Neurochemical Characterization of the TRPV1-Positive Nociceptive Primary Afferents Innervating Skeletal Muscles in the Rats

OBJECTIVE: Transient receptor potential vanilloid subfamily type 1 (TRPV1), a most specific marker of the nociceptive primary afferent, is expressed in peptidergic and non-pepetidergic primary afferents innervating skin and viscera. However, its expression in sensory fibers to skeletal muscle is not well known. In this study, we studied the neurochemical characteristics of TRPV1-positive primary afferents to skeletal muscles. METHODS: Sprague-Dawley rats were injected with total 20 microliter of 1% fast blue (FB) into the gastrocnemius and erector spinae muscle and animals were perfused 4 days after injection. FB-positive cells were traced in the L4-L5 (for gastrocnemius muscle) and L2-L4 (for erector spinae muscle) dorsal root ganglia. The neurochemical characteristics of the muscle afferents were studied with multiple immunofluorescence with TRPV1, calcitonin gene-related peptide (CGRP) and P2X(3). To identify spinal neurons responding to noxious stimulus to the skeletal muscle, 10% acetic acids were injected into the gastrocnemius and erector spinae muscles and expression of phospho extracellular signal-regulated kinase (pERK) in spinal cords were identified with immunohistochemical method. RESULTS: TRPV1 was expressed in about 49% of muscle afferents traced from gastrocnemius and 40% of erector spinae. Sixty-five to 60% of TRPV1-positive muscles afferents also expressed CGRP. In contrast, expression of P2X3 immnoreaction in TRPV1-positive muscle afferents were about 20%. TRPV1-positive primary afferents were contacted with spinal neurons expressing pERK after injection of acetic acid into the muscles. CONCLUSION: It is consequently suggested that nociception from skeletal muscles are mediated by TRPV1-positive primary afferents and majority of them are also peptidergic.

Degeneration of primary afferent terminals following brachial plexus extensive avulsion injury in rats / Degeneración de los terminales aferentes primarios de rata luego de lesión extensa por avulsión del plexo braquial

Important breakthroughs in the understanding regeneration failure in an injured CNS have been made by studies of primary afferent neurons. Dorsal rhizotomy has provided an experimental model of brachial plexus (BP) avulsion. This is an injury in which the central branches of primary afferents are disrupted at their point of entry into the spinal cord, bringing motor and sensory dysfunction to the upper limbs. In the present work, the central axonal organization of primary afferents was examined in control (without lesion) adult Wistar rats and in rats subjected to a C3-T3 rhizotomy. Specific sensory axon subtypes were recognized by application of antibodies to the calcitonin gene-related peptide (CGRP), the P2X3 purinoreceptor, the low-affinity p75-neurotrophin receptor and the retrograde tracer cholera toxin subunit beta (TCbeta ). Other subtypes weres labeled with the lectin Griffonia simplicifolia IB4. Using immunohistochemistry and high resolution light microscopy, brachial plexus rhizotomy in adult rats has proven a reliable model for several neural deficits in humans. This lesion produced different degrees of terminal degeneration in the several types of primary afferents which define sub-populations of sensitive neurons. Between the C6 and C8 levels of the spinal cord,,deafferentation was partial for peptidergic GCRP-positive fibers, in contrast with elimination of non peptidergic and myelinated fibers. Dorsal rhizotomy has provided an adequate experimental model to study sensory alterations such as acute pain and allodynia as well as factors that affect regeneration into the CNS., Therefore, the differential deafferentation response must be considered inr the evaluation of therapies for nociception (pain) and regeneration for brachial plexus avulsion. The anatomical diffierences among the primary afferent subtypes also affect their roles in normal and damaged conditions.

