Ann F. Sullivan

EVIDENCE FOR SPINAL N-METHYL-D-ASPARTATE RECEPTOR INVOLVEMENT IN PROLONGED CHEMICAL NOCICEPTION IN THE RAT

We used in vivo electrophysiology and a model of more persistent nociceptive inputs to monitor spinal cord neuronal activity in anaesthetised rats to reveal the pharmacology of enhanced pain signalling. The study showed that all responses were blocked by non-selective antagonism of glutamate receptors but a selective and preferential role of the N-methyl-d-aspartate (NMDA) receptor in the prolonged plastic responses was clearly seen. The work lead to many publications, initially preclinical but increasingly from patient studies, showing the importance of the NMDA receptor in central sensitisation within the spinal cord and how this could relate to persistent pain states. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

Subcutaneous formalin-induced activity of dorsal horn neurones in the rat: differential response to an intrathecal opiate administered pre or post formalin

&NA; Many studies of pain and nociception use short‐lasting acute stimuli which may have limited relevance to prolonged or chronic pain states. Using extracellular single‐unit recording in the dorsal horn of the rat lumbar spinal cord the present study examines the response of neurones to a long‐lasting nociceptive stimulus i.e., 50 &mgr;l 5% formalin injected into the corresponding receptive field in the ipsilateral hind paw, and modulation of this response by an opioid. Formalin produced a distinct biphasic excitatory response in all convergent neurones tested; an immediate acute or phasic peak of neuronal firing (mean maximum 22 spikes/sec) 0–10 min post injection, and a second more prolonged tonic excitatory response (mean maximum 12 spikes/sec) over a period 20–65 min after formalin. Cells only activated by innocuous stimuli were not excited by formalin indicating the involvement of C fibre afferents in the excitatory response of convergent neurones to formalin. Both the biphasic nature and the time course of the neuronal response are similar to those observed in behavioural studies. Intrathecal DAGO (Tyr‐D‐AlaGlyMePheGly‐ol), a potent and selective mu opioid receptor agonist, applied 20 min prior to formalin completely inhibited both peaks of excitation. Co‐administration of intrathecal naloxone with the agonist restored the biphasic response. By contrast, when the administration of naloxone was delayed to 2 min post formalin so that inhibition of the first peak by DAGO pretreatment occurred, there was no subsequent second peak of activity although antagonism of the opioid would have occurred. When DAGO was applied 2 min post formalin so the initial acute response occurred, the inhibitory effect of the agonist on the second peak was far less. Thus the relative ability of DAGO to modulate the biphasic excitatory response of cells to formalin depends on whether the agonist is administered prior to or after the formalin and the appearance of the second peak may depend on the presence of the first. These results are discussed in light of the role of these neurones in nociception, opioid effects and changes in neural systems following peripheral stimuli

Differential effects of excitatory amino acid antagonists on dorsal horn nociceptive neurones in the rat

The effects of two excitatory amino acid receptor antagonists gamma-D-glutamylglycine (DGG) and 2-amino-5-phosphonovaleric acid (APS), applied onto the spinal cord surface, were tested on the responses of dorsal horn nociceptive neurones in the anaesthetized rat. DGG is a non-selective antagonist at both the N-methyl-D-aspartate (NMDA) and non-NMDA receptors, whereas AP5 acid is selective for the NMDA receptor. DGG dose-dependently reduced the A and C fibre-evoked responses of neurones in all laminae of the dorsal horn and also inhibited the post-discharges of intermediate and deep neurones resulting from repeated C fibre stimulation. There was little difference in the effects of the antagonist on the intermediate neuronal population compared to superficial or deep cells in the dorsal horn. AP5 has little effect on C fibre-evoked activity in superficial cells but produced slight inhibitions of the C fibre-evoked responses and clear reductions in the post-discharge of the deep neurones. This contrasts with the excitatory effects of the antagonist on both types of responses in the intermediate cells. A fibre-evoked responses were unaffected by AP5. Taking into account the results with the two antagonists it appears that both A and C fibre-evoked responses of dorsal horn nociceptive neurones are mediated by non-NMDA receptors whilst the C fibre-evoked wind-up of deep dorsal horn cells involves the NMDA receptor which also seems to mediate, in a complex manner, C fibre responses of intermediate, presumed substantia gelatinosa neurones. The results are discussed with regard to nociceptive mechanisms in the dorsal horn.

Electrophysiological studies on the effects of intrathecal morphine on nociceptive neurones in the rat dorsal horn

&NA; We have studied the effects of intrathecal morphine on the responses of 38 dorsal horn neurones in the intact rat under halothane anaesthesia to A and C fibre electrical stimulation and to natural stimuli applied to their receptive fields. Morphine selectively reduced the C fibre and pinch evoked activity in a dose‐dependent naloxone‐reversible manner with an ED50 of 7 nmoles. The ‘wind‐up’ of neurones to repetitive stimulation was little altered except with the highest doses (50–150 nmoles) tested. By contrast, the A fibre evoked responses of the neurones were only slightly reduced by morphine and both the tactile responses and receptive field size to innocuous stimuli enhanced for certain cells. The results are discussed in relation to the spinal actions of opiates and their clinical applications.

Peripheral origins and central modulation of subcutaneous formalin-induced activity of rat dorsal horn neurones

Extracellular recordings of single convergent dorsal horn neurones in the spinal cord region L1-L3 were made in rats anaesthetised with halothane in a gaseous mix of N2O and O2. Subcutaneous formalin (5%, 50 microliters) has previously been found to produce a prolonged distinct biphasic response of dorsal horn convergent neurones in the same preparation. The present study demonstrates that the second period of neuronal excitation which occurs at about 20 min and lasts for at least another 40 min, could be abolished by lignocaine (2%, 50 microliters) injected into the site of the formalin injection (n = 5). The inhibition of formalin-evoked activity lasted for about 10-20 min and was followed by complete recovery of the neuronal response. The same dose of i.v. lignocaine had no effect on the formalin-induced neuronal activity (n = 4). Profound inhibitions of the second phase were also produced by tactile segmental stimulation and noxious stimuli applied to widespread areas of the body (diffuse noxious inhibitory controls). These findings are discussed with regard to the peripheral and central consequences of prolonged noxious stimuli.

