Excitatory Amino Acid Analogues Research Articles

From the Cover: Selective Enhancement of Domoic Acid Toxicity in Primary Cultures of Cerebellar Granule Cells by Lowering Extracellular Na+ Concentration.

Domoic acid (DOM) is an excitatory amino acid analog of kainic acid (KA) that acts through glutamic acid (GLU) receptors, inducing a fast and potent neurotoxic response. Here, we present evidence for an enhancement of excitotoxicity following exposure of cultured cerebellar granule cells to DOM in the presence of lower than physiological Na+ concentrations. The concentration of DOM that reduced by 50% neuronal survival was approximately 3 µM in Na+-free conditions and 16 µM in presence of a physiological concentration of extracellular Na+. The enhanced neurotoxic effect of DOM was fully prevented by AMPA/KA receptor antagonist, while N-methyl-D-aspartate-receptor-mediated neurotoxicity did not seem to be involved, as the absence of extracellular Na+ failed to potentiate GLU excitotoxicity under the same experimental conditions. Lowering of extracellular Na+ concentration to 60 mM eliminated extracellular recording of spontaneous electrophysiological activity from cultured neurons grown on a multi electrode array and prevented DOM stimulation of the electrical activity. Although changes in the extracellular Na+ concentration did not alter the magnitude of the rapid increase in intracellular Ca2+ levels associated to DOM exposure, they did change significantly the contribution of voltage-sensitive calcium channels (VScaCs) and the recovery time to baseline. The prevention of Ca2+ influx via VSCaCs by nifedipine failed to prevent DOM toxicity at any extracellular Na+ concentration, while the reduction of extracellular Ca2+ concentration ameliorated DOM toxicity only in the absence of extracellular Na+, enhancing it in physiological conditions. Our data suggest a crucial role for extracellular Na+ concentration in determining excitotoxicity by DOM.

Enhanced expression of WD repeat-containing protein 35 (WDR35) stimulated by domoic acid in rat hippocampus: involvement of reactive oxygen species generation and p38 mitogen-activated protein kinase activation

BackgroundDomoic acid (DA) is an excitatory amino acid analogue of kainic acid (KA) that acts via activation of glutamate receptors to elicit a rapid and potent excitotoxic response, resulting in neuronal cell death. Recently, DA was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in vitro. We have reported that WDR35, a WD-repeat protein, may mediate apoptosis in several animal models. In the present study, we administered DA to rats intraperitoneally, then used liquid chromatography/ion trap tandem mass spectrometry (LC-MS/MS) to identify and quantify DA in the brains of the rats and performed histological examinations of the hippocampus. We further investigated the potential involvement of glutamate receptors, ROS, p38 MAPK, and WDR35 in DA-induced toxicity in vivo.ResultsOur results showed that intraperitoneally administered DA was present in the brain and induced neurodegenerative changes including apoptosis in the CA1 region of the hippocampus. DA also increased the expression of WDR35 mRNA and protein in a dose- and time-dependent manner in the hippocampus. In experiments using glutamate receptor antagonists, the AMPA/KA receptor antagonist NBQX significantly attenuated the DA-induced increase in WDR35 protein expression, but the NMDA receptor antagonist MK-801 did not. In addition, the radical scavenger edaravone significantly attenuated the DA-induced increase in WDR35 protein expression. Furthermore, NBQX and edaravone significantly attenuated the DA-induced increase in p38 MAPK phosphorylation.ConclusionIn summary, our results indicated that DA activated AMPA/KA receptors and induced ROS production and p38 MAPK phosphorylation, resulting in an increase in the expression of WDR35 in vivo.

Enhanced neurogenesis in organotypic cultures of rat hippocampus after transient subfield-selective excitotoxic insult induced by domoic acid

