Effects of hypomagnesia on transmitter actions in neocortical slices.

1. The effects of hypomagnesia on the neuronal responses induced by iontophorectically applied acetylcholine, glutamate, N-methylaspartate (NMDA) and gamma-aminobutyric acid (GABA) were investigated using intracellular recording techniques in in vitro slices of sensorimotor cortex (guinea-pigs). 2. Perfusion with Mg-free media, with or without tetrodotoxin (TTX), induced a small hyperpolarization (approximately 4 mV) and a small decrease (approximately 10%) in the input resistance of neurones. During TTX-blockade of Na-spike genesis, spontaneous depolarizing waves of low frequencies were observed in neurones of slices under Mg-free conditions. 3. The effects of acetylcholine and to a lesser extent, GABA actions, were depressed in a dose-dependent, reversible manner by decreases in the [Mg2+] of the perfusing media. In neurones of slices that had been incubated in Mg-free artificial cerebrospinal fluid to ensure a maximal depletion, the responses to these transmitters were potentiated by each sequentially administered increase in extracellular [Mg2+]. The actions of NMDA were potentiated during perfusion of Mg-free media. However, the responses to glutamate, which may activate receptors for NMDA, were either depressed or unchanged under these conditions. 4. A regulatory role for external Mg cations in the responses of neocortical neurones to the transmitter substances, acetylcholine and GABA, can be inferred from these investigations which simulate hypomagnesemia. The dose-dependent depression of GABA actions by low extracellular [Mg2+] additionally provides a plausible mechanism that may contribute to the neuronal hyperexcitability that is observed during conditions of hypomagnesemia.     

Concentration-dependent isoflurane effects on depolarization-evoked glutamate and GABA outflows from mouse brain slices.

The synaptic concentrations of glutamate and gamma-aminobutyric acid (GABA) are modulated by their release and re-uptake. The effects of general anaesthetics on these two processes remain unclear. This study evaluates the effects of isoflurane, a clinically important anaesthetic, on glutamate and GABA release and re-uptake in superfused mouse cerebrocortical slices. Experiments consisted of two 1.5-min exposures to 40 mM KCl in 30 min intervals. During the second exposure, different concentrations of isoflurane with and without 0.3 mM L-transpyrrolidine-2,4-dicarboxylic acid (PDC, a competitive inhibitor of glutamate uptake transporter) or 1 mM nipecotic acid (a competitive inhibitor of GABA uptake transporter) were introduced. The ratios of the second to first KCl-evoked increases in glutamate and GABA were used to determine the isoflurane concentration-response curves. The results can be described as a sum of two independent processes, corresponding to the inhibitions of release and re-uptake, respectively. The EC50 values for the inhibitions of release and re-uptake were 295+/-16 and 805+/-43 microM for glutamate, and 229+/-13 and 520+/-25 microM for GABA, respectively. Addition of PDC did not significantly affect glutamate release but shifted the re-uptake curve to the left (EC50= 315+/-20 microM). Nipecotic acid completely blocked GABA uptake, rendering isoflurane inhibition of GABA re-uptake undetectable. Our data suggest that isoflurane inhibits both the release and re-uptake of neurotransmitters and that the inhibitions occur at different EC50's. For GABA, both EC50's are within the clinical concentration range. The net anaesthetic effect on extracellular concentrations of neurotransmitters, particularly GABA, depends on the competition between inhibition of release and that of re-uptake.     

The role of afferents to the locus coeruleus in the handling stress-induced increase in the release of noradrenaline in the medial prefrontal cortex: a dual-probe microdialysis study in the rat brain