El uso de las neuronas sensoriales primarias ha aportado avances en el entendimiento de las razones por las cuales falla la regeneración cuando el sistema nervioso central (SNC) es dañado. La rizotomía dorsal se puede usar como un modelo experimental de las lesiones por avulsión del plexo braquial, una lesión en la cual son desprendidas, en su punto de entrada en la médula espinal, las ramas centrales de los aferentes primarios causando una disfunción motora y sensorial grave e irreversible del miembro superior. En el presente trabajo, se examinó la organización central de los aferentes primarios en ratas Wistar adultas. Éstas fueron divididas en controles normales no lesionados y en animales rizotomizados entre los niveles cervical 3 y torácico 3 (C3-T3). Se estudió la deaferentación de los subtipos de axones sensoriales utilizando anticuerpos específicos contra el péptido relacionado con el gen de la calcitonina (CGRP), el receptor purinérgico (P2X3), el receptor de baja afinidad p75 para el factor de crecimiento nervioso (NGF) y contra la subunidad ®de la toxina de cólera (TCbeta ). Otro subtipo fue marcado con la lectina Griffonia simplicifolia IB4. La inmunohistoquímica y la microscopía óptica de alta resolución demostraron que el modelo animal de rizotomía completa del plexo braquial reproduce diversos déficit observados en las lesiones humanas. Esta lesión produce diferentes grados de degeneración terminal entre los diversos tipos de aferentes primarios que definen subpoblaciones de neuronas sensoriales. En los niveles de la médula espinal estudiados (entre C6 y C8), la deaferentación fue parcial para las fibras peptidérgicas GCRPpositivas, en contraste con la eliminación de las fibras no peptidérgicas y las mielinizadas. La rizotomía dorsal es un modelo experimental apropiado para estudiar las alteraciones sensoriales como el dolor agudo y la alodinia, así como los factores que podrían afectar la regeneración en el SNC. Por tanto, la respuesta de deaferentacion diferencial debe ser tenida en cuenta para la evaluación de terapias antinociceptivas y regenerativas tras la avulsión del plexo braquial. Se discute la anatomía de los subtipos de aferentes primarios y su papel en condiciones normales y después de la lesión.

Ultrastructural analysis of glutamate immunoreactive primary afferent terminals in the trigeminal nucleus principalis and trigeminal nucleus oralis of the rat / 대한해부학회지

The present study was aimed to investigate the ultrastructure of the primary afferent terminals and whether glutamate may be a transmitter in these terminals within the trigeminal nucleus principalis and oralis of the rat. Labeling of primary afferent terminals was performed by the injection of the CTB-HRP into the trigeminal ganglion. Ultrastructural analysis and assessment of the glutamate like immunoreactivity by the immunogold technique was performed with the 66 peroxidased labeled boutons in the nucleus principalis and 62 in the nucleus oralis. Labeled boutons were presynaptic to dendritic shafts of the secondary neurons and postsynaptic to the pleomorphic vesicles containing endings (p-endings). Most of the labeled boutons made synaptic contact with the dendritic shafts. A little labeled boutons in the nucleus oralis but no in the nucleus principalis was observed to make synaptic contact with the soma or proximal dendrite. Most of the labeled boutons made synaptic contact with one to three neurofiles, but labeled boutons showing complex synaptic connections, such as those with five or more neurofiles, were more in principalis than in oralis. The average diameter of p-endings were smaller than that of labeled boutons (p<0.05). The diameter of the postsynaptic dendritic shafts were smaller in nucleus principalis than in nucleus oralis, thus indicated that the labeled boutons made synaptic contact with more distal portion of the postsynaptic dendrite in the nucleus principalis than in the nucleus oralis. The gold particle density over the labeled boutons were significantly higher than that over the p-endings and average tissue particle density. They were ranged from 110 to 430% of the average tissue particle density. These findings indicate that synaptic connection of the primary afferent terminals is organized in different manner in nucleus principalis and oralis, and suggest that glutamate is involved as neuroactive substance in the primary afferent terminals of the trigeminal system.

THE DISTRIBUTION OF PRIMARY AFFERENT FIBERS FROM THE GREATER SPLANCHNIC NERVE TO THE SPINAL GRAY MATTER AND NUCLEUS GRACILIS IN THE CAT / 解剖学报

The central distribution of visceral primary afferent fibers from greater splanchnic nerve of the cat have been studied. Horseradish peroxidase (HRP) dissolved in distilled water was injected into the coeliac ganglion or greater splanchnic nerve unilaterally. The HRP was taken up by afferent fibers and transported across the spinal ganglia and then anterogradely as far as central terminals in the spinal cord and medulla. The HRP reaction-product within the central processes of the primary sensory neurons was detected ipsilaterally in the Lissauer's tract, dorsal funiculus and spinal gray matter. After entering the spinal cord via the spinal dorsal roots, the great majority of labeled fibers (viz. the central processes of primary sensory neurons) were found to run longitudinally within the zone of Lissauer, and only sparse fibers to ascend in the dorsal funiculus. The medial projection fibers (MPF) and lateral projection fibers (LPF) arose periodically from the labeled fiber bundle in the Lissauer's tract to form a thin shell surrounding both the medial and lateral sides of the dorsal horn. The terminal areas of the more prominent LPF were found at laminae I, V, VII and the region surrounding the central canal, whereas the MPF ended at the medial part of lamina V and lamina X. Some smaller fiber bundles from LPF were seen to enter the cell clusters of the intermediolateral nucleus, where these fibers divided rostrocaudally and ran parallel with the longitudinal dendritic branches of the intermediolateral cells. The few labeled fibers located at the dorsal funiculus were traced to the lower medulla and the distinct terminal area of them was situated in the ventrolateral part of the nucleus gracilis at the level below the obex.