Freezing eliminates a specific population of L-glutamate receptors in synaptic membranes

The binding of L-[3H]glutamate (L-Glu) to freeze-thawed synaptic membranes (SPMs) exhibited saturation kinetics, with Kd 507 nM and Bmax 6.99 pmol/mg protein. The effects of ions, the susceptibility to Triton X-100 and the pharmacological properties of the binding indicated that those sites detected in freeze-thawed SPMs were only of the Cl-/Ca2+-independent type. The Cl-/Ca2+-dependent (2-amino-4-phosphonobutyrate-sensitive) L-Glu binding sites which are additionally present in fresh SPMs are abolished by freezing.     

Immunostimulating effect of quinpirole,a D2 dopamine receptor agonist

Laboratory of Mechanisms of Neurochemical Modulation, Institute of Physiology, Siberian Branch, Academy of Medical Sciences of the USSR, Novosibirsk. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. P. Nikitin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 4, pp. 374–376, April, 1990.     

Effect of antagonists of excitatory amino acids on neurodegenerative action of quinolinic acid in vitro compared with their anticonvulsant action in situ

Laboratory of Experimental Neurocytology, Brain Institute, All-Union Mental Health Research Center, Academy of Medical Sciences of the USSR. Laboratory of Psychopharmacology, V. M. Bekhterev Leningrad Psychoneurologic Research Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR O. S. Andrianov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 8, pp. 190–193, August, 1989.     

Role of the NR2A/2B subunits of the N-methyl-D-aspartate receptor in glutamate-induced glutamic acid decarboxylase alteration in cortical GABAergic neurons in vitro

The vulnerability of brain neuronal cell subpopulations to neurologic insults varies greatly. Among cells that survive a pathological insult, for example ischemia or brain trauma, some may undergo morphological and/or biochemical changes that may compromise brain function. The present study is a follow-up of our previous studies that investigated the effect of glutamate-induced excitotoxicity on the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67)'s expression in surviving DIV 11 cortical GABAergic neurons in vitro [Monnerie and Le Roux, (2007) Exp Neurol 205:367-382, (2008) Exp Neurol 213:145-153]. An N-methyl-D-aspartate receptor (NMDAR)-mediated decrease in GAD expression was found following glutamate exposure. Here we examined which NMDAR subtype(s) mediated the glutamate-induced change in GAD protein levels. Western blotting techniques on cortical neuron cultures showed that glutamate's effect on GAD proteins was not altered by NR2B-containing diheteromeric (NR1/NR2B) receptor blockade. By contrast, blockade of triheteromeric (NR1/NR2A/NR2B) receptors fully protected against a decrease in GAD protein levels following glutamate exposure. When receptor location on the postsynaptic membrane was examined, extrasynaptic NMDAR stimulation was observed to be sufficient to decrease GAD protein levels similar to that observed after glutamate bath application. Blocking diheteromeric receptors prevented glutamate's effect on GAD proteins after extrasynaptic NMDAR stimulation. Finally, NR2B subunit examination with site-specific antibodies demonstrated a glutamate-induced, calpain-mediated alteration in NR2B expression. These results suggest that glutamate-induced excitotoxic NMDAR stimulation in cultured GABAergic cortical neurons depends upon subunit composition and receptor location (synaptic vs. extrasynaptic) on the neuronal membrane. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation and inhibition that is often observed after injury.     

JN403, in vitro characterization of a novel nicotinic acetylcholine receptor α7 selective agonist

This report describes the in vitro features of a novel selective nicotinic acetylcholine receptor (nAChR) α7 agonist, JN403, (S)-(1-Aza-bicyclo[2.2.2]oct-3-yl)-carbamic acid (S)-1-(2-fluoro-phenyl)-ethyl ester. JN403 was evaluated in a number of in vitro systems of different species, at recombinant receptors using radioligand binding, signal transduction and electrophysiological studies. When using [¹²⁵I] α-bungarotoxin (α-BTX) as a radioligand, JN403 has high affinity for human recombinant nAChR α7 (pK_D = 6.7). Functionally, JN403 is a partial and potent agonist at human nAChR α7. The compound stimulates calcium influx in GH3 cells recombinantly expressing the human nAChR with an pEC₅₀ of 7.0 and an E_max of 85% (compared to the full agonist epibatidine). In Xenopus oocytes expressing human nAChR α7 JN403 induces inward currents with an pEC₅₀ of 5.7 and an E_max of 55%. In both recombinant systems JN403 is a partial agonist and the agonistic effects are blocked after pre-administration of methyllycaconitine (MLA, 100 nM), a nAChR α7 antagonist. In functional calcium influx assays, JN403 displays a significantly lower potency for other subtypes of human nAChRs like α4β2, α3β4, α1β1γδ as well as 5HT₃ receptors when tested functionally as an antagonist (pIC₅₀ < 4.8) and is devoid of agonistic activity (pEC₅₀ < 4). Similarly, JN403 shows low binding activity at a wide panel of neurotransmitter receptors. Thus, JN403 is a potent and selective nAChR α7 agonist and will be a useful tool for the characterization of nAChR α7 mediated effects both in vitro and in vivo.     