New neurons are continuously generated in the hippocampus and may play an important role in many physiological and pathological conditions. Here we present evidence of cell proliferation and neurogenesis after a selective and transient excitotoxic injury to the hippocampal cornu ammonis 1 (CA1) area induced by low concentrations of domoic acid (DOM) in rat organotypic hippocampal slice cultures (OHSC). DOM is an excitatory amino acid analog to kainic acid that acts through glutamate receptors to elicit a rapid and potent excitotoxic response. Exposure of slice cultures to varying concentrations of DOM for 24 h induced dose-dependent neuronal toxicity that was independent of activation of classic apoptotic markers. Treatment with 2 μM DOM for 24 h caused a selective yet transient neurotoxic injury in the CA1 subfield of the hippocampus that appeared recovered after 7 days of incubation in a DOM-free medium and showed significant microgliosis but no sign of astrogliosis. The DOM insult (2 μM, 24 h) resulted in a significant upregulation of cell proliferation, as assessed by 5-bromo-2-deoxyuridine (BrdU) incorporation, and a concurrent increase of the neuronal precursor cell marker doublecortin (DCX) within the subgranular zone of the dentate gyrus and area CA1. Neurogenesis occurred primarily during the first week after termination of the DOM exposure. Our study shows that exposure of OHSC to concentrations of DOM below those required to induce permanent neurotoxicity can induce proliferation and differentiation of neural progenitor cells that may contribute to recovery from mild injury and to develop abnormal circuits relevant to disease.

Failure of σ-receptor ligands to reduce the excitatory actions of N-methyl-DL-aspartate on rat spinal neurons in-vivo

Haloperidol and (+)-3-PPP, compounds with known affinity for the sigma-receptor, have been examined for their ability to reduce the excitatory actions of N-methyl-DL-aspartate (NMDLA), quisqualate and kainate on rat spinal neurons in-vivo. The actions of (-)-3-PPP were also tested. Haloperidol was injected intravenously whereas the 3-PPP enantiomers were administered by microelectrophoresis. Haloperidol had little effect on excitations evoked by NMDLA, quisqualate or kainate whereas both (+)- and (-)-3-PPP usually enhanced, non-selectively, responses to all three excitatory amino acid analogues. The results support suggestions that phencyclidine (PCP)-like compounds with affinity for both PCP and sigma-receptors reduce neuronal excitations mediated by the N-methyl-D-aspartate (NMDA) receptor via a selective effect at the PCP site.

Glutamate Acting on N-Methyl-d-aspartate Receptors Attenuates Insulin-like Growth Factor-1 Receptor Tyrosine Phosphorylation and Its Survival Signaling Properties in Rat Hippocampal Neurons

Impairing intracellular signaling induced by survival factors and excess glutamate have recently been suggested to play important role in neurodegenerative processes. However, the underlying mechanism(s) and interrelationships between these factors mostly remain to be established. In the present study, we show that glutamate attenuates the tyrosine phosphorylation of the insulin-like growth factor-1 (IGF-1) receptor and the survival effect of IGF-1 (100 nm) in hippocampal cultured neurons. Pretreatment of cultured hippocampal neurons with glutamate concentration dependently inhibited the tyrosine phosphorylation of IGF-1 receptors as well as that of IRS-1 and Shc, two IGF-1 receptor adapter proteins. The effect of glutamate was also evident on the phosphorylation of Akt, as well as its upstream kinase PI3K/PDK1 and downstream targets, GSK3beta and FOXO3a. The inhibitory effect of glutamate (1 mm) was blocked by antagonists of the N-methyl-d-aspartate (NMDA) receptor, including MK801 (20 microm) and AP5 (100 microm), but not by blockers of other ionotropic or metabotropic glutamate receptor sub-types demonstrating the involvement of the NMDA receptor. This hypothesis is supported further by the observation that treatment with NMDA concentration dependently inhibited the activation and phosphorylation of IGF-1 receptors and downstream targets induced by IGF-1 (100 nm). These findings demonstrate that glutamate can block the effect of IGF-1 by decreasing IGF-1 receptor signaling and responsiveness, hence attenuating the survival properties of this trophic factor in neuronal cells. Our results also suggest a novel mechanism by which glutamate can reduce cell viability and induce neurotoxicity.

