Infrared spectroscopy of serum fails to identify early biomarker changes in an equine model of traumatic osteoarthritis.

OBJECTIVE: to determine the accuracy of infrared (IR)-based serum biomarker profiling to differentiate horses with early inflammatory changes associated with a traumatically induced model of equine carpal osteoarthritis (OA) from controls. METHOD: unilateral carpal OA was induced in 9 of 17 healthy Thoroughbred fillies, while the remainder served as sham operated controls. Serum samples were obtained before induction of OA (Day 0) and weekly thereafter until Day 63 from both groups. Films of dried serum were created, and IR absorbance spectra acquired. Following pre-processing, partial least squares discriminant analysis (PLSDA) and principal component analysis (PCA) were used to assess group and time differences and generate predictive models for wavenumber ranges 1300-1800 â€‹cm-1 and 2600-3700 â€‹cm-1. RESULTS: the overall correct classification rate when classifying samples by group (OA or Sham) was 52.7% (s.d. â€‹= â€‹12.8%), while it was 94.0% (s.d. â€‹= â€‹1.4%) by sampling Day. The correct classification results by group-sampling Day combinations with pre-intervention serum (Day 0) was 50.5% (s.d. â€‹= â€‹21.7%). CONCLUSION: with the current approach IR spectroscopic analysis could not differentiate serum of horses with induced carpal OA from that of controls. The high classification rate obtained by Day of sampling may reflect the effect of exercise on the biomarker profile. A longer study period (advanced disease) or naturally occurring disease may provide further information on the suitability of this technique in horses.

Longitudinal study of the effect of sporidesmin toxicity on lamb production and serum biochemistry in a flock of 46 Romney ewes using a standardised measure of liver damage.

AIMS: To evaluate the effect of sporidesmin toxicity on production outcomes and serum biochemistry analytes in mixed age Romney ewes, using a standardised measure of liver damage. METHODS: This was a prospective longitudinal study following 46 mixed age Romney ewes from sporidesmin intoxication in April 2019, to slaughter 8 months later. The ewes were blood-sampled up to eight times, with a panel of serum biochemistry tests performed on the final six samples. However, only gamma-glutamyl transferase (GGT) activity was measured in the first two samples collected at the end of sporidesmin intoxication and 2 weeks later. Body condition score, ewe weight and production data were also recorded. Using a standardised liver score, based on histology of liver samples collected at slaughter, ewes were assigned to one of three liver disease categories (LDC); low, middle, and high. These were then used as the outcome or predictor variables for statistical analyses. Finally, two separate decision tree models, using recursive partitioning (RP), were fitted to the biochemistry data and to the GGT data collected at FE outbreak, to predict ewes in the low LDC. RESULTS: There was no evidence of a difference for the effect of LDC on ewe weight (p = 0.86) with ewes, on average, gaining weight to weaning. Weaning percent, lamb rearing percent and ewe flock efficiency were lower in ewes with high LDC, and scanning-to-weaning lamb loss was significantly higher in sheep with high LDC (p = 0.02). Serum activities of GGT and glutamate dehydrogenase and concentration of globulin were significantly lower in sheep with low LDC than in sheep with middle or high LDC (p < 0.05). However, there was no evidence of a difference for the effect of LDC on other biochemistry variables (p > 0.05). The final RP model for the biochemistry data categorised ewes as low LDC if their GGT was <122 IU/L, 3 months after sporidesmin intoxication, or if their GGT was <514 IU/L, <18 days after sporidesmin intoxication. CONCLUSIONS AND CLINICAL RELEVANCE: Sheep with gross and histological evidence of severe sporidesmin-induced liver damage were able to maintain or gain body weight, suggesting that sporidesmin intoxication alone is not causative of poor body condition. Similarly, many of the serum biochemistry tests were not associated with evidence of liver damage. Lamb production was reduced in ewes with evidence of severe liver damage and the decision tree model showed promise as a basis to select ewes for culling.

[Hallucinations: Etiological analysis of children admitted to a pediatric emergency department]. / Hallucinations : analyse étiologique chez des enfants admis en service d'accueil des urgences.