This study was aimed to identify the neuronal pathways that mediate the handling stress-induced increase in the release of noradrenaline in the medial prefrontal cortex of the rat brain. For that purpose a microdialysis probe was implanted in the vicinity of the locus coeruleus and a second probe was placed in the ipsilateral medial prefrontal cortex. Receptor specific antagonists acting on the alpha(2)-adrenoceptor (50 microM idazoxan), GABA(A) (50 microM bicuculline), GABA(B) (100 microM (3, 4-Dichlorophenyl)methyl]propyl](diethoxymethyl) phosphonic acid; CGP 52432), acetylcholine (10 microM atropine), corticotropin releasing factor (CRF) (100 microM butyl-ethyl-[2,5-dimethyl-7-(2,4, 6-trimethyl-phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-amine; CP-154, 526), NMDA glutamate (300 microM (+/-)-3(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid; CPP) and non-NMDA glutamate receptors (500 microM 6,7-dinitroquinoxaline-2, 3-dione; DNQX) were infused into the locus coeruleus by retrograde dialysis, whereas extracellular noradrenaline was recorded in the ipsilateral medial prefrontal cortex. During infusion of the various compounds rats were gently handled for 10 min. Infusion of idazoxan potentiates the handling-induced increase in the release of noradrenaline in the medial prefrontal cortex. The infusions of, atropine, bicuculline, CGP 52432 and DNQX were without effect on the handling response. Infusion of the NMDA receptor antagonist CPP or the non-peptide CRF receptor antagonist CP-154,526 suppressed the stimulation of noradrenaline during stress. It is concluded that alpha(2)-adrenoceptors, NMDA glutamate receptors and CRF receptors modify the handling stress response of locus coeruleus neurones. The data suggest no major role for glutamatergic, GABAergic, or cholinergic afferents to the locus coeruleus in mediating the stress response.     

Effects of depressant amino acids and antagonists on an in vitro spinal cord preparation from the adult rat

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.     

Differential modulation of glutamatergic transmission by 3,5-dibromo-L-phenylalanine

An increasing body of evidence supports the hypothesis that diminished function of N-methyl-D-aspartate (NMDA) receptors and the associated increase in glutamate release and overstimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors are critical elements of the pathophysiology of schizophrenia. Here, we describe a halogenated derivative of the aromatic amino acid L-phenylalanine that 1) activates NMDA receptors, 2) depresses presynaptic glutamate release, and 3) blocks AMPA/kainate receptors. The experiments were conducted in rat cerebrocortical cultured neurons by using the patch-clamp technique. 3,5-Dibromo-L-phenylalanine (3,5-DBr-L-Phe) augmented NMDA miniature excitatory postsynaptic currents (mEPSCs) and activated the steady-state current, effects that were eliminated by NMDA receptor antagonists DL-2-amino-5-phosphonopentanoic acid and MK-801 (dizocilpine maleate; 5H-dibenzo[a,d]cyclohepten-5,10-imine). 3,5-DBr-L-Phe was a partial agonist at the glutamate-binding site of NMDA receptors with an EC50 of 331.6 +/- 78.6 microM and with an efficacy of 30.5 +/- 4.7% compared with NMDA. 3,5-DBr-L-Phe depressed both amplitude and frequency of AMPA/kainate mEPSCs. The IC50 of 3,5-DBr-L-Phe to inhibit AMPA/kainate mEPSC frequency was 29.4 +/- 4.3 microM. 3,5-DBr-L-Phe significantly decreased paired pulse depression of AMPA/kainate EPSCs and attenuated current activated by AMPA with higher efficacy at lower concentration of AMPA. 3,5-DBr-L-Phe neither affected GABA miniature inhibitory postsynaptic currents nor elicited action potentials. By enhancing NMDA receptor function, reducing glutamate release and blocking AMPA/kainate receptors 3,5-DBr-L-Phe represents a new type of polyvalent modulator of glutamatergic synaptic transmission with potential therapeutic applications.     

Interaction of group I mGlu and NMDA receptor agonists within the dorsal horn of the spinal cord of the juvenile rat

1. The modulatory effects of mGlu receptors on NMDA-induced potential changes in spinal motoneurones were studied in vitro. 2. Selective activation of mGlu5 receptors by 10 microM (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG; EC(50)=280 +/- 24 microM) did not produce any change in the ventral root potential. However, the same concentration of CHPG (10 min perfusion) significantly attenuated the NMDA-induced ventral root depolarization (VRD). The effect persisted for 10 min after washout. NMDA-induced responses returned to control in 30 min. Brief co-application of CHPG and NMDA did not alter the NMDA-induced response indicating lack of direct receptor interaction. 3. The attenuating effect of CHPG on the NMDA-induced VRD was inhibited by the mGluR5 receptor antagonist, 2-methyl-6-phenyl-ethynylpyridine (MPEP). 4. In the presence of CGP56433A, a GABA(B) receptor antagonist, the NMDA-induced VRD was unchanged. However, NMDA-induced responses were potentiated after 10 min co-application of CHPG and CGP56433A. 5. (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate ((2R,4R)-APDC), a group II mGlu receptor agonist did not attenuate the NMDA-induced response. 6. Under normal physiological conditions group I mGlu receptor agonists activate at least two populations of neurones: (1) GABA-ergic cells, which could release GABA and inhibit dorsal horn neurones, and (2) deep dorsal horn neurones/motoneurones which express NMDA receptors. Therefore, activation of mGlu5 receptors located on GABA-ergic interneurones could influence any direct potentiating interaction between mGlu5 and NMDA receptors in spinal cord and result in depression of the VRD. In the presence of a GABA(B) receptor antagonist, the direct synergistic interaction is unmasked. These data suggest that group I mGlu receptors provide a complex modulation of spinal synaptic processes.     

