Search

A series of Special Issues of Neuropharmacology celebrates the 40th anniversary of a seminal review on excitatory amino acid (EAA) receptors by two pioneers of the field – Dick Evans and Jeff Watkins. Brought together in the Department of Pharmacology at the University of Bristol in the 1970s, they forged a partnership that, through the synthetic chemistry prowess of Jeff Watkins, which provided novel agonists and antagonists for EAA receptors for Dick Evans's deft experimental studies, generated enormous insight into the multitude of actions of EAAs in the nervous system. Among many achievements from this time was not just the naming of the N-methyl- d -aspartate (NMDA) receptor, but also the demonstration of its antagonism by magnesium ions. Here, Dick and Jeff reflect upon those early halcyon days of EAA research, which, as these six 1 Special Issues of Neuropharmacology demonstrate, is very much alive and kicking. Bruno G. Frenguelli, Editor-in-Chief, Neuropharmacology.

The present study aimed to compare the actions of the selective κ opioid receptor agonist enadoline (CI-977) with morphine in order to see if there is a heterogeneity of opioid receptors between spinal reflex pathways. High (C- and A-fibre evoked activity) and low (A-fibres only) intensity electrical stimulation of dorsal roots in the neonatal rat hemisected spinal cord preparation in vitro was used to distinguish between synaptic activity measured in the corresponding ventral root. Enadoline selectively depressed the high intensity-evoked EPSP with an EC 50 of 7.6 nM ( n =7), contrasting with our previous finding in this preparation that morphine is an equipotent depressant of A- and C-fibre-mediated synaptic responses. The depressant effects of enadoline and morphine were reversed by naloxone giving apparent K d values of 14±3 nM ( n =4) for enadoline-induced and 4.2±1 nM ( n =4) for morphine-induced depression. These data suggest that activation of κ opioid receptors has a selective depressant action on C-fibre-mediated synaptic activity. Such a functional difference mediated at a subclass of opioid receptors has not been previously observed in an in vitro spinal preparation.

The potencies of two GABAB receptor antagonists P-[3-aminopropyl]- P-diethoxymethyl-phosphinic acid (CGP35348) and the novel compound 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P- benzyl-phosphinic acid (CGP55845) have been compared in an in vitro spinal cord preparation. They have been tested as antagonists of baclofen-induced depression of EPSCs of patch-clamped dorsal horn neurons following electrical stimulation of dorsal roots. Mean EC50 values for the depressant action of baclofen were increased by 50- and 140-fold respectively in the presence of CGP35348 (200 microM) (n = 5) and CGP55845 (100 nM) (n = 4). This potency of CGP55845 is > 1000-fold higher than that reported previously for other GABAB receptor antagonists.

The patch-clamp technique has been used to record synaptic responses, elicited by electrical stimulation of dorsal roots, in 28 single motoneurones of in vitro spinal cord preparations from neonate (P5 to P8) rats. The effects of (RS)-α-methyl-4-phosphonophenylglycine (MPPG) (200 μM), a potent antagonist at l-2-amino-4-phosphonobutanoate (AP4)-sensitive receptors, and (RS)-α-methyl-4-carboxyphenylglycine (MCPG) (500 μM), which is a less selective antagonist of mGluRs, were tested on EPSCs alone and as antagonists of AP4-induced depression of EPSCs. The ec50 for depression of EPSCs by AP4 (1.16 ± 0.12 μM, n = 8) was increased to 18.9 ± 0.7 μM (n = 6) by MPPG. MCPG (500 μM) had no significant effect on the depressant potency of AP4. Under control conditions, EPSCs had mean peak amplitudes of 983 pA ± 64 SEM and mean charge transferred of 306 ± 37 pC (n = 28). These values were increased significantly (p < 0.05) to 1168 ± 68 pA and 363 ± 39 pC by MPPG (n = 6), and 1150 ± 54 pA and 358 ± 33 pC (n = 6) by MCPG. There was no significant difference between the enhancement of the initial peak of the EPSCs (mean latency from stimulus artifact 5.9 ± 0.3 ms) and later components, suggesting mGluRs to be present on primary afferent terminals presynaptic to motoneurones as well as in pathways via interneurones. These results are consistent with the presence of at least two types of presynaptic mGluR that modulate release of glutamate in segmental pathways convergent onto motoneurones. These receptors appear to be activated by interstitial glutamate tonically present in the present preparations. © 1997 Elsevier Science Ltd. All rights reserved.