Children's hallucinations pose the dual challenge of diagnosing a possible medical emergency and a possible psychiatric disorder. PURPOSE: The main objective was to analyze the causes of such hallucinations in children presenting to a pediatric emergency department. MATERIAL AND METHODS: We conducted a single-center, observational, retrospective study including all children aged less than 15 years experiencing hallucinations and admitted to our tertiary level pediatric emergency department between 1 January 2007 and 31 December 2015. The data collected were demographic; medical: previous medical or psychiatric history, current medications, associated clinical or psychiatric symptoms, type and character of hallucinations, length and recurrence of hallucinatory phenomena; and other biological, radiological and neurological explorations. RESULTS: Sixty-eight patients were included (29 boys). The mean age was 9.1±3 years (range, 2-14 years and 10 months; median, 9.2 years). Admissions were seasonal with a bimodal distribution (a peak during springtime and another one during fall). Hallucinations were mainly visual (90%), acute (77%) and complex (63%). Visual hallucinations were associated with other types of hallucinations: auditory (n=17), somatosensory (n=7). Fifteen children had a psychiatric history and had already experienced hallucinatory phenomena (93%). Among 47 patients (69%), these hallucinations were associated with other symptoms: agitation (41%), headaches (28%), hyperthermia (21%) and negative symptoms of the schizophrenia spectrum (15%). On admission, 20 patients (29%) had one or more treatments under way (34 drugs, 41% known for hallucinogenic adverse effects). Neurological explorations were undertaken in half of the cases. Toxicological analysis prescribed in 19 children was positive in five cases (26%). Fifty-three percent of patients were hospitalized and 51 children received a specialized follow-up (by a neurologist and/or a psychiatrist). A nonpsychiatric origin of these hallucinations was diagnosed in 29 patients (43%): neurological causes (n=10), infectious diseases (n=10), intoxications (n=5) and a medication side effect (n=4). CONCLUSION: Hallucinations with a suspected underlying psychiatric cause differed on several factors: chronic duration (p=0.02), an onset after 10 years of age (p=0.004), previous identical episodes (p=0.014) and a parental psychiatric history (p=0.036), auditory hallucinations (p=0.0009), absence of fever (p=0.005), headaches (p=0.036) and the presence of negative symptoms of the schizophrenic spectrum (p=0.02).

Honeybee locomotion is impaired by Am-CaV3 low voltage-activated Ca2+ channel antagonist.

Voltage-gated Ca2+ channels are key transducers of cellular excitability and participate in several crucial physiological responses. In vertebrates, 10 Ca2+ channel genes, grouped in 3 families (CaV1, CaV2 and CaV3), have been described and characterized. Insects possess only one member of each family. These genes have been isolated in a limited number of species and very few have been characterized although, in addition to their crucial role, they may represent a collateral target for neurotoxic insecticides. We have isolated the 3 genes coding for the 3 Ca2+ channels expressed in Apis mellifera. This work provides the first detailed characterization of the honeybee T-type CaV3 Ca2+ channel and demonstrates the low toxicity of inhibiting this channel. Comparing Ca2+ currents recorded in bee neurons and myocytes with Ca2+ currents recorded in Xenopus oocytes expressing the honeybee CaV3 gene suggests native expression in bee muscle cells only. High-voltage activated Ca2+ channels could be recorded in the somata of different cultured bee neurons. These functional data were confirmed by in situ hybridization, immunolocalization and in vivo analysis of the effects of a CaV3 inhibitor. The biophysical and pharmacological characterization and the tissue distribution of CaV3 suggest a role in honeybee muscle function.

[Feeding disorders in infants and toddlers: At-risk situations and predisposing factors]. / Troubles du comportement alimentaire restrictifs du nourrisson et du jeune enfant : situations à risque et facteurs favorisants.