Trichloroethanol potentiation of gamma-aminobutyric acid-activated chloride current in mouse hippocampal neurones.

1. The action of 2,2,2-trichloroethanol on gamma-aminobutyric acid (GABA)-activated Cl- current was studied in mouse hippocampal neurones in tissue culture by use of whole-cell patch-clamp recording. 2. Trichloroethanol increased the amplitude of currents activated by 1 microM GABA or 0.1 microM muscimol. Trichloroethanol, 1-25 mM, potentiated current activated by 1 microM GABA in a concentration-dependent manner with an EC50 of 3.0 +/- 1.4 mM and a maximal response (Emax) of 576 +/- 72% of control. 3. Trichloroethanol potentiated currents activated by GABA concentrations < 10 microM, but did not increase the amplitude of currents activated by concentrations of GABA > or = 10 microM. Despite marked potentiation of currents activated by low concentrations of GABA, trichloroethanol did not significantly alter the EC50, slope, or Emax of the GABA concentration-response curve. 4. Trichloroethanol, 5 mM, potentiated GABA-activated current in neurones in which ethanol, 10-500 mM, did not. The effect of trichloroethanol was not altered by the putative ethanol antagonist, Ro 15-4513. Trichloroethanol did not potentiate currents activated by pentobarbitone. 5. In the absence of exogenous GABA, trichloroethanol at concentrations > or = 2.5 mM activated a current that appeared to be carried by Cl- as its reversal potential changed with changes in the Cl- gradient and as it was inhibited by the GABAA antagonists, bicuculline methiodide and picrotoxin. 6. Since trichloroethanol is thought to be the active metabolite of chloral hydrate and other chloral derivative anaesthetics, potentiation of the GABA-activated current in central nervous system neurones by trichloroethanol may contribute to the sedative/hypnotic effects of these agents.     

Actions of anaesthetics and avermectin on GABAA chloride channels in mammalian dorsal root ganglion neurones.

1. The gamma-aminobutyric acid (GABA)-mimetic actions of some anaesthetics and the antehelminthic avermectin B1a were examined on freshly isolated mammalian dorsal root ganglion (DRG) neurones by use of suction electrodes and a single electrode voltage clamp. 2. Pentobarbitone (60 microM-3 mM), chloralose (600 microM-1 mM), etomidate (10-100 microM), alphaxalone (10-60 microM) and avermectin (10-60 microM) directly activated chloride channels in GABA-sensitive DRG neurones. The agonist action was sensitive to block by bicuculline and picrotoxinin. 3. Steady-state current-voltage (I-V) curves for the anaesthetics were either linear, or rectified in the opposite direction to steady-state I-V curves obtained with GABA. Current relaxations in response to voltage jumps were also of the opposite direction. An extra surge of current ('bounce') was commonly observed on washout of some of these agonists. 4. Pentobarbitone was ineffective as an agonist at alkali pH (10.4 and 9.4), but was approximately twice as effective at acid (5.4) than at normal (7.4) pH values. 5. These results suggest that some anaesthetics and avermectin are capable of 'blocking' GABA channels in addition to activating them.     

The dissociative anaesthetics, ketamine and phencyclidine, selectively reduce excitation of central mammalian neurones by N-methyl-aspartate.