Regional Susceptibility to Domoic Acid in Primary Astrocyte Cells Cultured from the Brain Stem and Hippocampus

Domoic acid is a marine biotoxin associated with harmful algal blooms and is the causative agent of amnesic shellfish poisoning in marine animals and humans. It is also an excitatory amino acid analog to glutamate and kainic acid which acts through glutamate receptors eliciting a very rapid and potent neurotoxic response. The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM toxicity. Histopathology evidence indicates that in addition to neurons, the astrocytes were also injured. Electron microscopy data reported in this study further supports the light microscopy findings. Furthermore, the effect of DOM was confirmed by culturing primary astrocytes from the hippocampus and the brain stem and subsequently exposing them to domoic acid. The RNA was extracted and used for biomarker analysis. The biomarker analysis was done for the early response genes including c-fos, c-jun, c-myc, Hsp-72; specific marker for the astrocytes- GFAP and the glutamate receptors including GluR 2, NMDAR 1, NMDAR 2A and B. Although, the astrocyte-GFAP and c-fos were not affected, c-jun and GluR 2 were down-regulated. The microarray analysis revealed that the chemokines / cytokines, tyrosine kinases (Trk), and apoptotic genes were altered. The chemokines that were up-regulated included - IL1-alpha, IL-Beta, IL-6, the small inducible cytokine, interferon protein 10P-10, CXC chemokine LIX, and IGF binding proteins. The Bax, Bcl-2, Trk A and Trk B were all down-regulated. Interestingly, only the hippocampal astrocytes were affected. Our findings suggest that astrocytes may present a possible target for pharmacological interventions for the prevention and treatment of amnesic shellfish poisoning and for other brain pathologies involving excitotoxicity.

Regional Susceptibility to Domoic Acid in Primary Astrocyte Cells Cultured from the Brain Stem and Hippocampus

Domoic acid is a marine biotoxin associated with harmful algal blooms and is the causative agent of amnesic shellfish poisoning in marine animals and humans. It is also an excitatory amino acid analog to glutamate and kainic acid which acts through glutamate receptors eliciting a very rapid and potent neurotoxic response. The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM toxicity. Histopathology evidence indicates that in addition to neurons, the astrocytes were also injured. Electron microscopy data reported in this study further supports the light microscopy findings. Furthermore, the effect of DOM was confirmed by culturing primary astrocytes from the hippocampus and the brain stem and subsequently exposing them to domoic acid. The RNA was extracted and used for biomarker analysis. The biomarker analysis was done for the early response genes including c-fos, c-jun, c-myc, Hsp-72; specific marker for the astrocytes- GFAP and the glutamate receptors including GluR 2, NMDAR 1, NMDAR 2A and B. Although, the astrocyte-GFAP and c-fos were not affected, c-jun and GluR 2 were down-regulated. The microarray analysis revealed that the chemokines / cytokines, tyrosine kinases (Trk), and apoptotic genes were altered. The chemokines that were up-regulated included - IL1-a, IL-1B, IL-6, the small inducible cytokine, interferon protein IP-10, CXC chemokine LIX, and IGF binding proteins. The Bax, Bcl-2, Trk A and Trk B were all downregulated. Interestingly, only the hippocampal astrocytes were affected. Our findings suggest that astrocytes may present a possible target for pharmacological interventions for the prevention and treatment of amnesic shellfish poisoning and for other brain pathologies involving excitotoxicity

Regional Susceptibility to Domoic Acid in Primary Astrocyte Cells Cultured from the Brain Stem and Hippocampus

Domoic acid is a marine biotoxin associated with harmful algal blooms and is the causative agent of amnesic shellfish poisoning in marine animals and humans. It is also an excitatory amino acid analog to glutamate and kainic acid which acts through glutamate receptors eliciting a very rapid and potent neurotoxic response. The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM toxicity. Histopathology evidence indicates that in addition to neurons, the astrocytes were also injured. Electron microscopy data reported in this study further supports the light microscopy findings. Furthermore, the effect of DOM was confirmed by culturing primary astrocytes from the hippocampus and the brain stem and subsequently exposing them to domoic acid. The RNA was extracted and used for biomarker analysis. The biomarker analysis was done for the early response genes including c-fos, c-jun, c-myc, Hsp-72; specific marker for the astrocytes- GFAP and the glutamate receptors including GluR 2, NMDAR 1, NMDAR 2A and B. Although, the astrocyte-GFAP and c-fos were not affected, c-jun and GluR 2 were down-regulated. The microarray analysis revealed that the chemokines / cytokines, tyrosine kinases (Trk), and apoptotic genes were altered. The chemokines that were up-regulated included - IL1-a, IL-1B, IL-6, the small inducible cytokine, interferon protein IP-10, CXC chemokine LIX, and IGF binding proteins. The Bax, Bcl-2, Trk A and Trk B were all downregulated. Interestingly, only the hippocampal astrocytes were affected. Our findings suggest that astrocytes may present a possible target for pharmacological interventions for the prevention and treatment of amnesic shellfish poisoning and for other brain pathologies involving excitotoxicity

