Differential expression of mGluR5 metabotropic glutamate receptor mRNA by rat striatal neurons

The ability to control the degree and spatial distribution of cooling in biological tissues during a thermally mediated therapeutic procedure would be useful for several biomedical applications of lasers. We present a theory based on the solution of the heat conduction equation that demonstrates the feasibility of selectively cooling biological tissues. Model predictions are compared with infrared thermal measurements of in vivo human skin in response to cooling by a cryogen spurt. The presence of a boundary layer, undergoing a liquid-vapour phase transition, is associated with a relatively large thermal convection coefficient (approximately 40 kW m-2 K-1), which gives rise to the observed surface temperature reductions (30-40 degrees C). The degree and the spatial-temporal distribution of cooling are shown to be directly related to the cryogen spurt duration.     

AMPA glutamate receptor subunits are differentially distributed in rat brain

To demonstrate the regional, cellular and subcellular distributions of non-N-methyl-D-aspartate glutamate receptors in rat brain, we generated antipeptide antibodies that recognize the C-terminal domains of individual subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-preferring glutamate receptors (i.e. GluR1, GluR4, and a region highly conserved in GluR2, GluR3 and GluR4c). On immunoblots, antibodies detect distinct proteins with mol. wts ranging from 102,000 to 108,000 in homogenates of rat brain. Immunocytochemistry shows that glutamate receptor subunits are distributed abundantly and differentially within neuronal cell bodies and processes in cerebral cortex, basal ganglia, limbic system, thalamus, cerebellum and brainstem. The precise patterns and cellular localizations of glutamate receptor subunit immunoreactivities are unique for each antibody. In neocortex and hippocampus, pyramidal neurons express GluR1 and GluR2/3/4c immunoreactivities; many non-pyramidal, calcium-binding, protein-enriched neurons in cerebral cortex are selectively immunoreactive for GluR1. In striatum, the cellular localizations of GluR1, GluR2/3/4c and GluR4 immunoreactivities are different; in this region, GluR1 co-localizes with many cholinergic neurons but is only present in a minor proportion of nicotinamide adenine dinucleotide phosphate diaphorase-positive striatal neurons. GluR1 co-localizes with most dopaminergic neurons within the substantia nigra. In several brain regions, astrocytes show GluR4 immunoreactivity. Within the cerebellar cortex, cell bodies and processes of Bergmann glia express intense GluR4 and GluR1 immunoreactivities; perikarya and dendrites of Purkinje cells show GluR2/3/4c immunoreactivity but no evidence of GluR1 or GluR4. Ultrastructurally, GluR subunit immunoreactivities are localized within cell bodies, dendrites and dendritic spines of specific subsets of neurons and, in the case of GluR1 and GluR4, in some populations of astrocytes. This investigation demonstrates that individual AMPA-preferring glutamate receptor subunits are distributed differentially in the brain and suggests that specific neurons and glial cells selectively express glutamate receptors composed of different subunit combinations. Thus, the co-expression of all AMPA receptor subunits within individual cells may not be obligatory for the functions of this glutamate receptor in vivo.     

Expression and subcellular distribution of glutamate receptor subunits 2/3 in the developing cerebellar cortex

The expression and subcellular location of glutamate receptor subunits 2&3 was investigated in the developing postnatal cerebellum. Immunoblotting revealed that glutamate receptor subunits 2/3 is expressed in an identical pattern of immunoreactive bands of approximately 108 kDa from postnatal day zero to adult animals. Light microscopy showed that within the cerebellar cortex, GluR 2/3 immunoreactivity was essentially confined to Purkinje neurons. Strong immunostaining could be observed at postnatal days 1-3 within Purkinje cell bodies and primary dendrites. With ongoing development, the cell body and an increasingly elaborate dendritic tree was outlined by immunoreaction product. In adult animals, staining of Purkinje cell dendrites was patchy, and staining intensity of the cell body, in particular, was greatly reduced. Ultrastructural analysis revealed that during early postnatal development, immunoreaction product was localized to the cell membrane, but was not confined to postsynaptic densities. From the second postnatal week, glutamate receptor subunits 2/3 immunoreactivity was largely restricted to postsynaptic densities. These observations reveal a developmentally regulated refinement of the subcellular distribution of defining subunits of the AMPA-type glutamate receptor. The presence of membrane bond receptors prior to the formation of synapses also provides a rationale for the known transmitter-mediated modulation of Purkinje cell dendritogenesis.     

