Application Of Excitatory Amino Acids Research Articles

Electrophysiological and Morphological Studies of SOD1 Transgenic Mice: An Animal Model of ALS

Amyotrophic lateral sclerosis (ALS) is a disease where upper and lower motor neurons die, and it is often associated with mutations of superoxide dismutase 1 (SOD1). We have used mouse models to compare physiologic and morphologic characteristics of cervical motor neurons in wild-type and mutant animals. Slices of the cervical spinal cord were prepared from old wild-type and mutant G93A and G85R mice, and intracellular recordings of membrane potential, resistance and responses to application of excitatory neurotransmitters were studied. Some motor neurons were injected with Lucifer Yellow for morphological analysis. There were no significant differences between membrane potential in the SOD1 mutants and aged wild-type mice, but membrane resistance was somewhat higher in the mutant motor neurons. Dendrites of the mutant motor neurons were not responsive to ionophoretic application of excitatory amino acids, although the cell body responded strongly. In Lucifer-filled cells, the dendrites were found to disappear. Mutant motor neurons were sometimes spontaneously active. Responses of mutant motor neurons to perfused glutamate with varying calcium concentrations in the Ringer’s solution were different from those of the wild-type cells.

Inhibitory Regulation of Responses of Glutamate Receptors in Frog Motoneurons

Experiments on isolated frog spinal cords (Rana ridibunda) with intracellular recording of potentials from lumbar motoneurons were performed to study interactions between the effects of exogenous excitatory (glutamate and its agonists NMDA, AMPA, kainate) and inhibitory (GABA, glycine) amino acids. Application of glycine and GABA induced reductions in responses to application of glutamate to a mean of 45.8 ± 2.9% (n = 12) and 67.8 ± 3.9% (n = 16), responses to AMPA to 13.9 ± 4.3% (n = 9) and 32.1 ± 8.3% (n = 12), responses to KA to 36.8 ± 8.2% (n = 7) and 48.0 ± 11.8% (n = 6), responses to NMDA to 7.7 ± 3.5% (n = 9) and 18.1 ± 3.8% (n = 14) of control baseline levels respectively. Similar patterns were seen on sequential application of excitatory amino acids (EAA) after glycine and GABA and on application of a mixture of GABA and glycine with an EAA. The specific glycine receptor antagonist strychnine blocked the effect of suppression of EAA responses on the background of glycine but not GABA administration, while the GABAA receptor blocker gabazine decreased the effect of suppression of EAA responses induced by application of GABA. These results point to a difference in the inhibitory influences on excitatory reactions mediated by different types of glutamate receptors and particularly the predominance of suppression of excitatory reactions mediated by NMDA receptors and the relatively weak suppression of responses mediated by kainate receptors. The inhibitory effect of glycine was about twice that of GABA (at equal concentrations).

Cholinergic modulation of neuron spike responses to dendritic and somatic application of excitatory amino acids.

The effects of acetylcholine on the spike discharges of neurons induced by iontophoretic application of excitatory amino acids to the bodies and dendrites of cells were studied in 98 neurons in living slices of guinea pig parietal cortex. Acetylcholine applied microiontophoretically to both the bodies and dendrites facilitated improvements in the parameters of responses induced by dendritic activation, with significant decreases in latent periods and increases in the intensity and duration of responses. Thee effects were stably induced at distances of 300 microm from the body and lasted 1 min after exposure to acetylcholine ended. Responses induced by application of excitatory amino acids directly to the cell body did not change significantly in the presence of acetylcholine regardless of the point on the membrane at which they were applied. It is concluded that the predominant effect of acetylcholine is on the efficiency of dendrosomatic conduction.