Feeding disorders and food refusal can be found in 25% of infants, with a minority of them having an organic explanation. Failure to thrive and/or severe malnutrition are found in 3-5% of infants in this population. The objective of this study was to analyze the risk factors of feeding disorders in infants and children less than 3 years of age. This study was conducted from January 2011 to December 2014 and included 103 children, 57 with feeding and/or eating disorders and 46 healthy children considered to be normal eaters. Parents participated in a structured interview and completed a data sheet to record the mode of delivery, neonatal status, medical history, milk feeding, and medical treatment. Statistical analysis indicated that cesarean delivery, prematurity, neonatal diseases, history of eating disorders in the family, consumption of protein hydrolysates, and treatment with proton pump inhibitors were highly significant risk factors in children with eating disorders. In the present study, we showed that several prenatal and postnatal conditions or interventions were associated with the development of eating disorders in young children. Recommendations for future studies include identifying environmental risk factors and implementing prevention programs focused on family, caregivers, as well as healthcare professionals. The objective is to allow physicians to efficiently sort out the wide variety of conditions, categorize them for therapy, and when necessary refer patients to specialists in the field.

The dynamics of root meristem distribution in the soil.

Plants must develop efficient root architectures to secure access to nutrients and water in soil. This is achieved during plant development through a series of expansion and branching processes, mostly in the proximity of root apical meristems, where the plant senses the environment and explores immediate regions of the soil. We have developed a new approach to study the dynamics of root meristem distribution in soil, using the relationship between the increase in root length density and the root meristem density. Initiated at the seed, the location of root meristems in barley seedlings was shown to propagate, wave-like, through the soil, leaving behind a permanent network of roots for the plant to acquire water and nutrients. Data from observations on barley roots were used to construct mathematical models to describe the density of root meristems in space. These models suggested that the morphology of the waves of meristems was a function of specific root developmental processes. The waves of meristems observed in root systems of barley seedlings exploring the soil might represent a more general and fundamental aspect of plant rooting strategies for securing soil resources.

[Treatment of children with feeding disorders]. / Prise en charge des enfants présentant un refus d'alimentation.

The treatment of children presenting a refusal of feeding is multidisciplinary. This is why the point of view of various speakers were approached at the time of this round table. The gastroenterologists -pediatrician stressed the importance of the clinical data to support the diagnosis of a possible organic pathology and to evaluate the nutritional state. When the denutrition is proven, it is sometimes necessary to have recourse to an artificial enteral feeding. If this one must be prolonged a gastrostomy is preferred. The psychiatrics pointed out the oropharyngeal psychopathologies related to the various feeding disorders met during the childhood, including anorexia, the type of treatment being specific to each nosologic entity. The parent-child's observation in interaction is of primary importance for the diagnosis. The speech therapists evoked the importance of the knowledge of the various stages of maturation of the swallowing and the sensory character of this act to understand the bases of rehabilitation. This rehabilitation is long and does not have to neglect relational dynamics.

An endogenous adrenoceptor ligand potentiates excitatory synaptic transmission in cultured hippocampal neurons.

Noradrenergic inputs modulate hippocampal function via distinct receptors. In hippocampal neuronal cultures, mRNA expression of adrenoceptor subtypes is maintained from 1 day in vitro (DIV) to 22 DIV. Noradrenaline dose-dependently stimulates phosphoinositide (PI) breakdown in both immature and mature cultures through the activation of alpha1 receptors. At 22 DIV, basal PI breakdown depends on excitatory synaptic activity since it is decreased by tetrodotoxin or glutamate receptor antagonists. At 22 DIV, a similar decrease of basal PI breakdown is also observed with alpha1, alpha2 or beta adrenoceptor antagonists. These effects are not additive with that produced by tetrodotoxin. Adrenergic antagonists also strongly reduce spontaneous excitatory post-synaptic currents (sEPSC) as evidenced by whole cell recording. Therefore, in hippocampal cultures, excitatory transmission is modulated by a tonic activation of adrenoceptors probably produced by an endogenous ligand. Indeed, (i) the depletion of catecholamine pools by reserpine also decreases both basal PI metabolism and sEPSC; (ii) hippocampal neurons possess both tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase mRNAs, encoding enzymes required for catecholamine synthesis; and (iii) some hippocampal neurons show TH-immunoreactivity. TH-positive cells are also detected in E18 hippocampal sections. Thus, cultured hippocampal neurons synthesize and release an adrenergic-like ligand, which tonically potentiates excitatory synaptic transmission in mature cultures.

