Pd Kitchener

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.

Plasticity of cutaneous primary afferent projections to the spinal dorsal horn

Reorganization of the somatotopic map in the spinal dorsal horn may be elicited by a variety of deafferenting lesions, including transection of peripheral nerves or dorsal roots, or the application of neurotoxins. While such lesions give rise to a variety of neurochemical and morphological changes in the dorsal horn, collateral sprouting of intact primary afferents appears to be minimal. Recently, intraaxonal injection of neurobiotin has allowed visualization of the entire spinal arborization of single A beta primary afferent fibers in animals where the somatotopy of the relevant region of dorsal horn has also been mapped. In contrast to the somatotopic precision of the terminal fields of peripheral nerves suggested by transganglionic tracing, these studies have shown that afferents make connections many millimeters rostral and caudal to the region where their receptive field is represented in the somatotopic map. Intracellular recording from dorsal horn neurons has further shown that these long-ranging projections make functional, but weak, synaptic connections. Thus the functional somatotopic reorganization that follows nerve lesions in mature animals might be explained simply by an increased synaptic efficacy of these existing projections. In contrast to the negligible sprouting of intact A beta primary afferents, those undergoing axonal regeneration exhibit dense collateral sprouting into deafferented regions of the dorsal horn, particularly the superficial laminae, where the terminal arbors of many small (A delta and C) nociceptive afferent fibres degenerate following peripheral nerve lesions. The inappropriate connections made by these collateral sprouts may partly underlie the painful sequelae of nerve injury in man.

A quantitative approach to recording peristaltic activity from segments of rat small intestine in vivo

Abstract  We have developed methods that allow correlation of propulsive reflexes of the intestine with measurements of intraluminal pressure, fluid movement and spatio‐temporal maps of intestinal wall movements for the first time in vivo. A segment of jejunum was cannulated and set up in a Trendelenburg recording system while remaining connected to the vascular and nerve supply of the anaesthetized rat. The resting intraluminal pressure in intact intestine was 2–4 mmHg. Hydrostatic pressures of 2, 4, 8 and 16 mmHg were imposed. At a baseline pressure of 4 mmHg, propulsive waves generated pressures of 9 ± 1 mmHg, that progressed oral to anal at 2–5 mm s−1. Individual propulsive waves propelled 0.8 ± 0.4 mL of fluid. The frequency of propulsive waves increased with pressure, but peristaltic efficiency (mL per contraction) decreased with pressure increase between 4 and 16 mmHg. Atropine, as a bolus, transiently blocked peristalsis, but caused maintained block when infused. Hexamethonium blocked propulsive contractions. Inhibition of nitrergic transmission converted regular peristalsis to non‐propulsive contractions. These studies demonstrate the utility of an adapted Trendelenburg method for quantitative investigation of motility and pharmacology of enteric reflexes in vivo.

Functional and in situ hybridization evidence that preganglionic sympathetic vasoconstrictor neurons express ghrelin receptors

Agonists of ghrelin receptors can lower or elevate blood pressure, and it has been suggested that the increases in blood pressure are caused by actions at receptors in the spinal cord. However, this has not been adequately investigated, and the locations of neurons in the spinal cord that express ghrelin receptors, through which blood pressure increases may be exerted, are not known. We investigated the effects within the spinal cord of two non-peptide ghrelin receptor agonists, GSK894490 and CP464709, and two peptide receptor agonists, ghrelin and des-acyl ghrelin, and we used polymerase chain reaction (PCR) and in situ hybridization to examine ghrelin receptor expression. I.v. application of the non-peptide ghrelin receptor agonists caused biphasic changes in blood pressure, a brief drop followed by a blood pressure increase that lasted several minutes. The blood pressure rise, but not the fall, was antagonized by i.v. hexamethonium. Application of these agonists or ghrelin peptide directly to the spinal cord caused only a blood pressure increase. Des-acyl ghrelin had no significant action. The maximum pressor effects of agonists occurred with application at spinal cord levels T9 to T12. Neither i.v. nor spinal cord application of the agonists had significant effect on heart rate or the electrocardiogram. Ghrelin receptor gene expression was detected by PCR and in situ hybridization. In situ hybridization localized expression to neurons, including autonomic preganglionic neurons of the intermediolateral cell columns at all levels from T3 to S2. The numbers of ghrelin receptor expressing neurons in the intermediolateral cell columns were similar to the numbers of nitric oxide synthase positive neurons, but there was little overlap between these two populations. We conclude that activation of excitatory ghrelin receptors on sympathetic preganglionic neurons increases blood pressure, and that decreases in blood pressure caused by ghrelin agonists are mediated through receptors on blood vessels.

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.

The proliferative human monocyte subpopulation contains osteoclast precursors

IntroductionImmediate precursors of bone-resorbing osteoclasts are cells of the monocyte/macrophage lineage. Particularly during clinical conditions showing bone loss, it would appear that osteoclast precursors are mobilized from bone marrow into the circulation prior to entering tissues undergoing such loss. The observed heterogeneity of peripheral blood monocytes has led to the notion that different monocyte subpopulations may have special or restricted functions, including as osteoclast precursors.MethodsHuman peripheral blood monocytes were sorted based upon their degree of proliferation and cultured in macrophage colony-stimulating factor (M-CSF or CSF-1) and receptor activator of nuclear factor-kappa-B ligand (RANKL).ResultsThe monocyte subpopulation that is capable of proliferation gave rise to significantly more multinucleated, bone-resorbing osteoclasts than the bulk of the monocytes.ConclusionsHuman peripheral blood osteoclast precursors reside in the proliferative monocyte subpopulation.