Transmitter Sensitivity of Primary Afferent Cells of the Lamprey Lampetra fluviatilis Spinal Cord

Using dorsal sensory cells (primary afferents) of the spinal cord of the lamprey Lampetra fluviatilis, isolated by an enzymatic-mechanical method, their responses have been studied to application of excitatory amino acids (EAA), glutamate, aspartate, kainate, N-methyl-D-aspartate (NMDA), and of inhibitory amino acids, GABA and glycine, as well as of neuromodulator serotonin (5-hydroxytryptamine, 5-HT). The patch-clamp method was applied to fix either potential or current through membranes of studied cells. It was shown that of all the above substances, only NMDA and kainate could produce in dorsal cells depolarization, input current with maximal amplitude up to 16 nA, and action potential. The main attention in this work was paid to NMDA-current properties. The dose–effect curves were obtained; a blocking effect of specific antagonist of NMDA-receptors, 2-amino-5-phosphonovalerian acid (APV), was shown; passage of Ba ions through Ca2+-channels of dorsal sensory cell membranes at NMDA application was proven. It has been established that in a half of the studied cells under effect of NMDA there is an increase of the maximal amplitude of the potential-activated current through Ca2+-channels, on average, by 22.5 ± 10.5% (n = 21). It was suggested that this variation might be essential for an increase of amount of the transmitter released in synapses formed by processes dorsal sensory cells, while NMDA could be considered a modulator facilitating synaptic activity of these cells.

Subthreshold aortic nerve inputs to neurons in nucleus of the solitary tract.

Subthreshold aortic nerve (AN) inputs to neurons receiving a monosynaptic AN-evoked input (MSNs: respond to each of two AN stimuli separated by 5 ms) and neurons receiving a polysynaptic AN input (PSNs) in the nucleus of the solitary tract (NTS) were identified in anesthetized rats. In extracellular recordings from 24 MSNs and 49 PSNs, 12% of MSNs and 29% of PSNs only responded to AN stimulation during the application of excitatory amino acids. In intracellular recordings from 24 MSNs and 22 PSNs, 12% of MSNs and 14% of PSNs responded to AN stimulation with excitatory postsynaptic potentials that did not evoke action potential discharge. Reductions in arterial pressure produced minimal changes in the spontaneous discharge of suprathreshold AN-evoked neurons, suggesting that these neurons receive excitatory inputs from nonbaroreceptor sources. The results suggest that some baroreflex-related NTS neurons exist in a "reserve state and can be changed to an active state or vice versa. This will change the number of neurons involved in baroreflex circuits and provides a novel mechanism for regulating baroreflex function independently of alterations in peripheral afferent input.

Spontaneous and locomotor-related GABAergic input onto primary afferents in the neonatal rat.

The in vitro brain stem-spinal cord preparation of neonatal rats (0-5 days old) was used to examine the contribution of GABAA (gamma-aminobutyric acid) receptors to the spontaneous and locomotor-related antidromic firing in the dorsal roots of neonatal rats. Spontaneous bursts of antidromic discharges were generated by the underlying afferent terminal depolarizations reaching spiking threshold. The number of antidromic action potentials increased significantly in saline solution with Cl- concentration reduced to 50% of control. Bath application of the GABAA receptor antagonist bicuculline, at low concentrations (1-2 microM), or picrotoxin blocked the antidromic discharges in the dorsal roots almost completely. The increase in Cl- conductance was therefore mediated by an activation of GABAA receptors. Increasing the concentration of bicuculline to 10-20 microM never blocked these discharges further. On the contrary, in half of the preparations, the number of antidromic action potentials was higher in the presence of high concentrations of bicuculline (10-20 microM) than in the presence of picrotoxin or low concentrations of bicuculline. This suggests that bicuculline, at high concentrations, may have other effects, in addition to blocking GABAA receptors. Dorsal root firing was observed during fictive locomotion induced by bath application of excitatory amino acids and serotonin. A rhythmical pattern was often demonstrated. Bicuculline at low concentrations caused a decrease of the antidromic discharge whereas, at high concentrations, bursts of discharges appeared. A double-bath with a barrier built at the L3 level was then used to separate the mechanisms which generate locomotion from those mediating primary afferent depolarizations. Excitatory amino acids and serotonin were perfused in the rostral pool only. Decreasing the concentration of chloride in the caudal bath caused a sharp increase in the number of antidromic action potentials recorded from the L5 dorsal root. These discharges, which were modulated in phase with the locomotor rhythm, were blocked by bicuculline. These data demonstrate the existence of a locomotor-related GABAergic input onto primary afferent terminals in the neonatal rat.

