central terminals of a subpopulation of small-diameter primary sensory neurons in the rat and cat

The distribution of binding of the plant lectin soybean agglutinin to primary sensory neurons has been investigated in the rat and cat. Soybean agglutinin was found to bind to approximately 30% of neurons in the fourth, lumbar dorsal root ganglion of the rat and approximately 50% of neurons in the first, sacral dorsal root ganglion in the cat. Morphometric analysis of these dorsal root ganglia revealed that in both species those neurons which bind soybean agglutinin appear to be a subpopulation of the small-diameter cells. Electron microscopic analysis of the lumbar dorsal roots revealed that soybean agglutinin binds to the plasma membrane of a subpopulation of unmyelinated axons in both species. Myelinated axons did not bind the lectin. In the dorsal horn of the spinal cord, soybean agglutinin bound to components of Lissauers tract and the superficial laminae (laminae I and II) of the dorsal horn in both species. In the dorsal horn lectin binding was limited to lamina I and the outer portion of lamina II (sublamina IIo) in the cat, while in the rat lamina I and the entire dorsoventral extent of lamina II (sublaminae IIo and IIi) were labelled. Thirty days following dorsal rhizotomy, soybean agglutinin binding in the superficial dorsal horn, ipsilateral to the rhizotomy, was abolished in both species. These results show the plant lectin soybean agglutinin to be a histochemical marker for the soma and central terminals of a subpopulation of small-diameter sensory neurons in both the rat and cat. It is suggested that soybean agglutinin binding may be used in conjunction with immunohistochemistry to allow identification of putative neurotransmitters within, or in synaptic profiles associated with, the central terminals of small-diameter primary sensory neurons.

Soybean agglutinin binds to a subpopulation of primary sensory neurones in the cat

The binding of the plant lectin soybean agglutinin (SBA) to primary sensory neurones has been investigated in the rat. SBA binding was found in Lissauers tract and in laminae I and IIo of the dorsal horn at cervical, thoracic and lumbar levels. Morphometric analysis of the S1 dorsal root ganglia revealed that SBA binding was associated with the small diameter cell population, considered to be the cell bodies of unmyelinated afferent fibres (C-fibres). These findings suggest that SBA may be a useful ultrastructural marker for C-fibre terminals.

Transganglionic labelling of primary sensory afferents in the rat lumbar spinal cord: comparison between wheatgerm agglutinin and the I-B4 isolectin from Bandeiraea simplicifolia.

SummaryWe recently reported that the I-B4 isolectin fromBandeiraea simplicifolia could be used as a transganglionic neuronal tracer which appears to be selective for unmyelinated cutaneous afferents (C fibres) and their terminals in the superficial dorsal horn. As terminals in the superficial dorsal horn are also labelled by wheatgerm agglutinin, we sought to compare these two neuronal tracers. Three days after the injection of 1% wheatgerm agglutinin-HRP or 1% BSI-B4—HRP into the sciatic nerve of adult rats the lumbar spinal cord was processed for HRP reactivity. The majority of labelled structures was found in the superficial dorsal horn, with fewer labelled structures seen in the overlying white matter (including Lissauers tract). In wheatgerm agglutinin-HRP experiments most labelled structures were synaptic terminals (63%) and unmyelinated axons (32%). About 3% of wheatgerm agglutinin-HRP-labelled structures were fine myelinated fibres (which were found only in lamina I and outer lamina II) and about 2% of label was located in neuronal somata. In contrast, label from BSI-B4—HRP experiments was found only in synaptic terminals (37%) and unmyelinated axons (63%). Previous studies have shown that small diameter dorsal root ganglion neurons and their terminals in the superficial dorsal horn express a range of structurally related carbohydrates that contain binding sites for BSI-B4 or wheatgerm agglutinin or both. Comparison of the labelling patterns produced by the two transganglionic tracers in the present study suggests that unmyelinated sciatic afferents express wheatgerm agglutinin and BSI-B4 binding sites, but some thin myelinated afferents, and a distinct form of synaptic terminal in lamina I/II outer, express the wheatgerm agglutinin binding site and not the BSI-B4 binding site.