Selective innervation of NK1 receptor-lacking lamina I spinoparabrachial neurons by presumed nonpeptidergic Aδ nociceptors in the rat.

Fine myelinated (Aδ) nociceptors are responsible for fast, well-localised pain, but relatively little is known about their postsynaptic targets in the spinal cord, and therefore about their roles in the neuronal circuits that process nociceptive information. Here we show that transganglionically transported cholera toxin B subunit (CTb) labels a distinct set of afferents in lamina I that are likely to correspond to Aδ nociceptors, and that most of these lack neuropeptides. The vast majority of lamina I projection neurons can be retrogradely labelled from the lateral parabrachial area, and these can be divided into 2 major groups based on expression of the neurokinin 1 receptor (NK1r). We show that CTb-labelled afferents form contacts on 43% of the spinoparabrachial lamina I neurons that lack the NK1r, but on a significantly smaller proportion (26%) of those that express the receptor. We also confirm with electron microscopy that these contacts are associated with synapses. Among the spinoparabrachial neurons that received contacts from CTb-labelled axons, contact density was considerably higher on NK1r-lacking cells than on those with the NK1r. By comparing the density of CTb contacts with those from other types of glutamatergic bouton, we estimate that nonpeptidergic Aδ nociceptors may provide over half of the excitatory synapses on some NK1r-lacking spinoparabrachial cells. These results provide further evidence that synaptic inputs to dorsal horn projection neurons are organised in a specific way. Taken together with previous studies, they suggest that both NK1r(+) and NK1r-lacking lamina I projection neurons are directly innervated by Aδ nociceptive afferents.

Simultaneous identification of unmyelinated and myelinated primary somatic afferents by co-injection of isolectin B4 and Cholera toxin subunit B into the sciatic nerve of the rat.

Several studies have used the transganglionic tracers cholera toxin subunit B (CTb) and either Bandeiraea simplicifolia isolectin B4 (IB4) or wheat-germ agglutinin (WGA) to label myelinated and unmyelinated afferent fibres respectively. In this study, we aim to determine whether co-injection of CTb and either IB4 or WGA into the sciatic nerve of rat will selectively label myelinated and unmyelinated simultaneously. A double immunofluorescence approach was used to detect these tracers in dorsal root ganglia (DRGs) and afferent fibre terminals in the spinal cord. CTb- and IB4-labelled neurons were seen mainly in L4 and L5 DRGs, with CTb labelling detected primarily in large sized neurons and IB4 staining seen mainly in smaller cells. Only a minority of CTb labelled DRG neuron profiles (5.1%) were also labelled with IB4. In the spinal cord, IB4-labelling was largely confined to lamina II of spinal segments L3-L5, whereas CTb-labelled terminals were seen in all laminae but sparse in lamina II. Confocal microscopy showed no evidence for colocalisation of CTb and IB4 labelling in any terminals in laminae I-III. Although the central distribution of CTb labelling in laminae I and II inner-IV had the same rostro-caudal and medio-lateral coverage as IB4 labelling in spinal segments L3-L5, CTb labelling in ventral laminae (of putative proprioceptor afferents) extended between T12 and S1. Similar patterns of central labelling were found when CTb and WGA were injected together. We therefore concluded that this co-injection approach provides a reliable method to identify both myelinated and unmyelinated somatic primary afferents simultaneously.

Unmyelinated primary afferents from adjacent spinal nerves intermingle in the spinal dorsal horn: a possible mechanism contributing to neuropathic pain.

Peripheral nerve injury in animals can cause neuropathic pain often expressed in the form of hyperalgesia and allodynia. Spinal nerve ligation, in which the fifth and sixth lumbar (L5 and L6) or only the L5 spinal nerve is ligated and cut, is a model commonly used to produce neuropathic pain. The purpose of the present study was to test whether there is any anatomical evidence to support the suggestion that terminating unmyelinated (C) fibres of injured and adjacent uninjured nerves interact at the level of the spinal dorsal horn. Thus, in the first series of experiments, rats received injections of anterograde tracers, either wheat germ agglutinin (WGA) conjugated to horseradish peroxidase or Bandeiraea simplicifolia isolectin B4 (IB4), into the L4 or L5 spinal nerves. Results with both tracers showed that the central terminals of nerve L4 were concentrated in both L4 and L3 segments of the dorsal horn with clear although reduced levels of labelling in L2 and L5. Similarly, the central terminals of nerve L5 were found in both L5 and L4 again with less labelling in L3 and L6. These results suggest an intermingling of primary afferents of adjacent nerves at the level of the spinal dorsal horn. A second series of experiments was therefore conducted to test whether primary afferent terminals from adjacent nerves target the same neuronal elements in the regions of overlap. Consequently, additional rats were injected with WGA into the L5 spinal nerve and IB4 into the adjacent L4 spinal nerve. Double immunofluorescent staining and confocal microscopy revealed that IB4-labelled and WGA-labelled boutons, belonging to L4 and L5 spinal nerves, terminated in the same region within the L4 spinal segment. This suggests that neurons located in regions of overlap receive input from both L4 (intact) and L5 (injured) afferents. Consequently, spinal neurons located in regions of terminal overlap may show augmented responses to activation of the intact L4 nerve due to neuronal sensitisation resulting from injury to the adjacent L5 nerve. This may in part provide an anatomical basis for hyperalgesic reaction to injury.

Peripheral axotomy induces depletion of the vesicular glutamate transporter VGLUT1 in central terminals of myelinated afferent fibres in the rat spinal cord.

Myelinated primary afferent axons use glutamate as their principal neurotransmitter. We have shown previously that central terminals of myelinated tactile and proprioceptive afferents contain the vesicular glutamate transporter VGLUT1. Peripheral nerve injury is known to induce changes in the anatomy, neurochemistry, and physiology of primary afferents. In this study, we have examined the effect of peripheral axotomy on VGLUT1 expression in central terminals of myelinated afferents in laminae III-V and lamina IX of the rat spinal cord. Bilateral injections of cholera toxin B subunit (CTb) were made into the sciatic nerves of rats that had undergone unilateral sciatic nerve transection 1, 2, 4, or 8 weeks previously. Immunofluorescence staining and confocal microscopy were used to compare levels of VGLUT1 in CTb-labelled boutons on the intact and sectioned sides at each postoperative survival time. VGLUT1 was depleted from central terminals of transected myelinated afferents in rats injected with CTb 1 week after nerve section, and this depletion became more severe in animals with longer postaxotomy survival times. By 4 weeks, the level of VGLUT1 in CTb-labelled boutons in lamina IX was reduced by over 80% compared to that seen in intact (contralateral) afferents, while for boutons in laminae III-V, VGLUT1 levels were reduced by 50-70%. This suggests that loss of VGLUT1 is more severe in proprioceptive than cutaneous afferents. Depletion of VGLUT1 may lead to a decrease in levels of transmitter glutamate in these afferents and thus to a reduction in synaptic efficacy.