The preparation of an isolated hemisected spinal cord preparation, maintained in vitro, from mature (180-300 g body weight) rats is described. Sacral and coccygeal segments (S2-Co1) gave consistent ventral root reflexes (DR-VRP) from electrical stimulation of dorsal roots. The mean latency and amplitude of the fastest component in the ventral root reflex, at 25 degrees C, were 1.6 msec +/- 0.4 SE mean and 8.2 mV +/- 0.9 SE mean, respectively (28 preparations). This component was resistant to the NMDA antagonist, 2-amino-5-phosphonopentanoate (AP5) but was depressed markedly by kynurenate. A slow component of the ventral root reflex, which was sensitive to AP5 was enhanced and spontaneous AP5-sensitive synaptic potentials sensitive to AP5 appeared in the absence of magnesium ions. The excitant amino acids L-aspartate, L-glutamate, N-methyl-D-aspartate (NMDA), kainate and quisqualate produced dose-dependent depolarizing responses in the ventral roots. The relative depolarizing potencies +/- SE mean (N) of NMDA, kainate and quisqualate, relative to L-glutamate = 1, were 96 +/- 30 (6), 234 +/- 57 (6) and 145 +/- 40 (5), respectively. These properties, apart from reduced latency of synaptic responses, are similar to those described previously for preparations from immature animals. However, it will be easier with the mature preparation to selective activate high and low threshold primary afferents.

A mature sacrococcygeal in vitro spinal preparation from the rat has been used to demonstrate effects of neutral amino acids and their antagonists. gamma-Aminobutanoate (GABA), glycine and taurine (0.5-5 mM) produced dose-dependent depression of spontaneous paroxysmal activity generated in Mg2+ -free medium. The depressant effect of GABA was antagonised selectively by picrotoxin (25-50 microM) and the depressant effects of glycine and taurine were antagonised selectively by strychnine (0.2 microM). Glycine (0.5-5 mM) had a dose-dependent depolarizing action which was present at the central ends of isolated ventral roots. gamma-Aminobutanoate and taurine, had only weak depolarizing actions on ventral root fibres. Depolarizing responses to glycine showed a marked fading. Reduction in the fading appeared to be responsible for a paradoxical potentiation of glycine-induced depolarizations, which occurred in the presence of strychnine (0.2-2 microM). Strychnine (2-10 microM), picrotoxin (10-50 microM) or bicuculline (10 microM) had little or no effect on the amplitude, duration or latency of the monosynaptic component of ventral root reflexes evoked by supramaximal stimulation of dorsal roots (DR-VRP). However all three antagonists introduced slow, NMDA receptor mediated, components to these ventral root potentials. Picrotoxin and bicuculline, but not strychnine, reversibly depressed the dorsal root potential evoked from an adjacent dorsal root (DR-DRP). The depressant actions of 2-amino-5-phosphonopentanoate (AP5), kynurenate and 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) revealed both NMDA and non-NMDA receptor mediated components in the dorsal root potential.

Spinal motoneurones of the immature rat have been shown to possess cholinoceptors. Threshold depolarizing responses, recorded from the ventral root of the isolated hemisected spinal cord, were obtained at 1 μM carbachol and these responses were specifically antagonized by atropine (50 nM). Neostigmine (1 μM) potentiated responses to acetylcholine. These effects were observed in the presence of tetrodotoxin (0.1 μM). In the absence of tetrodotoxin, neostigmine (1 μM) produced a depolarization of motoneurones indicating the release of acetylcholine from nerve terminals within the preparation.

Urethane (50 mM) produced a non-selective antagonism of depolarizations evoked by excitant amino acids or carbachol recorded from ventral roots of isolated spinal cord preparations of the frog or immature rat. Depolarizing responses to substance P or eledoisin-related-peptide were either unaffected or potentiated by this concentration of urethane. The threshold level for depression of dorsal to ventral root transmission was 10 mM urethane and transmission was completely blocked at 70–100 mM urethane. It is suggested that post-junctional blockade of the actions of excitant amino acids may be important in the anaesthetic action of urethane.