Vanilloid receptor TRPV1-mediated phosphorylation of ERK in murine adjuvant arthritis.

OBJECTIVE: The vanilloid receptor transient receptor potential vanilloid 1 (TRPV1), expressed by sensory neurons that innervate joints, is implicated in arthritis but the mechanisms are not fully understood. One possibility is that downstream effects of activation of TRPV1 are mediated by the extracellularly-regulated kinase (ERK). ERK is phosphorylated (p-ERK) in sensory neurons in response to noxious stimuli and its inhibition has been found to be antinociceptive in several pain models. We here wanted to ascertain whether TRPV1 may contribute to the pain hypersensitivity and inflammation of arthritis via an ERK-mediated pathway. METHODS: We used a model of adjuvant-induced arthritis (AIA) of the ankle and investigated the changes in expression of p-ERK in sensory afferent neurons in dorsal root ganglia (DRG) and spinal dorsal horn of TRPV1-knockout (KO) mice, compared to wild-type (WT) mice of the same genetic background, using multiple immunofluorescence. RESULTS: Two to three weeks after inducing AIA in mice, the number of neurons in DRG and spinal cord that expressed p-ERK was significantly higher on the side of AIA than on the contralateral, vehicle-injected side. The fraction of p-ERK-positive neurons in the DRG that also expressed TRPV1 was increased, indicating that activation of ERK occurred preferentially in TRPV1-positive neurons. Moreover, TRPV1-KO mice had reduced activation of ERK in sensory neurons, compared to WT mice. These changes in expression of p-ERK correlated with changes in pain behavior and joint histopathology: TRPV1-KO mice had reduced nociceptive behavior and severity of arthritis, compared to WT mice. CONCLUSION: Our results support the idea that activation of ERK in primary afferent neurons is mediated, at least in part, by TRPV1. In the absence of TRPV1, the signs of arthralgia and histopathology in the mouse model of AIA are reduced. We conclude that TRPV1, expressed by neurons in the articular afferent pathway, contributes to the pathogenesis of arthritis via an ERK-mediated pathway.

Interference of biocytin with opioid-evoked hyperpolarization and membrane properties of rat spinal substantia gelatinosa neurons.

In our laboratory, preliminary whole-cell, tight seal recordings of rat spinal substantia gelatinosa neurons including biocytin in the patch pipette yielded a significantly smaller proportion of neurons hyperpolarized by selective opioid agonists compared with recordings without biocytin. Therefore, we investigated the effects of biocytin inclusion on opioid responses and other membrane properties during whole-cell, tight seal recordings of these neurons. The percentage of neurons hyperpolarized by mu-, delta(1)-, and kappa-selective opioids was significantly reduced when 1% but not < or =0.2% biocytin was included in the recording pipette, compared with neurons recorded without biocytin. However, a significantly higher proportion of neurons fired spontaneous action potentials with either 0.05-0.2 or 1% biocytin compared to no biocytin. Resting membrane potential, input impedance and the proportion of neurons displaying transient outward rectification were each significantly altered for neurons recorded with 1% but not 0.05-0.2% biocytin. These effects may be due to a relatively specific blockade of diverse potassium channel types. Because efficient labeling can be achieved with 0.1% biocytin with whole-cell recording, higher concentrations are contraindicated.

Spinal laminae I-II neurons in rat recorded in vivo in whole cell, tight seal configuration: properties and opioid responses.

Using the in vivo whole cell recording procedure described previously, we recorded 73 neurons in laminae I and II in the lumbar spinal cord of the rat. Input impedances averaged 332 MOmega, which indicated that prior sharp electrode recordings contained a significant current shunt. Characterization of the adequate stimuli from the excitatory hindlimb receptive field indicated that 39 of 73 neurons were nociceptive, 6 were innocuous cooling cells, 20 responded maximally to brush, and 8 cells were not excited by stimulation of the skin of the hindlimb. The locations of 15 neurons were marked with biocytin. Nociceptive neurons were mostly found in lamina I and outer II, cooling cells in lamina I, and innocuous mechanoreceptive cells were mostly found in inner II or in the overlying white matter. The mu-opioid agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-Enkephalin (DAMGO) hyperpolarized 7 of 19 tested neurons with a conductance increase. This hyperpolarization was reversed by naloxone in the neurons in which it was applied. DAMGO also decreased the frequency of spontaneous PSPs in 13 neurons, 7 of which were also hyperpolarized by DAMGO. Five of the seven hyperpolarized neurons were nociceptive, responding to both heat and mechanically noxious stimuli, whereas two responded to slow, innocuous brush. These results indicate that whole cell, tight seal recordings sample a similar population of lamina I and II neurons in the rat as those found with sharp electrode recordings in cat and monkey. They further indicate that DAMGO hyperpolarizes a subset of the nociceptive neurons that have input from both heat and mechanical nociceptors and that presynaptic DAMGO effects can be observed in nociceptive neurons that are not hyperpolarized by DAMGO.

Induction of fos-like immunoreactivity by electrocutaneous stimulation of the rat hindpaw.

