Boris A. Chizh

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.

Modulation of excitatory amino acid responses by tachykinins and selective tachykinin receptor agonists in the rat spinal cord

1 The effects of tachykinins and agonists selective for the three subtypes of neurokinin (NK) receptor have been tested on spinal neuronal responses both to the excitatory amino acids (EAAs) NMDA, AMPA and kainate, and to noxious heat stimuli. The agonists were applied by microiontophoresis in in vivo experiments in α‐chloralose‐anaesthetized, spinalized rats. 2 The NK1selective agonist, GR 73632, enhanced responses to all three EAAs similarly, whilst the NK2‐selective agonist, GR64349, reduced responses to AMPA and kainate without affecting those to NMDA, and the NK3 selective agonist, senktide, enhanced responses to AMPA and kainate. 3 The endogenous ligands substance P (SP) and neurokinin A (NKA) both enhanced responses to NMDA with little effect on responses to kainate, whereas neurokinin B (NKB) selectively enhanced responses to kainate without affecting those to NMDA. 4 The effects of GR73632 on EAA responses showed some differences between the dorsal and ventral horn, with more selectivity towards enhancement of NMDA responses in the ventral horn, but a smaller maximum effect. 5 Background activity was significantly enhanced by GR73632, GR64349, SP and NKA but not by senktide or NKB. GR73632 had the greatest effect on background firing, but this action was variable between cells and was related both to the location within the spinal cord and to the degree of spontaneous activity prior to GR73632 administration. 6 Responses to noxious heat were enhanced consistently only by NKA. 7 These data show that selective agonists for the tachykinin receptors are capable of modulating EAA responses differentially. SP, NKA and NKB appear to act via more than one receptor type when modulating EAA responses in vivo. This indicates that NK‐EAA interactions can be more specific than suggested hitherto, with the combined actions at NK1 and NK2 receptors biasing EAA responsiveness towards NMDA receptor mediated functions, whereas NK3 receptor activation would have the opposite effect. The physiological role of such interactions is likely to be complex.

A comparison of intravenous NBQX and GYKI 53655 as AMPA antagonists in the rat spinal cord

1 The effects of intravenous administration of two α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) antagonists were studied on responses of single neurones to iontophoretically applied excitatory amino acids. The tests were performed on spinal neurones in α‐chloralose anaesthetized, spinalized rats. 2 Both the quinoxaline, NBQX (2–16 mg kg−1) and the 2,3‐benzodiazepine, GYKI 53655 (2–8 mg kg−1) dose‐dependently decreased responses to AMPA. 3 Both compounds were short acting, with half‐recovery times of 15 min for NBQX and 7 min for GYKI 53655. 4 The selectivity for responses to AMPA over those to N‐methyl‐d‐aspartate (NMDA) was significantly poorer for systemic NBQX than for either systemic GYKI 53655 or iontophoretic NBQX, suggesting that systemic NBQX may be converted to a less selective metabolite. 5 GYKI 53655 is therefore likely to be a more valuable tool than NBQX for the study of AMPA receptor‐mediated processes in vivo.

Endogenous modulation of excitatory amino acid responsiveness by tachykinin NK1 and NK2 receptors in the rat spinal cord.

1 The effects of selective tachykinin (neurokinin, NK) NK1 and NK2 receptor antagonists have been examined on spinal neurones in α‐chloralose anaesthetized, spinalized rats. They were tested for effects on responses both to excitatory amino acids (EAA) and to noxious heat stimuli. They were also tested for their ability to reverse the actions of selective NK agonists. 2 The NK1selective antagonists GR82334 (peptide) and CP‐99,994 (non‐peptide), when applied by microiontophoresis, both reduced responses to kainate >AMPA> NMDA. Intravenous CP‐99,994 (3 mg kg−1) also reduced responses to kainate but had inconsistent effects on nociceptive responses. 3 GR82334, applied microiontophoretically, reduced the enhancement by the selective NK1 agonist, GR73632 of both responses to EAAs and background activity. Systemic CP‐99,994 (≤10 mg kg−1) failed to reverse the effects of GR73632. 4 The selective peptide NK2 antagonist, GR103537, had no consistent effects on responses to EAAs when applied by iontophoresis. In contrast, the non‐peptide NK2 antagonist, GR159897, administered systemically (0.5‐2 mg kg−1, i.v.) enhanced responses to kainate (but not NMDA); responses to noxious heat were enhanced only weakly. 5 Iontophoretically‐administered GR 103537 attenuated the effects of the NK2 agonist GR64349, which selectively reduced responses to kainate compared to those to NMDA. Systemically administered GR159897 (≤2 mg kg−1, i.v.) caused little antagonism of the effects of GR64349. 6 The data indicate that under these conditions the non‐peptide antagonists are not reliable at reversing the actions of selective NK agonists. 7 These results suggest that there is a tonic release of endogenous tachykinins that can modulate glutamatergic neurotransmission in the spinal cord. They provide further support for the hypothesis that release of endogenous NKs acting on NK1 and NK2 receptors can promote NMDA receptor mediated glutamatergic transmission.

