P. Max Headley

Excitatory amino acids and synaptic transmission: the evidence for a physiological function

For 30 years physiological techniques have been used to investigate excitatory amino acids as neurotransmitters. In the last ten years progress on the definition of receptor subtypes and the availability of more selective agonists and antagonists has fuelled physiological, neurochemical and histochemical approaches to elucidating the involvement of excitatory amino acids at synaptic sites throughout the vertebrate CNS. Here Max Headley and Sten Grillner assess the advances made in defining the roles of excitatory amino acids as functional transmitters, taking examples mainly from studies on the spinal cord, and comment on the limitations of the types of approach that are used in such studies.

NMDA receptor antagonists as analgesics: focus on the NR2B subtype

Ifenprodil and a group of related compounds are selective antagonists of NR2B-containing NMDA receptors. These compounds are antinociceptive in a variety of preclinical pain models and have a much lower side-effect profile compared with other NMDA receptor antagonists. It remains unclear whether the improved safety of these compounds is due to their subtype selectivity or to a unique mode of inhibition of the receptor. Human trials have so far confirmed the good tolerability of these subtype-selective NMDA receptor antagonists; however, whether they are as effective as other NMDA receptor antagonists in pain patients remains to be demonstrated.

Autoradiographic and electrophysiological evidence for excitatory amino acid transmission in the periaqueductal gray projection to nucleus raphe magnus in the rat

Selective retrograde labelling was used as an autoradiographic method to identify possible excitatory amino acid afferents to nucleus raphe magnus (NRM). Injections of 25-50 nl 10(-2) or 10(-3) M D-[3H]aspartate into the NRM resulted in prominent labelling of cells in ventrolateral mesencephalic periaqueductal gray (PAG). Electrophysiologically, stimulation in ventrolateral PAG excited cells in NRM with a latency of 2-12 ms. With many cells, microelectrophoretic application of the excitatory amino acid antagonists, kynurenate and gamma-D-glutamyl-glycine, resulted in a reversible reduction of the PAG-evoked response. Selective antagonists of N-methyl-D-aspartate (NMDA) were less effective. It is suggested that neurones in the ventrolateral PAG projecting to NRM utilize an excitatory amino acid or structurally related compound as a transmitter, and that this transmitter acts on receptors of the non-NMDA type.

Drug-induced rhythmical activity in the inferior olivary complex of the rat

Experiments have been performed on pentobarbitone anesthetized or decerebrated rats. The nature of the synchronous rhythmical activity which occurs in the inferior olive following the electrophoretic or systemic administration of harmaline, harmine, dihydro-beta-erythroidine and various other compounds, is described. Harmine was shown to reduce the late phase of biphasic unitary action potentials and to evoke massed synchronous rhythmical activity on which the units were superimposed. The beta-carboline was more effective than ACh or DL-homocysteate (DLH) in increasing cell discharge rates. Synchronized rhythmical activity was recorded more than 500 mum from the site of ejection of the rhythm-inducing drugs. Developed rhythmical activity reduced the size of antidromic field potentials, but antidromic invasion could reset the rhythm of submaximal rhythmical activity. The effects of ACh and DLH, glycine, GABA, NA, DA and 5-HT were tested on established rhythmical activity. Of these, 5-HT was the only compound which almost invariably antagonized the rhythm. A number of tryptamine derivatives and reported 5-HT antagonists, as well as parachlorophenylalanine, have been tested, but the results were largely inconclusive. The hypothesis is advanced that the drug-induced rhythm results from the inhibition of a tonic inhibitory serotonergic input. This antagonism releases an innate tendency of olivary cells to discharge both rhythmically and synchronously.

Membrane Characteristics and Synaptic Responsiveness of Superficial Dorsal Horn Neurons in a Slice Preparation of Adult Rat Spinal Cord