Stimulation of peripheral nerves activates the proto-oncogene c-fos, which in turn generates its gene product, Fos. Fos and Fos-like proteins are produced in the central nervous system in response to chemical, mechanical, thermal, and electrical manipulation. The present study demonstrated a relationship between the number of Fos-like-immunoreactive nuclei in the spinal dorsal horn and graded intensities of electrical stimulation applied to the hindpaws of anesthetized and unanesthetized rats. Stimulation levels within the range of 0.1 to 1.0 mA were chosen on the basis of parmeters previously determined in behavioral investigations of escape reactions. Focal stimulation at these intensities activates peripheral axons directly, but does not injure or traumatize peripheral tissues. There was no evidence of inflammation or edema as a result of the focal electrical stimulation. As the stimulation intensity increased, the number and distribution of Fos-like-labeled nuclei increased with respect to rostral-caudal and laminar orientation. The threshold for expression of Fos-like immunoreactivity was different for anesthetized and unanesthetized animals. For anesthetized animals, the number of labeled nuclei increased significantly from the control level only when 1.0 mA was applied. However, in unanesthetized animals, the pattern of labeling was statistically significant at 0.2 mA. The present study demonstrates that electrical stimulation can evoke the expression of Fos-like immunoreactivity by activating nociceptors in the absence of tissue injury, and that the use of anesthetics can modulate this expression.

Effects of anterolateral spinal lesions on escape responses of rats to hindpaw stimulation.

In order to determine the effects of spinal cord lesions on nociceptive sensitivity of rodents, methods were developed to assess the speed of operant escape responses to electrocutaneous stimulation (ES). ES was delivered across the dorsal and ventral surfaces of either hindpaw, producing a current path through deep tissues. In order to guide establishment of a range of stimulus intensities for this manner of stimulation, a preliminary human psychophysical experiment was conducted with stimulation between the dorsal and ventral surfaces of a finger. For the human subjects, detection thresholds averaged 0.13 mA, and thresholds for a sharp (but nonpainful) sensation were 0.42 mA. Levels of stimulation between these thresholds for detection and a sharp quality elicited sensations of tingle or itch. Thresholds for reports of pain averaged 0.67 mA. On the basis of these results, intensities of ES ranging from 0.05 to 1.0 mA were presented to the feet of rats that were trained to perform an escape response with one forelimb. Thresholds for escape averaged slightly less than 0.1 mA; responding was consistent at 0.4 mA; and response probability and speed were maximal at approximately 0.8 mA. Thus, the rats responded aversively at intensities below those rated as sharp or painful by the human subjects, but the speed of escape reached a plateau at intensities that were above pain threshold for the human subjects. Unilateral thoracic lesions of the lateral spinal column of rats produced a contralateral hypalgesia. Escape thresholds were elevated, and the speed of escape responses to all intensities was reduced. This effect depended upon interruption of axons in the middle and anterior portions of one lateral column, corresponding to the location of long ascending pathways for nociception, including the spinothalamic tract. The speed of escape responding increased over 20 weeks of postoperative testing of animals with the largest lesions. This confirms results obtained previously from monkeys (by means of a similar paradigm), and corresponds to clinical reports of humans who have received spinal lesions for control of intractable pain. Thus, the location and organization of nociceptive pathways in the spinal cord of rodents appear to be similar to those of primates, and similar adaptations occur following interruption of these pathways.

Serotoninergic and noradrenergic projections to the ventral posterolateral nucleus of the monkey thalamus.

In the present study, serotoninergic and noradrenergic varicosities were identified in the ventral posterolateral nucleus of the macaque monkey. Monoaminergic neurons projecting to the ventral posterolateral nucleus of the thalamus were identified by using retrograde labeling with horseradish peroxidase combined with immunocytochemical staining for serotonin or dopamine-beta-hydroxylase. The midbrain nucleus raphe dorsalis was the major site of origin for neurons providing a serotoninergic projection to the ventral posterolateral nucleus. A few retrogradely labeled serotonin-containing neurons were also observed in the central superior and the raphe pontis nuclei. Noradrenergic cells with projections to the thalamus were primarily located in the nucleus locus coeruleus with some projection neurons in the nucleus subcoeruleus, and the A5 catecholamine cell group of the pons.

Mapping study of serotoninergic input to diencephalic-projecting dorsal column neurons in the rat.

Several lines of evidence indicate that the processing of somatosensory information in the dorsal column nuclei (DCN) is subject to descending controls. Anatomical experiments have demonstrated projections to the DCN from the sensorimotor cerebral cortex and the reticular formation. Physiological studies have shown that the activity of DCN neurons can be altered following stimulation of the cerebral cortex, reticular formation, periaqueductal gray, or raphe nuclei. Recent biochemical and electrophysiological evidence suggests a serotoninergic modulation of DCN neurons. The present study identifies serotonin-containing contacts on cells in the DCN that project to the thalamus in the rat. Retrograde labeling of brainstem neurons by horseradish peroxidase demonstrated projections to the DCN from the nucleus reticularis paragigantocellularis lateralis and from several raphe nuclei, including nuclei raphe obscurus (RO), pallidus (RP), and magnus (RM). Double labeling with horseradish peroxidase and antibody for serotonin indicated that the RO, RP and RM are likely to be the sources of the serotoninergic projections to the DCN. Thus, the role of the serotoninergic output from the raphe nuclei includes modulation of activity in the DCN.