µ-Opioid receptor inhibition of substance P release from primary afferents disappears in neuropathic pain but not inflammatory pain.

Opiate analgesia in the spinal cord is impaired during neuropathic pain. We hypothesized that this is caused by a decrease in µ-opioid receptor inhibition of neurotransmitter release from primary afferents. To investigate this possibility, we measured substance P release in the spinal dorsal horn as neurokinin 1 receptor (NK1R) internalization in rats with chronic constriction injury (CCI) of the sciatic nerve. Noxious stimulation of the paw with CCI produced inconsistent NK1R internalization, suggesting that transmission of nociceptive signals by the injured nerve was variably impaired after CCI. This idea was supported by the fact that CCI produced only small changes in the ability of exogenous substance P to induce NK1R internalization or in the release of substance P evoked centrally from site of nerve injury. In subsequent experiments, NK1R internalization was induced in spinal cord slices by stimulating the dorsal root ipsilateral to CCI. We observed a complete loss of the inhibition of substance P release by the µ-opioid receptor agonist [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO) in CCI rats but not in sham-operated rats. In contrast, DAMGO still inhibited substance P release after inflammation of the hind paw with complete Freund's adjuvant and in naïve rats. This loss of inhibition was not due to µ-opioid receptor downregulation in primary afferents, because their colocalization with substance P was unchanged, both in dorsal root ganglion neurons and primary afferent fibers in the dorsal horn. In conclusion, nerve injury eliminates the inhibition of substance P release by µ-opioid receptors, probably by hindering their signaling mechanisms.

Selective knockdown of NMDA receptors in primary afferent neurons decreases pain during phase 2 of the formalin test.

The role of NMDA receptors (NMDARs) expressed by primary afferent neurons in nociception remains controversial. The aim of this study was to develop mice with a tissue selective knockdown of NMDARs in these neurons and to evaluate their behavioral responses to different types of painful stimuli. Mice with floxed NMDAR NR1 subunit gene (fNR1) were crossed with mice expressing Cre recombinase under the control of the peripherin promotor (Prph-Cre). Male Prph-Cre+ floxed NR1 mice were compared to Cre- littermates. Both quantitative RT/PCR and Western blotting indicated a ∼75% reduction in NR1 expression in dorsal root ganglia (DRG) extracts with no effect on NR1 expression in spinal cord, brain or the enteric nervous system. Immunocytochemistry with antibodies to NR1 revealed decreased staining in all size classes of DRG neurons. NMDA produced a detectable increase in [Ca2+]i in 60% of DRG neurons cultured from Cre- mice, but only 15% of those from Cre+ mice. Furthermore, the peak [Ca2+]i responses were 64% lower in neurons from Cre+ mice. There was no significant difference between Cre+ and Cre- mice in response latencies to the hotplate or tail withdrawal tests of thermal nociception, nor was there a difference in withdrawal thresholds to mechanical stimuli of the tail or paw. However, compared to Cre- littermates, Cre+ knockdown mice had a 50% decrease in the phase 2 response to formalin injection (P<0.001). There was no effect on phase 1 responses. These results suggest that NMDA receptors expressed by primary afferent nerves play an important role in the development of sensitized pain states.

NMDA receptors in primary afferents require phosphorylation by Src family kinases to induce substance P release in the rat spinal cord.