Cholinergic excitation of dendrites in neocortical neurons

Discharge patterns were studied in response to iontophoretic application of acetylcholine to the soma and dendrites of 128 neocortical pyramidal neurons of layer V. Extracellular recordings were obtained from slices of the guinea-pig parietal cortex. All responses found were excitatory and were better expressed in spontaneously firing cells than in silent ones. Sensitivity to acetylcholine was approximately the same at somatic and dendritic sites in all the cells. Activation of muscarinic receptors gave rise to firing patterns with equal latencies and intensities when applied to both soma and dendrites. The latter suggests that membrane excitation elicited in dendrites by binding of acetylcholine to muscarinic-cholinoreceptor is likely to propagate towards the soma through intracellular biochemical processes. Modulating effect of acetylcholine on output firing patterns, elicited by dendritic application of excitatory amino acids, included shortening of the somatic response latency and increase of response intensity and duration. We propose that, in contrast to glutamatergic excitation, spread of cholinergic excitation along dendrites involves intra-cellular chemical signalling and resulted in changing the electrical properties of dendrites all over their length.

Nitric oxide and cGMP modulate retinal glutamate receptors.

1. In the retina, as in other regions of the vertebrate central nervous system, glutamate receptors mediate excitatory chemical synaptic transmission and are a critical site for the regulation of cellular communication. In this study, retinal horizontal cells from the hybrid less were dissociated in cell culture, voltage clamped by the whole cell recording technique, and the currents evoked by application of excitatory amino acids recorded. 2. Responses to glutamate and its agonist kainate were reduced by approximately 50% in the presence of the nitric oxide (NO) donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine. The effect of these compounds was blocked by the NO scavenger hemoglobin. 3. This effect of NO donors on kainate currents could be mimicked by the application of a membrane permeable guanosine 3',5'-cyclic monophosphate (cGMP) analogue, 8-Br-cGMP. The NO effect was also blocked by application of the guanylate cyclase inhibitor LY-83583, and by a protein kinase G inhibitor peptide. 4. In H1-type horizontal cells, stimulation of endogenous nitric oxide synthase with L-arginine reduced kainate responses, whereas application of D-arginine had no effect. 5. This receptor modulation mechanism may act in concert with other pre- and postsynaptic mechanisms to modify horizontal cell synaptic function according to the adaptational state of the retina and also may protect horizontal cells from glutamate excitotoxicity.

Motor patterns for two distinct rhythmic behaviors evoked by excitatory amino acid agonists in the Xenopus embryo spinal cord

1. Mechanisms underlying the selective expression of different motor patterns in vertebrates are poorly understood. Immobilized, spinal Xenopus embryos are used here to examine the motor patterns evoked by various concentrations of excitatory amino acids. 2. Relatively low concentrations of N-methyl-D-aspartate (NMDA) (40-60 microM), kainate (7-8 microM), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) (5 microM) evoked motor root discharge characteristic of swimming. Brief applications of higher concentrations of kainate (20-40 microM), AMPA (25-30 microM), quisqualate (5 microM), and glutamate (1-4 mM) evoked sequences of a different motor pattern: struggling. This is characterized by a longer cycle period, increased burst duration, and a reversed longitudinal pattern of motor root discharge. The struggling pattern was never evoked by higher concentrations of NMDA (300-500 microM), but was evoked by 30 microM AMPA or 5 microM quisqualate in the presence of 50 microM D-2-amino-5-phosphonopentanoic acid. 3. Intracellular recordings from presumed spinal motoneurons showed different patterns of activity during agonist-evoked swimming and struggling. The patterns were like those described previously during sensory-evoked behavior. 4. Caudal applications of excitatory amino acids that produced struggling discharge did so only at caudal motor roots, whereas caudal applications of NMDA evoked swimming activity throughout the spinal cord. 5. During excitatory-amino-acid-evoked struggling, sensory Rohon-Beard neurons depolarized up to 7 mV, but did not fire. 6. The results show that expression of the struggling pattern, like swimming, is not critically dependent on sensory discharge. The results are also consistent with the idea that expression of the two very different motor patterns for swimming or struggling in this simple vertebrate preparation can be controlled by the level of excitation within the spinal motor circuitry, and need not involve the activity of a specific external neuromodulator.

