Inwardly rectifying and Ca2+-permeable AMPA-type glutamate receptor channels in rat neocortical neurons

Inwardly rectifying and Ca2+-permeable AMPA-type glutamate receptor channels in rat neocortical neurons. J. Neurophysiol. 78: 2592-2605, 1997. Current-voltage (I-V) relations and Ca2+ permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)type glutamate receptor channels were investigated in neurons of rat neocortex by using the whole cell patch-clamp technique in brain slices. To activate AMPA receptor channels, kainate was used as a nondesensitizing agonist. A patch pipette was filled with solution containing 100 mu M spermine to maintain the inward rectification of Ca2+-permeable AMPA receptor channels. Three types of responses to kainate were observed: type I response with outwardly rectifying I-V relation, type II response with I-V relation of marked inward rectification, and intermediate response with I-V relation of weaker inward rectification. Neurons with type I, type II and intermediate I-V relations were referred to as type I, type II, and intermediate neurons, respectively. Of a total of 223 recorded cells, 90 (40.4%) were type I, 129 (57.8%) intermediate, and 4 (1.8%) type II neurons. Properties of AMPA receptor channels were examined in the former two types of neurons. The value of PCa:PCs, the ratio of the permeability coefficients of Ca2+ and Cs+, was estimated from the reversal potentials of kainate responses in the outside-out patches bathed in Na+-free solution containing 100 mM Ca2+ according to the constant-field equation. They ranged from 0.05 to 0.10 (0.08 +/- 0. 02, mean +/- SD, n = 8) for type I neurons and from 0.14 to 1.29 (0. 60 +/- 0.37, n = 11) for the intermediate neurons. There was a close correlation between the inward rectification and the Ca2+ permeability in AMPA receptor channels in these neurons. Intermediate neurons stained with biocytin were nonpyramidal cells with ellipsoidal-shaped somata. Type I neurons had either triangular- or ellipsoidal-shaped somata. Excitatory postsynaptic currents (EPSCs) recorded in both type I and intermediate neurons had 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive fast and -2-amino-5-phosphonovalerate-sensitiveslow components. The I-V relation of the fast component exhibited inward rectification in the intermediate neuron, whereas that in the type I neuron showed slight outward rectification. The fast component of EPSCs in the intermediate neuron was suppressed more prominently (to 56 +/- 15% of the control, n = 12) than that in the type I neuron (to 78 +/- 6% of the control, n = 6) by bath application of 1 mM spermine. These results indicate that inwardly rectifying and Ca2+-permeable AMPA receptor channels are expressed in a population of neurons of rat neocortex and are involved in excitatory synaptic transmission.     

Excitation of rat subthalamic nucleus neurones in vitro by activation of a group I metabotropic glutamate receptor

The subthalamic nucleus (SThN) provides a glutamate mediated excitatory drive to several other component nuclei of the basal ganglia, thereby significantly influencing locomotion and control of voluntary movement. We have characterised functionally the metabotropic glutamate (mGlu) receptors in the SThN using extracellular single unit recording from rat midbrain slices. SThN neurones fired action potentials spontaneously at a rate of 10 Hz which was increased by the group I/II mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3 R-ACPD; 1-30 microM) and the group I selective agonist (S, R)-dihydroxyphenylglycine (DHPG; 1-30 microM). However, both the group II selective agonist (1S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; 1 microM) and the group III selective agonist (S)-2-amino-4-phosphonobutanoic acid (L-AP4; 10 microM) were without effect, indicating that the excitation was mediated by a group I mGlu receptor. The excitation caused by DHPG (3 microM) was reversed by co-application of the mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG; 500 microM). Thus a group I mGlu receptor mediates excitation of SThN neurones, and suggests a use for group I mGlu receptor ligands for treatment of both hypo- and hyperkinetic disorders of basal ganglia origin, such as Parkinson's disease and Huntington's disease.     