[Children also die]. / Les enfants meurent aussi.

Palliative care has recently been developed in adults. However, children die as well and quite a few articles have been published on this special issue. The object of this article is to summarize the available literature in order to call for the development of clinical policies and minimum standards adapted to French pediatrics.

Regulation of spontaneous inhibitory synaptic transmission by endogenous glutamate via non-NMDA receptors in cultured rat hippocampal neurons.

The regulation of gamma-aminobutyric acid (GABA)-mediated spontaneous inhibitory synaptic transmission by endogenously released glutamate was studied in cultured rat hippocampal neurons. After 7 days in vitro (DIV), both spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) could be detected. After 15 DIV, most postsynaptic spontaneous currents occurred as sEPSC/sIPSC sequences when recorded at a holding voltage of -30 mV. In the presence of the glutamate alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subtype antagonist LY303070, both the frequency and amplitude of sIPSC were strongly and reversibly reduced. The N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP5), had no effect on sIPSC while cyclothiazide strongly increased sIPSC frequency. Under blockade of AMPA receptors, the kainate- and GluR5-selective kainate receptor agonists, (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid) (ATPA) and (S)-5-iodowillardiine (5IWill), induced a large enhancement of the frequency of small-amplitude sIPSC which was blocked by the non-NMDA receptor antagonist, 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX). All of these effects were sensitive to tetrodotoxin (TTX). In the presence of LY303070 and TTX, kainate could induce a small inward current while GluR5 agonists had no effect. In the presence of NMDA and AMPA receptor antagonists, the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC) could restore sIPSC. When NBQX was used as an AMPA antagonist, the stimulatory effect of t-PDC was blocked while the group I metabotropic glutamate agonist, 3,5-dihydroxyphenylglycine (DHPG), induced a strong enhancement of sIPSC. Therefore, both AMPA and kainate receptors can regulate inhibitory synaptic transmission in cultured hippocampal neurons, the former by tonic activation, the latter when the glutamate concentration is increased by impairing glutonate uptake.

Kainate receptors are involved in synaptic plasticity.

The ability of synapses to modify their synaptic strength in response to activity is a fundamental property of the nervous system and may be an essential component of learning and memory. There are three classes of ionotropic glutamate receptor, namely NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid) and kainate receptors; critical roles in synaptic plasticity have been identified for two of these. Thus, at many synapses in the brain, transient activation of NMDA receptors leads to a persistent modification in the strength of synaptic transmission mediated by AMPA receptors. Here, to determine whether kainate receptors are involved in synaptic plasticity, we have used a new antagonist, LY382884 ((3S, 4aR, 6S, 8aR)-6-((4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-decahydro isoquinoline-3-carboxylic acid), which antagonizes kainate receptors at concentrations that do not affect AMPA or NMDA receptors. We find that LY382884 is a selective antagonist at neuronal kainate receptors containing the GluR5 subunit. It has no effect on long-term potentiation (LTP) that is dependent on NMDA receptors but prevents the induction of mossy fibre LTP, which is independent of NMDA receptors. Thus, kainate receptors can act as the induction trigger for long-term changes in synaptic transmission.

The GluR5 subtype of kainate receptor regulates excitatory synaptic transmission in areas CA1 and CA3 of the rat hippocampus.

Activation of kainate receptors depresses excitatory synaptic transmission in the hippocampus. In the present study, we have utilised a GluR5 selective agonist, ATPA [(RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid], and a GluR5 selective antagonist, LY294486 [(3SR,4aRS,6SR,8aRS)-6-([[(1H-tetrazol-5-y l)methyl]oxy]methyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3 -carboxylic acid], to determine whether GluR5 subunits are involved in this effect. ATPA mimicked the presynaptic depressant effects of kainate in the CA1 region of the hippocampus. It depressed reversibly AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor-mediated field excitatory postsynaptic potentials (field EPSPs) with an IC50 value of approximately 0.60 microM. The dual-component excitatory postsynaptic current (EPSC) and the pharmacologically isolated NMDA (N-methyl-D-aspartate) receptor-mediated EPSC were depressed to a similar extent by 2 microM ATPA (61 +/- 7% and 58 +/- 6%, respectively). Depressions were associated with an increase in the paired-pulse facilitation ratio suggesting a presynaptic locus of action. LY294486 (20 microM) blocked the effects of 2 microM ATPA on NMDA receptor-mediated EPSCs in a reversible manner. In area CA3, 1 microM ATPA depressed reversibly mossy fibre-evoked synaptic transmission (by 82 +/- 10%). The effects of ATPA were not accompanied by any changes in the passive properties of CA1 or CA3 neurones. However, in experiments where K+, rather than Cs+, containing electrodes were used, a small outward current was observed. These results show that GluR5 subunits comprise or contribute to a kainate receptor that regulates excitatory synaptic transmission in both the CA1 and CA3 regions of the hippocampus.