The interaction of two dissociative anaesthetics, ketamine and phencyclidine, with the responses of spinal neurones to the electrophoretic administration of amino acids and acetylcholine was studied in decerebrate or pentobarbitone-anaesthetized cats and rats. Both ketamine and phencyclidine selectively blocked excitation by N-methyl-aspartate (NMA) with little effect on excitation by quisqualate and kainate. Ketamine reduced responses to L-aspartate somewhat more than those of L-glutamate; the sensitivity of responses to these two putative transmitters was between that to NMA on one hand and that to quisqualate or kainate on the other. On Renshaw cells, ketamine and phencyclidine reduced responses to acetylcholine less than those to NMA but more than those to quisqualate or kainate. Dorsal root-evoked synaptic excitation of Renshaw cells was reduced to a greater extent than that following ventral root excitation. Intravenous ketamine, 2.5-20 mg/kg, and phencyclidine, 0.2-0.5 mg/kg, also selectively blocked excitation of neurones by NMA. Ketamine showed no consistent or selective effect on inhibition of spinal neurones by electrophoretically administered glycine or gamma-aminobutyricacid (GABA). The results suggest that reduction of synaptic excitation mediated via NMA receptors contributes to the anaesthetic/analgesic properties of these two dissociative anaesthetics.     

A quantitative study of the actions of excitatory amino acids and antagonists in rat hippocampal slices.

1. A quantitative pharmacological investigation of the actions of excitatory amino acids on hippocampal CA1 neurones has been made using a new slice preparation developed for grease gap recording; d.c. potential was measured across a grease barrier placed between alvear fibres and the bathing medium. 2. In Mg2+-free perfusate, N-methyl-D-aspartate (NMDA, 1-100 microM), quisqualate (1-500 microM), kainate (1-200 microM) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 1-100 microM) caused dose-dependent depolarizations. 3. The dose-response relationships were fitted to logistic expressions. The maximum responses to AMPA, NMDA and kainate were similar; their respective EC50 values were 5, 13 and 23 microM. Quisqualate had a smaller maximum; its EC50 value was 10 microM. The slopes of the dose-response relationships were different for the 4 agonists; the order of steepness of the slopes was NMDA greater than AMPA greater than kainate greater than quisqualate. 4. Similar amino acid-induced depolarizations were observed in slices of just the CA1 region or in whole slices bathed in tetrodotoxin. Isolated alvear fibres, however, were insensitive to the excitatory amino acids. 5. D-2-Amino-5-phosphonovalerate (APV, 50 microM) selectively and reversibly antagonized responses induced by NMDA (apparent pA2 = 5.21). 6. Kynurenic acid (1 mM) reversibly depressed responses to the three agonists tested. The dose-ratios for antagonism of AMPA, kainate and quisqualate were 6.9, 5.6 and 4.6 respectively. 7. This preparation has a different sensitivity profile to agonists from those of previously reported preparations of spinal cord, neocortex and cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anaesthetic drugs: linking molecular actions to clinical effects

The use of general anaesthetics has facilitated great advantages in surgery within the last 150 years. General anaesthesia is composed of several components including analgesia, amnesia, hypnosis and immobility. To achieve these components, general anaesthetics have to act via multiple molecular targets at different anatomical sites in the central nervous system. Much of our current understanding of how anaesthetics work has been obtained within the last few years on the basis of genetic approaches, in particular knock-out or knock-in mice. Anaesthetic drugs can be grouped into volatile and intravenous anaesthetics according to their route of administration. Common volatile anaesthetics induce immobility via molecular targets in the spinal cord, including glycine receptors, GABA(A) receptors, glutamate receptors, and TREK-1 potassium channels. In contrast, intravenous anaesthetics cause immobility almost exclusively via GABA(A) receptors harbouring beta3 subunits. Hypnosis is predominantly mediated by beta3-subunit containing GABA(A) receptors in the brain, whereas beta2 subunit containing receptors, which make up more than 50% of all GABA(A) receptors in the central nervous system, mediate sedation. At clinically relevant concentrations, ketamine and nitrous oxide block NMDA receptors. Unlike all other anaesthetics in clinical use they produce analgesia. Not only desired actions of anaesthetics, but also undesired side effects are linked to certain receptors. Respiratory depression involves beta3 containing GABA(A) receptors whereas hypothermia is largely mediated by GABA(A) receptors containing beta2 subunits. These recent insights into the clinically desired and undesired actions of anaesthetic agents provide new avenues for the design of drugs with an improved side-effect profile. Such agents would be especially beneficial for the treatment of newborn children, elderly patients and patients undergoing ambulatory surgery.     