Motor patterns and propulsion in the rat intestine in vivo recorded by spatio‐temporal maps

Abstract  We have used spatio‐temporal maps derived from video images to investigate propagated contractions of the rat small intestine in vivo. The abdomen, including an exteriorized segment of jejunum, was housed in a humid chamber with a viewing window. Video records were converted to spatio‐temporal maps of jejunal diameter changes. Intraluminal pressure and fluid outflow were measured. Contractions occupied 3.8 ± 0.2 cm of intestine and propagated anally at 3.1 ± 0.2 mm s−1 when baseline pressure was 4 mmHg. Contractions at any one point lasted 8.7 ± 0.6 s. Contractions often occurred in clusters; within cluster frequencies were 2.28 ± 0.04 min−1. Pressure waves, with amplitudes greater than about 9 mmHg, expelled fluid when the baseline pressure was 4 mmHg. In the presence ofl‐NAME, circular muscle contractions occurred at a high frequency, but they were not propagated. We conclude that video recording methods give good spatio‐temporal resolution of intestinal movement when applied in vivo. They reveal neurally‐mediated propulsive contractions, similar to those previously recorded from intestinal segments in vitro. The propagated contractions had speeds of propagation that were slower and frequencies of occurrence that were less than speeds and frequencies of slow waves in the rat small intestine.

Effects of the peripherally acting NK3 receptor antagonist, SB-235375, on intestinal and somatic nociceptive responses and on intestinal motility in anaesthetized rats

Abstract  We investigated the effects of the selective NK3 tachykinin receptor antagonist, SB‐235375, on noxious signalling from gut and skin and on intestinal motility in anaesthetized rats. We also measured penetrance into brain and spinal cord. Nociceptive responses in reaction to colorectal distension and skin pinch were assessed by recording the electromyogram (EMG) from the external oblique muscle (a visceromotor response). Motility was measured by recording intraluminal pressure waves during changes in baseline pressure in the jejunum. Colorectal compliance was assessed by measuring luminal pressure change during isovolumic distension. SB‐235375 (20 mg kg−1, by i.v. bolus) reduced the EMG response to colorectal distension by over 90%. The reduction was slow at onset, peaked at about 60 min, and lasted for over 2 h. Responses to noxious skin pinch were unchanged. Amplitudes of propulsive waves in the jejunum were slightly reduced, but their frequency of occurrence was unchanged. SB‐235375 decreased colorectal compliance by 5–10%. There was undetectable penetration of i.v. SB‐235375 into brain or spinal cord. We conclude that SB‐235375 acts peripherally to substantially reduce nociceptive signalling from colorectum without affecting noxious signalling from skin and with little effect on intestinal motility.

Development of motoneurons and primary sensory afferents in the thoracic and lumbar spinal cord of the South American opossum Monodelphis domestica.

The postnatal development of the primary sensory afferent projection to the thoracic (T4) and lumbar (L4) spinal cord of the marsupial species Monodelphis domestica was studied by using anterograde and retrograde neuronal tracers. Large numbers of primary afferents and motoneurons were labelled by application of the carbocyanine dye DiI into individual dorsal root ganglia (DRG) afferents in short‐term organ cultures. Dorsal root axons had entered the cord at birth, but most primary afferent innervation of the grey matter and the establishment of cytoarchitectural lamination occurs postnatally. In addition to ipsilateral projections, some primary afferents that projected to the dorsal horn extended across the midline into the equivalent contralateral regions of the grey matter. Similarly, motoneuron dendrites occasionally extended across midline and into the contralateral grey matter. The first fibres innervating the spinal cord project to the ventral horn and formed increasingly complex terminal arbours in the motor columns between P1 and P7. After P5 many afferents were seen projecting to the dorsal horn, with the superficial dorsal horn being the last region of the spinal grey to be innervated. Histochemical labelling with the lectin Griffonia simplicifolia indicated that C fibre primary afferents had arborised in the superficial dorsal horn by P14. The sequence of primary afferent innervation is thus similar to that described in the rat, but this sequence occurs over a period of several weeks in Monodelphis, compared with several days in the rat. J. Comp. Neurol. 414:423–436, 1999.

Sciatic axotomy compromises axonal transport of transganglionic tracer BSI-B4 from the soma to the central terminals of C fibre afferents.

The C afferent specific lectin BSI-B4 was used to examine the effects of sciatic axotomy on axonal transport by the C afferent subpopulation. From about 4 days after sciatic nerve lesion, BSI-B4 injected into the peripheral nerve is transported only as far as the neuronal cell bodies in the dorsal root ganglion. The previous demonstrations of A beta afferent terminal sprouting into lamina II, and the atrophy of lamina II terminals, in response to sciatic lesions may be related to the inability of C afferents to maintain transganglionic transport.