2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline and 2-amino-4-phosphonobutyric-acid

Information that defines the depth of activation of retinal neurons is useful in considering strategies for stimulation with a retinal prosthesis, or interpreting the results from human studies that have previously been performed. The purpose of this study was to test the assertion that electrical pulse durations >0.5 msec preferentially stimulate retinal neurons deep to the ganglion cell layer. Thirteen Dutch-belted rabbits (1.2-2.0 kg) were used in this study. A Goldmann-like dome was used to deliver photic stimuli to the retina to measure the electroretinogram (ERG) and the light-induced cortical potential (VECP). Then, a micromanipulator was used to position a 500 microm inner diameter bipolar electrode near the visual streak on the epi-retinal surface. Symmetric biphasic pulses (7-1600 microA; 0.25 msec and 2.0 msec pulses per phase; biphasic pulses delivered at 2 Hz) were delivered to the retina with a current source. Extra-dural electrodes were used to record electrical evoked cortical potentials (EECPs) over the occipital cortex by performing 50 consecutive computer-averaged stimulations. The effect on the EECP of sequential epi-retinal (i.e. return electrode on epi-retinal surface) vs. trans-retinal (i.e. return electrode behind sclera) stimulation was compared. The effect upon the ERG, VECP and EECP was then assessed after 2,3,dihydroxy-6-nitro-7-sulfamoyl-benzo-f-quinoxaline (NBQX) at 112 microM concentration, d-2-amino-7-phosphonoheptanoic acid (D-AP7) at 1200 microM concentration, and l-amino-4-phosphonobutyrate (APB) at 300 microM concentration were delivered into the vitreous cavity to selectively block neuronal input to the retinal ganglion cells. Median values were reported. The amplitudes of the light-induced ERG and VECP were markedly reduced by instillation of the intra-vitreal synaptic blocking drugs. By comparison, pharmacological blockade of input to the retinal ganglion cells did not significantly alter the threshold charge or amplitude of the electrically-induced cortical responses (P>0.05). For the electrical stimuli, there was no significant difference in threshold charge for the EECP for epi-retinal vs. trans-retinal stimulation (P>0.05). The amplitude of the EECP increased linearly with increasing charge using both 0.25 msec and 2.0 msec pulses, even after synaptic blockade of input to the ganglion cells. The lack of obvious degradation of cortical amplitudes after drug instillation indicates that neurons of the middle retina

Topics: 2-Amino-5-phosphonovalerate; Aminobutyrates; Animals; Electric Stimulation; Electroretinography; Evoked Potentials, Somatosensory; Evoked Potentials, Visual; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Neurons, Afferent; Quinoxalines; Rabbits; Retina; Retinal Ganglion Cells; Time Factors; Visual Cortex

Electroretinograms (ERGs) were recorded from the giant danio (Danio aequipinnatus) to study glutamatergic input mechanisms onto bipolar cells. Glutamate analogs were applied to determine which receptor types mediate synaptic transmission from rods and cones to on and off bipolar cells. Picrotoxin, strychnine, and tetrodotoxin were used to isolate the effects of the glutamate analogs to the photoreceptor-bipolar cell synapse. Under photopic conditions, the group III metabotropic glutamate receptor (mGluR) antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) only slightly reduced the b-wave, whereas the excitatory amino acid transporter (EAAT) blocker dl-threo-beta-benzyl-oxyaspartate (TBOA) removed most of it. Complete elimination of the b-wave required both antagonists. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) blocked the d-wave. Under scotopic conditions, rod and cone inputs onto on bipolar cells were studied by comparing the sensitivities of the b-wave to photopically matched green and red stimuli. The b-wave was >1 log unit more sensitive to the green than to the red stimulus under control conditions. In CPPG or l-AP4 (l-(+)-2-amino-4-phosphonobutyric acid, a group III mGluR agonist), the sensitivity of the b-wave to the green stimulus was dramatically reduced and the b-waves elicited by the 2 stimuli became nearly matched. The d-wave elicited by dim green stimuli, which presumably could be detected only by the rods, was eliminated by NBQX.. 1) cone signals onto on bipolar cells involve mainly EAATs but also mGluRs (presumably mGluR6) to a lesser extent; 2) rods signal onto on bipolars by mainly mGluR6; 3) off bipolar cells receive signals from both photoreceptor types by AMPA/kainate receptors.