Phaclofen-insensitive presynaptic inhibitory action of (+/-)-baclofen in neonatal rat motoneurones in vitro.

1. Intracellular recordings were made from antidromically identified motoneurones in transverse spinal cord slices from neonatal (12-16 day) rats. 2. Superfusion of (+/-)-baclofen (0.5-50 microM) reduced the excitatory postsynaptic potentials (e.p.s.ps) and inhibitory postsynaptic potentials (i.p.s.ps) evoked by dorsal root or dorsal root entry zone stimulation in a concentration-dependent manner; the calculated EC50 was 2.4 microM. Baclofen in comparable concentrations also reversibly eliminated spontaneously occurring e.p.s.ps and i.p.s.ps. 3. (-)-Baclofen was more effective as compared to baclofen in reducing the synaptic responses, whereas (+)-baclofen at concentrations as high as 50 microM was ineffective. 4. Baclofen (less than 5 microM) attenuated the synaptic responses without causing a significant change of passive membrane properties and depolarizations induced by exogenously applied glutamate. In addition to synaptic depression, baclofen (greater than 5 microM) caused a hyperpolarization associated with decreased membrane resistance in some of the motoneurones; the glutamate responses were also attenuated. 5. Baclofen reversibly depressed the spike after-hyperpolarization of the motoneurones. 6. GABA (1-10 mM) depressed synaptic transmission and depolarized or hyperpolarized motoneurones. While potentiated by the uptake inhibitor nipecotic acid, the synaptic depressant effect of GABA was not antagonized by bicuculline. 7. The synaptic depressant effect of baclofen was neither blocked by GABAA antagonists bicuculline and picrotoxin (10-50 microM) nor by the GABAB antagonist phaclofen (0.1-1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of inhalational general anaesthetics on native glycine receptors in rat medullary neurones and recombinant glycine receptors in Xenopus oocytes.

1. Glycine responses were studied under voltage clamp in Xenopus oocytes injected with cDNA encoding mammalian glycine receptor subunits and in rat medullary neurones. Bath application of glycine gave strychnine-sensitive currents which reversed close to the expected equilibrium potentials for chloride ions. The peak currents for the receptors expressed in oocytes fitted a Hill equation with EC50 = 215 +/- 5 microM and Hill coefficient nH = 1.70 +/- 0.05 (means +/- s.e. means). The peak currents from the receptors in medullary neurones fitted a Hill equation with EC50 = 30 +/- 1 microM and Hill coefficient nH = 1.76 +/- 0.08. The current-voltage relationship for the receptors expressed in oocytes showed strong outward rectification (with Vrev = -21 +/- 2 mV), while that for the glycine responses from the medullary neurones in symmetrical Cl- was linear (with Vrev = 3.2 +/- 0.6 mV). 2. Inhalational general anaesthetics, at concentrations close to their human minimum alveolar concentrations (MACs), potentiated responses to low concentrations of glycine. The potentiation observed with the recombinant receptors (between 60-22%) was approximately twice that found with the medullary neurones (between 40-80%). For both the recombinant receptors and the receptors in medullary neurones, the degree of potentiation increased in the order of methoxyflurane approximately sevoflurane < halothane approximately isoflurane approximately enflurane. There was no significant difference between the potentiations observed for the two optical isomers of isoflurane. 3. For both the recombinant and native receptors, isoflurane potentiated the currents in a dose-dependent manner at low concentrations of glycine, although at high glycine concentrations the anaesthetic had no significant effect on the glycine-activated responses. The major effect of isoflurane was to cause a parallel leftward shift in the glycine concentration-response curves. The glycine EC50 concentration for the recombinant receptors decreased from a control value of 215 +/- 5 microM to 84 +/- 7 microM glycine at 610 microM isoflurane, while that for the medullary neurones decreased from a control value of 30 +/- 1 microM to 18 +/- 2 microM glycine at the same concentration of isoflurane. The potentiation was independent of membrane potential. 4. Isoflurane also potentiated responses to taurine, a partial agonist at the glycine receptor. This was observed for receptors expressed in oocytes at both low and saturating concentrations of taurine. The EC50 concentration decreased from a control value of 1.6 +/- 0.2 to 0.9 +/- 0.1 mM taurine in the presence of 305 microM isoflurane, while the maximum response to taurine increased from 47 +/- 2 to 59 +/- 2% of the maximum response to glycine. 5. Glycine receptors, like other members of the fast ligand-gated receptor superfamily, are sensitive to clinically relevant concentrations of inhalational general anaesthetics. Effects at these receptors may, therefore, play some role in the maintenance of the anaesthetic state.     