Endbulb synapses in the anteroventral cochlear nucleus express a specific subset of AMPA-type glutamate receptor subunits

The anteroventral cochlear nucleus (AVCN) acts as the first relay center in the conduction of auditory information from the ear to the brain, and it probably performs a crucial role in sound localization. Auditory nerve input to the principal neurons of the AVCN, the spherical bushy cells, appears to be mediated by an excitatory amino acid such as glutamate, which acts at a specialized, large synaptic ending called an endbulb of Held. Presumably, endbulb synapses contain some specific combination of glutamate receptors to facilitate rapid neurotransmission of auditory signals. AMPA glutamate receptor composition at the endbulb synapses was examined with both light and electron microscope immunocytochemistry. Electron microscope localization of AMPA receptors was examined with two techniques, preembedding immunoperoxidase and postembedding immunogold, which provide maximum sensitivity and greatest accuracy, respectively. Dense and frequent labeling was seen with the AMPA receptor subunit antibodies GluR2/3 and GluR4, which were colocalized at the endbulb synapses. In contrast, immunolabeling with antibody to GluR2 was low. These data indicate that the major glutamate receptor at this synapse is an AMPA receptor made up mainly of GluR3 and GluR4 subunits. Receptors composed of these subunits display properties, such as calcium permeability and rapid desensitization, that facilitate their specialized functions in auditory information processing.     

Agmatine selectively blocks the N-methyl-D-aspartate subclass of glutamate receptor channels in rat hippocampal neurons

We investigated in rat hippocampus neurons whether 4-(aminobutyl)guanidine (agmatine), formed by decarboxylation of L-arginine by arginine decarboxylase and metabolized to urea and putrescine, can modulate the function of N-methyl-D-aspartate (NMDA) receptor channels. In cultured hippocampal neurons studied by whole-cell patch clamp, extracellular-applied agmatine produced a voltage- and concentration-dependent block of NMDA but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid nor kainate currents. Analysis of the voltage dependence of the block suggests that agmatine binds at a site located within the NMDA channel pore with a dissociation constant of 952 microM at 0 mV and an electric distance of 0.62. We also tested effects of several agmatine analogs. Arcaine (1,4-butyldiguanidine) also produced a similar voltage-dependent block of the NMDA current, whereas putrescine (1, 4-butyldiamine) had little effect, suggesting that the guanidine group of agmatine is the active moiety when blocking the NMDA channel. Moreover, spermine (an endogenous polyamine) potentiated the NMDA current even in the presence of blocker agmatine or arcaine, suggesting that the guanidine-containing compounds agmatine and arcaine interact with the NMDA channel at a binding site different from that of spermine. Our results indicate that in hippocampal neurons agmatine selectively modulates the NMDA subclass of glutamate receptor channels mediated by the interaction between the guanidine group and the channel pore. The results support other data that agmatine may function as an endogenous neurotransmitter/neuromodulator in brain.     

Distribution of L-type calcium channels in rat thalamic neurones

One major pathway for calcium entry into neurones is through voltage-activated calcium channels. The distribution of calcium channels over the membrane surface is important for their contribution to neuronal function. Electrophysiological recordings from thalamic cells in situ and after acute isolation demonstrated the presence of high-voltage activated calcium currents. The use of specific L-type calcium channel agonists and antagonists of the dihydropyridine type revealed an about 40% contribution of L-type channels to the total high-voltage-activated calcium current. In order to localize L-type calcium channels in thalamic neurones, fluorescent dihydropyridines were used. They were combined with the fluorescent dye RH414, which allowed the use of a ratio technique and thereby the determination of channel density. The distribution of L-type channels was analysed in the three main thalamic cell types: thalamocortical relay cells, local interneurones and reticular thalamic neurones. While channel density was highest in the soma and decreased significantly in the dendritic region, channels appeared to be clustered differentially in the three types of cells. In thalamocortical cells, L-type channels were clustered in high density around the base of dendrites, while they were more evenly distributed on the soma of interneurones. Reticular thalamic neurones exhibited high density of L-type channels in more central somatic regions. The differential localization of L-type calcium channels found in this study implies their predominate involvement in the regulation of somatic and proximal dendritic calcium-dependent processes, which may be of importance for specific thalamic functions, such as those mediating the transition from rhythmic burst activity during sleep to single spike activity during wakefulness or regulating the relay of visual information.     

Thermodynamic characteristics of hafnium and tantalum diboride in a wide temperature range

Translated from Poroshkovaya Metallurgiya, No. 2(314), pp. 60–64, February, 1989.     