Topics: Aminobutyrates; Anesthetics, Local; Animals; Aspartic Acid; Cyclopentanes; Dose-Response Relationship, Radiation; Drug Interactions; Electroretinography; Excitatory Amino Acid Agonists; GABA Antagonists; Glycine; Glycine Agents; Neurons; Photic Stimulation; Picrotoxin; Quinoxalines; Receptors, Metabotropic Glutamate; Retina; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Strychnine; Synaptic Transmission; Tetrodotoxin; Visual Pathways; Zebrafish

Previous studies have demonstrated that microinjection of the putative group III metabotropic glutamate receptor (mGluR) agonist, l(+)-2-amino-4-phosphonobutyric acid (L-AP4), into the nucleus tractus solitarius (NTS) produces depressor and sympathoinhibitory responses. These responses are significantly attenuated by a group III mGluR antagonist and may involve ionotropic glutamatergic transmission. Alternatively, a previous report in vitro suggests that preparations of L-AP4 may nonspecifically activate NMDA channels due to glycine contamination (Contractor A, Gereau RW, Green T, and Heinemann SF. Proc Natl Acad Sci USA 95: 8969-8974, 1998). Therefore, the present study tested whether responses to L-AP4 specifically require the N-methyl-D-aspartate (NMDA) receptor and whether they are due to actions at the glycine site on the NMDA channel. To test these possibilities in vivo, we performed unilateral microinjections of L-AP4, glycine, and selective antagonists into the NTS of urethane-anesthetized rats. L-AP4 (10 mM, 30 nl) produced sympathoinhibitory responses that were abolished by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (AP-5, 10 mM) but were unaffected by the non-NMDA antagonist 6-nitro-7-sulfamobenzoquinoxaline-2,3-dione (NBQX, 2 mM). Microinjection of glycine (0.02-20 mM) failed to mimic sympathoinhibitory responses to L-AP4, even in the presence of the inhibitory glycine antagonist, strychnine (3 mM). Strychnine blocked pressor and sympathoexcitatory actions of glycine (20 mM) but failed to reveal a sympathoinhibitory component due to presumed activation of NMDA receptors. The results of these experiments suggest that responses to L-AP4 require NMDA receptors and are independent of non-NMDA receptors. Furthermore, although it is possible that glycine contamination or other nonspecific actions are responsible for the sympathoinhibitory actions of L-AP4, our data and data in the literature argue against this possibility. Thus we conclude that responses to L-AP4 in the NTS are mediated by an interaction between group III mGluRs and NMDA receptors. Finally, we also caution that nonselective actions of L-AP4 should be considered in future studies.

Topics: 2-Amino-5-phosphonovalerate; Aminobutyrates; Animals; Cardiovascular System; Excitatory Amino Acid Agonists; Glycine; Glycine Agents; Male; Microinjections; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Solitary Nucleus; Strychnine

Depolarization could be evoked in slices of rat cingulate cortex by the normally non-excitatory compound L-2-amino-4-phosphonobutyrate (L-AP4) if the slices had been sensitized by exposure to quisqualate. The magnitude of the response to L-AP4 was dependent on the concentrations of both L-AP4 and quisqualate and was inhibited by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antagonism. A series of phenylglycine analogues were capable of evoking similar dose-dependent depolarizations in the rat cingulate cortex following quisqualate sensitization, the most potent being (S)-4-carboxy-3-hydroxyphenylglycine. If the superfusate collected during application of (S)-4-carboxy-3-hydroxyphenylglycine to a quisqualate-sensitized slice was administered to a slice not previously exposed to quisqualate, a small depolarization was obtained. All the compounds shown to be capable of evoking the quisqualate-sensitized response showed affinity for the L-AP4 uptake site whilst having no affinity at ionotropic glutamate receptors and different profiles of activity at metabotropic glutamate receptors. None of the compounds was active at the metabotropic glutamate 4a receptor. There was a statistically significant correlation between a compound's effectiveness in inhibiting [3H]DL-AP4 uptake into rat cortical synaptosomes and its potency in evoking quisqualate-sensitized depolarization. It is concluded that this response may be the result of hetero-exchange between L-AP4 ligands and quisqualate.

