Excitatory Amino Acid Transmission Research Articles

Effects of interleukin-2 on various models of experimental epilepsy in DBA/2 mice.

The effects of interleukin-2 (IL-2) on various models of experimental epilepsy were studied after intracerebroventricular administration in DBA/2 mice, a strain genetically susceptible to sound-induced seizures. Convulsions were induced by physical stimulus (sound of 109 dB. 12-16 kHz) or by chemical compounds (bicuculline, cephazolin or kainate). The present study demonstrated that human recombinant IL-2 (hr-IL-2) and mouse recombinant IL-2 (mr-IL-2) not only did not antagonize audiogenic seizures in DBA/2 mice but increased the incidence of seizures after the highest doses studied. In addition, hr-IL-2 and mr-IL-2 dose dependently facilitated sound-induced seizures at subthreshold sound exposure (83 dB). Pretreatment with monoclonal rat-antimouse IL-2 antibodies significantly affected the changes of occurrence of audiogenic seizures in DBA/2 mice induced by mr-IL-2. In addition, pretreatment with anti-IL-2 receptor monoclonal antibodies (anti-Tac) was able to completely antagonize or reduce the effects of IL-2 on audiogenic seizures. The effects of mr-IL-2 were also studied in two different models of epilepsy: the bicuculline and cephazolin models, due to impairment of GABAergic transmission, and the kainate model, due to an increase in excitatory amino acid transmission. In all models, mr-IL-2 demonstrated to facilitate the seizures induced by these chemoconvulsants. Since the proconvulsant properties of IL-2 were antagonized by specific monoclonal antibodies, we suggest that some epileptic phenomena may be linked to stimulation of IL-2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of excitatory amino acid transmitters on hypothalamic corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP) release in vitro: implications in pituitary-adrenal regulation

The effect of excitatory amino acid (EAA) on the release of CRH and AVP - two major neurohumoral activators of the hypothalamic-pituitary-adrenal axis - was studied by in vitro perifusion of hypothalamic organ explants with various concentrations of EAA receptor agonists and antagonists. The agonists l-glutamate (GLU), kainic acid (KAIN) and l-aspartate (ASP) significantly decreased CRH release, while N-methyl- d-aspartate (NMDA) and quisqualic acid (QUIS) did not affect this parameter. AVP release was significantly stimulated by ASP and NMDA, decreased by KAIN and QUIS, and not influenced by GLU. Co-perifusion with equimolar concentrations of ASP and the selective NMDA receptor antagonist d-α-aminoadipic acid (aAA) partially diminished the effect of ASP on AVP release, but failed to affect ASP-induced suppression of CRH secretion. The broad-spectrum EAA receptor antagonist kynurenic acid (KYN) completely abolished ASP effects on CRH and AVP release in vitro. The results suggest that EAA transmitters might participate in the regulation of hypothalamic-pituitary-adrenal axis by differentially affecting the release of the two major ACTH secretagogues. In addition, EAA effects on hypothalamic CRH and AVP secretion appear to employ more than one subtype of amino acid receptors.

Place of newer antiepileptic drugs in the treatment of epilepsy.

There are several new antiepileptic drugs undergoing extensive clinical investigation. Five new drugs--vigabatrin, lamotrigine, gabapentin, felbamate and oxcarbazepine--appear to be the most widely tested and promising agents. Vigabatrin is most effective in drug-resistant partial epilepsy. Vigabatrin is also effective in infantile spasms, but seems to have negative effects on myoclonic epilepsies and absence seizures. Lamotrigine and felbamate seem to be effective in partial epilepsy and in Lennox-Gastaut syndrome. In addition, lamotrigine and felbamate seem to have efficacy in idiopathic generalised epilepsies. Oxcarbazepine appears to be equally as effective as carbamazepine, but less toxic. Gabapentin has few adverse effects and has efficacy in some patients with drug-resistant partial epilepsy. Some of the new antiepileptic drugs modify excitatory or inhibitory amino acid transmission, but some of them may employ new, still unknown mechanisms of action. Depending on the mechanism of action, the therapeutic effectiveness of the antiepileptic drugs may differ in specific epileptic syndromes. Future antiepileptic drugs may thus give us the possibility to design rational polypharmacy for individual patients by combining agents with different spectra of effectiveness. Considering the goal of good tolerability in the development of the new antiepileptic drugs, polypharmacy with these agents is not expected to increase adverse effects significantly.