Regulation of phosphoinositide turnover in neonatal rat cerebral cortex by group I- and II- selective metabotropic glutamate receptor agonists

1. The interactive effects of different metabotropic glutamate (mGlu) receptor subtypes to regulate phosphoinositide turnover have been studied in neonatal rat cerebral cortex and hippocampus by use of agonists and antagonists selective between group I and II mGlu receptors. 2, The group II-selective agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC; 100 microM) had no effect on basal total inositol phosphate ([3H]-InsPx) accumulation (in the presence of Li+) in myo-[3H]-inositol pre-labelled slices, but enhanced the maximal [3H]-InsPx response to the group I-selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) by about 100% in both hippocampus and cerebral cortex. In cerebral cortex the enhancing effect of 2R,4R-APDC occurred with respect to the maximal responsiveness and had no effect on EC50 values for DHPG (-log EC50 (M): control, 5.56+/-0.05; +2R,4R-APDC, 5.51+/-0.08). 2R,4R-APDC also caused a significant enhancement of the DHPG-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass response over an initial 0-300 s time-course. 3. The enhancing effects of 2R,4R-APDC on DHPG-stimulated [3H]-InsPx accumulation were observed in both the presence and nominal absence of extracellular Ca2+, and irrespective of whether 2R,4R-APDC was added before, simultaneous with, or subsequent to DHPG. Furthermore, increasing the tissue cyclic AMP concentration up to 100 fold had no effect on DHPG-stimulated Ins(l,4,5)P3 accumulation in the absence or presence of 2R,4R-APDC. 4. 2R,4R-APDC and (2S, 1'R, 2'R, 3'R)-2-(2,3-dicarboxylcyclopropyl)glycine (DCG-IV), the latter agent in the presence of MK-801 to prevent activation of NMDA-receptors, each inhibited forskolin-stimulated cyclic AMP accumulation by about 50%, with respective EC50 values of 1.3 and 0.04 microM (-log EC 50 (M): 2R,4R-APDC, 5.87+/-0.09; DCG-IV, 7.38+/-0.05). In the presence of DHPG (30 microM), 2R,4R-APDC and DCG-IV also concentration-dependently increased [3H]-InsPx accumulation with respective EC50 values of 4.7 and 0.28 microM (-log EC50 (M): 2R,4R-APDC, 5.33+/-0.04; DCG-IV, 6.55+/-0.09) which were 3-7 fold rightward-shifted relative to the adenylyl cyclase inhibitory responses. 5. The group II-selective mGlu receptor antagonist LY307452 (30 microM) caused parallel rightward shifts in the concentration-effect curves for inhibition of forskolin-stimulated adenylyl cyclase, and enhancement of DHPG-stimulated [3H]-InsPx accumulation, by 2R,4R-APDC yielding similar equilibrium dissociation constants (KdS, 3.7+/-1.1 and 4.1+/-0.4 microM respectively) for each response. 6. The ability of 2R,4R-APDC to enhance receptor-mediated [3H]-InsPx accumulation appeared to be agonist-specific; thus although DHPG (100 microM) and the muscarinic cholinoceptor agonist carbachol (10 microM) stimulated similar [3H]-InsPx accumulations, only the response to the former agonist was enhanced by co-activation of group II mGlu receptors. 7. These data demonstrate that second messenger-generating phosphoinositide responses stimulated by group I mGlu receptors are positively modulated by co-activation of group II mGlu receptors in cerebral cortex and hippocampus. The data presented here are discussed with respect to the possible mechanisms which might mediate the modulatory activity, and the physiological and pathophysiological significance of such crosstalk between mGlu receptors.     

