Helen H. Willcockson

The effect of stimulus duration on noxious-stimulus induced c-fos expression in the rodent spinal cord

C-fos is a proto-oncogene that is expressed within some neurons following depolarization. The protein product, fos, has been proposed as an anatomical marker for neuronal activity following noxious peripheral stimulation. However, the literature on noxious-stimulus induced fos expression contains several puzzling observations on the time course and laminar distribution of neuronal labeling within the spinal cord. This study has analyzed the effect of stimulus duration on the expression of fos-like immunoreactivity (FLI) within the spinal cord of anesthetized rats. In order to examine the time course of fos expression following brief periods of stimulation, we required a type of stimulus that was intense enough to activate nociceptors but that did not produce tissue damage. We have therefore employed pulsed, high intensity electrical stimulation, with stimulus durations ranging from 3 s to 24 h. The results indicate that stimulus duration has a profound effect upon the number of labeled cells, the intensity of neuronal labeling, the laminar pattern of FLI, and the time course of fos expression. Brief stimulation periods induce relatively few and relatively lightly labeled neurons, located predominantly within the most superficial laminae of the dorsal horn. Maximal immunoreactivity appears approximately 2 h after stimulation has ceased, and disappears within hours. Continuous stimulation produces many more labeled cells, darker labeling, and FLI within both dorsal and ventral laminar regions. Maximal FLI is seen after approximately 4.5 h of continuous stimulation, with reduction in the number of labeled cells thereafter. These data indicate that the results of any study employing c-fos as a marker for neuronal activity may be affected by the duration of the exciting stimulus.

Influence of the vanilloid receptor TRPV1 on the activation of spinal cord glia in mouse models of pain

Although activation of spinal glia has been implicated in the development of pathological pain, the mechanisms underlying glial activation are not fully understood. One such mechanism may be triggered by reaction to neuroactive substances released from central axons of sensory afferents. The vanilloid receptor TRPV1, a nonselective cation channel in nociceptive sensory afferents, mediates the release of neurotransmitters, such as glutamate and CGRP in the dorsal horn, which can subsequently activate glia. To test the hypothesis that activation of spinal glia is mediated, at least in part, by TRPV1, we studied the expression of markers for microglia (ionized calcium-binding adapter molecule 1, Iba1) and astrocytes (glial fibrillary acidic protein, GFAP) in the spinal cord of TRPV1 knockout mice (KO) vs. wild-type mice (WT) in models of acute (intraplantar capsaicin), inflammatory (adjuvant-induced arthritis, AIA), and neuropathic pain (partial sciatic nerve ligation, PSNL). We found that (i) naïve KO mice had denser immunostaining for both Iba1 and GFAP than naive WT mice; (ii) the immunostaining for Iba1 increased significantly in treated mice, compared to naïve mice, 3 days after capsaicin and 7-14 days after AIA or PSNL, and was significantly greater in WT than in KO mice 3 days after capsaicin, 7-14 days after AIA, and 7 days after PSNL; and iii) the immunostaining for GFAP increased significantly in treated mice, compared to naïve mice, 3 days after capsaicin and 14-21 days after AIA or PSNL, and was significantly greater in WT than in KO mice 14 days after AIA or PSNL. Our results suggest that TRPV1 plays a role in the activation of spinal glia in mice with nociceptive, inflammatory, and neuropathic pain.

The effect of fentanyl on c-fos expression in the trigeminal brainstem complex produced by pulpal heat stimulation in the ferret