The function of N-methyl-d-aspartate (NMDA) receptors in primary afferents remains controversial, in particular regarding their ability to evoke substance P release in the spinal cord. The objective of this study was, first, to confirm that substance P release evoked by NMDA is mediated by NMDA receptors in primary afferent terminals. Second, we investigated whether these NMDA receptors are inactivated in some conditions, which would explain why their effect on substance P release was not observed in some studies. Substance P release was induced in spinal cord slices and measured as neurokinin 1 (NK1) receptor internalization in lamina I neurons. NMDA (combined with d-serine) induced NK1 receptor internalization with a half of the effective concentration (EC50) of 258 nM. NMDA-induced NK1 receptor internalization was abolished by the NK1 receptor antagonist L-703,606, confirming that is was caused by substance P release, by NMDA receptor antagonists (MK1801 and ifenprodil), showing that it was mediated by NMDA receptors containing the NR2B subunit, and by preincubating the slices with capsaicin, showing that the substance P release was from primary afferents. However, it was not affected by lidocaine and omega-conotoxin MVIIA, which block Na+ channels and voltage-dependent Ca2+ channels, respectively. Therefore, NMDA-induced substance P release does not require firing of primary afferents or the opening of Ca2+ channels, which is consistent with the idea that NMDA receptors induce substance P directly by letting Ca2+ into primary afferent terminals. Importantly, NMDA-induced substance P release was eliminated by preincubating the slices for 1 h with the Src family kinase inhibitors PP1 and dasatinib, and was substantially increased by the protein tyrosine phosphatase inhibitor BVT948. In contrast, PP1 did not affect NK1 receptor internalization induced by capsaicin. These results show that tyrosine-phosphorylation of these NMDA receptors is regulated by the opposite actions of Src family kinases and protein tyrosine phosphatases, and is required to induce substance P release.

Substance P release and neurokinin 1 receptor activation in the rat spinal cord increase with the firing frequency of C-fibers.

Both the firing frequency of primary afferents and neurokinin 1 receptor (NK1R) internalization in dorsal horn neurons increase with the intensity of noxious stimulus. Accordingly, we studied how the pattern of firing of primary afferent influences NK1R internalization. In rat spinal cord slices, electrical stimulation of the dorsal root evoked NK1R internalization in lamina I neurons by inducing substance P release from primary afferents. The stimulation frequency had pronounced effects on NK1R internalization, which increased up to 100 Hz and then diminished abruptly at 200 Hz. Peptidase inhibitors increased NK1R internalization at frequencies below 30 Hz, indicating that peptidases limit the access of substance P to the receptor at moderate firing rates. NK1R internalization increased with number of pulses at all frequencies, but maximal internalization was substantially lower at 1-10 Hz than at 30 Hz. Pulses organized into bursts produced the same NK1R internalization as sustained 30 Hz stimulation. To determine whether substance P release induced at high stimulation frequencies was from C-fibers, we recorded compound action potentials in the sciatic nerve of anesthetized rats. We observed substantial NK1R internalization when stimulating at intensities evoking a C-elevation, but not at intensities evoking only an Adelta-elevation. Each pulse in trains at frequencies up to 100 Hz evoked a C-elevation, demonstrating that C-fibers can follow these high frequencies. C-elevation amplitudes declined progressively with increasing stimulation frequency, which was likely caused by a combination of factors including temporal dispersion. In conclusion, the instantaneous firing frequency in C-fibers determines the amount of substance P released by noxious stimuli.

Acute inflammation induces segmental, bilateral, supraspinally mediated opioid release in the rat spinal cord, as measured by mu-opioid receptor internalization.

The objective of this study was to measure opioid release in the spinal cord during acute and long-term inflammation using mu-opioid receptor (MOR) internalization. In particular, we determined whether opioid release occurs in the segments receiving the noxious signals or in the entire spinal cord, and whether it involves supraspinal signals. Internalization of neurokinin 1 receptors (NK1Rs) was measured to track the intensity of the noxious stimulus. Rats received peptidase inhibitors intrathecally to protect opioids from degradation. Acute inflammation of the hind paw with formalin induced moderate MOR internalization in the L5 segment bilaterally, whereas NK1R internalization occurred only ipsilaterally. MOR internalization was restricted to the lumbar spinal cord, regardless of whether the peptidase inhibitors were injected in a lumbar or thoracic site. Formalin-induced MOR internalization was substantially reduced by isoflurane anesthesia. It was also markedly reduced by a lidocaine block of the cervical-thoracic spinal cord (which did not affect the evoked NK1R internalization) indicating that spinal opioid release is mediated supraspinally. In the absence of peptidase inhibitors, formalin and hind paw clamp induced a small amount of MOR internalization, which was significantly higher than in controls. To study spinal opioid release during chronic inflammation, we injected complete Freund's adjuvant (CFA) in the hind paw and peptidase inhibitors intrathecally. Two days later, no MOR or NK1R internalization was detected. Furthermore, CFA inflammation decreased MOR internalization induced by clamping the inflamed hind paw. These results show that acute inflammation, but not chronic inflammation, induces segmental opioid release in the spinal cord that involves supraspinal signals.