Serotonin promotes the differentiation of glutamate neurons in organotypic slice cultures of the developing cerebral cortex

The monoamines serotonin (5-HT), noradrenaline (NA), and dopamine (DA), which are present in the developing brain apparently before they assume their neurotransmitter functions, are regarded as strong candidates for a role in the maturation of the cerebral cortex. Here we sought to investigate their effects on the generation and differentiation of cortical cell types. Slice cultures, prepared from the cortices of embryonic day (E) 14, E16, and E19 rat fetuses, were kept in defined medium or in defined medium plus 5-HT for 7 d. E16 cortices were also exposed to NA or DA for the same period. At the end of this period, the proportions of the neuronal [glutamate (Glu)-, GABA-, calbindin-, calretinin-labeled], glial (GFAP), and neuroepithelial (nestin) cell types were estimated for all conditions. We found that in E16 cultures, application of 5-HT, but not of NA or DA, significantly increased the proportion of Glu-containing neurons without affecting the overall neuronal population or the proportions of any other cell types. A similar effect was observed in co-cultures of E16 cortex with slices through the midbrain raphe nuclei of E19 rats. The total amount of cortical Glu, as measured with HPLC, was also increased in these co-cultures. To investigate whether the effect of 5-HT was the result of changes in cell proliferation, we exposed slices to bromodeoxyuridine (BrdU) and found that the proportion of BrdU-labeled cells was similar in the 5-HT-treated and control slices. These results indicate that 5-HT promotes the differentiation of cortical Glu-containing neurons without affecting neuroepithelial cell proliferation.     

Differential regulation of GABA(A) receptor gene expression by ethanol in the rat hippocampus versus cerebral cortex

Previous research has shown that chronic ethanol consumption dramatically alters GABA(A) receptor alpha1 and alpha4 subunit gene expression in the cerebral cortex and GABA(A) receptor alpha1 and alpha6 subunit gene expression in the cerebellum. However, it is not yet known if chronic ethanol consumption produces similar alterations in GABA(A) receptor gene expression in other brain regions. One brain region of interest is the hippocampus because it has recently been shown that a subset of GABA(A) receptors in the hippocampus is responsive to pharmacologically relevant concentrations of ethanol. Therefore, we directly compared the effects of chronic ethanol consumption on GABA(A) receptor subunit gene expression in the hippocampus and cerebral cortex. Furthermore, we investigated whether the duration of ethanol consumption (14 or 40 days) would influence regulation of GABA(A) receptor gene expression in these two brain regions. Chronic ethanol consumption produced a significant increase in the level of GABA(A) receptor alpha4 subunit peptide in the hippocampus following 40 days but not 14 days. The relative expression of hippocampal GABA(A) receptor alpha1, alpha2, alpha3, beta(2/3), or gamma2 was not altered by either period of chronic ethanol exposure. In marked contrast, chronic ethanol consumption for 40 days significantly increased the relative expression of cerebral cortical GABA(A) receptor alpha4 subunits and significantly decreased the relative expression of cerebral cortical GABA(A) receptor alpha1 subunits. This finding is consistent with previous results following 14 days of chronic ethanol consumption. Hence, chronic ethanol consumption alters GABA(A) receptor gene expression in the hippocampus but in a different manner from that in either the cerebral cortex or the cerebellum. Furthermore, these alterations are dependent on the duration of ethanol exposure.     

Agmatine selectively blocks the N-methyl-D-aspartate subclass of glutamate receptor channels in rat hippocampal neurons

