The medial amygdaloid nucleus is involved in the cardiovascular pathway activated by noradrenaline into the lateral septal area of rats

We have previously reported that noradrenaline (NA) microinjected into the lateral septal area (LSA) caused pressor and bradicardic responses that were mediated by vasopressin release into the circulation through the paraventricular nucleus of hypothalamus (PVN). Although PVN is the final structure involved in the cardiovascular responses caused by NA in the LSA, there is no evidence of direct connections between these areas, suggesting that some structures could be links in this pathway. In the present study, we verified the effect of reversible synaptic inactivation of the medial amygdaloid nucleus (MeA), bed nucleus of stria terminalis (BNST) or diagonal band of Broca (DBB) with Cobalt Chloride (CoCl2) on the cardiovascular response to NA microinjection into the LSA of unanesthetized rats. Male Wistar rats had guide cannulae implanted into the LSA and the MeA, BNST or DBB for drug administration, and a femoral catheter for blood pressure and heart rate recordings. Local microinjection of CoCl2 (1 mm in 100 nL) into the MeA significantly reduced the pressor and bradycardic responses caused by NA microinjection (21 nmol in 200 nL) into the LSA. In contrast, microinjection of CoCl2 into the BNST or DBB did not change the cardiovascular responses to NA into the LSA. The results indicate that synapses within the MeA, but not in BNST or DBB, are involved in the cardiovascular pathway activated by NA microinjection into the LSA.

Purinergic and glutamatergic interactions in the hypothalamic paraventricular nucleus modulate sympathetic outflow

P2X receptors are expressed on ventrolateral medulla projecting paraventricular nucleus (PVN) neurons. Here, we investigate the role of adenosine 5′-triphosphate (ATP) in modulating sympathetic nerve activity (SNA) at the level of the PVN. We used an in situ arterially perfused rat preparation to determine the effect of P2 receptor activation and the putative interaction between purinergic and glutamatergic neurotransmitter systems within the PVN on lumbar SNA (LSNA). Unilateral microinjection of ATP into the PVN induced a dose-related increase in the LSNA (1 nmol: 38 ± 6 %, 2.5 nmol: 72 ± 7 %, 5 nmol: 96 ± 13 %). This increase was significantly attenuated by blockade of P2 receptors (pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid, PPADS) and glutamate receptors (kynurenic acid, KYN) or a combination of both. The increase in LSNA elicited by L-glutamate microinjection into the PVN was not affected by a previous injection of PPADS. Selective blockade of non-N-methyl-D-aspartate receptors (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt, CNQX), but not N-methyl-D-aspartate receptors (NMDA) receptors (DL-2-amino-5-phosphonopentanoic acid, AP5), attenuated the ATP-induced sympathoexcitatory effects at the PVN level. Taken together, our data show that purinergic neurotransmission within the PVN is involved in the control of SNA via P2 receptor activation. Moreover, we show an interaction between P2 receptors and non-NMDA glutamate receptors in the PVN suggesting that these functional interactions might be important in the regulation of sympathetic outflow

Modeling and simulations of some anisotropic soft-matter systems: from biaxial to chiral materials

We have modeled various soft-matter systems with molecular dynamics (MD) simulations. The first topic concerns liquid crystal (LC) biaxial nematic (Nb) phases, that can be possibly used in fast displays. We have investigated the phase organization of biaxial Gay-Berne (GB) mesogens, considering the effects of the orientation, strength and position of a molecular dipole. We have observed that for systems with a central dipole, nematic biaxial phases disappear when increasing dipole strength, while for systems characterized by an offset dipole, the Nb phase is stabilized at very low temperatures. In a second project, in view of their increasing importance as nanomaterials in LC phases, we are developing a DNA coarse-grained (CG) model, in which sugar and phosphate groups are represented with Lennard-Jones spheres, while bases with GB ellipsoids. We have obtained shape, position and orientation parameters for each bead, to best reproduce the atomistic structure of a B-DNA helix. Starting from atomistic simulations results, we have completed a first parametrization of the force field terms, accounting for bonded (bonds, angles and dihedrals) and non-bonded interactions (H-bond and stacking). We are currently validating the model, by investigating stability and melting temperature of various sequences. Finally, in a third project, we aim to explain the mechanism of enantiomeric discrimination due to the presence of a chiral helix of poly(gamma-benzyl L-glutamate) (PBLG), in solution of dimethylformamide (DMF), interacting with chiral or pro-chiral molecules (in our case heptyl butyrate, HEP), after tuning properly an atomistic force field (AMBER). We have observed that DMF and HEP molecules solvate uniformly the PBLG helix, but the pro-chiral solute is on average found closer to the helix with respect to the DMF. The solvent presents a faster isotropic diffusion, twice as HEP, also indicating a stronger interaction of the solute with the helix.