Suppressive effects of intrathecal granulocyte colony-stimulating factor on excessive release of excitatory amino acids in the spinal cerebrospinal fluid of rats with cord ischemia: role of glutamate transporters

Recently, the hematopoietic factor, granulocyte colony-stimulating factor (G-CSF), has been shown to exhibit neuroprotective effects in CNS injuries. Our previous study demonstrated that intrathecal (i.t.) G-CSF significantly improved neurological defects in spinal cord ischemic rats. Considerable evidence indicates that the release of excessive amounts of excitatory amino acids (EAAs) plays a critical role in neuron injury induced by ischemic insult. In the present study, we used a spinal cord ischemia-microdialysis model to examine whether i.t. G-CSF exerted antiexcitotoxicity effects in a rat model of spinal cord ischemia. I.t. catheters and a microdialysis probe were implanted in male Wistar rats. The results revealed that spinal cord ischemia-induced neurological defects were accompanied by a significant increase in the concentration of EAAs (aspartate and glutamate) in the spinal dialysates from 30 min to 2 days after reperfusion. I.t administration of G-CSF immediately after the performance of surgery designed to induce ischemia led to a significant reduction in ischemia-induced increases in the levels of spinal EAAs. Moreover, i.t. G-CSF also brought about a significant reduction in the elevation of spinal EAA concentrations induced by exogenous i.t. administration of glutamate (10 μl of 500 mM). I.t. G-CSF attenuated spinal cord ischemia-induced downregulation of expression of three glutamate transporters (GTs), glial transporter Glu–Asp transporter (GLAST), Glu transporter-1 (GLT-1), and excitatory amino acid carrier 1 (EAAC1) protein 48 h after spinal cord ischemic surgery. Immunohistofluorescent staining showed that i.t. G-CSF significantly upregulated expression of the three GTs in the gray matter of the lumbar spinal cord from 3 to 24 h after injection. We propose that i.t. G-CSF possesses an ability to reduce the extent of spinal cord ischemia-induced excitotoxicity by inducing the expression of glutamate transporters.     

Effect of prenatal alcohol exposure on3H-diazepam binding to cerebral cortical synaptic membranes at various stages of postnatal development

Laboratory of Neurochemical Mechanisms of the Conditioned Reflex, Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. S. Rusinov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 105, No. 5, pp. 558–561, May, 1988.     

NMDA receptor antagonists reduce medial,but not lateral,perforant path-evoked EPSPs in dentate gyrus of rat hippocampal slice

Summary NMDA receptor antagonists produced differential effects on medial and lateral perforant path-evoked excitatory postsynaptic potentials (EPSPs) recorded in the dentate gyrus molecular layer of hippocampal slices. D-(-)-2-amino-5-phosphonovaleric acid (D(-)APV) and 3-[(±)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP) significantly reduced the peak amplitude and total area, but not the initial negative slope, of the medial perforant path-evoked EPSP. Neither antagonist affected any component of the lateral perforant path-evoked EPSP. In contrast, population spikes evoked by stimulation of either pathway were depressed.     

The effect of Ca2+ on the pharmacological specificity of [3H]glutamate binding sites in rat hippocampus

The inhibition of [3H]glutamate binding to rat hippocampal membranes by various compounds was measured. L-Glutamic, L-2-amino-4-phosphonobutyric and 4-fluoroglutamic acids had similar IC50 values (2.12 microM, 4.98 microM and 3.90 microM respectively). D-Glutamate and D-2-amino-4-phosphonobutyrate were less potent than L-glutamate (IC50S 18.9 microM and 46.5 microM respectively) and L-glutamate diethylester (IC50 519 microM) was the weakest inhibitor tested. Addition of 10 mM CaCl2 caused a 2.27-fold increase in displaceable binding but did not alter the IC50 values of any of the inhibitors tested.     

The effects of development of a food-related operant reflex on the receptor binding of glutamate in the rat brain

Receptor binding of glutamate was studied in the striatum, hippocampus, and cerebral cortex of rats with different abilities to acquire an operant food-related reflex in a Skinner box. The striatum of rapidly-learning rats and rats unable to learn showed significantly higher levels of glutamate binding than controls were not trained in the Skinner box (p<0.05). Striatal receptor binding of glutamate in slow-learning rats was lower than that in rapidly-learning rats and rats which were unable to learn (p<0.05). In the hippocampus, all groups of rats (rapidly-learning, slow-learning, and those unable to learn) showed increased receptor binding of glutamate as compared with controls (p<0.05), in the cerebral cortex, there was a significant decrease in glutamate binding as compared with controls in all groups of animals subjected to training (p<0.05). Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 84, No. 9, pp. 913–919, September, 1998.     