Synthesis of Excitatory Amino Acid Analogues

A general route to excitatory amino acid analogues has been developed as exemplified by the synthesis of A-1 and A-2. The key reactions involved were a Negishi coupling of Jackson’s organozinc reagent with vinyl bromide 8 and subsequent ring closure of 15 and 16 using the Mitsunobu reaction.

The appearance and maturation of excitatory and inhibitory neurotransmitter sensitivity during retinal regeneration of the adult newt

Using living slice preparations from newt retinas at different stages of regeneration, we examined the time course of appearance and maturation of neurotransmitter-induced currents with whole-cell patch-clamp methods. Neurons from which currents were recorded were identified by Lucifer Yellow fills. All progenitor cells examined at the regenerating retinas did not express any voltage-gated Na + currents and responsiveness to excitatory amino acid analogues (AMPA and NMDA) and inhibitory amino acids (GABA and glycine). Voltage-gated Na + currents were first detected in premature ganglion cells with round cell body located at the most proximal level of the ‘intermediate-II’ regenerating retina. AMPA- GABA- and glycine-induced currents were simultaneously observed in many premature ganglion cells expressing Na + channels, but not all, suggesting that the onset of the Na + channels is slightly earlier than that of excitatory and inhibitory amino acid receptors in regeneration. NMDA-evoked currents were first observed in the ‘intermediate-III’ regenerating retina just before the synaptogenesis. Pharmacological properties and reversal potential values of the excitatory and inhibitory amino acid responses did not change substantially between regenerating ganglion cells and mature ganglion cells, while rectification properties of current–voltage relations for AMPA and NMDA responses were somewhat different between them.

L-Quisqualic acid transport into hippocampal neurons by a cystine-sensitive carrier is required for the induction of quisqualate sensitization

A brief exposure of hippocampal slices to L-quisqualic acid sensitizes CA1 pyramidal neurons 30–250-fold to depolarization by two classes of excitatory amino acid analogues: (1) those whose depolarizing effects are rapidly terminated following washout, e.g. L-2-amino-4-phosphonobutanoic acid ( L-AP4) and L-2-amino-6-phosphonohexanoic acid ( L-AP6) and (2) those whose depolarizing effects persist following washout, e.g. L-aspartate-β-hydroxamate ( L-AβH). This process has been termed quisqualate sensitization. In this study we directly examine the role of amino acid transport systems in the induction of quisqualate sensitization. We report that L-quisqualate is a low-affinity substrate ( K M=0.54 mM) for a high capacity ( V max=0.9 nmol (mg protein) −1 min −1) Na +-dependent transport system(s) and a high-affinity substrate ( K M=0.033 mM) for a low-capacity ( V max=0.051 nmol (mg protein) −1 min −1) transporter with properties similar to the cystine/glutamate exchange carrier, System x c −. We present evidence that suggests that System x c − participates in quisqualate sensitization. First, simultaneous application of L-quisqualate and inhibitors of System x c −, but not inhibitors of Na +-dependent glutamate transporters, prevents the subsequent sensitization of hippocampal neurons to phosphonates or L-AβH. Second, L-quisqualic acid only sensitizes hippocampal neurons to other substrates of System x c −, including cystine. Third, immunocytochemical analysis of L-quisqualate uptake demonstrates that only inhibitors of System x c − inhibit the highly concentrative uptake of L-quisqualate into a widely dispersed group of GABAergic hippocampal interneurons. We conclude that quisqualate sensitization is a direct consequence of the unique interaction of various excitatory amino acids, namely L-quisqualate, cystine, and phosphonates, with the exchange carrier, System x c −. Therefore, the results of this study have important implications for the mechanism by which L-quisqualate, and other substrates of this transporter which are also excitatory amino acid agonists (such as glutamate and β- N-oxalyl- L-α,β-diaminopropionic acid, β- L-ODAP) may trigger neurotoxicity.