Rhythmical oral-motor activity recorded in an in vitro brainstem preparation.

The present study employed the neonatal rat isolated brainstem preparation to determine whether oral-motor rhythmical activity, a substrate for the complex behaviors of suckling and chewing, could be elicited in vitro by path application of excitatory amino acids (EAAs). Bath application of EAA agonists (kainate [KA], [+/-]-a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid [AMPA], N-methyl-D, L-aspartate [NMA]), in conjunction with the gamma-aminobutyric acid antagonist bicuculline, either failed to induce rhythmic activity (n = 17 preparations) or induced a low-amplitude, low-frequency burst discharge (< 1 Hz, n = 10 preparations) from the motor branches of the trigeminal nerves when the brainstem was contiguous from the spinomedullary junction to the superior colliculus. Burst activity was in most cases bilaterally synchronous. However, when a discrete coronal transection was made at the level of the facial colliculus, between the trigeminal and facial motor nuclei, the rhythmic bursts produced by the resultant 3- to 5-mm block of tissue following bath application of EAA agonists increased in amplitude and frequency (4-8 Hz, n = 35). Application of 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), a non-N-methyl-D-aspartate (non-NMDA) receptor antagonist, blocked the rhythm induced by non-NMDA receptor agonist (n = 4) but was less effective in suppressing NMA-induced rhythmicity. In contrast, D, L-2-amino-5-phosphonovaleric acid (APV) blocked by both NMA-induced (n = 5) and, in most cases, KA-induced (n = 5) rhythmicity, suggesting an essential role for NMA receptors in production of EAA-induced rhythmical oral-motor activity in the neonatal rat. The present data demonstrate that a narrow, bilaterally distributed region of brainstem surrounding the trigeminal motor nucleus contains sufficient neuronal circuitry for the production of oral-motor rhythmogenesis.

Changes of extracellular calcium concentration induced by application of excitatory amino acids in the human neocortex in vitro

The influence of the glutamate subreceptor agonists N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) on cortical field potentials and on changes in extracellular free calcium concentration ([Ca 2+] o) was tested on human neocortical slices (eleven from nine different patients). The tissue used was a small portion of that which is normally removed for the treatment of a brain tumor. [Ca 2+] o and field potentials were measured by Ca 2+-selective microelectrodes. Local pressure-microejection of NMDA (100 μmol/1)- and AMPA (1 mmol/1)-induced negative field potentials with maximal amplitudes of 0.9 ± 0.1 mV (11 slices, mean ± S.E.M.) and 1.0 ± 0.1 mV (nine slices), respectively. The negative field potentials induced by NMDA were accompanied by monophasic decreases of [Ca 2+] o (0.8 ± 0.1 mmol/l, nine slices). AMPA elicited no (three slices) or only minor decreases of [Ca 2+] o (0.2 ± 0.1 mmol/l, five slices). The responses to the glutamate subreceptor agonists NMDA and AMPA were reversibly depressed by adding their specific antagonists DL-2-amino-5-phosphonovalerate (APV, 100 μmol/1, six slices) and 6-cyano-7-nitroquinoxalin-2,3-dion (CNQX, 5 μmol/1, four slices), respectively. The results correspond to findings in animal experiments and are consistent with the interpretation that in the human neocortex the Ca 2+ permeability of channels gated by NMDA is higher than those gated by AMPA.