Altered distribution of Galphah/type 2 transglutaminase following catecholamine deprivation is associated with depression of adrenoreceptor signal transduction in cultured ventricular zone germinal cells.

Type 2 transglutaminase (Tg), which catalyzes the covalent cross-linking of cytoplasmic proteins during apoptosis, also functions as the alpha subunit of a heterodimeric G-protein (Gh) which can activate phospholipase C-delta1 during the signal transduction pathway linked to alpha1-adrenoreceptors. Continued stimulation of rat forebrain ventricular zone (VZ) germinal cells with the alpha1-agonist phenylephrine during development in vitro suppresses apoptosis and promotes DNA synthesis [Pabbathi et al., Brain Res., 760, 1997, 22-33]. Immunocytochemistry with a monoclonal antibody to Galphah/Tg reveals that alpha1-agonist deprivation during culture of VZ cells in the presence of a protein synthesis inhibitor results after 20 h in a loss of peripheral distribution of the protein and an increase in the reaction product of Tg in the cytoplasm of cells undergoing apoptosis. Using photoaffinity labelling, we observed reduced GTP binding to Galphah/Tg in phenylephrine-deprived cultures. Formation of inositol triphosphate (IP3) and intracellular Ca2+ transients occurred in the presence of phenylephrine. In cultures grown in phenylephrine-deprived conditions in the presence of protein synthesis inhibitor, both the IP3 response and the amplitude and duration of Ca2+ transients were reduced. These results show that loss of signal transduction coincides with the onset of transglutaminase activity in VZ cells during a period when cell survival is reduced following withdrawal of alpha1-agonist, and support the hypothesis that Tg/Galphah could be implicated in both signal transduction and programmed cell death.

The synaptic activation of kainate receptors.

L-Glutamate, the principal excitatory neurotransmitter in the vertebrate central nervous system, acts on three classes of ionotropic glutamate receptors, named after the agonists AMPA, NMDA and kainate. AMPA receptors are known to mediate fast synaptic responses and NMDA receptors to mediate slow synaptic responses at most excitatory synapses in the brain. Kainate receptors are formed from a separate set of genes (GluR5-7, KA-1 and KA-2) and are widely distributed throughout the brain. They are implicated in epileptogenesis and cell death. However, the physiological functions of kainate receptors are not known. The development of 2,3-benzodiazepine antagonists that are selective for AMPA receptors enables kainate receptors to be specifically activated by exogenous ligands, such as kainate. Here we demonstrate that high-frequency stimulation of mossy fibres in rat hippocampal slices, in the presence of the highly selective AMPA receptor antagonist GYKI 53655 plus NMDA- and GABA-receptor antagonists, activates an inward current in CA3 neurons that has a pharmacology typical of kainate receptors. The finding that kainate receptors can be activated synaptically adds to the diversity of information transfer at glutamatergic synapses.

Catecholaminergic regulation of proliferation and survival in rat forebrain paraventricular germinal cells.