We investigated in rat hippocampus neurons whether 4-(aminobutyl)guanidine (agmatine), formed by decarboxylation of L-arginine by arginine decarboxylase and metabolized to urea and putrescine, can modulate the function of N-methyl-D-aspartate (NMDA) receptor channels. In cultured hippocampal neurons studied by whole-cell patch clamp, extracellular-applied agmatine produced a voltage- and concentration-dependent block of NMDA but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid nor kainate currents. Analysis of the voltage dependence of the block suggests that agmatine binds at a site located within the NMDA channel pore with a dissociation constant of 952 microM at 0 mV and an electric distance of 0.62. We also tested effects of several agmatine analogs. Arcaine (1,4-butyldiguanidine) also produced a similar voltage-dependent block of the NMDA current, whereas putrescine (1, 4-butyldiamine) had little effect, suggesting that the guanidine group of agmatine is the active moiety when blocking the NMDA channel. Moreover, spermine (an endogenous polyamine) potentiated the NMDA current even in the presence of blocker agmatine or arcaine, suggesting that the guanidine-containing compounds agmatine and arcaine interact with the NMDA channel at a binding site different from that of spermine. Our results indicate that in hippocampal neurons agmatine selectively modulates the NMDA subclass of glutamate receptor channels mediated by the interaction between the guanidine group and the channel pore. The results support other data that agmatine may function as an endogenous neurotransmitter/neuromodulator in brain.     

AMPA receptor subunits are differentially expressed in parvalbumin- and calretinin-positive neurons of the rat hippocampus

Recent studies suggest a functional diversity of native alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor channels (AMPARs). In several types of interneurons, AMPARs are characterized by higher Ca2+ permeability and faster kinetics than AMPARs in principal cells. We studied the expression profile of AMPAR subunits in the hippocampal parvalbumin (PV)- and calretinin (CR)-positive cells, which represent different populations of non-principal cells. To this end, non-radioactive in situ hybridization with AMPAR subunit specific cRNAs was combined with immunocytochemistry for PV or CR. Double-immunolabelling using antibodies against AMPAR subunits and PV or CR was also performed. PV-containing neurons represent a fairly homogeneous population of cells expressing high levels of GluR-A and GluR-D mRNAs, moderate levels of GluR-C and low levels of GluR-B mRNAs in all the examined regions of hippocampus. The vast majority of CR-containing cells have a much lower expression of GluR-A, -C and -D mRNA than PV-positive neurons, although similarly featuring low levels of GluR-B mRNA. Only a subpopulation of CR-containing cells, the spiny neurons of the dentate gyrus and CA3 region of the hippocampus were characterized by a strong expression of GluR-A and -D subunit mRNAs. The differential pattern found for the AMPAR subunit mRNA expression was confirmed by immunocytochemistry at protein level. Despite the common feature of low GluR-B subunit expression, PV- and CR-containing interneurons differ with respect to the density and combination of their expressed AMPAR subunits. The different combination of subunits might subserve different properties of the AMPA channels featured by these cell types, with implications for the functioning of the hippocampal network.     

Differential distribution of (GABA)A receptor subunits on bulbospinal serotonergic and nonserotonergic neurons of the ventromedial medulla of the rat

Spinally projecting neurons of the ventromedial medulla (VMM) compose an important efferent pathway for the modulation of nociception. These neurons receive a substantial gamma-aminobutyric acid (GABA)-ergic input, but the GABA receptor that mediates this input is unknown. This study examined the distribution of GABA(A) receptor alpha1 and alpha3 subunits in serotonergic and nonserotonergic neurons of the VMM that project to the dorsal horn in the rat. A pledget of Gelfoam soaked in Fluoro-Gold was placed at the thoracolumbar junction of the spinal cord to label spinally projecting neurons. Alternate sections of the medulla were then incubated with a mixture of antisera to either serotonin and the alpha1 subunit, or to serotonin and the alpha3 subunit of the GABA(A) receptor. Nearly 30% of spinally projecting neurons in the VMM were immunoreactive for the alpha1 subunit. A similar percentage of spinally projecting neurons in the VMM were immunoreactive for the alpha3 subunit, although diffuse cellular labeling combined with intense staining of processes in the neuropil precluded a rigorous semi-quantitative estimation of this population. No alpha1-subunit-immunoreactive neurons colocalized serotonin. In contrast, serotonergic neurons were immunoreactive for the alpha3 subunit. However, these double-labeled neurons were a modest percentage of the serotonergic population. A small percentage of spinally projecting serotonergic neurons was immunoreactive for the alpha3 subunit. These results suggest that significant numbers of spinally projecting serotonergic and nonserotonergic neurons of the VMM possess GABA(A) receptors that differ in their respective subunit compositions and that both classes of neurons may mediate the antinociception produced by the microinjection of GABA(A) receptor antagonists in the VMM.     