The effects of chlormethiazole on single unit activity in rat brain; interactions with inhibitory and excitatory neurotransmitters.

Extracellular recordings were made of single unit activity in the brainstem of urethane anaesthetized rats. Drugs were applied by microiontophoresis from multibarrelled micropipettes or administered intraperitoneally. Chlormethiazole (CMZ) caused a decrease in spontaneous firing rate when applied with high currents (greater than 40 nA). When applied with lower currents CMZ did not cause changes in firing rate, but enhanced the inhibitory effects of gamma-aminobutyric acid (GABA), muscimol and glycine in a dose-dependent manner. The inhibitory actions of acetylcholine were not affected. Excitatory responses to glutamate and acetylcholine were unaffected by applications of CMZ which caused potentiation of GABA, muscimol and glycine. When applied at higher currents CMZ caused a decrease in the response to glutamate. Intraperitoneal administration of CMZ (50-600 mumol kg-1) also enhanced responses to microiontophoretically applied GABA, muscimol and glycine. These results are compared with those reported for other anticonvulsant drugs and possible mechanisms of action of CMZ are discussed.     

Operational characteristics of somatostatin receptors mediating inhibitory actions on rat locus coeruleus neurones.

1. In order to characterize somatostatin (SRIF) receptor inhibiting spontaneous firing of rat locus coeruleus neurones, and their transduction mechanism(S), extracellular recordings were obtained from a pontine slice preparation of rat brain containing the locus coeruleus (LC). LC neurones were identified by electrophysiological and pharmacological properties; spontaneous firing (characteristically 0.5-5 Hz) was reversibly and concentration-dependently inhibited by exogenously applied noradrenaline. 2. Spontaneous firing of LC neurones was reversibly and concentration-dependently inhibited by SRIF and the N-terminally extended form, somatostatin-28 (SRIF-28), with EC50 values of 15.1 and 19.4 nM, respectively. The synthetic SRIF analogues (octreotide, MK-678, BIM-23027 and L-362,855) also caused concentration-dependent inhibition of LC neurone firing with a rank order of agonist potencies compatible with actions at a receptor resembling the recombinant sst2 receptor. The putative sst3 selective agonist, BIM-23056, was without agonist or antagonist effect. 3. Addition of 100 nM desipramine significantly increased the efficacy of exogenously applied noradrenaline (EC50 values, 2.96 and 0.13 microM, absence and presence of desipramine, respectively) but did not significantly affect SRIF-induced inhibition (EC50 values, 15.6 and 8.0 nM, respectively). Furthermore, application of phenoxybenzamine (3 microM) abolished responses to NA, but did not affect responses to SRIF (EC50 = 14.1 nM). 4. Application of the cyclic AMP analogue, 8-bromoadenosine-cyclic monophosphate (8-Br-cyclic AMP; 500 microM), significantly increased the spontaneous firing rate of all neurones tested (223 +/- 24% over basal rate). Concentration-effect curves for SRIF constructed in the absence and presence of 8-Br-cyclic AMP had similar threshold concentrations, maxima and EC50 values. 5. Incubation of pontine slices in a modified artificial CSF containing 500 ng ml-1 pertussis toxin (PTX) for 18 h prior to extracellular recording affected neither the spontaneous firing of LC neurones, nor the inhibitory responses to muscimol (EC50 2.2 and 1.2 microM, absence and presence of PTX). However, inhibitory responses to SRIF were markedly attenuated. 6. We conclude that the inhibitory actions of SRIF on spontaneous firing of LC neurones are mediated directly by activation of somatodendritic SRIF receptors, and not indirectly by release of noradrenaline. The SRIF receptors involved appear to couple via a pertussis toxin sensitive G-protein, and elicit their response by a mechanism apparently independent of inhibition of cyclic AMP formation. The agonist profile of several selective and novel SRIF analogues suggests the identity of this receptor to be similar to the recombinant sst2 receptor.     