Intracellular recordings have been made from neurons of the superficial dorsal horn in slices of the lumbar and thoracic spinal cord of young adult rats. Three broad categories of neurons could be distinguished on the basis of their firing patterns to intracellular current pulses and their afterhyperpolarizations (AHP); there was no detectable difference in the regional distribution of the three types. Category 1 cells were characterized by maintained firing to intracellular depolarizing current pulses, brief action potential durations and polyphasic AHPs. Category 2 cells showed spike adaptation, without spike attenuation, during intracellular current pulses, and had monophasic AHPs. Category 3 cells fired only 1 or 2 spikes to maintained depolarizing pulses and had smaller monophasic AHPs than category 2 neurons. Spontaneous excitatory and inhibitory postsynaptic potential (epsp and ipsp) activity was seen with psp durations varying widely. Low intensity electrical stimulation of afferent fibres, or of superficial white matter, resulted in polyphasic epsps and/or ipsps. The spike discharge in response to such afferent inputs correlated with the membrane properties of the cells, such that the synaptic responses of category 1 neurons were usually bursts of spikes, whereas category 2 and 3 neurons either failed to fire or fired only a single spike. These results in adult rat spinal cord suggest that the discharge pattern within synaptic sensory responses of superficial dorsal horn neurons is determined by postsynaptic membrane properties as well as by the pattern of the afferent input.

Actions of kainate and AMPA selective glutamate receptor ligands on nociceptive processing in the spinal cord

Kainate receptors expressing the GluR5 subunit of glutamate receptor are present at high levels on small diameter primary afferent neurones that are considered to mediate nociceptive inputs. This suggests that GluR5 selective ligands could be novel analgesic agents. The role of kainate receptors on C fibre primary afferents has therefore been probed using three compounds that are selective for homomeric GluR5 receptors. The agonist, ATPA, and the antagonists, LY294486 and LY382884, have been tested in four models of nociception: responses evoked by noxious stimulation of the periphery have been recorded electrophysiologically (1) from hemisected spinal cords from neonatal rats in vitro, (2) from single motor units in adult rats in vivo, (3) from dorsal horn neurones in adult rats in vivo, and (4) in hotplate tests with conscious mice. In some protocols comparisons were made with the AMPA selective antagonist GYKI 53655. The agonist ATPA reduced nociceptive reflexes in vitro, but failed to have effects in vivo. In all tests, the GluR5 antagonists reduced nociceptive responses but only at doses that also affected responses to exogenous AMPA. The AMPA antagonist reduced nociceptive responses at doses causing relatively greater reductions of responses to exogenous AMPA. The results indicate that GluR5 selective ligands do reduce spinal nociceptive responses, but they are not strongly analgesic under these conditions of acute nociception.

Reversal by naloxone of the spinal antinociceptive actions of a systemically-administered NSAID

1 Possible interactions between non‐steroidal anti‐inflammatory drugs (NSAIDs) and endogenous opioids were examined in electrophysiological experiments in α‐chloralose anaesthetized spinalized rats without or with carrageenan‐induced acute inflammation of one hindpaw. Spinal reflex responses, monitored as single motor unit discharges, were elicited by noxious pinch and electrical stimuli. 2 The μ‐opioid agonist, fentanyl, was an effective depressant of reflexes under all conditions (ED50 6–14 μg kg−1, i.v.). In rats without peripheral inflammation the NSAID, flunixin, a niflumic acid derivative, had only a small effect that was not dose‐dependent. However, in animals with unilateral inflammation, flunixin reduced spinal reflexes evoked both by noxious pinch stimuli (that activate peripheral nociceptors; ID50 4 mg kg−1, i.v.) and by electrical stimuli (that bypass nociceptor endings; ID50 6.5–11 mg kg−1, i.v.), indicating that it has a central site of action at doses comparable to those used clinically. 3 The opioid antagonist, naloxone (1 mg kg−1, i.v.), reversed all actions of fentanyl. It did not reverse the small effects that flunixin had in rats without inflammation, showing that the NSAID is not a direct opioid agonist. In rats with carrageenan‐induced inflammation of the hindpaw, however, naloxone fully reversed or prevented the antinociception by flunixin, but not that by the α2‐adrenoceptor agonist, medetomidine. 4 We conclude that under conditions of peripheral inflammation and the resultant central changes, the NSAID, flunixin, has antinociceptive actions that are mediated by endogenous opioids acting within the spinal cord.