We have investigated the possible role of alpha1-adrenoreceptors in regulating the germination of progenitor cells cultured from embryonic rat neocortex. High binding levels of the alpha1-selective radioligand 3[H]prazosin were detected in the forebrain of the rat embryo at E13, and the greatest density of binding sites was localized to the ventricular and subventricular zones. Catecholamine-containing axon terminals were present in these zones in the same period. Germinal neuroepithelial cells retained specific 3[H]prazosin binding in culture. Approximately 25% of cells in culture displayed complex intracellular Ca2+ transients in response to phenylephrine, many of which were abolished with the alpha1B antagonist, chloroethylclonidine. Cultures exhibited concentration-dependent catecholamine stimulation of DNA synthesis mediated by alpha1 receptors in serum-limited conditions. Neuroepithelial cells were labelled via their ventricular processes by intraventricular injection of Fast blue in E13 embryos prior to transfer of the neocortex to dissociated cell culture. Many of labelled cells were present in culture in germinal foci. Some cells which migrated from these foci underwent apoptosis, as determined by TUNEL in situ hybridization. During a transitory period of up to 48 h in culture, alpha1-adrenoreceptor activation by phenylephrine or noradrenaline increased the number of surviving cells. Apoptosis was observed in vivo in both ventricular and subventricular zones of the neocortex from E13 to E15 in increasing numbers. We propose that both the supply of noradrenaline to forebrain germinal cells, and the expression of alpha1-adrenoreceptors on their surface could act to determine whether they die or continue to proliferate.

The synaptic activation of the GluR5 subtype of kainate receptor in area CA3 of the rat hippocampus.

Two new compounds (LY293558 and LY294486), that antagonize homomeric human GluR5 receptors, were examined against responses mediated by kainate receptors in the CA3 region of rat hippocampal slices. Both compounds (applied at a concentration of 10 microM) antagonized reversibly currents induced by 200 nM kainate. They also antagonized reversibly the synaptic activation of kainate receptors, evoked by high-frequency stimulation of mossy fibres, in the presence of NMDA and AMPA receptor antagonists. These results show that GluR5 subunits are likely to contribute to a kainate receptor on CA3 neurones that mediates responses to both kainate and synaptically-released L-glutamate.

Cadmium rapidly and irreversibly blocks presynaptic phospholipase C-linked metabotropic glutamate receptors.

Calcium ions (Cd2+) inhibit inositol phosphate (IP) formation elicited by glutamate (GLU) or K+ ions, without affecting carbachol (Carb)-induced IP response in 8-day-old rat forebrain synaptoneurosomes and synaptosomes. On the contrary, Cd2+ was almost ineffective in blocking GLU- and K(+)-responses in hippocampal neurones in culture. The mechanism of Cd2+ inhibition was thus examined in synaptoneurosomes. Extensive washing of synaptoneurosomes pretreated for 1, 5, 15, or 30 min by 100 microM Cd2+ did not modify the inhibitory effect of Cd2+ on GLU-, K(+)- and A23187-evoked IP formation or its lack of effect on Carb response. The later addition of a high affinity Cd2+ chelator (100 microM), N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) also did not reverse the inhibitory effect. TPEN, however, penetrates into synaptoneurosomes and efficiently displaces Cd2+ from the Fura-2-Cd2+ complex as shown by Fura-2 fluorescence recordings. TPEN is not easily removed from the intracellular space, as demonstrated by its ability to still block Cd(2+)-induced Fura-2 fluorescence increase after extensive washing. Pretreatment of synaptoneurosomes by this chelator did not prevent Cd2+ inhibition of GLU-induced IP formation. These data indicate that Cd2+ ions rapidly, irreversibly and extracellularly inhibit GLU-elicited IP formation in synaptoneurosomes or synaptosomes, but not in hippocampal neurones in culture. It is speculated that Cd2+ ions could allow one to distinguish the activity of presynaptic metabotropic glutamate receptors (mGLURs) linked to phosphoinositide metabolism from that of mGLURs located postsynaptically.

Intra- vs extracellular calcium regulation of neurotransmitter-stimulated phosphoinositide breakdown.