α2-Adrenoceptor modulation of nociception in rat spinal cord: location, effects and interactions with morphine

The effects of intrathecal clonidine alone and prior to intrathecal morphine were studied on electrically evoked A beta and C fibre activity in the dorsal horn of the halothane-anaesthetised rat. Clonidine reduced C fibre-evoked activity in a dose-dependent manner, to a maximum 52% inhibition which was reversed by rauwolscine and yohimbine but not naloxone. High doses of clonidine also produced small inhibitions of A fibre-evoked activity. Clonidine potentiated the inhibitory action of intrathecal morphine on electrically evoked C fibre activity but not A fibre activity. In addition, the location of alpha 2-adrenoceptor and opiate binding sites in consecutive sections of rat lumbar cord was investigated using in vitro autoradiography with selective ligands, and it was demonstrated that both opiate and alpha 2-receptor types are present within the same superficial layers of the dorsal horn of the same animal. The results indicate that alpha 2-adrenoceptors and opiate receptors can interact in the modulation of nociceptive transmission in rat spinal cord.

Alterations in neuronal excitability and the potency of spinal mu, delta and kappa opioids after carrageenan-induced inflammation

&NA; These electrophysiological results show that the development of inflammation following peripheral injection of carrageenan into the paw is accompanied by alterations in the magnitude of the C‐fibre evoked response of multireceptive dorsal horn neurones. The evoked response of the dorsal horn cells was found to either increase or decrease in the 3 h following the carrageenan injection, and the direction of this change was related to the degree of wind‐up exhibited by the cell. Regardless of whether a cell was facilitated or inhibited by carrageenan, mu, delta and kappa opioids applied topically onto the spinal cord (equivalent to an intrathecal injection) exhibited increased antinociceptive potency. This increased effectiveness was especially marked for the mu opioid, morphine, which showed a 30‐fold increase in potency. Interestingly the facilitations seen with the lowest doses of the mu and kappa opioids in this model in normal animals were absent after carrageenan. In addition, a very low dose of spinal naloxone caused a small but significant reduction in the C‐fibre evoked responses. These results demonstrate that following peripheral inflammation, functional changes develop in both spinal transmission and modulatory systems. Alterations in the antinociceptive potency of opioid agonists occurs, with the mu agonist, morphine, showing the greatest change.

Intrathecal opioids, potency and lipophilicity

&NA; To determine the relationship between potency and lipophilicity for intrathecal opioids, morphine, normorphine, pethidine and methadone were studied in an electrophysiological model in the rat. Dose‐response curves were constructed for the opioid effects on C fibre evoked activity of dorsal horn nociceptive neurones following intrathecal application of each opioid, and the ED50 values were correlated with lipid solubility. A significant inverse correlation was found (P = 0.002; r = 0.998) so that the most lipid soluble drugs were the least potent. The possible mechanism of this relationship, the implications for spinal opioid use and the effect of different administration routes on the relationship between lipid solubility and potency are considered.

Opioid receptor subtypes in the rat spinal cord: electrophysiological studies with μ- and δ-opioid receptor agonists in the control of nociception

We have compared the ability of selective mu- and delta-opiate agonists to modulate nociceptive transmission at the level of the rat dorsal horn using electrophysiological approaches. Single-unit extracellular recordings were made from neurones in the lumbar dorsal horn of the intact rat under halothane anaesthesia. Neurones could be activated by both A- and C-fibre electrical stimulation (and by natural innocuous and noxious stimuli). Agonists were applied directly onto the cord in a volume of 50 microliters. The intrathecal administration of 3 agonists, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) (mu-selective) (2 X 10(-3)-10 nmol) Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET) (mu/delta) (7 X 10(-4)-70 nmol), and cyclic Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) (delta) (2 X 10(-2)-100 nmol) produced dose-dependent inhibitions of C-fibre-evoked neuronal activity whilst A-fibre activity was relatively unchanged. DAGO produced near-maximal inhibitions which could be completely reversed by naloxone (1.5 nmol) whilst DPDPE causes less marked inhibitions which could only be partially reversed by naloxone (1.5-13.5 nmol). DTLET produced effects intermediate to those of DAGO and DPDPE. The results suggest that both mu- and delta-opioid receptors can modulate the transmission of nociceptive information in the rat spinal cord.

The antinociceptive actions of dexmedetomidine on dorsal horn neuronal responses in the anaesthetized rat

The actions of the selective alpha 2-adrenoceptor agonist dexmedetomidine were examined on the nociceptive C and innocuous A beta fibre-evoked responses of dorsal horn neurones to transcutaneous electrical stimulation in the intact anaesthetized rat. C fibre-evoked responses were dose dependently reduced by intrathecal dexmedetomidine--to a maximum 86 +/- 6% inhibition by 10 micrograms of the agonist. The ED50 for inhibition of C fibre responses was estimated to be 2.5 micrograms. A beta-evoked responses were inhibited to a lesser degree--a maximum 54 +/- 8% inhibition after 10 micrograms dexmedetomidine. The antinociceptive effects of dexmedetomidine were reversed by the alpha 2-adrenoceptor antagonist atipamezole and the opioid antagonist naloxone. The results are discussed with reference to adrenergic and opioid mechanisms in the spinal cord.