Calcitonin receptor-like receptor and receptor activity modifying protein 1 in the rat dorsal horn: localization in glutamatergic presynaptic terminals containing opioids and adrenergic alpha2C receptors.

Calcitonin gene-related peptide (CGRP) is abundant in the central terminals of primary afferents. However, the function of CGRP receptors in the spinal cord remains unclear. CGRP receptors are heterodimers of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1). We studied the localization of CRLR and RAMP1 in the rat dorsal horn using well-characterized antibodies against them, which labeled numerous puncta in laminae I-II. In addition, RAMP1 was found in cell bodies, forming patches at the cell surface. The CRLR- and RAMP1-immunoreactive puncta were further characterized using double and triple labeling. Colocalization was quantified in confocal stacks using Imaris software. CRLR did not colocalize with primary afferent markers, indicating that these puncta were not primary afferent terminals. CRLR- and RAMP1-immunoreactive puncta contained synaptophysin and vesicular glutamate transporter-2 (VGLUT2), showing that they were glutamatergic presynaptic terminals. Electron microscopic immunohistochemistry confirmed that CRLR immunoreactivity was present in axonal boutons that were not in synaptic glomeruli. Using tyramide signal amplification for double labeling with the CRLR and RAMP1 antibodies, we found some clear instances of colocalization of CRLR with RAMP1 in puncta, but their overall colocalization was low. In particular, CRLR was absent from RAMP1-containing cells. Many of the puncta stained for CRLR and RAMP1 were labeled by anti-opioid and anti-enkephalin antibodies. CRLR and, to a lesser extent, RAMP1 also colocalized with adrenergic alpha(2C) receptors. Triple label studies demonstrated three-way colocalization of CRLR-VGLUT2-synaptophysin, CRLR-VGLUT2-opioids, and CRLR-opioids-alpha(2C) receptors. In conclusion, CRLR is located in glutamatergic presynaptic terminals in the dorsal horn that contain alpha(2C) adrenergic receptors and opioids. Some of these terminals contain RAMP1, which may form CGRP receptors with CRLR, but in others CRLR may form other receptors, possibly by dimerizing with RAMP2 or RAMP3. These findings suggest that CGRP or adrenomedullin receptors modulate opioid release in the dorsal horn.

N-methyl-D-aspartate receptors and large conductance calcium-sensitive potassium channels inhibit the release of opioid peptides that induce mu-opioid receptor internalization in the rat spinal cord.