Differential effect of morphine on dopaminergic neurons in frontal cortex and striatum of the rat

Lactate dehydrogenase-5 and creatine kinase from rabbit muscle were labeled by coupling with N-hydroxysuccinimidyl 3-(4'-hydroxy-[3',5'-125I]diiodophenyl)propionate. After purification, the analytical recovery of catalytically-active labeled enzyme averaged 90% for lactate dehydrogenase, 81% for creatine kinase. The labeled enzymes were injected intravenously into rabbits and disappearance from plasma of catalytic activity and radioactivity was measured. The disappearance curves for lactate dehydrogenase-5 differed considerably from those observed with the enzyme labeled by direct iodination. The discrepancy was due to rapid hydrolysis in vivo of the labeled amide-enzyme linkage, because about 50% of the injected radioactivity appeared in the urine as 125I-labeled 3-(4'-hydroxy-3',5'-diiodophenyl)propionic acid within 4-8 h of injection. Similar outputs were observed after administration of this acid to rabbits. The free acid was also detected in the urines of rabbits within 4-8 h of the intravenous injection of creatine kinase labeled similarly. We conclude that this method of labeling is unsuitable for preparing radioactive enzymes for study of their catabolism.     

Involvement of a cyclic-AMP pathway in group I metabotropic glutamate receptor responses in neonatal rat cortex

3,5-Dihydroxyphenylglycine (DHPG), (S)-3-hydroxyphenylglycine and (S)-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG) stimulated phosphoinositide hydrolysis in neonatal rat cortical slices, but with lower maximal effect, in comparison with 2S,1'S,2'S-2-(2'-carboxycyclopropyl)glycine (L-CCG I) or (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid (1S,3R-ACPD). DHPG, 1S,3R-ACPD, and S-4C3HPG also evoked a rapidly desensitizing increase in [Ca2+]i in cortical layers of neonatal brain slices. (R,S)-alpha-methyl-4-tetrazolyl-phenylglycine (MTPG), and (R,S)-alpha-methyl-4-phosphono-phenylglycine (MPPG) inhibited the increase of phosphoinositide hydrolysis elicited by 1S,3R-ACPD but not that by R,S-DHPG. In contrast, the selective group II receptor agonist (1S,2S,5R,6S)-2-amino-bicyclo-[3.1.0]-hexane-2,6-dicarboxylate (LY 354740) potentiated the response of R,S-DHPG. Finally, 8-(4-chlorophenylthio)-cAMP, a membrane permeant analogue of cAMP, reversed the stimulatory effect of 1S,3R-ACPD and S-4C3HPG on phosphoinositide hydrolysis and [Ca2+]i mobilization, without affecting the response induced by R,S-DHPG. These data suggest that, in neonatal rat cortex, the activation of group II metabotropic glutamate receptors potentiates the phosphoinositide hydrolysis and [Ca2+]i responses mediated by group I metabotropic glutamate receptors.     

Effects of oxidants and glutamate receptor activation on mitochondrial membrane potential in rat forebrain neurons

Both glutamate and reactive oxygen species have been implicated in excitotoxic neuronal injury, and mitochondria may play a key role in the mediation of this process. In this study, we examined whether glutamate-receptor stimulation and oxidative stress interact to affect the mitochondrial membrane potential (delta psi). We measured delta psi in rat forebrain neurons with the ratiometric fluorescent dye JC-1 by using laser scanning confocal imaging. Intracellular oxidant levels were measured by using the oxidation-sensitive dyes 2',7'-dichlorodihydrofluorescein (DCFH2) and dihydroethidium (DHE). Application of hydrogen peroxide (0.3-3 mM) or 1 mM xanthine/0.06 U/ml xanthine oxidase decreased delta psi in a way that was independent of the presence of extracellular Ca2+ and was not affected by the addition of cyclosporin A, suggesting the presence of either a cyclosporin A-insensitive form of permeability transition, or a separate mechanism. tert-Butylhydroperoxide (730 microM) had less of an effect on delta psi than hydrogen peroxide despite similar effects on intracellular DCFH2 or DHE oxidation. Hydrogen peroxide-, tert-butylhydroperoxide-, and superoxide-enhanced glutamate, but not kainate, induced decreases in delta psi. The combined effect of peroxide or superoxide plus glutamate was Ca2+ dependent and was partially inhibited by cyclosporin A. These results suggest that oxidants and glutamate depolarize mitochondria by different mechanisms, and that oxidative stress may enhance glutamate-mediated mitochondrial depolarization.     