Low doses of NMDA receptor antagonists synergistically increase the anticonvulsant effect of the AMPA receptor antagonist NBQX in the kindling model of epilepsy.

Excitatory amino acid transmitters are involved in the initiation of seizures and their propagation. Most attention has been directed to synapses using N-methyl-D-aspartate (NMDA) receptors, although more recent evidence indicates potential roles for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors as well. In the present experiments in amygdala-kindled rats, i.e. a model of partial epilepsy, competitive and uncompetitive NMDA antagonists exerted only weak anticonvulsant effects, whereas the AMPA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX) potently increased focal seizure threshold and inhibited seizure spread from the focus. These effects of NBQX were dramatically increased by pretreatment with low doses of NMDA antagonists, whereas adverse effects of NBQX were not potentiated. These data suggest that both non-NMDA and NMDA receptors are critically involved in the kindled state, and that combinations of AMPA and NMDA receptor antagonists provide a new strategy for treatment of epileptic seizures.

Chronic administration of a nitric oxide synthase inhibitor, N omega-nitro-L-arginine, and drug-induced increase in cerebellar cyclic GMP in vivo.

N omega-nitro-L-arginine (NG-nitro-L-arginine) is a potent nitric oxide synthase inhibitor which crosses the blood brain barrier and does not undergo extensive metabolism in vivo. In this study, effect of chronic pretreatment of N omega-nitro-L-arginine (75 mg/kg, i.p., twice daily for 7 days) on the harmaline- (100 mg/kg, s.c.), picrotoxin- (4 mg/kg, s.c.), pentylenetetrazole- (50 mg/kg, i.p.), and L-glutamic acid- (400 micrograms/10 microliters/mouse, i.c.v.) induced increase in cerebellar cGMP was assessed. All the four drugs produced significant increase in cerebellar cGMP in vehicle pretreated control animals. Cerebellar cGMP increased induced by harmaline, picrotoxin, and L-glutamic acid was attenuated in N omega-nitro-L-arginine pretreated animals. These results indicate that in vivo cerebellar cGMP levels are increased by the prototype excitatory amino acid receptor agonist, L-glutamic acid and also by the drugs which augment the excitatory amino acid transmission. Furthermore, parenteral chronic administration of N omega-nitro-L-arginine blocks NO synthase in the brain and hence cerebellar cGMP response in chronic N omega-nitro-L-arginine treated animals could be used as a tool to assess the physiological functions of nitric oxide in vivo.

Excitatory and inhibitory synaptic processing in the accessory olfactory system of the female rat