Effects of N-ethylmaleimide and forskolin on glutamate release from rat hippocampal slices. Evidence that prejunctional adenosine receptors are linked to N-proteins,but not to adenylate cyclase

In the present experiments we have examined the effect of N-ethylmaleimide (NEM) on the release of [³H] glutamate from rat hippocampal slices. Pretreatment of slices with NEM in a concentration between 50 μM and 200 μM, can inhibit the GTP-binding protein (N) that transmitts receptor signals into inhibitions of adenylate cyclase, without affecting the N_s-protein, that transmits signals into stimulation of the cyclase, or the cyclase. The adenosine receptor agonist R-phenylisopropyladenosine (R-PIA, I μM) caused an approximately 50% inhibition of the evoked [³H] glutamate release. This effect was completely prevented by NEM treatment, which did not affect basal or stimulated release of the amino acid. By contrast, the effect of R-PIA was unaffected by adding an adenylate cyclase stimulator (forskolin 1 μM) and a phosphodiesterase inhibitor (rolipram, ZK 62.711, 30 μM) which raised the cyclic AMP content of the slices approximately 1 o-fold. In conclusion, these results suggest that the adenosine receptor that mediates prejunctional inhibition of glutamate release is coupled to a protein similar to the N₁-protein, but that another effector than adenylate cylase is involved.,     

Sleep-promoting action of IIK7, a selective MT2 melatonin receptor agonist in the rat

Several novel melatonin receptor agonists, in addition to various formulations of melatonin itself, are either available or in development for the treatment of insomnia. Melatonin is thought to exert its effects principally through two high affinity, G-protein coupled receptors, MT₁ and MT₂, though it is not known which subtype is responsible for the sleep-promoting action. The present study used radiotelemetry to record EEG and EMG in un-restrained freely moving rats to monitor the sleep-wake behaviour and examined the acute sleep-promoting activity of an MT₂ receptor subtype selective melatonin analog, IIK7. IIK7 is a full agonist at the MT₂ receptor subtype but a partial agonist at the MT₁ receptor and has ∼90-fold higher affinity for MT₂ than MT₁. Like melatonin, IIK7 (10 mg/kg i.p.) significantly reduced NREM sleep onset latency and transiently increased the time spent in NREM sleep, but did not alter REM sleep latency or the amount of REM sleep. An analysis of the EEG power spectrum showed no change in delta (1–4 Hz) or theta activity (5–8 Hz) following IIK7 administration. Core body temperature was slightly decreased (∼0.3 °C) by IIK7 compared to vehicle-treated rats. The acute and transient changes in the sleep-wake cycle mimic the changes seen with melatonin and suggest that its sleep-promoting activity is mediated by activation of the MT₂ receptor subtype.     

Intraseptal infusion of selective and competitive glutamate receptor agonist NMDA and antagonist d-2-amino-5-phosphonopentanoic acid spectral implications for the physostigmine-induced hippocampal theta rhythm in urethane-anesthetized rats

Theta () rhythm may be mediated, at least in part, by a glutamate neurotransmitter. Thus, in the present study, it was hypothesized that the septum glutamatergic NMDA receptor subtype may be involved in the modulation of physostigmine-induced rhythm. To test this hypothesis, we analyzed, in the urethane-anesthetized rat, the effects of septum application of NMDA and d-2-amino-5-phosphonopentanoic acid (AP5), selective and competitive NMDA agonist and antagonist, respectively, on the spectral characteristics of hippocampal rhythm elicited by intravenous injection of a anticholinesterase agent, physostigmine. A low dose (16 nmol) of AP5 did not significantly affect EEG recordings, whereas a high dose (50.75 nmol) resulted in significant decreases in phase (-61.8%) at frequency, peak power (-64.2%), and absolute power of the low-frequency band (-67%). These electroencephalographic alterations, which appeared at 50.75 nmol AP5, were amplified following application of massive doses of the drug (121.8 nmol, n=1, and 162 nmol, n=1). Amplification, however, was slight and the waves remained clearly detectable. On the other hand, the infusion of NMDA resulted in a significant increase in frequency (+25%) of this rhythm, but this effect was completely antagonized by prior local administration of 16 nmol AP5. Our data suggest that the septal NMDA receptors exert subtle modulatory influences on the septohippocampal cells involved in physostigmine-induced wave production, which has not been reported elsewhere: tonic with respect to both low-frequency band power and phase, and phasic with respect to frequency. Our data also indicate that the septum may be a sensitive action site for exogenously administered glutamatergic drugs.