Metabotropic glutamate receptor in GHRH and beta-endorphin neurones of the hypothalamic arcuate nucleus

Growth hormone-releasing hormone (GHRH) and beta-endorphin are mainly synthesized in neurones of the hypothalamic arcuate nucleus. Arcuate neurones also contain both ionotropic and metabotropic glutamate receptors. The aim of present study was to investigate whether glutamate receptors are present in GHRH and beta-endorphin containing nerve cells of this hypothalamic area. Using double-label immunocytochemistry as well as the mirror technique, we found that almost all GHRH and beta-endorphin immunoreactive arcuate neurones contain the metabotropic glutamate receptor la. The observations provide morphological evidence for the view that glutamate, which appears to be a major excitatory neurotransmitter in the hypothalamus, may directly stimulate GHRH and beta-endorphin neurones of the medial hypothalamus.     

Temporal characteristics of capsaicin desensitization and stimulus-induced recovery in the oral cavity

It was recently discovered that capsaicin desensitization of the tongue can be temporarily reversed during recurrent stimulation ("stimulus-induced recovery," SIR). The effects of concentration and the temporal pattern of stimulation on desensitization and SIR were studied in four experiments. In Experiment 1, three different concentrations of capsaicin (3.3, 33, or 330 microM) were delivered to the tongue in blocks of twelve stimuli at the rate of one per minute (treatment block) followed by a block of 20 exposures to 33 microM capsaicin (test block). The 33- and 330-microM treatments both caused significant desensitization, but by the end of the test block recovery was substantial only for the 33-microM condition. Experiment 2 provided a second test of the effect of concentration while also exploring whether intermittent stimulation was a requirement for SIR, i.e., stimuli were refreshed on the tongue every six minutes rather than every 60 s. SIR occurred but was pronounced only when the concentration of the test stimulus equaled or exceeded that of the treatment stimulus. In the third experiment we delivered capsaicin in a candy formulation to examine SIR under conditions in which stimulation was not interrupted at all. Although desensitization was evident during the first few minutes of succeeding exposures to the candy, peak irritation was unchanged throughout nine exposures over 3 days. In the final experiment, we returned to intermittent stimulation to examine the effect of lengthening ISI on SIR. The results showed that SIR remained virtually complete until ISI was lengthened to 60 s. We conclude that SIR is a robust phenomenon that is maximized by rapid exposure to capsaicin in a concentration at least as high as that of the desensitizing stimulus. The implications of these findings for hypotheses about the mechanism of SIR, its function in endogenous chemonociception, and for clinical use of capsaicin as a topical analgesic are discussed.     

Regional difference of high voltage-activated Ca2+ channels in rat CNS neurones

The diversity of high voltage-activated (HVA) Ca2+ channels in rat CNS neurones was investigated with the nystatin perforated patch recording configuration. The neurones were freshly dissociated from rat substantia nigra, ventromedial hypothalamus, tuberomammillary nucleus, nucleus tractus solitarius, hippocampal CA1 region and cerebellum. Five different types of HVA Ca2+ channels were distinguished pharmacologically; dihydropyridine sensitive L-type, omega-conotoxin-GVIA sensitive N-type, omega-agatoxin-IVA sensitive P-type, omega-conotoxin-MVIIC sensitive Q-type, and R-type which is insensitive to these organic Ca2+ antagonists. The results showed clearly that the five subtypes of HVA Ca2+ channels differ considerably in their distribution among various CNS regions.     

Nociceptin receptor coupling to a potassium conductance in rat locus coeruleus neurones in vitro