The accessory olfactory system constitutes a sensory system specifically involved in regulating neuroendocrine function and reproductive behavior. The chemosensitive structure of this system, the vomeronasal organ, exclusively innervates the accessory olfactory bulb, which in turn projects via monoor disynaptic pathways to a limited number of regions implicated in endocrine and sexual function, including the amygdala and hypothalamus. The present study investigated synaptic processing between the different levels of this sensory system, with particular focus on the input from the accessory olfactory bulb to the medial amygdala and the reciprocal connections between the medial amygdala and the ventromedial hypothalamus. Extracellular single-unit recordings were obtained from medial amygdala neurons in anesthetized female rats to study the synaptic responses elicited by stimulation of the olfactory bulb and hypothalamus. Locally applied agonists and antagonists of amino acid neurotransmitters were tested for their ability to mimic and block these synaptic responses in an attempt to identify the neurotransmitters involved in the stimulated pathways. Electrical stimulation of the accessory olfactory bulb induced orthodromic responses in 88% of 288 medial amygdala neurons, with 67% showing orthodromic inhibition and 21% showing excitation as the shortest-latency response. Many cells displayed multiphasic responses with both orthodromic excitation and inhibition. In separate experiments, main olfactory bulb stimulation also produced excitatory (39%) or inhibitory (50%) orthodromic responses in medial amygdala neurons ( n = 105), but the latency and distribution of responses were significantly different than with accessory olfactory bulb stimulation. Stimulation of the ventromedial hypothalamus also had prominent excitatory (29%) or inhibitory (59%) orthodromic actions on the majority of amygdala neurons. Out of 288 cells tested with both accessory olfactory bulb and ventromedial hypothalamus stimulation, 79% were orthodromically responsive to both areas, whereas only 3% responded to neither area. Fourteen (6%) medial amygdala neurons were antidromically activated from the ventromedial hypothalamus, and 11 of these 14 cells were simultaneously orthodromically responsive to accessory olfactory bulb stimulation, indicating that the accessory olfactory bulb can influence medial amygdala neurons that project directly to the hypothalamus. Iontophoretic application of drugs to the medial amygdala revealed that glutamate and GABA consistently mimicked the orthodromic excitatory and inhibitory responses, respectively, from both the accessory olfactory bulb and ventromedial hypothalamus. In some cases, ejection of bicuculline could block or reverse excitation of the orthodromic inhibitory responses, whereas kynurenic acid could eliminate the orthodromic excitation, indicating that excitatory and inhibitory amino acid transmitters are at least partially involved in these pathways. These findings form a framework for determining the synaptic mechanisms underlying information processing in the accessory olfactory system and olfactory modulation of reproductive function.

Transmitters in the developing and senescent human brain.

During development and throughout adult life, modeling of CNS structure and function occurs as a result of experience. Transmitters play a central role in this mechanism both directly and indirectly (through control of neurotrophin expression) by governing synapse formation, elimination or consolidation. Cholinergic and excitatory amino acid transmitter system activities have been examined in postmortem human brain obtained from normal individuals varying from the prenatal period to old age. Whereas glutamate NMDA receptor binding (measured using MK801) was not substantially altered across the postnatal period, dramatic and differing patterns of choline acetyltransferase (ChAT) activity were evident. Thus, in the cerebellum, ChAT activity was 10-fold higher in fetal compared to adult individuals whereas in the hippocampus there was little or no activity in the fetus and activity rose postnatally to reach a maximum in middle age and then declined to half that level by the tenth decade. Acetylcholinesterase (AChE) histochemical reactivity paralleled the developmental pattern for ChAT in the hippocampus and adjacent cortex with respect to fiber reactivity. These findings indicate that cholinergic synaptic plasticity may be restricted to the prenatal period in cerebellum but occur in both the postnatal period and throughout adult life in the hippocampus and cortex, a concept consistent with the temporal and regional expression of cholinoneurotrophins (NGF and related peptides). Vulnerability of the hippocampus and cortex to age-related pathology such as beta-amyloidosis and neuritic plaque formation may relate to the extended period of cholinergic synaptic sculpting in these areas.

Brief calcium transients evoked by glutamate receptor agonists in rat dorsal horn neurons: fast kinetics and mechanisms.