We have previously shown that Fos-like immunoreactivity (Fos-LI) is evoked in the brainstem of ferrets following stimulation of pulpal A delta and C fibers originating from the maxillary canine. This study evaluated the effects of the mu-opioid receptor agonist fentanyl on Fos expression evoked by noxious thermal stimulation of the right maxillary and mandibular canines in pentobarbital/chloral hydrate anesthetized adult male ferrets. Pulpal heating evoked Fos expression in two distinct regions of the spinal trigeminal nuclear complex: the transitional region between subnucleus interpolaris and caudalis (Vi/Vc) and within the subnucleus caudalis (Vc). More Fos positive cells were expressed in both regions ipsilateral to the site of stimulation compared with the contralateral side (P < 0.05, ANOVA). Pretreatment with fentanyl significantly and dose-dependently suppressed the number of Fos positive cells in both the Vi/Vc transitional region and Vc (P < 0.05, ANOVA). The suppressive effect of fentanyl on Fos expression was blocked by the intravenous administration of naloxone, an opioid antagonist, indicating a specific opioid receptor effect. In addition, opioid receptor antagonism with naloxone alone enhanced Fos expression in Vi/Vc and Vc in response to heat stimulation. The administration of naloxone without heat stimulation failed to evoke Fos expression in Vi/ Vc and Vc. These findings suggest that the activation of trigeminal Vi/Vc and Vc neurons by noxious dental heat stimulation is controlled by a naloxone sensitive endogenous opioid system as indicated by Fos expression. Collectively, these results suggest that neuronal populations in Vi/Vc and Vc regions may contribute to pain responses to noxious dental stimulation and these responses can be modulated by both endogenous and exogenous opioids.

Receptor activity modifying proteins (RAMPs) interact with the VPAC2 receptor and CRF1 receptors and modulate their function

Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon‐like peptide 1 receptor (GLP‐1 receptor), the human vasoactive intestinal polypeptide/pituitary AC‐activating peptide 2 receptor (VPAC2) and the type 1 corticotrophin releasing factor receptor (CRF1) has been examined.

Presynaptic low- and high-affinity kainate receptors in nociceptive spinal afferents

Abstract Presynaptic ionotropic glutamate receptors are increasingly attributed a role in the modulation of sensory input at the first synapse of dorsal root ganglion (DRG) neurons in the spinal dorsal horn. Central terminals of DRG neurons express AMPA and NMDA receptors whose activation modulates the release of glutamate, the main transmitter at these synapses. Previous work, with an antibody that recognizes all low‐affinity kainate receptor subunits (GluR5, 6, 7), provided microscopic evidence of presynaptic kainate receptors in unidentified primary afferent terminals in superficial laminae of the spinal dorsal horn (Hwang SJ, Pagliardini S, Rustioni A, Valtschanoff JG. Presynaptic kainate receptors in primary afferents to the superficial laminae of the rat spinal cord. J Comp Neurol 2001; 436: pp. 275–289). We show here that, although all such subunits may be expressed in these terminals, GluR5 is the subunit most readily detectable at presynaptic sites in sections processed for immunocytochemistry. We also show that the high‐affinity kainate receptor subunits KA1 and KA2 are expressed in central terminals of DRG neurons and are co‐expressed with low‐affinity receptor subunits in the same terminals. Quantitative data show that kainate‐expressing DRG neurons are about six times more likely to express the P2X3 subunit of the purinergic receptor than to express substance P. Thus, nociceptive afferents that express presynaptic kainate receptors are predominantly non‐peptidergic, suggesting a role for these receptors in the modulation of neuropathic rather than inflammatory pain.

Fetal-derived adrenomedullin mediates the innate immune milieu of the placenta.

The remodeling of maternal uterine spiral arteries (SAs) is an essential process for ensuring low-resistance, high-capacitance blood flow to the growing fetus. Failure of SAs to remodel is causally associated with preeclampsia, a common and life-threatening complication of pregnancy that is harmful to both mother and fetus. Here, using both loss-of-function and gain-of-function genetic mouse models, we show that expression of the pregnancy-related peptide adrenomedullin (AM) by fetal trophoblast cells is necessary and sufficient to promote appropriate recruitment and activation of maternal uterine NK (uNK) cells to the placenta and ultimately facilitate remodeling of maternal SAs. Placentas that lacked either AM or its receptor exhibited reduced fetal vessel branching in the labyrinth, failed SA remodeling and reendothelialization, and markedly reduced numbers of maternal uNK cells. In contrast, overexpression of AM caused a reversal of these phenotypes with a concomitant increase in uNK cell content in vivo. Moreover, AM dose-dependently stimulated the secretion of numerous chemokines, cytokines, and MMPs from uNK cells, which in turn induced VSMC apoptosis. These data identify an essential function for fetal-derived factors in the maternal vascular adaptation to pregnancy and underscore the importance of exploring AM as a biomarker and therapeutic agent for preeclampsia.