1. In this study we have examined the effects of nociceptin, an endogenous ligand for the opioid-like receptor ORL1 on the membrane properties of rat locus coeruleus (LC) neurones in vitro, using intracellular and whole cell patch clamp recording. 2. When locus coeruleus neurones were voltage clamped to -60 mV, application to nociceptin caused an outward current in all cells examined (n = 49), with an EC50 of 90 nM. Neither the potency nor the maximal effect of nociceptin was altered in the presence of the peptidase inhibitors, bestatin (20 microM) or thiorphan (2 microM). 3. The outward currents caused by nociceptin in 2.5 mM extracellular K+ reversed polarity at -123 mV, more negative than the predicted K+ reversal potential of -105 mV. Increasing extracellular K+ to 6.5 mM resulted in a shift of the reversal potential of +25 mV, a shift consistent with a K+ conductance. The conductance activated by nociceptin showed mild inward rectification. 4. Application of a high concentration of nociceptin (3 microM) occluded the current produced by simultaneous application of high concentrations of Met-enkephalin (10 microM), (3 microM) somatostatin and UK 14304 (3 microM), indicating that nociceptin activated the same conductance as mu-opioid and somatostatin receptors and alpha 2-adrenoceptors. 5. The actions of nociceptin were weakly antagonized by the opioid antagonist, naloxone, with pKb's estimated from 2 cells of -4.23 and -4.33. The mu-opioid antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2, 1 microM), the opioid antagonist, nalorphine (30 microM) or the somatostatin antagonist, CPP (cyclo(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr[Bz1]) 3 microM) did not affect the nociceptin-induced current. 6. Dynorphin A (microM), another putative endogenous ligand for ORL1, caused a robust outward current in locus coeruleus neurones that was, however, completely antagonized by moderate concentrations of naloxone (300 nM-1 microM). 7. Continuous application of nociceptin (3 microM) resulted in a decrease of the outward current to a steady level of 70% of the maximum response with a t1/2 of 120s. Desensitization was largely homologous because simultaneous application of Met-enkephalin (30 microM) during the desensitized period of the nociceptin response resulted in an outward current that was 92% of control responses to Met-enkephalin in the same cells. Conversely, continuous application of Met-enkephalin (30 microM) resulted in a decrease of Met-enkephalin current to a steady level that was 54% of the initial current. During this desensitized period application of nociceptin (3 microM) resulted in a current that was 78% of the control responses to nociceptin in the same cells. 8. Thus nociceptin potently activates an inwardly rectifying K+ conductance in locus coeruleus neurones, with a pharmacological profile consistent with activation of the ORL1 receptor. Dynorphin A does not appear to be a ligand for ORL1 in rat locus coeruleus neurones.     

Distribution and developmental regulation of metabotropic glutamate receptor 7a in rat brain

Ligation of the TCR-CD3 complex initiates a cascade of tyrosine phosphorylation that results in T cell activation. Initial activation of tyrosine kinases depends on the phosphorylation of activation motifs on CD3 chains. We previously found that a 90-kDa protein was tyrosine phosphorylated upon TCR cross-linking and the induction of the phosphorylation was dependent on the structure of the CD3 complex. In this study, we further characterized p90 phosphorylation. Phosphorylation of p90 was induced only by stimulation through the TCR-CD3 complex but not by other kinds of stimulation including CD28- or hydrogen peroxide-mediated activation and was dynamically regulated. Phosphorylated p90 was associated with the TCR-CD3 complex upon T cell activation. In a normal T cell population, thymocytes but not splenic T cells induced the tyrosine phosphorylation of p90 upon TCR cross-linking. These results suggest that p90 is a novel phosphoprotein associated with the TCR-CD3 complex and may play a role in TCR signaling during thymocyte differentiation.     

In vivo and in vitro evaluation of AMPA receptor antagonists in rat hippocampal neurones and cultured mouse cortical neurones

The effects of four glutamate receptor antagonists on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)- and N-methyl-D-aspartate (NMDA)-responses were evaluated using both in vitro and in vivo electrophysiological techniques: whole cell patch-clamp recordings from cultured mouse cortical neurones and microiontophoresis in the rat hippocampus. The compounds tested were NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline), GYKI 52466 (1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5H-2,3-benzodiaze pine), PNQX (pyrido[3, 4-f]quinoxaline-2,3-dione, 1,4,7,8,9,10-hexahydro-9-methyl-6-nitro-, methanesulfonate), NS377 (7-ethyl-5-phenyl-1,6,7,8-tetrahydro-1,7-diaza-as-indacene-2 ,3-dione), and MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenz(a,d)cycloheptene-5,10-imine hydrogen maleate). In vitro, the IC50 values (in microM) for inhibition of AMPA-evoked inward currents were approximately 0.4 for NBQX, approximately 7.5 for GYKI 52466, approximately 1 for PNQX and approximately 15 for NS377. PNQX and NS377 also inhibited NMDA-induced currents with IC50 values at approximately 5 and approximately 18 microM, respectively, while NBQX at 60 microM and GYKI 52466 at 100 microM had only weak effects. The ED50 values in micromol/kg i.v. for inhibition of AMPA-evoked hippocampal neuronal spike activity in vivo were approximately 32 for NBQX, approximately 19 for GYKI 52466, approximately 17 for PNQX and approximately 11 for NS377 with efficacy values (maximal inhibition) between 71% and 81%. The ED50 values (in [Lmol/kg i.v.) and efficacy values for inhibition of NMDA-evoked hippocampal neuronal spike activity were approximately 28 with an efficacy of 61% for NBQX, approximately 16 with 35% for PNQX and approximately 6 with 61% for NS377. GYKI 52466 did not significantly affect NMDA responses, whereas MK-801 showed NMDA specificity in vivo.     