1. The calcium indicator dye, indo-1, was used to analyse the receptor-specific mechanisms of intracellular calcium ion ([Ca2+]i) responses evoked by excitatory amino acid (EAA) stimulation of dorsal horn neurons. Measurements of somal changes in [Ca2+]i were made on a subsecond time scale under conditions designed to allow membrane potential to mediate interactions between agonist-gated channels and voltage-gated calcium channels (VGCCs). 2. Voltage-gated calcium channels were activated in a receptor-independent manner using elevated extracellular [K+]. The concentration-dependence of K(+)-evoked [Ca2+]i transients was steep and variable among cells, with a mean maximal [Ca2+]i response of 1400 nM and a rapid maximal rate of rise. These data indicate that VGCCs provide a high-capacity route for Ca2+ entry that is very sensitive to small changes in membrane potential. 3. Stimulation of non-NMDA receptors using the non-desensitizing agonist kainate also evoked large [Ca2+]i responses (mean, 840 nM) that were predominantly due to indirect activation of VGCCs. However, in 60% of neurons tested, a component of the [Ca2+]i transient evoked by kainate at concentrations above 10 microM was not blocked by the potent VGCC blocker, lanthanum (La3+). The La(3+)-resistant [Ca2+]i responses to kainate rose exponentially, required extracellular Ca2+, and were caused neither by evoked release of EAA transmitters nor by reversal of Na(+)-Ca2+ exchange. These responses may be mediated by a Ca(2+)-permeable conformation of non-NMDA receptors and can also be evoked by quisqualate, (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and glutamate. 4. Non-NMDA receptors were activated in a desensitizing manner using quisqualate or AMPA. Quisqualate evoked small [Ca2+]i transients (210 nM) with a slow rate of rise. Typically, above 3 microM quisqualate, the size of the responses decreased, reflecting desensitization of the receptor. Responses to quisqualate were blocked by removal of extracellular Ca2+ indicating that mobilization of intracellular Ca2+ stores does not occur in the majority of dorsal horn neurons. However, trans-(+-)-1-amino-1,3-cyclopentane dicarboxylic acid (trans-ACPD) was occasionally able to evoke modest Ca2+ release. 5. Activation of the Ca(2+)-permeable NMDA receptors evoked [Ca2+]i transients that were large (780 nM), with a moderate rate of rise, and that generally achieved a maximum amplitude at NMDA concentrations around 300 microM. 6. Glutamate was used to examine [Ca2+]i responses to the activation of mixed EAA receptor subtypes by an endogenous ligand.(ABSTRACT TRUNCATED AT 400 WORDS)

The endogenous lectin RL-29 is transynaptically induced in dorsal horn neurons following peripheral neuropathy in the rat

Neurons containing immunoreactivity to the endogenous lactose-binding lectin, RL-29, were examined in the L4 segment of the spinal cord of rats with an experimental neuropathy. The cells appeared by 5 days and were also present at 14 and 28 days postoperatively. All neurons were found in the dorsal horn ipsilateral to the injury. The neurons were multipolar and the reaction product revealed the morphology of the primary and secondary dendrites and some axons. Most of the neurons were located in the reticulated region of the dorsal horn, corresponding to Rexed's lamina V. In 14-day neuropathy animals treated with the NMDA-receptor antagonist MK-801, the number of RL-29 cell profiles observed was significantly reduced. Double labeling experiments revealed that spinothalamic tract neurons did not contain RL-29. The results suggest that the neuropathic injury produces a long term, transynaptic change in a subpopulation of dorsal horn neurons, that is mediated by excitatory amino acid transmitters acting at NMDA receptors.

Quantitative localization of AMPA/kainate and kainate glutamate receptor subunit immunoreactivity in neurochemically identified subpopulations of neurons in the prefrontal cortex of the macaque monkey

Excitatory amino acid transmission has been proposed as the principal synaptic mechanism for distribution of information through corticocortical and thalamocortical pathways. The following study utilized a double labeling paradigm, using antibodies that recognize non-NMDA ionotropic glutamate receptor subunits and other neuronal markers, to further define, quantitatively, the subclasses of neurons that contain immunoreactivity for the AMPA/kainate and kainate receptor subunits in the monkey prefrontal cortex. Double labeling with an antibody that recognizes common epitopes in AMPA/kainate subunits GluR2 and GluR3 (GluR2/3) in combination with an antibody that recognizes the kainate receptor subunits GluR5, GluR6, and GluR7 (GluR5/6/7) demonstrated that immunoreactivity for these two receptor classes was highly colocalized in a great majority of the pyramidal neurons in this region but present in only a minority of neurochemically identified subclasses of GABAergic interneurons. Furthermore, GluR2/3 immunoreactivity had principally a somatic distribution whereas GluR5/6/7 labeling was predominately found in the perikarya and/or particular dendritic domains. In contrast, intense GluR1 labeling was observed in a small subpopulation of interneurons and low GluR1 immunoreactivity was present in many other cortical neurons. These results demonstrate that there is a high degree of specificity in the distribution of AMPA/kainate and kainate receptor-class proteins to subclasses of neurons within the neocortex. A neuron's combination of excitatory amino acid receptor subunits may regulate its response to excitatory inputs and further defines the role of identified subclasses of neurons in the complex circuitry of the cerebral cortex and may also indicate the basis for the apparent cellular selectivity of excitotoxic degenerative processes.