Responses of cortical neurons to local application of excitatory amino acids to their dendrites and soma

The efficacy of excitation induced by iontophoretic application of excitatory amino acids to the soma or different parts of the dendritic tree has been compared in experiments performed on parietal cortex slices. Spike activity was recorded extracellularly from single nerve cells of layer V. In total, the responses of 125 neurons were analyzed. Upon application of glutamate and aspartate to the neuronal soma and the majority of dendrites, latencies of excitatory responses did not exceed 500 msec. In 18% of cases, neuronal responses to transmitter application to basal and apical dendrites had longer (2–3 sec) latencies. The maximum intensity of responses was observed when excitatory amino acids had been applied to the soma or proximal parts of dendrites. If applied at a distance of over 100 µm to basal and 300 µm to apical dendrites, glutamate and aspartate elicited cellular responses whose intensity was 2–3 times lower than that of the responses induced by application to the soma. The maximum distances at which somatic spike responses could be recorded were 350 µm and 800 µm for basal and apical dendrites, respectively. Different latencies of the responses to somatic and dendritic applications of excitatory amino acids in some neurons, as well as high efficacy of responses to stimulation of remote parts of dendritic tree, may indicate nonidentity of electrical properties of dendritic and somatic membranes.

A computer-based model for realistic simulations of neural networks. II. The segmental network generating locomotor rhythmicity in the lamprey.

1. To analyze the function of the spinal interneuronal network generating locomotion in the lamprey CNS, a vertebrate model system, we performed computer simulations with realistic model neurons possessing the essential properties of their biological counterparts. 2. The segmental network has been simulated by modeling experimentally established types of neurons with their specific membrane properties and synaptic interconnections. Fictive locomotor activity, which can be experimentally induced by elevating the background excitability by bath application of excitatory amino acids, was simulated by opening membrane conductances for kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors. Kainate/AMPA receptor activation induced a rhythm in the middle and upper part of the physiological burst frequency range, whereas NMDA receptor activation evoked bursting in the lower part of the range, which corresponds well to earlier experimental findings. 3. Several factors contributing to the termination of the burst were studied and their interaction was assessed in simulations of the network. 1) The summation of spike afterhyperpolarizations (late AHPs), leading to adaptation of the discharge, acts as a primary burst-terminating factor at lower rates of kainate/AMPA-induced bursting, and it also interacts with the NMDA-induced oscillatory membrane properties during slow rhythmicity. 2) The termination of the depolarized NMDA plateau is another important factor during NMDA-evoked rhythmicity. 3) The synaptic inhibition from lateral interneurons to the interneurons mediating reciprocal inhibition is important at higher rates of kainate/AMPA-induced bursting. 4. The mechanism of action of 5-hydroxytryptamine (5-HT) on the lamprey segmental network was further investigated by simulation. 5-HT is known to lower the burst frequency during fictive locomotion and also to decrease the conductance through the Ca(2+)-dependent K+ channels, and thereby the size of the late AHP that follows the action potential. Decreasing this conductance in the network simulations resulted in a lesser amount of AHP summation and thereby less frequency adaptation during the burst, longer bursts, and a lower locomotor frequency. Thus the selective action of 5-HT on the Ca(2+)-dependent K+ channels, and hence on the AHP, can account for the modulatory effect on the fictive locomotor rhythm seen experimentally. 5. The results demonstrate that the present simulation of the segmental network can account for essential features of the motor pattern seen experimentally during lamprey locomotion.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential modulation by dopamine of responses evoked by excitatory amino acids in human cortex.