Effects of cholinergic overstimulation on isoflurane potency and efficacy in cortical and spinal networks

In scenarios of terrorist attacks with organophosphorus compounds it appears likely that medical aid is required by victims not only suffering from the intoxication but also from physical trauma. These subjects may have to undergo surgical interventions, raising the need for anaesthesia. This prompts the question of how anaesthetic agents work in intoxicated patients. Organophosphates block acetylcholinesterase activity, thereby inducing excessive cholinergic overstimulation in the central nervous system. As the neocortex and spinal cord are important substrates for general anaesthetics, we investigated to what extent cholinergic overstimulation affects the potency and efficacy of the commonly used volatile anaesthetic isoflurane in depressing action potential activity of cortical and spinal neurons. We first quantified the effects of isoflurane in the absence of acetylcholine by performing extracellular voltage recordings in cultured tissue slices. Isoflurane induced a concentration-dependent decrease of neuronal activity in neocortical (EC(50)=0.43+/-0.08 MAC) and spinal slices (EC(50)=0.41+/-0.03 MAC). At concentrations above 1.5 MAC, the anaesthetic almost completely depressed action potential firing in both preparations. Next, we studied the effects of acetylcholine (10microM) in the absence of isoflurane. Acetylcholine approximately doubled spontaneous activity in neocortical and spinal slices. When applying isoflurane together with acetylcholine, different interactions between these agents were observed in neocortical and spinal networks. Acetylcholine significantly reduced both the potency and efficacy of the anaesthetic in neocortical (efficacy 83%; EC(50)=1.16+/-0.02 MAC) but not in spinal (efficacy 100%; EC(50)=0.41+/-0.04 MAC) slices. Our results indicate that cholinergic overstimulation increases the requirement for anaesthetic agents in patients suffering from organophosphorus poisoning via enhancing neuronal background activity of neocortical and spinal neurons and in addition via decreasing drug potency and efficacy in the cortex. Raising anaesthetic concentrations into a high-dose range may not be an appropriate alternative to compensate the increased excitability, since high concentrations of anaesthetics may worsen cardiac abnormalities and hemodynamic instability frequently observed in these patients.     

Pregnenolone and its sulphate modulate GABAA-receptor-mediated contractile responses in the guinea-pig isolated ileum

Pregnenolone and its sulphate had concentration-dependent dual actions on gamma-aminobutyric acid (GABAA)-receptor-mediated ileal contractile responses, enhancing at 0.001-1 microM but inhibiting at 10 microM. There was a sinistral shift of the GABA dose-response curve in the presence of 0.01 microM pregnenolone, with a significant potentiation over the lower concentration range of GABA (3-30 microM), whilst 10 microM pregnenolone depressed the curve by 50% without a significant change in the EC50, in a manner resembling non-competitive inhibition with picrotoxin. Pregnenolone and its sulphate evidently have modulatory actions at ileal GABAA-receptor complexes in keeping with those seen using neurochemical studies in the central nervous system.     

Equilibrium constants for (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) acting at recombinant NR1/NR2A and NR1/NR2B N-methyl-D-aspartate receptors: Implications for studies of synaptic transmission

We have quantified the effects of the N-methyl-d-aspartate (NMDA) receptor antagonist (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) at rat recombinant N-methyl-D-aspartate receptor (NR)1/NR2A and NR1/NR2B NMDA receptors expressed in Xenopus laevis oocytes. We observed no difference in the steady-state levels of inhibition produced by NVP-AAM077 when it was either preapplied or coapplied with glutamate. The IC50 values for NVP-AAM077 acting at NR1/NR2A NMDA receptors were, as expected, dependent on the glutamate concentration used to evoke responses, being 31 +/- 2 nM (with glutamate at its EC50 concentration) and 214 +/- 10 nM (at 10 times the EC50 concentration). Schild analysis confirmed that the antagonism produced by NVP-AAM077 at NR1/NR2A NMDA receptors was competitive and gave an estimate of its equilibrium constant (K(B)) of 15 +/- 2 nM. Furthermore, Schild analysis of an NMDA receptor carrying a threonine-to-alanine point mutation in the NR2A ligand binding site indicated that NVP-AAM077 still acted in a competitive manner but with its K(B) increased by around 15-fold. At NR1/NR2B NMDA receptors, NVP-AAM077 displayed reduced potency. An IC50 value of 215 +/- 13 nM was obtained in the presence of the EC50 concentration of glutamate (1.5 microM), whereas a value of 2.2 +/- 0.14 microM was obtained with higher (15 microM) glutamate concentrations. Schild analysis gave a K(B) for NVP-AAM077 at NR2B-containing receptors of 78 +/- 3 nM. Finally, using a kinetic scheme to model "synaptic-like" activation of NMDA receptors, we show that the difference in the equilibrium constants for NVP-AAM077 is not sufficient to discriminate between NR2A-containing or NR2B-containing NMDA receptors.     