Extracellular neuroactive amino acids in the rat striatum during ischaemia: comparison between penumbral conditions and ischaemia with sustained anoxic depolarisation.

Changes in the extracellular levels of excitatory and inhibitory amino acid transmitters were studied in the rat striatum during penumbral ischaemia using intracerebral microdialysis. Effects of penumbral forebrain ischaemia were compared with those of ischaemia with sustained anoxic depolarisation and K+ (100 mM). Comparisons were also made between different groups of animals at 2 and 24 h after dialysis probe implantation. The K+ stimulus did not provoke any release of excitatory amino acids in the 24-h group, probably reflecting a decrease of functional synapses adjacent to the probe. During 30 min of penumbral ischaemia, excitatory amino acids did not reach critical concentrations in the extracellular fluid, and increases in levels of inhibitory/modulatory amino acids were similar. On the other hand, severe transient ischaemia resulted in massive synchronous release of many neuroactive excitatory and inhibitory compounds, in both the 2- and 24-h groups. These and other data suggest that changes during severe ischaemia may arise from both neurotransmitter and metabolic pools. It is concluded that ischaemic damage in the penumbra may not be related to extracellular neuroactive amino acid changes generated within this region.

Purkinje cell survival is differentially regulated by metabotropic and ionotropic excitatory amino acid receptors

We previously reported that trophic factors and neurotransmitters in concert regulate survival of cultured cerebellar Purkinje cells. In particular, excitatory amino acid (EAA) transmitters and NGF increased survival, whereas neither alone was effective. In the present studies, we sought to identify molecular mechanisms through which EAAs participate in the survival-promoting interaction. Initially, we characterized the potential role of ionotropic EAA receptors by exposing cultures to the antagonists MK-801, D-2-amino-5-phosphonovaleric acid, and 6,7-dinitroquinoxalinedione. Each increased cell number, suggesting that endogenous ionotropic activity decreased survival. To determine whether metabotropic EAA receptor stimulation modulates survival, the metabotropic agonist ACPD ([1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid; 1 microM) was tested. ACPD alone had no effect on survival. However, simultaneous exposure to ACPD and NGF significantly increased Purkinje number. Moreover, this increase in survival was blocked by L-AP3 [L(+)-2-amino-3-phosphonopropionic acid; 1 microM], a putative antagonist of certain metabotropic responses. L-AP3 also reduced cell number in the absence of exogenous EAA. Thus, endogenous metabotropic stimulation is normally necessary for survival. In sum, these studies reveal a novel mechanism whereby an excitatory neurotransmitter shapes neural development by simultaneous trophic and regressive actions that are, respectively, mediated by metabotropic and ionotropic EAA receptors.

Dihydropyridine-sensitive calcium current mediates neurotransmitter release from bipolar cells of the goldfish retina

The release of neurotransmitter is evoked by activation of the Ca current (ICa) at presynaptic terminals. Though multiple types of ICa have been reported in various cells, little is known about the properties of presynaptic ICa in the vertebrate CNS. The aim of this article is to identify the type of ICa involved in the release of neurotransmitter from retinal bipolar cells. Bipolar cells with a large axon terminal were isolated enzymatically from the goldfish retina, and studied by the following techniques: (1) recordings of ICa in the whole-cell recording configuration, (2) visualization of intracellular free Ca2+ concentration ([Ca2+]i) with the Fura-2 imaging system, and (3) real-time electrophysiological bioassay of released excitatory amino acid transmitter by a voltage-clamped horizontal cell isolated from the catfish retina. The only ICa found in bipolar cells was the high-voltage-activated, dihydropyridine-sensitive type. This result supports the recent study by Heidelberger and Matthews (1992). When ICa was activated by a short depolarizing pulse, a rapid increase of [Ca2+]i was restricted to the axon terminal. A much slower and smaller increase of [Ca2+]i was sometimes observed at the cell body, probably due to the diffusion of intracellular free Ca2+ from the axon terminal. The increase of [Ca2+]i was completely suppressed by nicardipine, suggesting that Ca2+ entered through dihydropyridine-sensitive Ca channels located mainly at the axon terminal. Activating ICa of the bipolar cell evoked a transmitter-induced current in the excitatory amino acid probe (i.e., the catfish horizontal cell). Both currents were suppressed concomitantly by nifedipine but not by omega-conotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Dorsal root and dorsal column mediated synaptic inputs to reticulospinal neurons in lampreys: involvement of glutamatergic, glycinergic, and GABAergic transmission.