Toxicity of valproic acid in mice with decreased plasma and tissue carnitine stores

The aim of this study was to investigate whether a decrease in carnitine body stores is a risk factor for valproic acid (VPA)-associated hepatotoxicity and to explore the effects of VPA on carnitine homeostasis in mice with decreased carnitine body stores. Therefore, heterozygous juvenile visceral steatosis (jvs)(+/-) mice, an animal model with decreased carnitine stores caused by impaired renal reabsorption of carnitine, and the corresponding wild-type mice were treated with subtoxic oral doses of VPA (0.1 g/g b.wt./day) for 2 weeks. In jvs(+/-) mice, but not in wild-type mice, treatment with VPA was associated with the increased plasma activity of aspartate aminotransferase and alkaline phosphatase. Furthermore, jvs(+/-) mice revealed reduced palmitate metabolism assessed in vivo and microvesicular steatosis of the liver. The creatine kinase activity was not affected by treatment with VPA. In liver mitochondria isolated from mice that were treated with VPA, oxidative metabolism of l-glutamate, succinate, and palmitate, as well as beta-oxidation of palmitate, were decreased compared to vehicle-treated wild-type mice or jvs(+/-) mice. In comparison to vehicle-treated wild-type mice, vehicle-treated jvs(+/-) mice had decreased carnitine plasma and tissue levels. Treatment with VPA was associated with an additional decrease in carnitine plasma (wild-type mice and jvs(+/-) mice) and tissue levels (jvs(+/-) mice) and a shift of the carnitine pools toward short-chain acylcarnitines. We conclude that jvs(+/-) mice reveal a more accentuated hepatic toxicity by VPA than the corresponding wild-type mice. Therefore, decreased carnitine body stores can be regarded as a risk factor for hepatotoxicity associated with VPA.

Evidence for glutamatergic ganglion cells in the avian retina

This dissertation presents structural, immunochemical and neurochemical evidence for glutamatergic retinotectal synaptic transmission, augmenting and extending previous physiological and anatomical studies. The evidence is especially striking when the laminar patterns of ($\sp3$H) L-glutamate receptor binding, ($\sp3$H) L-glutamate high affinity uptake (HAU) and glutamate immunoreactivity (GLIR) of the dorsal tectum are compared. All show high activity in the tectal SGFS, with a peak in the most superficial laminae of SGFS followed by dip in the b-c region, and a second broad peak in deeper SGFS. Uptake and immunoreactivity bear a stronger resemblance to one another than either does to receptor binding, consistent with the fact that HAU and GLIR are localized in the same structures: glutamatergic terminals, intrinsic cell bodies and their processes. Receptor binding, as attested by the lack of enucleation effects, is a marker of postsynaptic receptors. In summary, these results are consistent with the hypothesis that most of the retinal projection to the optic tectum is glutamatergic: (1) A glutamate/aspartate HAU system exists in the superficial laminae, and it is dependent upon an intact retinal input, as shown developmentally and by retinal ablation; (2) Glutamate-like immunoreactivity appears in retinorecipient tectal regions (partially responsive to enucleation), in cell bodies of retinal ganglion cells and displaced ganglion cells, and in a non-tectal ganglion cell projection, the ectomammilary nucleus; (3) Sodium-independent glutamate receptor binding (which remains unchanged by enucleation) is most intense in the retinorecipient regions of the tectum and the ectomammilary nucleus. This binding is pharmacologically typical of a CNS sensory structure, being dominated by the quisqualate/kainate receptor subclass. Thus, as with other sensory systems, a portion of the retinotectal projection has been shown to include glutamatergic afferents with the distribution and properties expected of the primary projection ^

The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons.