Coupling of sympathetic and somatic motor outflows from the spinal cord in a perfused preparation of adult mouse in vitro.

1 The relationship between sympathetic and somatic motor outflows from thoraco‐lumbar spinal cord was investigated in a novel arterially perfused trunk‐hindquarters preparation of adult mouse. 2 Ongoing activity was present in both somatic motor (obturator, sciatic or femoral nerves) and sympathetic outflows (either renal nerve or abdominal sympathetic chain). Sympathetic activity was rhythmic with bursts frequencies of 0.6‐2.2 Hz. No obvious rhythmic activity was found in the somatic motor outflow. There were periods during which sympathetic and somatic motor activity were correlated. 3 Addition of NMDA (20‐80 μM) to the perfusate elicited repetitive burst discharges in the somatic motor outflow which were sometimes rhythmic. The frequency of these burst discharges/rhythmic activity varied between preparations but in all cases increased with increasing NMDA concentration. 4 NMDA induced burst discharges in the sympathetic outflow. This bursting activity was of the same frequency as the somatic motor outflow and the two were coupled as revealed by correlation analysis. Periods of coupling persisted for up to 3 min. 5 Administration of hexamethonium (300 μM), to block sympathetic ganglionic transmission, had no effect on the somatic motor activity but severely attenuated sympathetic nerve discharge. 6 The thoraco‐sacral cord therefore has the neuronal machinery necessary for generating and coupling activity in somatic motor and sympathetic outflows. Our findings indicate a dynamic control over the degree of coupling. We discuss that the synchronization of these neural outflows reflects either coupling between two independent mechanisms or the presence of a common synaptic driver impinging on both somatic motor and sympathetic neurones.

Thyrotropin-releasing hormone (TRH)-induced facilitation of spinal neurotransmission: a role for NMDA receptors.

Thyrotropin-releasing hormone (TRH) is known to enhance spinal reflexes and modulate NMDA receptors in supraspinal areas. We have investigated the relationship between TRH and NMDA receptors in the spinal cord of alpha-chloralose-anaesthetized spinalized rats. TRH was tested (a) on dorsal horn neurone responses to iontophoretic NMDA, AMPA and kainate and (b) on spinal reflexes evoked by noxious pinch and electrical stimulation (2 Hz) shown to involve NMDA receptor activation. TRH given i.v. (0.5 mg/kg) or iontophoretically selectively potentiated neuronal responses to NMDA. TRH (0.5-2 mg/kg) also dose-dependently increased single motor unit reflex responses. The NMDA antagonist ketamine (2 mg/kg i.v.) abolished these TRH effects; ketamine reduced single motor unit (SMU) reflex responses more effectively when administered after TRH than in pre-TRH control tests. These results indicate that TRH-induced facilitation of spinal sensory transmission involves NMDA receptor activation.

An arterially-perfused trunk-hindquarters preparation of adult mouse in vitro

We describe a preparation of arterially-perfused spinal cord with attached hindquarters, taken from adult mouse. This is the first preparation of adult mammalian spinal cord tissue to have the advantages of an in vitro approach whilst retaining intact intraspinal circuitry, sensory inputs, and somatic and sympathetic segmental outputs. The functional integrity of the preparation has been demonstrated by the motor and sympathetic reflexes that can readily be evoked by peripheral noxious thermal, mechanical and electrical stimuli, and also by bladder distension. The mechanical stability of the preparation allows intracellular recordings to be made from spinal dorsal or ventral horn neurones. The intact connectivity permits synaptic responses to be evoked by stimulation of functionally-defined peripheral sensory receptors. The preparation is relatively quick to set up and remains viable for more than 6 h. This model offers the opportunity to perform complex electrophysiological and pharmacological studies on functionally characterised synaptic responses of mature spinal neurones. The choice of the mouse will furthermore permit studies to be performed on genetically mutant strains.