Endogenous opioids in the spinal cord play an important role in nociception, but the mechanisms that control their release are poorly understood. To simultaneously detect all opioids able to activate the mu-opioid receptor, we measured mu-opioid receptor internalization in rat spinal cord slices stimulated electrically or chemically to evoke opioid release. Electrical stimulation of the dorsal horn in the presence of peptidase inhibitors produced mu-opioid receptor internalization in half of the mu-opioid receptor neurons. This internalization was rapidly abolished by N-methyl-D-aspartate (IC50=2 microM), and N-methyl-D-aspartate antagonists prevented this effect. mu-Opioid receptor internalization evoked by high K+ or veratridine was also inhibited by N-methyl-D-aspartate receptor activation. N-methyl-D-aspartate did not affect mu-opioid receptor internalization induced by exogenous endomorphins, confirming that the effect of N-methyl-D-aspartate was on opioid release. We hypothesized that this inhibition was mediated by large conductance Ca2+-sensitive K+ channels BK(Ca2+). Indeed, inhibition by N-methyl-D-aspartate was prevented by tetraethylammonium and by the selective BK(Ca2+) blockers paxilline, penitrem A and verruculogen. Paxilline did not increase mu-opioid receptor internalization in the absence of N-methyl-D-aspartate, indicating that it does not produce an increase in opioid release unrelated to the inhibition by N-methyl-d-aspartate. The BK(Ca2+) involved appears to be a subtype with slow association kinetics for iberiotoxin, which was effective only with long incubations. The BK(Ca2+) opener NS-1619 also inhibited the evoked mu-opioid receptor internalization, and iberiotoxin prevented this effect. We concluded that Ca2+ influx through N-methyl-D-aspartate receptors causes the opening of BK(Ca2+) and hyperpolarization in opioid-containing dorsal horn neurons, resulting in the inhibition of opioid release. Since mu-opioid receptors in the dorsal horn mediate analgesia, inhibition of spinal opioid release could contribute to the hyperalgesic actions of spinal N-methyl-D-aspartate receptors.

GABA(A) receptor facilitation of neurokinin release from primary afferent terminals in the rat spinal cord.

Our goal was to test the following hypotheses: 1) GABA(A) receptors facilitate neurokinin release from primary afferent terminals; 2) they do this by suppressing an inhibitory effect of GABA(B) receptors; 3) the activation of these two receptors is controlled by the firing frequency of primary afferents. We evoked neurokinin release by stimulating the dorsal root attached to spinal cord slices, and measured it using neurokinin 1 receptor (NK1R) internalization. Internalization evoked by root stimulation at 1 Hz (but not at 100 Hz) was increased by the GABA(A) receptor agonists muscimol (effective concentration of drug for 50% of the increase [EC50] 3 microM) and isoguvacine (EC50 4.5 microM). Internalization evoked by root stimulation at 100 Hz was inhibited by the GABA(A) receptor antagonists bicuculline (effective concentration of drug for 50% of the inhibition [IC50] 2 microM) and picrotoxin (IC50 243 nM). Internalization evoked by incubating the root with capsaicin (to selectively recruit nociceptive fibers) was increased by isoguvacine and abolished by picrotoxin. Therefore, GABA(A) receptors facilitate neurokinin release. Isoguvacine-facilitated neurokinin release was inhibited by picrotoxin, low Cl-, low Ca2+, Ca2+ channel blockers and N-methyl-D-aspartate receptor antagonists. Bumetanide, an inhibitor of the Na(+)-K(+)-2Cl- cotransporter, inhibited isoguvacine-facilitated neurokinin release, but this could be attributed to a direct inhibition of GABA(A) receptors. The GABA(B) agonist baclofen inhibited NK1R internalization evoked by 100 Hz root stimulation (IC50 1.5 microM), whereas the GABA(B) receptor antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP-55845) increased NK1R internalization evoked by 1 Hz root stimulation (EC50 21 nM). Importantly, baclofen inhibited isoguvacine-facilitated neurokinin release, and CGP-55845 reversed the inhibition of neurokinin release by bicuculline. In conclusion, 1) GABA(B) receptors located presynaptically in primary afferent terminals inhibit neurokinin release; 2) GABA(A) receptors located in GABAergic interneurons facilitate neurokinin release by suppressing GABA release onto these GABA(B) receptors; 3) high frequency firing of C-fibers stimulates neurokinin release by activating GABA(A) receptors and inhibiting GABA(B) receptors, whereas low frequency firing inhibits neurokinin release by the converse mechanisms.