The amino acid L-glutamate is a neurotransmitter that mediates fast neuronal excitation in a majority of synapses in the central nervous system. Glutamate stimulates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. While activation of NMDA receptors has been implicated in a variety of neurophysiologic processes, excessive NMDA receptor stimulation (excitotoxicity) is thought to be primarily responsible for neuronal injury in a wide variety of acute neurological disorders including hypoxia-ischemia, seizures, and trauma. Very little is known about endogenous molecules and mechanisms capable of modulating excitotoxic neuronal death. Saturated N-acylethanolamides like palmitoylethanolamide accumulate in ischemic tissues and are synthesized by neurons upon excitatory amino acid receptor activation. Here we report that palmitoylethanolamide, but not the cognate N-acylamide anandamide (the ethanolamide of arachidonic acid), protects cultured mouse cerebellar granule cells against glutamate toxicity in a delayed postagonist paradigm. Palmitoylethanolamide reduced this injury in a concentration-dependent manner and was maximally effective when added 15-min postglutamate. Cannabinoids, which like palmitoylethanolamide are functionally active at the peripheral cannabinoid receptor CB2 on mast cells, also prevented neuron loss in this delayed postglutamate model. Furthermore, the neuroprotective effects of palmitoylethanolamide, as well as that of the active cannabinoids, were efficiently antagonized by the candidate central cannabinoid receptor (CB1) agonist anandamide. Analogous pharmacological behaviors have been observed for palmitoylethanolamide (ALI-Amides) in downmodulating mast cell activation. Cerebellar granule cells expressed mRNA for CB1 and CB2 by in situ hybridization, while two cannabinoid binding sites were detected in cerebellar membranes. The results suggest that (i) non-CB1 cannabinoid receptors control, upon agonist binding, the downstream consequences of an excitotoxic stimulus; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for CB2-like receptors on granule cells; and (iii) activation of such receptors may serve to downmodulate deleterious cellular processes following pathological events or noxious stimuli in both the nervous and immune systems.

Role of sodium and potassium ions in regulation of glucose metabolism in cultured astroglia.

Effects of increasing extracellular K+ or intracellular Na+ concentrations on glucose metabolism in cultures of rat astroglia and neurons were examined. Cells were incubated in bicarbonate buffer, pH 7.2, containing 2 mM glucose, tracer amounts of [14C]deoxyglucose ([14C]dGlc), and 5.4, 28, or 56 mM KCl for 10, 15, or 30 min, and then for 5 min in [14C]dGlc-free buffer to allow efflux of unmetabolized [14C]dGlc. Cells were then digested and assayed for labeled products, which were shown to consist of 96-98% [14C]deoxyglucose 6-phosphate. Increased K+ concentrations significantly raised [14C]deoxyglucose 6-phosphate accumulation in both neuronal and mixed neuronal-astroglial cultures at 15 and 30 min but did not raise it in astroglial cultures. Veratridine (75 microM), which opens voltage-dependent Na+ channels, significantly raised rates of [14C]dGlc phosphorylation in astroglial cultures (+20%), and these elevations were blocked by either 1 mM ouabain, a specific inhibitor of Na+,K(+)-ATPase (EC 3.6.1.37), or 10 microM tetrodotoxin, which blocks Na+ channels. The carboxylic sodium ionophore, monensin (10 microM), more than doubled [14C]dGlc phosphorylation; this effect was only partially blocked by ouabain and unaffected by tetrodotoxin. L-Glutamate (500 microM) also stimulated [14C]dGlc phosphorylation in astroglia--not through N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor mechanisms but via a Na(+)-dependent glutamate-uptake system. These results indicate that increased uptake of Na+ can stimulate energy metabolism in astroglial cells.

Amino acid, peptide and drug transport across monolayers of human intestinal (CAC0-2) cells in vitro

The properties of Caco-2 monolayers were compared on aluminium oxide and nitrocellulose permeable-supports. On nitrocellulose, Caco-2 cells displayed a higher rate of taurocholic acid transport than those cultured on aluminium oxide inserts. In addition, Caco-2 cells grown on these two inserts were not comparable with respect to cell morphology, cell numbers and transepithelial electrical resistance. The low adsorption potential of the aluminium oxide inserts, particularly for high molecular weight or lipophilic ligands, offers a distinct advantage over nitrocellulose inserts for drug transport studies. The carrier-mediated uptake and transport of the imino acid (L-proline) and the acidic amino acids (L-aspartate and L-glutamate) have been studied. At pH7.4, L-proline uptake is mediated via an A-system carrier. Elevated uptake and transport under acidic conditions occurs by activation of a distinct carrier population. Acidic amino acid transport is mediated via a X-AG system. The flux of baclofen, CGP40116 andCGP40117 across Caco-2 monolayers was described by passive transport. The transport of three peptides, thyrotrophin-releasing hormone, SQ29852 and cyclosporin were investigated. Thyrotrophin-releasing hormone transport acrossCaco-2 monolayers was characterised by a minor saturable (carrier-mediated,approximately 25%) pathway, superimposed onto a major non-saturable (diffusional)pathway. SQ29852 uptake into Caco-2 monolayers is described by a major saturable mechanism (Km = 0.91 mM) superimposed onto a minor passive component.However, the initial-rate of SQ29852 transport is consistent with a passive transepithelial transport mechanism. These data highlight the possibility that itsbasolateral efflux is severely retarded such that the passive paracellular transportdictates the overall transepithelial transport characteristics. In addition, modelsuitable for investigating the transepithelial transport of cyclosporin A has been developed. A modification of the conventional Caco-2 model has been developed which has a calcium-free Ap donor-solution and a Bl receiver-solution containing the minimumcalcium concentration required to maintain monolayer integrity (100 μM). The influence of calcium and magnesium on the absorption of [14C]pamidronate was evaluated by comparing its transport across the conventional and minimum calciumCaco-2 models. Ap calcium and magnesium ions retard the Ap-to-Bl flux of pamidronate across Caco-2 monolayers. The effect of self-emulsifying oleic acid-Tween 80 formulations on Caco-2monolayer integrity has been investigated. Oleic acid-Tween 80 (1 0:1) formulations produced a dose-dependent disruption of Caco-2 monolayer integrity. This disruption was related to the oleic acid content of the formulation.