Role of non-NMDA receptors in osmotic and glutamate stimulation of vasopressin release: effect of rapid receptor desensitization

Previous studies demonstrated that the increase in vasopressin (VP) release and induction of VPmRNA content by osmotic stimulation was blocked by kynurenic acid, a non-specific antagonist of excitatory amino acid (EAA) receptors. In order to identify the type of EAA receptor involved, perifused explants of the hypothalamo-neurohypophyseal system (HNS) were exposed to a ramp increase in osmolality (40 mOsm over 6 h achieved by increasing NaCl) in the presence and absence of 10 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX), an antagonist of non-n-methyl-d-aspartate (NMDA) excitatory amino acid receptors. Vasopressin release and VP mRNA content were significantly increased by exposure to the osmotic stimulus. 6,7-dinitroquinoxaline-2,3-dione inhibited osmotically stimulated VP release (F=16.65, P=0.0008) without significantly reducing basal release. It also prevented the osmotically stimulated increase in VP mRNA content (P <0.05). Although these results implicated glutamate, the primary endogenous ligand for EAA receptors, in the regulation of VP, exogenous glutamate was ineffective in stimulating VP release from HNS explants in either low-Mg2+ or Mg2+-replete medium. However, blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor desensitization with cyclothiazide (100 microM) caused a marked increase in VP release in response to 100 microM glutamate, and blockade of kainate receptor desensitization with concanavalin A resulted in a small, but significant increase in VP release in response to 1 mM glutamate. These results support a role for non-NMDA receptor activation in osmotic regulation of VP release.     

A comparison of the effects of selective metabotropic glutamate receptor agonists on synaptically evoked whole cell currents of rat spinal ventral horn neurones in vitro

1. Whole cell synaptic currents were recorded under voltage clamp from a total of 54 ventral horn neurones held near to their resting potential by the patch clamp technique in immature rat spinal cord preparations in vitro. Twenty eight neurones were identified, by antidromic invasion from ventral roots, as motoneurones. Excitatory postsynaptic currents (e.p.s.cs) of peak amplitude -480 pA +/- 66 s.e. mean and -829 +/- 124 pA were evoked respectively from the unidentified ventral horn neurones and the motoneurones in response to maximal activation of the segmental dorsal root. 2. The e.p.s.cs were depressed reversibly by the metabotropic glutamate agonists 1S3S-1-aminocyclopentane-1,3-dicarboxylate (1S3S-ACPD) (EC50 17.1 microM +/- 0.3 s.e. mean, n = 14) and L-2-amino-4-phosphonobutanoate (L-AP4) (EC50 = 2.19 +/- 0.19 microM, n = 15). Since both agonists independently produced more than 90% depression it is likely that the receptors that mediate their effects are present on the same presynaptic terminals. 3. When the Mg2+ concentration was raised from 0.75 mM to 2.75 mM together with the addition of 50 microM D-2-amino-5-phosphonopentanoate (AP5), a treatment which would increase the proportion of monosynaptic component in the e.p.s.c. the concentration-effect plots for both 1S3S-ACPD (EC50 1.95 +/- 0.4 microM, n = 8) and L-AP4 (EC50 0.55 +/- 0.20 microM, n = 7) were shifted to the left, suggesting that monosynaptic e.p.cs of primary afferents to ventral horn neurones are more susceptible to L-AP4 and 1S3S-ACPD than are other synapses in polysynaptic pathways. 4. lS3S-ACPD (20 and 50 microM) also caused mean sustained inward currents of 95 +/- 31 pA (n = 6) and248 +/- 49 pA (n = 10) respectively. In the combined presence of AP5 (50 microM) and Mg2+ (2.75 mM) themean response to 50 microM lS3S-ACPD was reduced to 106+/- 18 pA (n = 4). In the presence of tetrodotoxin(1 microM) the corresponding value was 48 +/- 6 pA (n = 4). Similar sustained inward currents produced by N-methyl-D-aspartate (NMDA) were almost abolished to < 10 pA in the presence of AP5 and 2.75 mMMg2+. In the presence of tetrodotoxin the maximum inward current produced by NMDA was undiminished. Thus a large component of the excitatory action of lS3S-ACPD was mediated at non-NMDA receptors both directly at the patch-clamped neurones and indirectly by synaptic relay.     