NMDA受体不同亚单位分布造成肌萎缩侧索硬化中运动神经元选择性易损

目的:研究NMDA受体不同亚单位NR2A和NR2B分布与肌萎缩侧索硬化中运动神经元选择性易损的关系.方法:首先在离体培养的脑组织片中用谷氦酸转运抑制剂造成兴奋毒性损伤,观察在同等损伤条件下运动皮层和梨状区皮层是否出现有差异的神经元数量减少,然后用Western blot方法测定运动皮层和梨状区皮层的NR2A和NR2B的蛋白含量,证明选择性运动皮层损伤与NR2A和NR2B的不同分布方式有关,为进一步明确NR2A和NR2B在运动神经元谷氨酸慢性损伤中的作用不同,在离体培养的皮层运动神经元和脑组织片中分别抑制NR2A和NR2B,观察对运动神经元产生的影响.结果:用谷氨酸转运抑制剂THA 100 μmol/L损伤脑片2周后,运动皮层神经元的死亡率高于梨状区皮层,分别为64.50%±2.19%和30.43%±4.75%(P＜0.01).Western blot结果表明NR2B/NR2A比值在运动皮层明显高于梨状区皮层,分别为0.87±0.09和0.47±0.09(P＜0.01).抑制剂保护作用试验中,NR2B抑制剂显示出保护作用,离体培养的皮层运动神经元对照组、谷氨酸损伤组、谷氨酸+NR2A抑制剂组和谷氨酸+NR2B抑制剂组的运动神经元死亡率分别为6.85%±1.47%、47.48%±5.75%、45.76%±8.09%和18.10%±3.11%,脑片对照组、THA损伤组、THA+NR2A抑制剂组和THA+NR2B抑制剂组的运动神经元死亡率分别为12.49%±2.09%、100%、110.87%±15.76%和35.13%±5.32%.结论:NR2A和NR2B在运动神经元慢性谷氨酸损伤中所起的作用不同,NR2B在运动皮层的高分布造成肌萎缩侧索硬化中运动神经元的选择性易损.     

Inwardly rectifying and Ca2+-permeable AMPA-type glutamate receptor channels in rat neocortical neurons

Inwardly rectifying and Ca2+-permeable AMPA-type glutamate receptor channels in rat neocortical neurons. J. Neurophysiol. 78: 2592-2605, 1997. Current-voltage (I-V) relations and Ca2+ permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)type glutamate receptor channels were investigated in neurons of rat neocortex by using the whole cell patch-clamp technique in brain slices. To activate AMPA receptor channels, kainate was used as a nondesensitizing agonist. A patch pipette was filled with solution containing 100 mu M spermine to maintain the inward rectification of Ca2+-permeable AMPA receptor channels. Three types of responses to kainate were observed: type I response with outwardly rectifying I-V relation, type II response with I-V relation of marked inward rectification, and intermediate response with I-V relation of weaker inward rectification. Neurons with type I, type II and intermediate I-V relations were referred to as type I, type II, and intermediate neurons, respectively. Of a total of 223 recorded cells, 90 (40.4%) were type I, 129 (57.8%) intermediate, and 4 (1.8%) type II neurons. Properties of AMPA receptor channels were examined in the former two types of neurons. The value of PCa:PCs, the ratio of the permeability coefficients of Ca2+ and Cs+, was estimated from the reversal potentials of kainate responses in the outside-out patches bathed in Na+-free solution containing 100 mM Ca2+ according to the constant-field equation. They ranged from 0.05 to 0.10 (0.08 +/- 0. 02, mean +/- SD, n = 8) for type I neurons and from 0.14 to 1.29 (0. 60 +/- 0.37, n = 11) for the intermediate neurons. There was a close correlation between the inward rectification and the Ca2+ permeability in AMPA receptor channels in these neurons. Intermediate neurons stained with biocytin were nonpyramidal cells with ellipsoidal-shaped somata. Type I neurons had either triangular- or ellipsoidal-shaped somata. Excitatory postsynaptic currents (EPSCs) recorded in both type I and intermediate neurons had 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive fast and -2-amino-5-phosphonovalerate-sensitiveslow components. The I-V relation of the fast component exhibited inward rectification in the intermediate neuron, whereas that in the type I neuron showed slight outward rectification. The fast component of EPSCs in the intermediate neuron was suppressed more prominently (to 56 +/- 15% of the control, n = 12) than that in the type I neuron (to 78 +/- 6% of the control, n = 6) by bath application of 1 mM spermine. These results indicate that inwardly rectifying and Ca2+-permeable AMPA receptor channels are expressed in a population of neurons of rat neocortex and are involved in excitatory synaptic transmission.     