Synaptosomal and vesicular accumulation of l-glutamate, l-aspartate and d-aspartate

We examined the vesicular accumulation of the excitatory amino-acid (EAA) neurotransmitters, l-glutamate and l-aspartate, together with the non-metabolisable EAA analogue d-aspartate. Synaptosomes derived from whole brain were incubated in various concentrations of [ 3H]-amino acids under conditions to facilitate vesicular turnover. Synaptosomes were then lysed in hypotonic medium and vesicles immunoprecipitated with monoclonal anti-synaptophysin antibodies coupled to sepharose beads. Using this method, saturable vesicular accumulation was observed for [ 3H]- l-glutamate, [ 3H]- l-aspartate, and [ 3H]- d-aspartate but not for the excitatory amino acid receptor ligands [ 3H]-AMPA or [ 3H]-kainate. Vesicular accumulation ( t 1/2=7.45 min) was markedly slower than synaptosomal accumulation ( t 1/2=1.03 min) and was substantially reduced at 4°C. Maximal accumulation of [ 3H]- l-glutamate, [ 3H]- l-aspartate, and [ 3H]- d-aspartate was estimated to be 98, 68, and 112 pmol/mg of synaptosomal protein, respectively, and uptake affinities 1.6, 3.4, and 2.1 mM, respectively. Maximal accumulation of [ 3H]- l-glutamate was non-competitively inhibited by both 100 μM unlabeled l-aspartate and 100 μM d-aspartate, suggesting that all are accumulated into a common vesicular pool by different transporters.

Dose-dependent antidepressive effect of new analogues of excitatory amino acids in forced swimming test

Dose-dependent antidepressive effect of new analogues of excitatory amino acids in forced swimming test

AV3V lesions attenuate the cardiovascular responses produced by blood-borne excitatory amino acid analogs.

Systemic injections of the excitatory amino acid (EAA) analogs, kainic acid (KA) and N-methyl-D-aspartate (NMDA), produce a pressor response in conscious rats that is caused by a centrally mediated activation of sympathetic drive and the release of arginine vasopressin (AVP). This study tested the hypothesis that the tissue surrounding the anteroventral part of the third ventricle (AV3V) plays a role in the expression of the pressor responses produced by systemically injected EAA analogs. Specifically, we examined whether prior electrolytic ablation of the AV3V region would affect the pressor responses to KA and NMDA (1 mg/kg iv) in conscious rats. The KA-induced pressor response was smaller in AV3V-lesioned than in sham-lesioned rats (11 +/- 2 vs. 29 +/- 2 mmHg; P < 0.05). After ganglion blockade, KA produced a pressor response in sham-lesioned but not AV3V-lesioned rats (+27 +/- 3 vs. +1 +/- 2 mmHg; P < 0.05). The KA-induced pressor response in ganglion-blocked sham-lesioned rats was abolished by a vasopressin V1-receptor antagonist. Similar results were obtained with NMDA. The pressor response to AVP (10 ng/kg iv) was slightly smaller in AV3V-lesioned than in sham-lesioned ganglion-blocked rats (45 +/- 3 vs. 57 +/- 4 mmHg; P < 0.05). This study demonstrates that the pressor responses to systemically injected EAA analogs are smaller in AV3V-lesioned rats. The EAA analogs may produce pressor responses by stimulation of EAA receptors in the AV3V region, or the AV3V region may play an important role in the expression of these responses.

Primary cortical glial reaction versus secondary thalamic glial response in the excitotoxically injured young brain: Microglial/macrophage response and major histocompatibility complex class I and II expression

The excitatory amino acid analog, N-methyl- d-aspartate, was injected intracortically into nine-day-old rats. Resulting axon-sparing lesions in the developing sensorimotor cortex, which secondarily affect thalamic neurons that become deprived of cortical targets, provide an experimental model for the study of the glial response in distantly affected areas. The microglial/macrophage response was studied using tomato lectin histochemistry and major histocompatibility complex I and II immunocytochemistry. Blood-brain barrier integrity was evaluated. In the cortical lesion site, where blood-brain barrier breakdown occurs, the rapid microglial response was restricted to the degenerating area. Microglial changes were first seen at 4 h post-injection, peaking at days 3–5. Reactive microglia changed morphology, increased tomato lectin binding and expressed major histocompatibility complex I. Additionally, some cells expressed major histocompatibility complex II. In the secondarily affected thalamus, the microglial response was not as pronounced as in the cortex, was first seen at 10 h post-injection and peaked at days 3–5. Reactive microglia showed a bushy morphology, were intensely lectin positive and expressed major histocompatibility complex I. The exceptional response of the nine-day-old brain to cortical lesions makes this model an interesting tool for studying the implications of microglial major histocompatibility factor expression in still enigmatic processes such as wound healing and plasticity.