Inhibition by propofol (2,6 di-isopropylphenol) of the N-methyl-D-aspartate subtype of glutamate receptor in cultured hippocampal neurones

1. The effects of propofol (2,6 di-isopropylphenol) on responses to the selective glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and kainate, were investigated in cultured hippocampal neurones of the mouse. Whole cell and single channel currents were recorded by patch-clamp techniques. Drugs were applied with a multi-barrel perfusion system. 2. Propofol produced a reversible, dose-dependent inhibition of whole cell currents activated by NMDA. The concentration of propofol which induced 50% of the maximal inhibition (IC50) was approximately 160 microM. The maximal inhibition was incomplete leaving a residual current of about 33% of the control response. This inhibitory action of propofol was neither voltage- nor use-dependent. 3. Analysis of the dose-response relation for whole cell NMDA-activated currents indicated that propofol caused no significant change in the apparent affinity of the receptor for NMDA. 4. Outside-out patch recordings of single channel currents evoked by NMDA (10 microM) revealed that propofol (100 microM) reversibly decreased the probability of channel opening but did not influence the average duration of channel opening or single channel conductance. 5. Whole-cell currents evoked by kainate (50 microM) were insensitive to propofol (1 microM-1 mM). 6. These results indicate that propofol inhibits the NMDA subtype of glutamate receptor, possibly through an allosteric modulation of channel gating rather than by blocking the open channel. Depression of NMDA-mediated excitatory neurotransmission may contribute to the anaesthetic, amnesic and anti-convulsant properties of propofol.     

Involvement of a cyclic-AMP pathway in group I metabotropic glutamate receptor responses in neonatal rat cortex

3,5-Dihydroxyphenylglycine (DHPG), (S)-3-hydroxyphenylglycine and (S)-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG) stimulated phosphoinositide hydrolysis in neonatal rat cortical slices, but with lower maximal effect, in comparison with 2S,1'S,2'S-2-(2'-carboxycyclopropyl)glycine (L-CCG I) or (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid (1S,3R-ACPD). DHPG, 1S,3R-ACPD, and S-4C3HPG also evoked a rapidly desensitizing increase in [Ca2+]i in cortical layers of neonatal brain slices. (R,S)-alpha-methyl-4-tetrazolyl-phenylglycine (MTPG), and (R,S)-alpha-methyl-4-phosphono-phenylglycine (MPPG) inhibited the increase of phosphoinositide hydrolysis elicited by 1S,3R-ACPD but not that by R,S-DHPG. In contrast, the selective group II receptor agonist (1S,2S,5R,6S)-2-amino-bicyclo-[3.1.0]-hexane-2,6-dicarboxylate (LY 354740) potentiated the response of R,S-DHPG. Finally, 8-(4-chlorophenylthio)-cAMP, a membrane permeant analogue of cAMP, reversed the stimulatory effect of 1S,3R-ACPD and S-4C3HPG on phosphoinositide hydrolysis and [Ca2+]i mobilization, without affecting the response induced by R,S-DHPG. These data suggest that, in neonatal rat cortex, the activation of group II metabotropic glutamate receptors potentiates the phosphoinositide hydrolysis and [Ca2+]i responses mediated by group I metabotropic glutamate receptors.     

Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N-methyl-D-aspartate receptor antagonist actions