Changes of mitochondrial cytochrome c oxidase and FoF1 ATP synthase subunits in rat cerebral cortex during aging

The contents of subunits I, II/III, and IV of cytochrome c oxidase and of subunits alpha, beta and gamma of FoF1 ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, and 26 months of age were analyzed by western blot. Age-related changes in the content of subunits, either of mitochondrial or nuclear origin, were observed. All the cytochrome c oxidase (COX) subunits examined showed an age-related increase from 2-month-old rats up to 24 months with a decrease at the oldest age (26 months). The same pattern of age-dependent changes was observed for gamma ATP synthase, while the alpha and beta subunits increased progressively up to 26 months.     

Muscarinic M1 receptor mediated inhibition of GABA release from rat cerebral cortex

Presynaptic modulation of [3H]GABA release was examined using rat cerebral cortical slices. In vitro addition of carbachol, a muscarinic receptor agonist, resulted in a significant suppression of the release of [3H]GABA evoked by high potassium (50 mM) stimulation in a dose dependent manner, while noradrenaline, isoproterenol, dopamine, 5-hydroxytryptamine, histamine and glutamic acid had no significant effect on the evoked release of [3H]GABA. This suppressive effect of carbachol was antagonized invariably by atropine. Furthermore, it was found that the suppressive action of carbachol could be antagonized by pirenzepine, a selective M1 muscarinic receptor antagonist, but not by AF-DX 116 and 4-DAMP, M2 and M3 receptor antagonists, respectively. These results suggest that the release of GABA from cerebral cortical GABA neurons may be modulated by presynaptic M1 muscarinic receptor.     

Differential localization of phosphoinositide-linked metabotropic glutamate receptor (mGluR1) and the inositol 1,4,5-trisphosphate receptor in rat brain

The type 1 metabotropic glutamate receptor (mGluR1) is through to act via the phosphoinositide (PI) system with the associated formation of inositol 1,4,5-trisphosphate (IP3) and Ca2+ release. Utilizing immunohistochemistry and in situ hybridization, we have localized protein and mRNA, respectively, for the mGluR1 and the IP3 receptor (IP3R). We have also localized glutamate-linked PI turnover by autoradiography with 3H-cytidine. We observe a striking contrast in localizations of mGluR1 and IP3R both for protein and mRNA. For instance, mGluR1 occurs in the apparent absence of IP3R in neurons of the stratum oriens of the CA1 hippocampus, islands of Calleja, anterodorsal nucleus of thalamus, lateral nucleus of hypothalamus, and the granular cell layer and the deep nuclei of cerebellum. mGluR1 actions in these brain regions may primarily be mediated through the protein kinase C limb of the PI system, as they contain moderate amounts of 3H-phorbol ester binding. The subthalamic nucleus, red nucleus, and Darkshevich's nucleus, which possess high levels of mGluR1, are devoid of both IP3R immunoreactivity and 3H-phorbol ester binding. These reciprocal localizations suggest that mGluR1 actions in many brain areas may not primarily involve IP3, reflecting instead influences on protein kinase C or other second messengers.     