Acute N-Methyl-D,L-Aspartate Administration Stimulates the Luteinizing Hormone Releasing Hormone Pulse Generator in the Ovine Fetus

To assess whether fetal luteinizing hormone releasing hormone (LH-RH) neurosecretory neurons have the capacity to respond to an exogenous stimulus, a synthetic excitatory amino acid analogue, N-methyl-D-L-aspartate (NMDA; 15 mg/kg), was given rapidly intravenously to 8 chronically catheterized fetuses (130–142 days of gestation; term 147 ± 3 days). All 8 fetuses exhibited a rise in plasma ovine luteinizing hormone (oLH) and ovine follicle-stimulating hormone (oFSH) within 5 min. The mean maximal increments of oLH (2.25 ± 0.36 ng/ml) and oFSH (1.21 ± 0.32 ng/ml) were significantly greater than in 6 normal saline-injected controls (oLH p < 0.0002; oFSH p < 0.03). The secretion of ovine prolactin (oPRL) and ovine growth hormone (oGH) was unaffected. LH-RH (5 μg) evoked a greater oLH response (p < 0.0009) and a greater oFSH response (p < 0.03) than NMDA (n = 6). Desensitization of the fetal gonadotrope by a potent LH-RH agonist, D-Trp⁶Pro⁹NEt-LH-RH (10 μg/day i.v. × 4 days), abolished the fetal oLH and the oFSH response to NMDA (n = 5). Moreover, D,L-2-amino-5-phosphonovalerate, a specific competitive antagonist for the NMDA receptor, completely inhibited the fetal oLH and oFSH response to NMDA, whereas D-L-2-amino-5-phosphonovalerate alone did not affect the plasma oLH or oFSH levels, the gonadotropin response to LH-RH, or the release of oGH or oPRL (n = 3). In primary ovine fetal pituitary cell cultures, NMDA (10^–10 to 10^–6 M) had no effect on oLH, oFSH, oGH, or oPRL secretion, whereas LH-RH stimulated oLH (10^–8 M; p < 0.0004) and oFSH (10^–8 M; p < 0.0001) release, evidence that NMDA did not have a direct pituitary effect. The results suggest that NMDA induces oLH and oFSH secretion by stimulation of the fetal LH-RH pulse generator and is mediated by central NMDA receptors. Fetal LH and FSH secretion and the response to LH-RH decrease in late gestation in the ovine and human fetus. The relative importance of sex steroid dependent and sex steroid independent central nervous system inhibition in this developmental change is unclear. It appears that central neural inhibition in addition to sex steroid negative feedback contributes to the decrease in fetal gonadotropin concentrations in late gestation. NMDA did not affect fetal oGH or oPRL secretion.

Effects of estradiol and exogenous insulin-like growth factor I (IGF-I) on the IGF-I axis during growth hormone inhibition and antagonism.