The responses of human neocortical neurons to iontophoretic application of excitatory amino acids and their modulation by dopamine (DA) were studied in vitro. Brain slices were obtained from children undergoing surgery for intractable epilepsy. Application of N-methyl-D-aspartate (NMDA) to the slices induced slow depolarizations accompanied by decreased input conductances and sustained action potentials in cortical neurons. Glutamate produced rapid depolarizations and firing with few changes in input conductances. Quisqualate also induced depolarization and firing, but input conductances increased during the rising phase of the membrane depolarization. Iontophoretic application of DA alone produced no change in membrane potential or input conductance. However, when DA was applied in conjunction with the excitatory amino acids, it produced contrasting effects. With either bath application of DA or when iontophoresis of DA preceded application of NMDA, the amplitude of the membrane depolarizations and the number of action potentials were increased, whereas the latency of these responses decreased. In contrast, DA decreased the amplitude of the depolarizations and the number of action potentials evoked by glutamate or quisqualate. The fact that DA affects responses to NMDA and glutamate or quisqualate in opposite directions is of considerable importance to the understanding of cellular mechanisms of neuromodulation and the role of DA in cognitive processing and in epilepsy.

Pharmacological properties of excitatory amino acid induced changes in extracellular calcium concentration in rat hippocampal slices.

We have studied extracellular ionic changes induced by iontophoretic application of excitatory amino acids in rat hippocampal slices. In contrast to kinetics of changes in [Ca2+]o, kinetics of changes in [K+]o, [Na+]o, [Cl-]o as well as in extracellular space size were comparable for different glutamate receptor agonists. Thus, alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA), quisqualate (quis), and kainate caused reductions in [Ca2+]o followed by an increase of [Ca2+]o above baseline, whereas glutamate, aspartate, N-methyl-D-aspartate (NMDA), and DL-homocysteic acid caused only reductions in [Ca2+]o. After blocking the NMDA receptors with ketamine and 2-amino-5- phosphonovaleric acid (2-APV), glutamate-induced decreases in [Ca2+]o were followed by an overshoot. Reduction of the transmembrane Na+ gradient by lowering [Na+]o, blocking of the Na(+)-K+ ATPase by lowering [K+]o, and application of ouabain blocked the overshoots after quis application, whereas vanadate, a blocker of the Ca(2+)-Mg2+ ATPase, had no effects. Lithium enhanced the reductions in [Ca2+]o and blocked the overshoots. Amiloride also reduced the overshoots. All organic Ca2+ entry blockers diminished reductions of [Ca2+]o but increased the overshoots. Inorganic Ca2+ antagonists had variable effects. Ni2+ had similar effects as the organic Ca2+ entry blockers while Cd2+ reduced both the [Ca2+]o decreases as well as the subsequent overshoots. Co2+ had initially a similar action as Ni2+. With prolonged application, [Ca2+]o decreases became augmented and, during wash, overshoots could no longer be elicited. We suggest that the overshoots in [Ca2+]o are due to a combined effect of extracellular space shrinkage and activation of the Na+/Ca2+ exchangers. This would imply that NMDA receptor activation blocks extrusion of Ca2+ from the cells. We tested the hypothesis that quis-induced intracellular Ca2+ release and extrusion of Ca2+ from the cells contributed to the overshoots. Dantrolene was without effect on the quis-induced signals, while ryanodine reduced the overshoots. Caffeine on the other hand diminished the [Ca2+]o decreases with no effects on the overshoots. To test for possible second messenger routes by which NMDA receptor activation might slow Ca2+ extrusion from cells, we investigated the effects of arachidonic acid and N-monomethyl-D- arginine on the quis-induced signals. While these agents reduced decreases in [Ca2+]o, they had no clear effects on the overshoots. Thus a possible route by which NMDA receptor activation may affect Ca2+ extrusion from cells has still to be elucidated.

Evidence that non-NMDA receptors are involved in the excitatory pathway from the pedunculopontine region to nigrostriatal dopaminergic neurons.