This study was aimed at characterizing the inputs from dorsal roots and dorsal columns to reticulospinal neurons within the posterior rhombencephalic reticular nucleus in the lamprey. The in vitro isolated brainstem and spinal cord preparation was used. Microstimulation of dorsal roots and columns on both sides induced, in identified reticulospinal neurons, synaptic responses which consisted of large IPSPs mixed with excitation, particularly from stimulation on the ipsilateral side. When the spinal cord was selectively exposed to kynurenic acid or to Ca2+ free Ringer's containing 2mM Mn2+, synaptic responses to stimulation of dorsal roots and columns were not modified, whereas the same responses were abolished when the brainstem was exposed selectively to kynurenic acid, thus suggesting that the responses were carried by long fibres ascending directly to the brainstem. The excitatory and inhibitory synaptic responses are relayed by interneurons located in the brainstem. The ascending excitatory inputs to inhibitory interneurons and, most likely, also to excitatory interneurons, use excitatory amino acid transmission. Inhibitory responses were abolished by adding the glycinergic antagonist strychnine (5 microM) to the physiological solution, thus suggesting that inhibitory interneurons use glycine transmission. The synaptic transmission was depressed by (-)-baclofen, a GABAB agonist, probably acting at a presynaptic site. Taken together, the present results suggest that dorsal root and dorsal column stimulations give rise to disynaptic inhibition and excitation of reticulospinal neurons mediated by excitatory and inhibitory amino acid transmission via brainstem interneurons.

NMDA receptor-mediated rhythmic bursting activity in rat supraoptic nucleus neurones in vitro.

1. Intracellular recordings were obtained from 112 supraoptic nucleus magnocellular neurosecretory cells (MNCs) in superfused explants of rat hypothalamus maintained in vitro. The effects of glutamate receptor agonists and antagonists were examined at 32-34 degrees C. 2. In control solutions, spontaneously active (> 5 Hz) phasic or continuous neurones showed interspike interval distributions slightly skewed toward short intervals, but did not feature pauses in the 0.4-2 s range. Current injection to alter the rate of cell discharge shifted the histograms according to the mean firing rate, but failed to induce intermittent pauses in the 0.4-2 s range. 3. Application of N-methyl-D-aspartate (NMDA) induced a mode of firing in which bimodal interspike interval distributions reflected a high incidence of clusters of short interspike intervals (0.5-1.5 s) recurring every 1-3 s. In contrast, firing evoked by application of D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA) was not associated with a clustering of impulse discharge. 4. The putative endogenous excitatory amino acid transmitters L-glutamate, L-aspartate and quinolinate all mimicked the effects of NMDA. Clustered spiking responses to these agents were reversibly blocked by D,L-2-amino-5-phosphono-valerate (APV), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In contrast, the non-NMDA receptor ligands kainate and quisqualate caused CNQX-sensitive increases in firing rate, but these responses were not associated with the appearance of clustered activity. 5. When applied to cells showing negative resting potentials (< -70 mV), or to neurones hyperpolarized by current injection, responses to NMDA consisted of rhythmic (approximately 1 Hz) voltage oscillations associated with bursts of spike discharge. In the presence of TTX, NMDA could induce subthreshold voltage oscillations in the absence of action potentials. 6. Application of a voltage clamp to potentials between -75 and -55 mV during rhythmic bursting responses failed to reveal any rhythmic oscillation of the membrane current. In all cases, rhythmic bursting activity resumed upon returning to the current-clamp mode. 7. Rhythmic bursting responses to NMDA application were abolished in Mg(2+)-free solutions, suggesting that the voltage dependence of NMDA channels served to promote regenerative voltage changes throughout the cycle. The NMDA-induced current itself, however, did not appear to decrease with time, suggesting that a distinct, outward current, was necessary to initiate the repolarizing phase of each cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructural and immunocytochemical study of lamina II islet cells in rat spinal dorsal horn