The dependence on Ca2+ of basal, glutamate- and carbachol-stimulated phosphoinositide (PI) turnover was studied on 8-day old rat brain synaptoneurosomes. For that purpose, intracellular and extracellular Ca2+ concentrations were buffered by bis-(alpha-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, in its tetra(acetoxymethyl)-ester form (BAPTA-AM) and in its free acid form (BAPTA), respectively. The effects of both forms of the calcium chelator intracellular and extracellular Ca2+ buffering on intracellular and extracellular Ca2+ concentration ([Ca2+]i and [Ca2+]e) were determined with fluorimetric assay using fura2, either in its acetoxymethyl ester form (fura2-AM) or in its free acid form. Intracellular chelation of Ca2+ ions with BAPTA-AM induced a dose-dependent reduction of the [Ca2+]i. Basal inositol phosphate (IP) formation was slightly affected by this [Ca2+]i buffering, while glutamate and carbachol stimulations of PI hydrolysis were similarly diminished. Chelation of extracellular Ca2+ ions with BAPTA produced a reduction of both [Ca2+]e and [Ca2+]i. Basal IP accumulation was maximally inhibited by 50%. The carbachol-induced PI hydrolysis was completely inhibited in the presence of 200 microM BAPTA, while a substantial residual glutamate-elicited IP response remained (40% of the control response). It is concluded that [Ca2+]i of synaptoneurosomes is not critical for basal and neurotransmitter-stimulated IP formation, whilst [Ca2+]e is critical. Glutamate may, in part, stimulate PI breakdown in a Ca(2+)-insensitive way.

Regulation of glutamate release by presynaptic kainate receptors in the hippocampus.

Most reported actions of kainate are mediated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptors. Here we report that, unlike AMPA which stimulates, kainate elicits a dose-dependent decrease in L-glutamate release from rat hippocampal synaptosomes and also depresses glutamatergic synaptic transmission. Brief exposure to kainate inhibited Ca(2+)-dependent [3H]L-glutamate release by up to 80%. Inhibition was reversed by kainate antagonists but not by the AMPA-selective non-competitive antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466). A corresponding reversible kainate-evoked depression of NMDA (N-methyl-D-aspartate) receptor-mediated excitatory postsynaptic currents (e.p.s.cs) was observed when AMPA receptors were blocked by GYKI 52466. The synaptic depression was preceded by a brief period of enhanced release and a small inward current was also observed. The effects of kainate were unaffected by metabotropic glutamate (mGlu), GABAA, GABAB, glycine and adenosine receptor antagonists. These results indicate that glutamate release can be modulated directly by kainate autoreceptors.

A modulation of glutamate-induced phosphoinositide breakdown by intracellular pH changes.

The influence of intracellular pH (pHi) changes on the formation of inositol phosphate metabolites (IPs) produced by glutamatergic stimulation was studied in 8-day-old rat brain synaptoneurosomes. For this purpose pHi was measured using 2',7'-bis-(2-carboxyl)-5,6-carboxyfluorescein (BCECF) fluorimetric assay in parallel with the basal and receptor-mediated formations of inositol monophosphate (IP1) and inositol bisphosphate (IP2). We found that glutamate (1 mM), which induces a transient acidification (delta pH = -0.05), produces an identical accumulation of IP1 and IP2. K+ (30 mM), which provokes an alkalinization of the internal medium (delta pH = +0.22), mainly leads to the formation of IP1 metabolites. Paired combinations of glutamate with 1, 5 and 10 mM NH4+ finally result in an alkalinization of the intrasynaptoneurosomal medium. These combinations produce a strong decrease of the IP2 level concomitant with an increase of the IP1 formation, compared to the levels of IP1 and IP2 evoked by glutamate alone. The total amount of IPs (IP1 + IP2) produced by these combinations is not different from that obtained with glutamate alone. Paired combinations of carbachol with NH4+ produce an identical alkalinization to that produced by NH4+ alone. These combinations produce an increased IP1 accumulation, while the IP2 formation is slightly decreased. When the internal medium is acidified by diminishing the external concentration of Na+, the ratio IP1/IP2 produced after metabotropic glutamate receptor (mGluR) activation is shifted to lower values, while it is not affected for the muscarinic stimulation. These data suggest that the mGluR-associated pathway in synaptoneurosomes is sensitive to pHi shifts, while the muscarinic receptor-associated pathway is less altered when pHi is manipulated. It may be proposed that pH-sensitive inositol phosphate dephosphorylating systems, i.e. phosphatases, are associated with mGluRs in this preparation.