Extracellular single-neuron recordings were obtained from electrophysiologically identified nigrostriatal neurons in chloral hydrate anesthetized rats, in order to test the hypothesis that excitatory amino acid receptors are involved in responses of these neurons to electrical stimulation of the pontine region where the pedunculopontine nucleus (PPN) is located. The effects of iontophoretic application of excitatory amino acids and their antagonists as well as of cholinergic antagonists were tested on the fast orthodromic excitation of nigrostriatal neurons evoked by stimulation of the PPN region. The N-methyl-D-aspartate (NMDA) receptor antagonist D-alpha-aminoadipic acid as well as the cholinergic receptor antagonists mecamylamine and atropine failed to suppress the synaptic excitation of nigral neurons. The NMDA receptor antagonist DL-2-amino-5-phosphonovalerate exerted a weak depressant action on the synaptic response in a few neurons only. On the contrary, the broad spectrum antagonists of excitatory amino acid receptors kynurenic acid and gamma-D-glutamyl-amino-methyl-sulphonate were found to block simultaneously both the synaptic excitation and the neuronal responses to iontophoretic pulses of glutamate while leaving unaffected the neuronal responses to local application of acetylcholine or carbachol. The competitive antagonist of non-NMDA receptors 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline suppressed the synaptic excitation at ejection currents which antagonized neuronal responses to quisqualate and kainate. These results suggest that PPN excitatory fibers synapsing onto pars compacta nigrostriatal neurons utilize an excitatory amino acid as a synaptic transmitter acting preferentially on non-NMDA receptors.

Depression of purine induced inhibition during NMDA receptor mediated activation of hippocampal pyramidal cells — an iontophoretic study

Single pyramidal cells in the rat hippocampal slice preparation were stimulated by iontophoretic application of excitatory amino acids and acetylcholine. The purine adenosine 5′-monophosphate (AMP), applied iontophoretically, readily depressed acetylcholine stimulated cell firing, was less effective on quisqualic acid stimulated cells and virtually ineffective during stimulation by N-methyl- D,L-aspartate (NMA). Inhibition could be restored if the AMP ejection current was increased 3-fold. In contrast, the inhibitory neurotransmitter γ-aminobutyric acid (GABA) exerted a comparable level of inhibition under all 3 neuroexcitants. These data support previously published results which suggest that purine mediated inhibition may be reduced during NMDA receptor channel activation. This may have important implications for the action of adenosine during seizures and ischaemic events as well as neuronal phenomena such as long term potentiation.

Effects of excitatory amino acid antagonists on synaptic transmission in the ampullae of Lorenzini of the skate Raja clavata

1. The effects of excitatory amino acid antagonists on synaptic transmission in the ampullae of Lorenzini of the skate Raja clavata were studied. 2. At concentrations of 10(-3) to 10(-6) M. L-glutamic acid diethylester (GDEE) and L-glutamic acid dimethylester (GDME) decreased the resting afferent discharge frequency as well as the electrically evoked activities and depressed the responses to application of excitatory amino acids. 3. D-alpha-Aminoadipic acid (AA) and 2-amino-4 phosphonobutyric acid (APB) had practically no effect either on resting afferent discharge or on evoked afferent activity. 4. 2-Amino-5-phosphonovaleric acid (APV) reduced the resting afferent discharge and electrically evoked activity in the afferent fibres. APV blocked N-methyl-D-aspartate (NMDA) induced responses at a lower concentration than those induced by aspartate,. 5. Responses caused by NMDA were Mg 1(2+)-dependent; those to quisqualate (Q) application depended to a lesser degree on the Mg2+ concentration. 6. Cis-2,-3-piperidinedicarboxylic acid (PDA) blocked both the resting afferent discharge and the electrically evoked activity; it also reversibly blocked the postsynaptic, amino acid-induced responses. The L-glutamate (L-GLU) response was more resistant to the blockade than the L-aspartate (L-ASP) response. 7. Both kynurenic acid (KEN) and gamma-D-glutamylglycine (DGG) reduced the resting and evoked activities in the afferent fibres. L-ASP was more resistant to blockade by KEN than L-GLU. 8. These observations suggest a prominent physiological role of quisqualate-, kainate- and NMDA-preferring receptors in the ampullae of Lorenzini of Raja clavata.