Neurokinin release produced by capsaicin acting on the central terminals and axons of primary afferents: relationship with N-methyl-D-aspartate and GABA(B) receptors.

Capsaicin stimulates neurokinin release in the spinal cord when applied both centrally and peripherally. To determine whether these two actions have different mechanisms, we measured neurokinin 1 receptor (NK1R) internalization in rat spinal cord slices elicited by incubating the whole slice or just the dorsal root with capsaicin. NK1R internalization produced by incubating the slices with capsaicin was abolished by the NK1R antagonist RP-67580, by the vanilloid receptor 1 (VR1) antagonist capsazepine, and by eliminating Ca(2+) from the medium, but was not affected by the Na(+) channel blocker lidocaine. Therefore, the internalization was due to neurokinin release mediated by Ca(2+) entry through VR1 receptors, but did not require the firing of action potentials. Incubating the root with capsaicin produced NK1R internalization in the ipsilateral dorsal horn that was abolished when capsazepine or lidocaine was included in, or when Ca(2+) was omitted from, the medium surrounding the root. Therefore, the internalization was mediated by Ca(2+) entry in the axons through VR1, and required firing of action potentials. The efficacy of capsaicin when applied to the root (36+/-3%) was lower than when applied to the slice (91+/-3%), but its potency was the same (0.49 microM and 0.37 microM, respectively). We also investigated whether presynaptic N-methyl-D-aspartate (NMDA) and GABA(B) receptors modulate these two actions of capsaicin. Neither the NMDA receptor blocker MK-801 nor the GABA(B) agonist baclofen decreased NK1R internalization produced by 1 microM capsaicin applied to the slices, but they inhibited the internalization produced by 0.3 microM capsaicin applied to the slices or 1 microM capsaicin applied to the root. Therefore, capsaicin can produce neurokinin release from primary afferents 1) by a direct action on their central terminals and 2) by increasing the firing of action potentials on their axons. The first effect largely bypasses other modulatory mechanism, but the second does not.

Relationship between capsaicin-evoked substance P release and neurokinin 1 receptor internalization in the rat spinal cord.

The relationship between substance P release and the activation of its receptor in the spinal cord remains unclear. Substance P release is usually measured by radioimmunoassay, whereas the internalization of the neurokinin 1 (NK1) receptor has been used to assess its activation by noxious stimuli. Our objective was to compare substance P release and NK1 receptor internalization produced by capsaicin in rat spinal cord slices. Superfusion of the slices with capsaicin for 3 min produced a gradual increase in substance P release that peaked 3-7 min afterward, and then decreased to baseline levels. The concentration-response curve for capsaicin was biphasic, with concentrations above 10 microM producing significantly less release. The effective concentration for 50% of response (EC(50)) for capsaicin, calculated from its stimulatory phase, was 2.3 microM. However, the potency of capsaicin to elicit NK1 receptor internalization in the same slices was one order of magnitude higher (EC(50)=0.37 microM) in lamina I, probably because NK1 receptors become saturated at relatively low concentrations of substance P. The potency of capsaicin to produce internalization was progressively lower in lamina III (EC(50)=1.9 microM) and lamina IV (EC(50)=14.5 microM), suggesting that neurokinins released in laminae I-II become diluted as they diffuse to the inner dorsal horn. To study the correlation between these two measures, we plotted substance P release against NK1 receptor internalization and fitted a saturation binding function to the points. The correlation was good for laminae I (R(2)=0.82) and III (R(2)=0.78), but it was poor (R(2)=0.35) for lamina IV because NK1 receptor internalization kept on increasing at high concentrations of capsaicin, whereas substance P release decreased. In conclusion, amounts of substance P able to activate NK1 receptors may fall under the threshold of detection of radioimmunoassay. Conversely, radioimmunoassay often detects levels of substance P release well over those required to saturate NK1 receptors in the superficial dorsal horn, but that may be able to activate these receptors in nearby regions of the spinal cord.