Implication du glutamate et de l'oxyde nitrique dans la modulation de la dépression synaptique à court terme à la jonction neuromusculaire de Rana pipiens

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, l-tryptophan

The crystal structure of anthranilate synthase (AS) from Serratia marcescens, a mesophilic bacterium, has been solved in the presence of its substrates, chorismate and glutamine, and one product, glutamate, at 1.95 Å, and with its bound feedback inhibitor, tryptophan, at 2.4 Å. In comparison with the AS structure from the hyperthermophile Sulfolobus solfataricus, the S. marcescens structure shows similar subunit structures but a markedly different oligomeric organization. One crystal form of the S. marcescens enzyme displays a bound pyruvate as well as a putative anthranilate (the nitrogen group is ambiguous) in the TrpE subunit. It also confirms the presence of a covalently bound glutamyl thioester intermediate in the TrpG subunit. The tryptophan-bound form reveals that the inhibitor binds at a site distinct from that of the substrate, chorismate. Bound tryptophan appears to prevent chorismate binding by a demonstrable conformational effect, and the structure reveals how occupancy of only one of the two feedback inhibition sites can immobilize the catalytic activity of both TrpE subunits. The presence of effectors in the structure provides a view of the locations of some of the amino acid residues in the active sites. Our findings are discussed in terms of the previously described AS structure of S. solfataricus, mutational data obtained from enteric bacteria, and the enzyme's mechanism of action.

Rôle du glutamate dans l'effet de l'amphétamine sur la récompense et l'activité locomotrice chez le rongeur

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Implication du glutamate 346 de NHE1 dans le transport du Na⁺ et l'interaction avec les inhibiteurs

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Rôle du glutamate dans l'effet de l'amphétamine sur la récompense et l'activité locomotrice chez le rongeur

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Implication du glutamate 346 de NHE1 dans le transport du Na⁺ et l'interaction avec les inhibiteurs

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Effects of oral supplementation with glutamine and alanyl-glutamine on glutamine, glutamate, and glutathione status in trained rats and subjected to long-duration exercise

Objective: We investigated the effect of supplementation with the dipeptide L-alanyl-L-glutamine (DIP) and a solution containing L-glutamine and L-alanine, both in the free form, on the plasma and tissue concentrations of glutamine, glutamate, and glutathione (GSH) in rats subjected to long-duration exercise. Methods: Rats were subjected to sessions of swim training. Twenty-one days before sacrifice, the animals were supplemented with DIP (1.5 g/kg, n = 6), a solution of free L-glutamine (1 g/kg) and free L-alanine (0.61 g/kg; GLN + ALA, n = 6), or water (CON, n = 6). Animals were sacrificed before (TR, n = 6) or after (LD, n = 6) long-duration exercise. Plasma concentrations of glutamine, glutamate, glucose, and ammonia and liver and muscle concentrations of glutamine, glutamate, and reduced and oxidized (GSSG) GSH were measured. Results: Higher concentrations of plasma glutamine were found in the DIP-TR and GLN + ALA-TR groups. The CON-LD group showed hyperammonemia, whereas the DIP-LD and GLN + ALA-LD groups exhibited lower concentrations of ammonia. Higher concentrations of glutamine, glutamate, and GSH/GSSG in the soleus muscle and GSH and GSH/GSSG in the liver were observed in the DIP-TR and GLN + ALA-TR groups. The DIP-LD and GLN + ALA-LD groups exhibited higher concentrations of GSH and GSH/GSSG in the soleus muscle and liver compared with the CON-LD group. Conclusion: Chronic oral administration of DIP and free GLN + ALA before long-duration exercise represents an effective source of glutamine and glutamate, which may increase muscle and liver stores of GSH and improve the redox state of the cell. (C) 2009 Published by Elsevier Inc.