In order to compare the ultrastructure of GABA-immunoreactive and nonimmunoreactive islet cells in lamina II of the rat dorsal horn, a combined ultrastructural and immunocytochemical study of nine Golgi-stained neurones was performed. Cell bodies of these neurones were tested with antiserum to GABA, and in most cases with antiserum to glycine, while parts of the cell body and dendritic tree were examined with the electron microscope. Four of the neurones had cell bodies that were immunoreactive with GABA antiserum, and 2 of these were also glycine-immunoreactive, while 2 were not. Cell bodies of the remaining five neurones were not immunoreactive with GABA antiserum, nor, in the 3 cases tested, with glycine antiserum. Three of the GABA-immunoreactive cells possessed vesicle-containing dendrites and were presynaptic at dendrodendritic synapses, whereas no vesicles were observed in the dendrites of any of the neurones that were not GABA-immunoreactive. The axon of one of the nonimmunoreactive cells was found with the electron microscope. It gave rise to boutons that contained round agranular vesicles and a few dense-cored vesicles. Three synapses formed by this axon were identified and all were asymmetric. No obvious differences were detected in the types of profile that were presynaptic to GABA-immunoreactive and nonimmunoreactive cells. These results suggest that GABAergic islet cells are a source of presynaptic dendrites in lamina II of the rat and that some presynaptic dendrites contain GABA and glycine, while others contain GABA without glycine. The nonimmunoreactive islet cells presumably represent a distinct functional class of neurones and some of these may release an excitatory amino acid transmitter, possibly in addition to one or more neuropeptides.

Effect of excitatory amino acid receptor antagonists on apomorphine-, oxytocin- and ACTH-induced penile erection and yawning in male rats

The effect of excitatory amino acid receptor antagonists, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate ((+)-MK-801), (±)-3-(2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and (±)-2-amino-4-phosphonobutanoic acid (AP-4), on penile erection and yawning induced by subcutaneous apomorphine (80 μg/kg), intracerebroventricular (i.c.v.) oxytocin (30 ng) and adrenocorticotropin (ACTH)-(1–24) (10 μg) was studied in male rats. Intraperitoneal (0.1–0.4 mg/kg) and i.c.v. (10–50 μg) (+)-MK-801 prevented dose dependently the penile erection and yawning induced by the three drugs. The (+)-MK-801 effect coincided with the appearance of head weaving, body rolling, hyperlocomotion and ataxia. Haloperidol (0.5 mg/kg i.p.) antagonized the prevention by (+)-MK-801 of oxytocin responses. Penile erection but not yawning was also prevented by high, but not low doses of CPP and CNQX, which impaired motor performance, AP-4 was ineffective at all doses tested. The above compounds were ineffective when injected into the paraventricular nucleus of the hypothalamus, the brain area where apomorphine and oxytocin act to induce penile erection and yawning. The results suggest that excitatory amino acid transmission is not involved in the expression of penile erection and yawning induced by the above compounds.

Simultaneous measurement by HPLC of the excitatory amino acid transmitter candidates homocysteate and homocysteine sulphinate supports a predominant astrocytic localisation.

Primary cultures of mouse cerebral cortex neurons, cerebellar granule cells and cortical astrocytes were maintained in vitro for respectively 8-10, 7-10 and 21-24 days. Following these times, amino acids were extracted from the cells by use of ice-cold 70% (v/v) ethanol and the extracts lyophilised. The lyophilised extracts when resuspended were subjected to reverse-phase high performance liquid chromatographic (HPLC) analysis for detection of free amino acids. Samples of cell culture growth medium and water blanks were treated in a similar manner. Identification of L-homocysteate (HCA) and L-homocysteine sulphinate (HCSA) was undertaken by matching retention times with regard to external standards and by 'spiking' cell extracts with authentic compounds. On this basis, HCA and HCSA were consistently detectable in astrocytes at levels of, respectively, 72.3 +/- 33.7 pmol/mg protein (n = 24) and 49.4 +/- 28.7 pmol/mg protein (n = 24). However, in neurons, a peak corresponding to HCSA could not be detected above the background noise, while the area of the peak corresponding to HCA was always greater than, but not significantly different from, that of the background noise present in water blanks. HCA and HCSA were not detectable in the serum used for preparation of the cell culture growth medium. Taken together, these findings indicate a predominant localisation of HCA and HCSA in astrocytes which, at least in culture, appear to possess the metabolic machinery necessary for synthesising and storing these amino acids without any neuronal influence.