Spinal antinociceptive actions of μ-and κ-opioids: the importance of stimulus intensity in determining selectivity between reflexes to different modalities of noxious stimulus

1 In electrophysiological experiments in spinalized rats, μ‐ and κ‐opioids were tested intravenously on the responses of single motoneurones to electronically controlled, alternating noxious heat and noxious pinch stimuli. The effects of μ‐ and κ‐opioids were compared with those of the general anaesthetic α‐chloralose and the dissociative anaesthetic/PCP ligand ketamine. 2 The κ‐opioids U‐50,488 (0.5–16 mg kg−1 i.v.) and tifluadom (0.05‐1.6 mg kg−1 i.v.) had very similar actions to the μ‐opioid fentanyl (0.5–16 μg kg−1 i.v.). Thus all three agonists reduced thermal and mechanical nociceptive reflexes in parallel and in a dose‐dependent manner, but only so long as neuronal responses to the alternating stimuli elicited similar excitability levels in the neurone under study. Ketamine (0.5–16 mg kg−1 i.v.) had similar actions to the opioids whereas α‐chloralose (20 mg kg−1 i.v.) had very little effect on neuronal responsiveness. 3 Apparently ‘selective’ depressions by both μ‐ and κ‐opioids could be orchestrated by a deliberate mismatch of the intensities of alternating noxious heat and pinch stimuli; as measured by neuronal firing rate, the weaker of the responses to either type of stimulus was invariably reduced to a greater degree. 4 Similar ‘selectivity’ could be demonstrated for both μ‐ and κ‐ligands when the weaker and stronger responses were of the same modality, being applied by the same pincher device but with alternating applied force. 5 It is concluded that the ‘selective’ spinal actions of κ‐opioids seen in non‐thermal over thermal behavioural models of nociception is likely to be related to the relative intensities, rather than the modalities, of the noxious stimuli used. The validity of the interpretation of results obtained in such behavioural studies is discussed.

The effects of sham and full spinalization on the systemic potency of μ- and κ-opioids on spinal nociceptive reflexes in rats

1 Flexor withdrawal reflexes to noxious mechanical pinch stimuli were recorded as single motor unit activity in α‐chloralose anaesthetized rats, by means of tungsten bipolar electrodes inserted per‐cutaneously into hindlimb flexor muscles. The relative spinal and supraspinal contributions to μ‐ and κ‐opioid agonists in inhibiting these spinal reflexes, together with possible potency changes elicited by surgical trauma, were examined by comparing their relative potencies in spinally unoperated, sham spinalized and spinalized rats. 2 The noxious stimuli, which were of comparable intensity in the three groups, elicited similar mean firing rates of the motor units in all groups. This indicates that the excitability levels in the reflex pathway were not greatly affected by either sham or actual spinalization. 3 The μ‐agonists morphine and fentanyl, and the κ‐agonist U‐50,488H, inhibited the reflexes in a dose‐dependent manner, when administered intravenously in a log2 cumulative dose regime. 4 The surgery of sham spinalization had little effect on the potency of morphine and fentanyl, whereas it doubled the potency of U‐50,488. 5 Spinalization did not affect the potency of morphine. In contrast it decreased the potency of fentanyl 2–4 fold and that of U‐50,488 approximately 6 fold. 6 The effects of all agonists were antagonized by naloxone. Dose‐dependence studies indicating that antagonism of U‐50,488H required about 5 times the dose of naloxone that antagonized morphine. 7 The data suggest that surgical trauma to the spinal column and/or dura mater triggers supraspinal mechanisms that significantly enhance the potency of κ‐ but not μ‐agonists. 8 It is concluded that most of the effects of systemic morphine on spinal reflexes are mediated, under all three conditions tested, by direct effects in the spinal cord. In contrast, the inhibition of reflexes by U‐50,488H is mediated at both spinal and supraspinal levels, the latter being enhanced in the presence of surgical trauma. The differences between morphine and fentanyl remain unexplained.

Exposure of rat spinal neurones to NMDA, AMPA and kainate produces only short-term enhancements of responses to noxious and non-noxious stimuli

The ability of excitatory amino acids (EAAs) to modulate nociceptive and non-nociceptive responses was tested on spinal neurones of the anaesthetized rat. NMDA (N-methyl-D-aspartate), AMPA ((RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate) and kainate were applied by iontophoretic ejection to increase the background firing rate of each cell to approximately 25 spikes/s. Responses to noxious heat and pinch and innocuous tap stimuli were enhanced to similar degrees by all three EAAs and returned to control immediately following termination of EAA ejection. This result shows that, whilst NMDA does enhance synaptic responses of spinal neurones, this effect is little or no greater than for AMPA or kainate. Furthermore, the rapid recovery of nociceptive responses indicates that more than NMDA receptor activation alone is required to induce longer-term enhancement of nociceptive responses (hyperalgesia).