Ionotropic and metabotropic glutamate receptor agonist-induced [Ca2+]i increase in isolated rat supraoptic neurons

Although a number of studies have shown that various free fatty acids (FFAs) and monoacylglycerides (MGs) have bactericidal properties in vitro, the role of these compounds in vivo has not been determined. This study evaluated the antibacterial properties of medium-chain MGs and FFAs for different bacterial enteropathogens with an in-vitro bacterial killing assay and an in-vivo model of intestinal colonisation. Incubation of test bacteria with medium-chain MGs for 4 h led to 100-10,000-fold reductions in numbers of viable cells of Vibrio cholerae, Salmonella typhi, Shigella sonnei and enterotoxigenic Escherichia coli (ETEC). Lauric acid was the only medium-chain FFA to show comparable in-vitro bactericidal activity. The ability of dietary MGs to reduce or eliminate bacterial colonisation of the intestinal tract was evaluated in mice that were predisposed to bacterial colonisation by treatment with streptomycin (STR+). Mice were treated with streptomycin, challenged intragastrically with V. cholerae or ETEC, and given monocaprin (C10:0 MG) either concurrently or as part of the daily diet. Control mice given STR+ without MGs and challenged with V. cholerae or ETEC showed high numbers of challenged bacteria in gastrointestinal contents by 1 h after administration. Concurrent administration of V. cholerae and C10:0 MG (2.5 mg/ml) caused > 1000-fold reduction in numbers of V. cholerae recovered from the gastrointestinal tracts of STR+ mice. Concurrent administration of C10:0 MG with ETEC did not cause a reduction in the number of viable ETEC present in the intestinal tract of STR+ mice. Administration of C10:0 MG in the diet had no effect on the number of viable V. cholerae or ETEC associated with caecal or ileal tissue of STR+ mice when C10:0 MG in the diet was started 1 day before, the same day, or 2 days after bacterial challenge. Collectively, these results suggested that dietary MGs may prevent intestinal colonisation by bacterial enteropathogens if administered at the time of exposure, but have little effect on established intestinal infections.     

Expression of glutamate receptor subunit mRNAs in gonadotropin-releasing hormone neurons during the sexual maturation of the female rat

Strongyloides stercoralis (SS) is endemic in tropical and subtropical areas worldwide and in the southeastern United States. The lifecycle of SS is both unique and complex. Human infection begins with the penetration of skin by filariform larvae that migrate hematogenously to the lungs. Larvae then ascend the airway, are swallowed, and mature in the gut. Unlike other nematodes, SS can autoinfect the same host and persist for decades. Categorization of infection includes acute, chronic-uncomplicated, and disseminated forms. Clinical manifestations depend on the particular organs involved. Fifteen to thirty percent of chronically infected people may be asymptomatic. On the other hand, SS may cause the adult respiratory distress syndrome, septic shock, and death. The diagnosis of SS infection is suspected in patients from endemic areas who have blood eosinophilia, and gastrointestinal or pulmonary symptoms. A definitive diagnosis is established by demonstration of SS larvae in stool, body fluids, or tissues. A presumptive diagnosis of SS infection can be achieved by serology. Thiabendazole is the mainstay of treatment, but repeat doses may be necessary if the parasite is not initially eradicated. The low incidence of disseminated SS in areas endemic for both SS and AIDS is surprising and unexplained.     

Real-time detection of GABA-induced synaptic glutamate release in cultured rat cortex

Glutamate is an important neurotransmitter in synaptic transmission. There are no methods, however, for continuous measurement of glutamate concentration at high temporal and spatial resolutions. We have developed a novel electrochemical detection method for the on-line measurement of glutamate release with nanomolar resolution in real time. Using this method, GABA was found to have a modulatory action on the synaptic glutamate release in cultured rat cortical cells. This synaptic modulation largely depends on the GABAA receptor and could be a key not only in neural development, but also in signal transduction in the brain. Our detection method is ideal for investigating such synaptic glutamate responses because of its higher sensitivity and real-time measurement capability.