Electrogenic uptake contributes a major component of the depolarizing action of L-glutamate in rat hippocampal slices.

1. A grease-gap technique has been used to measure d.c. potentials, in response to the application of excitatory amino acids and electrical stimulation of the Schaffer collateral-commissural pathway, in the CA1 region of rat hippocampal slices. The actions of L-glutamate (L-Glu) have been quantified and compared to those of structurally related compounds. 2. Perfusion of L-Glu (90s applications) depolarized the tissue with a threshold of approximately 50 microM and a maximum response in excess of 10 mM. L-Aspartate (L-Asp) produced a similar dose-response relationship. By comparison N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) were more potent excitants, producing dose-dependent depolarizations over the range 2-50 microM. 3. Application of the agonists depressed the amplitude of electrically-evoked synaptic responses; an effect that presumably reflects depolarization of neuronal tissue. However, for a given agonist-induced d.c. potential. L-Glu or L-Asp caused smaller depressions of synaptic responses than did either NMDA or AMPA. 4. The combined application of 50 microM D-2-amino-5-phosphonopentanoate (AP5) and 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) substantially depressed synaptic responses and antagonized responses to NMDA and AMPA producing mean (+/- s.e.) dose-ratios of 12.2 +/- 1.2 and 7.0 +/- 0.8, respectively. However, these compounds produced minimal antagonism of responses to L-Glu and L-Asp (dose-ratios of 1.5 +/- 0.1 and 1.5 +/- 0.2, respectively). 5. Responses to the stereoisomers of homocysteate (HCA) were compared over the range 50 microM to 10 mM. D-HCA was approximately 3.6 times more potent than L-HCA and was antagonized to a greater extent by the combined application of 50 microM AP5 and 10 microM CNQX; the dose ratios were 8.7 + 0.8 and 5.1 + 0.9 for the D- and L- isomers, respectively. 6. The application of high doses of an excitant (e.g., 50mM L-Glu or 5mM D-HCA) caused an irreversible loss of sensitivity to NMDA and AMPA and abolished synaptic transmission. Responses to the other excitants were depressed by this excitotoxic lesion in the following order: D-HCA > L-HCA > L-Glu = LAsp. In slices treated in this manner, L-Glu, L-Asp and L-HCA produced very similar dose-response curves. 7. Some slices were unresponsive to NMDA, AMPA and electrical stimulation from the onset of the experiment but had sensitivity to L-Glu, L-Asp and L-HCA similar to that of slices that had received an excitotoxic lesion. 8. Slices that were experimentally lesioned, such that they became unresponsive to synaptic stimulation, AMPA and NMDA, were depolarized by a variety of compounds when tested at 5 mm. The D- and L- enantiomers of Asp and Glu produced similar responses and were slightly more active than quisqualate and threo-hydroxy-L-aspartate (THLA). Glycine, L-serine, D-serine, L-alanine and 4-aminobutanoate (GABA) elicited responses 15-30% of the size, while L-lysine, alpha-amino-isobutanoate and L-proline produced depolarizations of less than 10% of the size of those induced by 5mM L-Glu. NMDA and dihydrokainate were either inactive or induced small negative shifts in the d.c. potential. 9. We conclude that when hippocampal slices are perfused with L-Glu (or L-Asp) the main source of the depolarization is due to an interaction with an electrogenic carrier system.

Prolonged electrophysiological maturation of transplanted hippocampal neurons

The CA1 region of the rat hippocampal formation was lesioned by transient forebrain ischemia and subsequently repopulated with dispersed fetal hippocampal neurons. Using the hippocampal slice preparation, 7–12 months post-transplantation, intracellular recordings were made during synaptic activation and extracellular calcium measurements were made during iontophoretic application of excitatory amino acids. The data indicate that the developmental period of the transplanted neurons is prolonged with respect to N-methyl- d-aspartate (NMDA) receptor-mediated responses and may be involved in maintaining calcium dependent developmental processes such as fiber outgrowth.