1. The block by ifenprodil of voltage-activated Ca2+ channels was investigated in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mM K+ (high-[K+]o) in Fura-2-loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole-cell voltage-clamp. The effects of ifenprodil on voltage-activated Ca2+ channels were compared with its antagonist actions on N-methyl-D-aspartate- (NMDA) evoked responses in the same neuronal preparations. 2. Rises in [Ca2+]i evoked by transient exposure to high-[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine-sensitive Ca2+ channels, with smaller contributions from nifedipine-resistant, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 microM) reversibly attenuated high-[K+]o-evoked rises in [Ca2+]i with an IC50 value of 17 +/- 3 microM, compared with an IC50 value of 0.7 +/- 0.1 microM for the reduction of rises in [Ca2+]i evoked by 20 microM NMDA. Tested in the presence of nifedipine 10 microM, ifenprodil (1-50 microM) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+]o-evoked rise in [Ca2+]i with an IC50 value of 13 +/- 4 microM. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage-activated Ca2+ channels. 3. Application of the polyamine, spermine (0.25-5 mM), produced a concentration-dependent reduction of rises in [Ca2+]i evoked by high-[K+]o. The antagonist effects of ifenprodil 20 micro M on high-[K+]0-evoked rises in [Ca2+]. were attenuated by spermine 0.25 mM but not by putrescine 1 or 5 mM. In contrast,spermine 0.1 mM increased rises in [Ca2+]i evoked by NMDA and enhanced the ifenprodil (5 micro M) block of NMDA-evoked rises in [Ca2+]i.4. Similar results were obtained in mouse cultured hippocampal pyramidal neurones under whole-cell voltage-clamp. Ifenprodil attenuated both the peak and delayed whole-cell IB. with an IC% value of 18 +/- 2 micro M, whilst it attenuated steady-state NMDA-evoked currents with an IC50 of 0.8 +/- 0.2 micro M. Block of IBa by ifenprodil 10 JaM was rapid in onset, fully reversible and occurred without change in thecurrent-voltage characteristics of Ba. The ifenprodil block of IBa was enhanced on membrane depolarization and was weakly dependent on the frequency of current activation. Spermine 0.1 mM potentiated control NMDA-evoked currents but attenuated IB,. In agreement with the microspectrofluorimetric studies, co-application of spermine produced a small enhancement of the inhibitory effect of ifenprodil 10 micro M on NMDA-evoked responses whereas the reduction of I4 by ifenprodil 10 micro M in the presence of spermine was less than expected if the inhibitory effects of ifenprodil and spermine on IBa were simply additive.5. The results indicate that ifenprodil blocks high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones. Although the Ca2+ channel blocking actions of ifenprodil are observed at higher concentrations than those associated with NMDA antagonist activity, Ca2+ channel blockade may contribute, at least in part, to the established neuroprotective and anticonvulsant properties of the compound.     

Localization of ionotropic and metabotropic glutamate receptors in distinct neuronal elements of the rat substantia nigra

PURPOSE: The contingent valuation method (CVM) is a survey-based approach for eliciting consumer's monetary valuations for programme benefits for use in cost-benefit analysis (CBA). We used the conceptual framework of O'Brien and Gafni (1996) to classify and critically appraise health care CVM studies. METHODS: Search of computerized health care and economic citation databases (e.g. MEDLINE, ECONLIT) and manual search for papers published between 1984 1996 reporting primary data valuing health programme benefits in monetary units by CVM using willingness-to-pay (WTP) or accept (WTA). We classified studies using both empirical (i.e. who was surveyed and how) and conceptual criteria (i.e. which measure of consumer utility was measured and why). RESULTS: 48 CVM studies were retrieved; the majority (42) undertook money valuation in the context of cost benefit analysis (CBA), with the remainder being pricing/demand studies. Among the 42 CBA studies, the consumer utility being measured (i.e. compensating (CV) vs. equivalent variation (EV) was explicitly stated in only three (7%) studies). WTP was measured in 95% of studies and WTA in 5%. By cross-tabulation, 42 (91%) studies were designed as WTP/CV, two (4%) were WTP/EV, two (4%) were WTA/CV and no studies used WTA/EV. Most studies were administered by mail (52%) with 38% being in-person interviews. Value elicitation techniques included open-ended questions (38%), payment cards (19%) discrete choice questions (26%) or bidding games (29%). Some form of construct validation tests, particularly associations between WTP and income, were done in 21 studies (50%). CONCLUSIONS: (i) The number of health care CVM studies is growing rapidly and the majority are done in the context of CBA; (ii) there is wide variation among health care CVM studies in terms of the types of questions being posed and the elicitation formats being used; (iii) classification and appraisal of the literature is difficult because reporting of methods and their relationship with the conceptual framework of CBA is poor; (iii) the applicability to health care of the CVM guidelines issued by the National Oceanic and Atmospheric Administration (NOAA) panel for environmental economics is unclear.