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. 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 Protodioscin 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 Protodioscin NK1r+ and NK1r-lacking lamina I projection neurons are directly innervated by Aδ nociceptive afferents. test was used to determine whether there was a significant difference in the proportions of NK1r+ and NK1r-lacking projection neurons that received contacts from CTb-labelled boutons. Mann-Whitney tests were used to compare densities of contacts from different types of axonal bouton onto these 2 different populations of projection neurons. 3 3.1 VGLUT2 and neuropeptide expression by CTb boutons in lamina I After injection of CTb into the sciatic nerve CTb-immunoreactive boutons were densely distributed throughout the sciatic territory in the deep part of the dorsal horn extending ventrally from lamina IIi and in addition there was a sparser plexus of labelled boutons Protodioscin in lamina I in the corresponding region as described in several previous studies [23 43 49 58 69 (Fig. 1a). The distribution of staining for VGLUT2 CGRP and substance P was the same as that described previously [2 15 16 24 34 58 and in all cases immunostaining was detected throughout the full thickness of the sections. Fig. 1 Expression of neuropeptides and VGLUT2 by CTb-labelled primary afferents in lamina I. (a) Low-magnification view of the upper part of the dorsal horn showing the general distribution of CTb-labelled profiles seen in a transverse section. Arrows point … Consistent with our previous report [58] we found that the majority of CTb-labelled boutons in lamina I (mean 75%; Table 2) were VGLUT2-immunoreactive although the strength of immunostaining varied considerably between boutons. Because some Aδ nociceptors are peptidergic [25 26 and many peptidergic primary afferent terminals in the dorsal horn do not have detectable levels of VGLUT2 [24 32 58 we tested whether the CTb+/VGLUT2? boutons in lamina I corresponded to peptidergic terminals. Although numerous boutons containing Protodioscin CGRP were observed in lamina I only 11.4% of the CTb boutons in this lamina showed CGRP immunoreactivity and most of these (75.9%) were also VGLUT2+ (Table 2 Fig. 1b to e). The remaining 24.1% of CGRP+ boutons (ie those that lacked VGLUT2) constituted 2.7% of all CTb-labelled boutons (24.1% of 11.4%) and therefore accounted for only about 10% of the CTb boutons that lacked VGLUT2. Substance P was found in an even lower proportion of PDGFB CTb-labelled boutons (mean 2.3%) and all of these were CGRP-immunoreactive (Fig. 1e inset). The mean z-axis lengths Protodioscin of nonpeptidergic CTb boutons with and without VGLUT2 was 2.72?±?0.65?μm and 2.69?±?0.54?μm respectively (n?=?40 boutons in each case). The corresponding values for CTb boutons with CGRP but not substance P and for those with both peptides were 2.68?±?0.58?μm and 2.71?±?0.56?μm respectively (n?=?20 boutons in each case). These values did not differ significantly (ANOVA test). Fig. 2 Example of a Fluorogold injection site. (a b) Dark field (DF) transmitted and fluorescent views of a section through the brainstem of one of the rats used in this study. The Fluorogold (FG) has spread throughout the entire lateral parabrachial area (LPb) … Table 3 Analysis of contacts onto different types of projection neuron. During this part of the study we observed that those NK1r-lacking projection cells that.