Sulphur-containing excitatory amino acid-evoked Ca 2+-independent release of d-[ 3H]aspartate from cultured cerebellar granule cells: The role of glutamate receptor activation coupled to reversal of the acidic amino acid plasma membrane carrier

Sulphur-containing excitatory amino acid transmitter candidates (500 μM) stimulated the Ca 2+independent efflux of exogenously-supplied d-[ 3H]aspartate from primary cultures of cerebellar granule cells superfused continuously with HEPES-buffered saline containing CoCl 2 (1 mM) in place of CaC 2. The stimulated release of d-[ 3H]aspartate was markedly attenuated by 200 μM 6,7-dinitroquinoxalinedione, a concentration at which the antagonist inhibits both non- N-methyl- d-aspartate and N-methyl- d-aspartate ionotropic excitatory amino acid receptors. The Ca 2+-independent component of evoked release was also markedly attenuated and, in some cases, abolished by removing NaCl from the superfusion medium. Furthermore, when 700 μM dihydrokainate (demonstrated herein as a mixed/non-competitive inhibitor of the high-affinity dicarboxylic amino acid transporter in cultured granule cells) was included in the superfusion medium, stimulated efflux of d-[ 3H]aspartate was reduced by between 15–78% of the control response; the extent of inhibition varying with the agonist employed. In contrast, agents which act as competitive inhibitors of the plasma membrane carrier in granule cells, e.g. β-methylene- d, l-aspartate. potentiated the release of d-[ 3H]aspartate in a synergistic manner. Taken together, these findings are consistent with a mechanism for the Ca 2+-independent release of d-[ 3H]aspartate that is mediated predominantly by activation of excitatory amino acid receptors resulting in a reversal of the high-affinity dicarboxylic amino acid transport system. Although the physiological relevance of such non-vesicular release from the cytosol remains obscure and is still a matter of some debate, this mode of release may be of pathological significance.

The effect of visual experience on development of NMDA receptor synaptic transmission in kitten visual cortex

We have studied the effect of dark rearing on the development of excitatory amino acid transmission in 6-week-old kittens. In normal kittens, the NMDA component of the visual response decreases between 3 and 6 weeks of age for cells located in layers IV, V, and VI (Fox et al., 1991). Dark rearing to 6 weeks of age prevents this decrease. Subsequent exposure to light allows the decrease to proceed. Ten days in the light after 6 weeks in the dark was sufficient to decrease the NMDA component of the visual response to the same levels seen in light-reared animals of the same age. Comparison of the effect of the non-NMDA antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione with the NMDA antagonist aminophosphonovalerate showed that the changes were due to the relative contributions of NMDA and non-NMDA receptors to the visual response rather than the overall contribution of glutamate receptors. We also studied the receptive field properties of the cells in the various groups of kittens. Cells given 4 d in the light after 6 weeks in the dark showed increased direction selectivity but little change in response firing rate. After 10 d in the light, visual responses did show some recovery toward adult values, but neither average firing rates nor the proportion of direction-selective cells reached the levels found in normal 6-week-old animals, contrary to the suggestion that a short period in the light can reverse the effect of dark rearing completely. These results show that the decrease in the NMDA component of the visual response seen during normal development of the cortex is caused by visual experience. Changes in NMDA receptors and developmental events such as geniculocortical afferent segregation and acquisition of orientation tuning covary as a function of visual experience rather than age, strongly suggesting that NMDA receptors are involved in experience-dependent developmental processes.