Topics: Aminobutyrates; Animals; Cerebral Cortex; Dose-Response Relationship, Drug; Electrophysiology; Excitatory Amino Acid Antagonists; Glycine; Gyrus Cinguli; Osmolar Concentration; Piperazines; Quinoxalines; Quisqualic Acid; Rats; Tritium

(S)-Bromohomoibotenic acid [(S)-BrHIbo] stereoselectively antagonized glutamate-stimulated phosphoinositide (PI) hydrolysis in baby hamster kidney (BHK) cells expressing mGluR1a in a competitive manner with an IC50 of 250 microM. However, (S)-BrHIbo did not inhibit (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD]-induced PI hydrolysis in rat hippocampal slices (S)- or (R)-BrHIbo did not show any effects on forskolin-stimulated cAMP-formation in BHK cells expressing mGluR2 or mGluR4 but did displace [3H]2-amino-4-phosphonobutyrate ([3H]AP4) binding from rat corticalmembranes with high affinities (IC50 = 1.0 microM and 1.1 microM, respectively). These data suggest that (S)-BrHIbo may interest with multiple PI-coupled glutamate receptors, however, at concentrations that are several fold higher than for displacement of [3H]AP4 binding from rat cortical membranes.

Topics: Aminobutyrates; Animals; Binding, Competitive; Cell Line; Cerebral Cortex; Cricetinae; Cyclic AMP; Cycloleucine; Dose-Response Relationship, Drug; Glutamic Acid; Hippocampus; Hydrolysis; Ibotenic Acid; Kidney; Membranes; Phosphatidylinositols; Quinoxalines; Receptors, AMPA; Receptors, Metabotropic Glutamate; Stereoisomerism

The pharmacological specificity of the mGluR1 alpha subtype of the metabotropic glutamate receptor (mGluR) was examined in a cloned baby hamster kidney cell line (BHK-ts13) measuring [3H]glutamate binding and inositol phosphate (PI) hydrolysis. PI-hydrolysis was maximally stimulated by quisqualate (1112 +/- 105% of basal), glutamate (1061 +/- 70% of basal), ibotenate (1097 +/- 115% of basal) and beta-N-methylamino-L-alanine (BMAA) (1010 +/- 104% of basal). In contrast, the maximal stimulation of PI-hydrolysis by (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (t-ACPD) was only 673 +/- 78% of the basal level. The relative order of potency was quisqualate > glutamate > ibotenate > t-ACPD > BMAA. Agonist-stimulated PI-hydrolysis was attenuated (25 +/- 4% inhibition) by L-2-amino-3-phosphonopropionic acid and partially blocked (44 +/- 7%) by pertussis toxin treatment. Saturation binding studies with [3H]glutamate on membranes prepared from BHK-ts13 cells expressing the mGluR1 alpha subtype showed that glutamate binds to a single affinity state of this receptor with a limited capacity (Kd = 296 nM, Bmax = 0.8 pmol/mg protein). In competition experiments, [3H]glutamate was displaced by quisqualate, glutamate, ibotenate, t-ACPD and BMAA with a rank order of potency similar to that found for stimulation of PI-hydrolysis.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amino Acids, Diamino; Aminobutyrates; Animals; Binding, Competitive; Cell Line; Cell Membrane; Clone Cells; Cricetinae; Cyanobacteria Toxins; Dizocilpine Maleate; Glutamates; Glutamic Acid; Ibotenic Acid; Inositol Phosphates; Kidney; Kinetics; Neurotoxins; Pertussis Toxin; Quinoxalines; Quisqualic Acid; Receptors, Glutamate; Recombinant Proteins; Transfection; Virulence Factors, Bordetella