Cellular uptake disguises action of L-glutamate on N-methyl-D-aspartate receptors. With an appendix: diffusion of transported amino acids into brain slices.

Pharmacological properties of the guanosine 3'5'-cyclic monophosphate (cyclic GMP) responses to excitatory amino acids and their analogues were compared in slices and dissociated cells from the developing rat cerebellum maintained in vitro. The intention was to determine the extent to which cellular uptake might influence the apparent properties of receptor-mediated actions of these compounds. In slices, the potencies of the weakly (or non-) transported analogues, N-methyl-D-aspartate (NMDA) and kainate (KA) (EC50 = 40 microM each) were higher than those of the transported amino acids, D- and L-aspartate (EC50 = 250 microM and 300 microM) and D- and L-glutamate (EC50 = 540 microM and 480 microM). Quisqualate (up to 300 microM) failed to increase cyclic GMP levels significantly. The sensitivity of agonist responses to the NMDA receptor antagonist, DL-2-amino-5-phosphonovalerate (APV), was in the order NMDA greater than L-aspartate greater than L-glutamate, KA. In dissociated cells, L-glutamate was 280 fold more potent (calculated EC50 = 1.7 microM). L- and D-aspartate (calculated EC50 = 13 microM) and D-glutamate (EC50 = 130 microM) were also more effective than in slices. The potencies of NMDA and KA were essentially unchanged. Responses to NMDA, L-glutamate and L-aspartate under these conditions were equally sensitive to inhibition by APV but the response to KA remained relatively resistant to this antagonist. The implications of these results are that, in slices, cellular uptake is responsible for (i) the dose-response curves to L-glutamate, L- and D-aspartate bearing little or no relationship to the true (or relative) potencies of these amino acids; (ii) the potency of APV towards the actions of transported agonists acting at NMDA receptors being reduced and (iii) a differential sensitivity to APV of responses to L-glutamate and L-aspartate being created, the consequence being that a potent action of L-glutamate on NMDA receptors is disguised. These conclusions are supported by theoretical considerations relating to the diffusion of transported amino acids into brain slices, as elaborated in the Appendix.     

N-methyl-D-aspartate exposure blocks glutamate toxicity in cultured cerebellar granule cells.

Exposure of cultured cerebellar granule cells to glutamate results in a concentration-dependent (EC50 = 22.7 +/- 0.4 microM) and delayed (24-72 hr) neurotoxicity, which is blocked by the specific N-methyl-D-aspartate (NMDA) receptor antagonists 2-amino-5-phosphovalerate and MK-801 but is unaffected by the non-NMDA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione. Although glutamate toxicity in these cells is mediated by the NMDA subtype of glutamate receptor, pretreatment of cerebellar granule cells with subtoxic concentrations of NMDA markedly antagonizes the neurotoxic actions of glutamate, with an IC50 of 55 +/- 4 microM. The neuroprotective effect of NMDA requires a preincubation time of approximately 120 min to be fully manifested and does not require the presence of NMDA during glutamate exposure. These data demonstrate that NMDA receptors mediate both neurotoxicity and neuroprotection in cerebellar granule cells. Among four glutamate receptor agonists tested (NMDA, quisqualate, ibotenate, and kainate), only NMDA was able to provide a robust neuroprotection against glutamate toxicity. Quisqualate was neither neurotoxic nor neuroprotective, whereas ibotenate, which was nontoxic by itself, induced a small degree of neuroprotection. In contrast, kainate, which was neurotoxic to cerebellar granule cells, also provided considerable neuroprotection against glutamate toxicity. Because preincubation of cerebellar granule cells with NMDA fails to alter NMDA receptor-mediated phosphoinositide hydrolysis or the specific binding of [3H]MK-801 to NMDA receptors, it appears that the neuroprotective effects of NMDA are not due to NMDA receptor desensitization.