tetramethylrhodamine and Pain

Blockade of Ca2+-permeable AMPA receptors in the rat spinal cord diminishes the development of hyperalgesia and allodynia associated with peripheral injury. Cobalt uptake studies reveal that Ca2+-permeable AMPA receptors are expressed by some substance P receptor-expressing (NK1R+) neurons in lamina I, as well as other neurons throughout the superficial dorsal horn. Selective elimination of NK1R+ neurons in lamina I and lamina III/IV of the dorsal horn also suppresses development of hyperalgesia and allodynia. These observations raise the possibility that Ca2+-permeable AMPA receptors contribute to excitatory synaptic drive onto the NK1R+ neurons associated with allodynia and hyperalgesia. The first synapse in the pain pathway is the glutamatergic excitatory drive from the primary afferent fibres onto dorsal horn neurons. Therefore, we tested whether Ca2+-permeable AMPA receptors are located on lamina I and lamina III/IV NK1R+ neurons postsynaptic to primary afferent fibres, using inward rectification and polyamine toxins for receptor identification. We examined three different populations of dorsal horn neurons; lamina I NK1R+ neurons, including projection neurons, and non-NK1R+ (NK1R-) neurons including interneurons, and lamina III/IV NK1R+ neurons, believed to contribute to the low-threshold mechanosensory pathway. The majority of synapses in all three groups had rectification indices less than 1.0 and greater than 0.4, indicating that the AMPA receptors at these synapses are a mixture of Ca2+-permeable and -impermeable forms. Lamina III/IV NK1R+ neurons and lamina I NK1R- neurons have a significantly higher proportion of postsynaptic Ca2+-permeable AMPA receptors than lamina I NK1R+ neurons. Thus synaptically positioned Ca2+-permeable AMPA receptors directly contribute to low-threshold sensory afferent drive into the dorsal horn, and can mediate afferent input onto interneurons such as GABAergic neurons. These receptors also contribute to high-threshold primary afferent drive onto NK1R+ neurons in the superficial dorsal horn, but do so less consistently.

Topics: Animals; Benzodiazepines; Calcium; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Fluorescent Dyes; In Vitro Techniques; Pain; Patch-Clamp Techniques; Permeability; Posterior Horn Cells; Rats; Receptors, AMPA; Rhodamines; Staining and Labeling; Substance P; Synapses; Synaptic Transmission; TRPV Cation Channels

Serotoninergic projections to the spinal dorsal horn are implicated in the modulation of nociceptive transmission. However, morphological evidence indicating that serotoninergic projection fibers make synapses on nociceptive neurons in the medullary dorsal horn is still meager. Thus, we examined whether axonal varicosities with serotonin (5-HT)-like immunoreactivity (5-HT-LI) might make synapses on nociceptive projection neurons in the caudal spinal trigeminal nucleus (Vc) of the rat. Projection neurons were retrogradely labeled with tetramethylrhodamine-dextran amine (TMR-DA) or wheat germ agglutinin-horseradish peroxidase (WGA-HRP) that was injected into the parabrachial or thalamic region. Vc neurons in which c-fos protein-like immunoreactivity (Fos-LI) was induced by subcutaneous injection of formalin into the lip were considered nociceptive. Vc neurons in direct contact with axonal varicosities that bind isolectin I-B4 were also considered nociceptive. Triple labeling for 5-HT, TMR-DA, and Fos as well as that for 5-HT, TMR-DA, and I-B4 were done by using the immunofluorescence and fluorescence histochemical techniques. Confocal laser-scanning microscopy revealed that axonal varicosities with 5-HT-LI were in close apposition to TMR-DA-labeled neurons showing Fos-LI in lamina I and the outer part of lamina II (lamina IIo), and that both axonal varicosities with 5-HT-LI and those binding I-B4 were in close apposition to single neuronal profiles labeled with TMR-DA. The presumed nociceptive neuronal profiles in close apposition to axon terminals with 5-HT-LI were mainly those of laminae I and II neurons as well as dendrites of lamina III neurons. Electron microscopy confirmed that axon terminals with 5-HT-LI and those with I-B4 binding activity in laminae I and II made synapses on somatic and dendritic profiles that were labeled with WGA-HRP. The results indicate that serotoninergic neurons project directly on nociceptive projection neurons in the Vc.

Topics: Animals; Antibody Specificity; Axons; Coloring Agents; Fluorescent Antibody Technique; Formaldehyde; Lectins; Male; Microscopy, Electron; Neurons; Nociceptors; Pain; Proto-Oncogene Proteins c-fos; Rats; Rats, Wistar; Rhodamines; Serotonin; Trigeminal Nucleus, Spinal; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate