The processing of sensory, including nociceptive, information in spinal dorsal horn is critically modulated by spinal GABAergic neurones. For example, blockade of spinal GABA A receptors leads to pain evoked by normally innocuous tactile stimulation (tactile allodynia) in rats. GABAergic dorsal horn neurones have been classified neurochemically and morphologically, but little is known about their physiological properties. We used a transgenic mouse strain coexpressing enhanced green fluorescent protein (EGFP) and the GABA-synthesizing enzyme GAD67 to investigate the properties of a subgroup of GABAergic neurones. Immunohistochemistry showed that EGFP-expressing neurones accounted for about one-third of the GABAergic neurones in lamina II of the spinal dorsal horn. They constituted a neurochemically rather heterogeneous group where 27% of the neurones coexpressed glycine, 23% coexpressed parvalbumin and 14% coexpressed neuronal nitric oxide synthase (nNOS). We found almost no expression of protein kinase Cγ (PKCγ) in EGFP-labelled neurones but a high costaining with PKCβII (78%). The whole-cell patch-clamp technique was used to intracellularly label and physiologically characterize EGFP-and non-EGFP-expressing lamina II neurones in spinal cord slices. Sixty-two per cent of the EGFP-labelled neurones were islet cells while the morphology of non-EGFP-labelled neurones was more variable. When stimulated by rectangular current injections, EGFP-expressing neurones typically exhibited an initial bursting firing pattern while non-EGFP-expressing neurones were either of the gap or the delayed firing type. EGFP-expressing neurones received a greater proportion of monosynaptic input from the dorsal root, especially from primary afferent C-fibres. In conclusion, EGFP expression defined a substantial but, with respect to the measured parameters, rather inhomogeneous subgroup of GABAergic neurones in spinal lamina II. These results provide a base to elucidate the functional roles of this subgroup of GABAergic lamina II neurones, e.g. for nociception.

It is functionally important to differentiate between primary afferent neurons with A-fibers, which are nociceptive or nonnociceptive, and C-fibers, which are mainly nociceptive. Neurochemical markers such as neurofilament 200 (NF200), substance P (SP), and isolectin B4 (IB4) have been useful to distinguish between A- and C-fiber neurons. However, the expression patterns of these markers change after peripheral nerve injury, so that it is not clear whether they still distinguish between fiber types in models of neuropathic pain. We identified neurons with Abeta-, Adelta-, and C-fibers by their conduction velocity (corrected for utilization time) in dorsal root ganglia taken from mice after a chronic constriction injury (CCI) of the sciatic nerve and control mice, and later stained them for IB4, SP, calcitonin gene-related peptide (CGRP), NF200, and neuropeptide Y (NPY). NF200 remained a good marker for A-fiber neurons, and IB4 and SP remained good markers for C-fiber neurons after CCI. NPY was absent in controls but was expressed in A-fiber neurons after CCI. After CCI, a group of C-fiber neurons emerged that expressed none of the tested markers. The size distribution of the markers was investigated in larger samples of unidentified dorsal root ganglion neurons and, together with the results from the identified neurons, provided only limited evidence for the expression of SP in Abeta-fiber neurons after CCI. The extent of up-regulation of NPY showed a strong inverse correlation with the degree of heat hyperalgesia.

Sensory nerve fibers differ not only with respect to their sensory modalities and conduction velocities, but also in their relative roles for pain hypersensitivity. It is presently largely unknown which types of sensory afferents contribute to various forms of neuropathic and inflammatory pain hypersensitivity. Vesicular glutamate transporter 3-positive (VGluT3

Adequate pain sensitivity requires a delicate balance between excitation and inhibition in the dorsal horn of the spinal cord. This balance is severely impaired in neuropathy leading to enhanced pain sensations (hyperalgesia). The underlying mechanisms remain elusive. Here we explored the hypothesis that the excitatory drive to spinal GABAergic neurons might be impaired in neuropathic animals. Transgenic adult mice expressing EGFP under the promoter for GAD67 underwent either chronic constriction injury of the sciatic nerve or sham surgery. In transverse slices from lumbar spinal cord we performed whole-cell patch-clamp recordings from identified GABAergic neurons in lamina II. In neuropathic animals rates of mEPSC were reduced indicating diminished global excitatory input. This downregulation of excitatory drive required a rise in postsynaptic Ca2+. Neither the density and morphology of dendritic spines on GABAergic neurons nor the number of excitatory synapses contacting GABAergic neurons were affected by neuropathy. In contrast, paired-pulse ratio of Aδ- or C-fiber-evoked monosynaptic EPSCs following dorsal root stimulation was increased in neuropathic animals suggesting reduced neurotransmitter release from primary afferents. Our data indicate that peripheral neuropathy triggers Ca2+-dependent signaling pathways in spinal GABAergic neurons. This leads to a global downregulation of the excitatory drive to GABAergic neurons. The downregulation involves a presynaptic mechanism and also applies to the excitation of GABAergic neurons by presumably nociceptive Aδ- and C-fibers. This then leads to an inadequately low recruitment of inhibitory interneurons during nociception. We suggest that this previously unrecognized mechanism of impaired spinal inhibition contributes to hyperalgesia in neuropathy.

Due to its distinct pharmacological profile and lower incidence of adverse events compared with other opioids, buprenorphine is considered a safe option for pain and substitution therapy. However, despite its wide clinical use, little is known about the synaptic effects of buprenorphine in nociceptive pathways. Here, we demonstrate dose-dependent, bimodal effects of buprenorphine on transmission at C-fiber synapses in rat spinal cord dorsal horn in vivo. At an analgesically active dose of 1500 g⅐kg Ϫ1, buprenorphine reduced the strength of spinal C-fiber synapses. This depression required activation of spinal opioid receptors, putatively 1 -opioid receptors, as indicated by its sensitivity to spinal naloxone and to the selective 1 -opioid receptor antagonist naloxonazine. In contrast, a 15,000-fold lower dose of buprenorphine (0.1 g⅐kg Ϫ1 ), which caused thermal and mechanical hyperalgesia in behaving animals, induced an enhancement of transmission at spinal C-fiber synapses. The ultra-low-dose buprenorphine-induced synaptic facilitation was mediated by supraspinal naloxonazine-insensitive, but CTOP-sensitive -opioid receptors, descending serotonergic pathways, and activation of spinal glial cells. Selective inhibition of spinal 5-hydroxytryptamine-2 receptors (5-HT 2 Rs), putatively located on spinal astrocytes, abolished both the induction of synaptic facilitation and the hyperalgesia elicited by ultra-low-dose buprenorphine. Our study revealed that buprenorphine mediates its modulatory effects on transmission at spinal C-fiber synapses by dose dependently acting on distinct -opioid receptor subtypes located at different levels of the neuraxis.