In adult female monkeys, serum concentrations of insulin-like growth factor I (IGF-I) are decreased by estradiol replacement, whereas levels of IGF-binding protein-3 (IGFBP-3) are increased. Furthermore, chronic IGF-I supplementation elevates serum IGFBP-3 despite a suppression of GH. To better understand how estradiol and IGF-I affect the IGF-I axis, a series of three studies was conducted to examine how estradiol and GH interact to affect the IGF-I axis and how IGF-I regulates IGFBP-1 and -3 during GH inhibition or receptor antagonism in adult female rhesus monkeys. In Exp 1, adult ovariectomized females were studied during a 28-day baseline condition and a 28-day treatment condition in which females received a constant s.c. infusion of a somatostatin analogue (octreotide, Sandoz; SSa; 6 microg/kg x day) with a 14-day washout period separating the two conditions. Within each 28-day phase, females were studied for 14 days with no estradiol replacement and for 14 days with estradiol replacement (3 microg/kg x day, s.c.). Treatment with estradiol and SSa alone significantly lowered serum IGF-I compared with baseline. In contrast, estradiol and SSa given in combination resulted in a significant increase in serum IGF-I. Serum IGFBP-3 was significantly increased by estradiol and the combination of estradiol and SSa. The response of serum GH to the acute administration of the excitatory amino acid analogue, n-methyl-D,L-aspartic acid (5 microg/kg, i.v.) was not differentially affected by any of the treatments. In Exp 2, the effects of a GH receptor antagonist (Trovert, Sensus Corp.) was assessed in ovariectomized, young adult, treated females (GHa; 1.0 mg/kg, s.c., weekly) and compared with that in untreated cohorts (Con) during 3 weeks of no estradiol and 3 weeks of estradiol replacement (3 microg/kg x day, s.c.). Serum IGF-I and IGFBP-3 were significantly suppressed in GHa compared with Con females. In Con females, estradiol replacement significantly decreased serum IGF-I and increased serum IGFBP-3. In contrast, estradiol replacement significantly elevated both serum IGF-I and IGFBP-3 in GHa females. In Exp 3, the effects of acute IGF-I administration (110 microg/kg, s.c.) were assessed during baseline conditions and during treatment with either GHa (1.0 mg/kg, s.c., weekly) or SSa (16 microg/kg, s.c. infusion) in young adult females during no estradiol replacement and during estradiol replacement (3 microg/kg x day, s.c.). Acute IGF-I administration produced a similar net increase in serum IGF-I during baseline and GHa or SSa treatment. Although serum IGFBP-3 was significantly reduced by both GHa and SSa, acute treatment with IGF-I produced a significant elevation in IGFBP-3, peaking by 3 h after treatment before returning to baseline at 7 h. Estradiol replacement elevated serum IGFBP-1 under baseline conditions as well as during GHa and SSa treatments. However, changes in serum insulin in response to the feeding patterns during the acute treatment with IGF-I, predicted changes in serum IGFBP-1. As GH secretion was inhibited during SSa, acute IGF-I had little effect on serum GH. Although acute IGF-I significantly suppressed serum GH by 3 h after treatment during baseline, the hypersecretion of GH during GHa treatment was unaffected by acute IGF-I. In conclusion, the results of the present analysis indicate that the effects of estradiol in postadolescent females on serum IGF-I are dependent on GH status, whereas estradiol consistently elevates serum IGFBP-3. Furthermore, acute IGF-I increases serum IGFBP-3 in females even during GH inhibition or receptor antagonism. Although overall serum concentrations of IGFBP-1 are elevated by estradiol and may be differentially affected by IGF-I treatment, acute changes in IGFBP-1 are more a consequence of changes in serum insulin in response to food intake. Taken together, these data suggest that IGFBP-3 is regulated by factors in addition to GH and that IGF-I can affect its own bioavailabi

Neurophysiology of glutamatergic signalling and anthelmintic action in Ascaris suum: pharmacological evidence for a kainate receptor.

Electrophysiological and pharmacological techniques were used to study glutamatergic signalling in the parasitic nematode, Ascaris suum. Glutamate or kainate injections into whole worms produced a paralysed quasi-static posture similar to the waveform in behaving worms. The DE2 motorneuron class is a primary target. Several glutamatergic substances produced pronounced conductance increases and depolarization in DE2; domoate and kainate were the most potent agonists tested. Glutamate responses and spontaneous excitatory post-synaptic potentials in DE2 were reversibly blocked in sodium-free saline. DE2 sensitivity to exogenous glutamate was sustained during block of synaptic transmission suggesting that glutamatergic receptors are located on DE2 neurons. The glutamate-induced response was localized to the DE2 dendrite, coincident with the synapses responsible for spontaneous potentials in DE2. Steady-state potentials reached during glutamate superfusion were similar to the reversal potentials for both the spontaneous post-synaptic potentials and glutamate, also suggesting that these potentials may be glutamatergic. Non-N-methyl-D-aspartate receptor antagonists partially blocked spontaneous DE2 excitatory potentials and responses elicited by exogenous glutamate and kainate. This glutamatergic pathway may play a role in nematode locomotory behaviour and account for the paralysing anthelmintic action of excitatory amino acid analogues like kainate and domoate.