Ionotropic glutamate receptors are expressed in GABAergic terminals in the rat superficial dorsal horn.

Ionotropic glutamate receptors (IGR), including NMDA, AMPA, and kainate receptors, are expressed in terminals with varied morphology in the superficial laminae (I–III) of the dorsal horn of the spinal cord. Some of these terminals can be identified as endings of primary afferents, whereas others establish symmetric synapses, suggesting that they may be γ‐aminobutyric acid (GABA)‐ergic. In the present study, we used confocal and electron microscopy of double immunostaining for GAD65, a marker for GABAergic terminals, and for subunits of IGRs to test directly whether IGRs are expressed in GABAergic terminals in laminae I–III of the dorsal horn. Although colocalization is hard to detect with confocal microscopy, electron microscopy reveals a substantial number of terminals immunoreactive for GAD65 also stained for IGRs. Among all GAD65‐immunoreactive terminals counted, 37% express the NMDA receptor subunit NR1; 28% are immunopositive using an antibody for the GluR2/4 subunits of the AMPA receptor; and 20–35% are immunopositive using antibodies for the kainate receptor subunits GluR5, GluR6/7, KA1, or KA2. Terminals immunoreactive for IGR subunits and GAD65 establish symmetric synapses onto dendrites and perikarya and can be presynaptic to primary afferent terminals within both type 1 and type 2 synaptic glomeruli. Activation of presynaptic IGR may reduce neurotransmitter release. As autoreceptors in terminals of Aδ and C afferent fibers in laminae I–III, presynaptic IGRs may play a role in inhibiting nociception. As heteroreceptors in GABAergic terminals in the same laminae, on the other hand, presynaptic IGRs may have an opposite role and even contribute to central sensitization and hyperalgesia. J. Comp. Neurol. 486:169–178, 2005.

Decoy Receptor CXCR7 Modulates Adrenomedullin-Mediated Cardiac and Lymphatic Vascular Development

Atypical 7-transmembrane receptors, often called decoy receptors, act promiscuously as molecular sinks to regulate ligand bioavailability and consequently temper the signaling of canonical G protein-coupled receptor (GPCR) pathways. Loss of mammalian CXCR7, the most recently described decoy receptor, results in postnatal lethality due to aberrant cardiac development and myocyte hyperplasia. Here, we provide the molecular underpinning for this proliferative phenotype by demonstrating that the dosage and signaling of adrenomedullin (Adm, gene; AM, protein)-a mitogenic peptide hormone required for normal cardiovascular development-is tightly controlled by CXCR7. To this end, Cxcr7(-/-) mice exhibit gain-of-function cardiac and lymphatic vascular phenotypes that can be reversed upon genetic depletion of adrenomedullin ligand. In addition to identifying a biological ligand accountable for the phenotypes of Cxcr7(-/-) mice, these results reveal a previously underappreciated role for decoy receptors as molecular rheostats in controlling the timing and extent of GPCR-mediated cardiac and vascular development.

AMPA and NMDA glutamate receptors are found in both peptidergic and non-peptidergic primary afferent neurons in the rat

Two distinct classes of nociceptive primary afferents, peptidergic and non-peptidergic, respond similarly to acute noxious stimulation; however the peptidergic afferents are more likely to play a role in inflammatory pain, while the non-peptidergic afferents may be more characteristically involved in neuropathic pain. Using multiple immunofluorescence, we determined the proportions of neurons in the rat L4 dorsal root ganglion (DRG) that co-express AMPA or NMDA glutamate receptors and markers for the peptidergic and non-peptidergic classes of primary afferents, substance P and P2X3, respectively. The fraction of DRG neurons immunostained for the NR1 subunit of the NMDA receptor (40%) was significantly higher than that of DRG neurons immunostained for the GluR2/3 (27%) or the GluR4 (34%) subunits of the AMPA receptor. Of all DRG neurons double-immunostained for glutamate receptor subunits and either marker for peptidergic and non-peptidergic afferents, a significantly larger proportion expressed GluR4 than GluR2/3 or NR1 and in a significantly larger proportion of P2X3- than SP-positive DRG neurons. These observations support the idea that nociceptors, involved primarily in the mediation of neuropathic pain, may be presynaptically modulated by GluR4-containing AMPA receptors.

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.