Quinolinic acid-induced lesions of the rat striatum: Quantitative autoradiographic binding assessment

Injection of the excitatory amino-acid analog quinolinic acid into the striatum of rats produces neuropathological and neurochemical alterations that are reminiscent of those observed in Huntington/s disease. In the present study, we evaluated quinolinic acid-induced striatal changes using quantitative autoradiographic binding assays for [3H]MK-801-labeled NMOA receptors, [3H]SCH 23390-labeled dopamine 0 7 and [3 H]sulpiride-Iabeled dopamine O2 receptors, [3H]CGS 21680-labeled adenosine A2a receptors, [3H]mazindol-Iabeled dopamine uptake sites, [3H]hemicholinium-3-labeled high affinity choline uptake sites and [3H]PK 11195-labeled peripheral-type benzodiazepine binding sites, as markers of different cellular populations of the striatum. We found that decrease in [3H]MK 801 and [3H]SCH 23390 binding, and increase in [3H]PK 11195 binding were the most significant alterations induced by the intrastriatal injection of quinolinic acid. Concentrations of [3H]CGS 21680 and [3H]hemicholinium-3 bindings were also decreased, however, to a lesser extent, and [3H]sulpiride binding was not significantly affected. Quinolinic acid also produced an increase in [3H]mazindol binding. We tested the specificity of the N-methyl-o-aspartate receptor-mediated mechanism of quinolinic acid neurotoxicity using MK 801 pretreatment, an N-methyl-D-aspartate receptor antagonist, and it prevented all quinolinic acid-induced binding changes. Because anticholinergic drugs were proposed to prevent the neurotoxic side-effects of MK 80 1, we also tested the effect of scopolamine pretreatment and found that it altered neither the neurotoxicity induced by quinolinic acid nor the neuroprotective effect ofMK 801. [Neural Res 1998; 20: 46-56]

FASTING-INDUCED SUPPRESSION OF HYPOTHALAMIC-PITUITARY-GONADAL AXIS IN THE ADULT RHESUS MONKEY: EVIDENCE FOR INVOLVEMENT OF EXCITATORY AMINO ACID NEUROTRANSMITTERS

The present study was designed to examine whether acute food-restriction in non-human primates, suppresses hypothalamic-pituitary-testicular (HPT) axis via alterations in the excitatory amino acid (EAA) neurotransmitter-utilizing drive to the GnRH neuron. This was achieved indirectly by comparing the plasma testosterone (T) responses to administration of an excitatory amino acid analogue, N-methyl-D,L-aspartic acid (NMA), in acutely fasted and normal fed monkeys. A set of 4 chair-restrained adult male rhesus monkeys, was assigned to the following treatments: a) normal feeding, b) one-day fasting (omission of morning and afternoon meals), c) normal feeding+NMA (15 mg/kg BW) and d) one-day fasting+NMA (15 mg/kg BW). Starting 1 h after the provision or omission of the afternoon meal, frequent blood sampling was initiated at 15-min intervals for a period of 3-h. NMA was administered as an iv bolus 1 h after start of the sampling. Secretion of T was affected (P<0.005) by the treatments. A peak in T was evident during the first h of the sampling in fed but not fasted monkeys. Mean 3-h T concentrations were suppressed (P<0.001) by the fasting. Administration of NMA in fasting conditions resulted into an acute stimulation of T secretion in 2 of the 4 monkeys. However, mean 60-min post-NMA T concentrations were greater (P<0.05) than those prevailing during the same period in fasted animals not given NMA. In contrast, all 4 fed-monkeys showed significant T elevations in plasma immediately following the NMA challenge and mean T levels during the 60-min post-NMA period were higher (P<0.05) than those in fed animals not injected with NMA, at a comparable time. Testosterone area under the curve for the 2-h post-NMA period was greater (P<0.05) in fed-than in fasted-monkeys. These results indicate that although NMA can stimulate GnRH release both in fed and short-term fasting conditions, the response appears to be suppressed in the later situation suggesting that fasting-induced suppression of the HPT axis in the adult male rhesus monkey may involve a reduction in the sensitivity of GnRH neurons to the EAA neurotransmission.