Role of desensitization of AMPA receptors on the neuronal viability and on the [Ca2+]i changes in cultured rat hippocampal neurons

We investigated the role of desensitization of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptors on the neurotoxicity and on the [Ca2+]i changes induced by kainate or by AMPA in cultured rat hippocampal neurons. The neuronal viability was evaluated either by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, or by analysis of cell morphology. Short-term exposure of the neurons to kainate or AMPA (30 min) was not toxic, but the exposure for 24 h to the excitotoxic drugs caused a concentration-dependent neurotoxic effect which was prevented by LY 303070, a noncompetitive AMPA receptor antagonist. In the presence of cyclothiazide (CTZ), kainate or AMPA was toxic (30 min exposure), or the toxic effect was significantly enhanced (24 h exposure), but in this case LY 303070 did not completely protect the cells against kainate-induced toxicity. The alterations in the [Ca2+]i caused by kainate or AMPA showed a great cell-to-cell variability. LY 303070 completely or partially inhibited the responses stimulated by kainate. CTZ differentially affected the responses evoked by kainate or AMPA. In the majority of hippocampal neurons, CTZ did not potentiate, or only slightly potentiated, the kainate-stimulated responses but in 11% of neurons there was a great potentiation. In AMPA-stimulated neurons, the responses were slightly or greatly potentiated in the majority of neurons, but not in all of them. The results show that AMPA and kainate may be toxic, depending on the time of exposure and on the blockade of the desensitization of the AMPA receptors. Overall, our results clearly show that there exist different populations of hippocampal neurons with different sensitivities to kainate, AMPA, CTZ and LY 303070. Moreover, the effects of CTZ on both [Ca2+]i alterations and neurotoxicity are not fully correlated.     

Role of desensitization and subunit expression for kainate receptor–mediated neurotoxicity in murine neocortical cultures

The neurotoxic actions of kainate and domoate were studied in cultured murine neocortical neurons at various days in culture and found to be developmentally regulated involving three components of neurotoxicity: (1) toxicity via indirect activation of N‐methyl‐d ‐aspartate (NMDA) receptors, (2) toxicity mediated by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptors, and (3) toxicity that can be mediated by kainate receptors when desensitization of the receptors is blocked. The indirect action at NMDA receptors was discovered because (5R,10S)‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine (MK‐801), an NMDA receptor antagonist, was able to block part of the toxicity. The activation of NMDA receptors is most likely a secondary effect resulting from glutamate release upon kainate or domoate stimulation. 1‐(4‐Aminophenyl)‐3‐methylcarbamyl‐4‐methyl‐3,4‐dihydro‐7,8‐ethylenedioxy‐5H‐2,3benzodiazepine (GYKI 53655), a selective AMPA receptor antagonist, abolished the remaining toxicity. These results indicated that kainate‐ and domoate‐mediated toxicity involves both the NMDA and the AMPA receptors. Pretreatment of the cultures with concanavalin A to prevent desensitization of kainate receptors led to an increased neurotoxicity upon stimulation with kainate or domoate. In neurons cultured for 12 days in vitro a small but significant neurotoxic effect was observed when stimulated with agonist in the presence of MK‐801 and GYKI 53655. This indicates that the toxicity is produced by kainate receptors in mature cultures. Examining the subunit expression of the kainate receptor subunits GluR6/7 and KA2 did, however, not reveal any major change during development of the cultures. J. Neurosci. Res. 55:208–217, 1999. © 1999 Wiley‐Liss, Inc.     

Sub-micromolar increase in [Ca(2+)](i) triggers delayed exocytosis of ATP in cultured astrocytes

Astrocytes release a variety of transmitter molecules, which mediate communication between glial cells in the brain and modulate synaptic transmission. ATP is a major glia-derived transmitter, but the mechanisms and kinetics of ATP release from astrocytes remain largely unknown. Here, we combined epifluorescence and total internal reflection fluorescence microscopy to monitor individual quinacrine-loaded ATP-containing vesicles undergoing exocytosis in cultured astrocytes. In resting cells, vesicles exhibited three-dimensional motility, spontaneous docking and release at low rate. Extracellular ATP application induced a Ca(2+)-dependent increase in the rate of exocytosis, which persisted for several minutes. Using UV flash photolysis of caged Ca(2+), the threshold [Ca(2+)](i) for ATP exocytosis was found to be approximately 350 nM. Subthreshold [Ca(2+)](i) transients predominantly induced vesicle docking at plasma membrane without subsequent release. ATP exocytosis triggered either by purinergic stimulation or by Ca(2+) uncaging occurred after a substantial delay ranging from tens to hundreds of seconds, with only approximately 4% of release occurring during the first 30 s. The time course of the cargo release from vesicles had two peaks centered on 相似文献   

[Ca^2＋]i change in hippocampal neurons influenced by preconditioning of etomidate fat emulsion following cerebral ischemia in rats

BACKGROUND: It is known that intravenous anesthetic etomidate fat emulsion has cerebral protection. Now many scholars focus on the research of its cerebral protection from molecular biology, but the mechanism of cerebral protection is still fully unclear. OBJECTIVE: To observe the influence of etomidate fat emulsion on the [Ca2+]i in hippocampal neurons during the transient cerebral ischemia injury in rats. DESIGN: Randomized controlled observation. SETTING: Weifang Medical College. MATERIALS: This study was carried out in the functional laboratory of Weifang Medical College between October 2005 and March 2006. Twenty-four male healthy Wistar rats, aged 3 to 4 months, were involved. Etomidate fat emulsion was provided by the limited company of En-hua Medical Bloc in Jiangsu Province (code of H20020511) and the other agents and materials were provided by Laboratory Center of Weifang Medical College. METHODS: The 24 Wistar rats were randomized into 3 groups: sham-operation group, model group and etomidate preconditioning group, with 8 rats in each. Rat models of transient cerebral ischemia injury were made by the ligation of bilateral carotid arteries combined with descending blood pressure in the latter two groups. Before ischemia (ligation of bilateral common carotid artery), rats in the etomidate preconditioning group were intraperitoneally injected with 12 mg/kg etomidate fat emulsion and then persistently intraperitoneally injected with etomidate fat emulsion at 1.0 mg/kg per minute. Rats in the model group were not administrated. Rats in the sham-operation group were only performed bilateral common carotid artery isolation. When rats were modeled, their brain tissues were quickly taken out and detected. MAIN OUTCOME MEASURES: Change of the fluorescence pixel value of the [Ca2+]i in each group by the laser scanning confocal microscope. RESULTS: Twenty-four rats were involved in the final analysis. Fluorescence pixel value in the sham-operation group was in the low level. Fluorescence pixel value in the model group was significantly higher than that in the sham-operation group (P < 0.01). Fluorescence pixel value in the etomidate preconditioning group was significantly lower than that in the model group (P < 0.01). CONCLUSION: The protection of etomidate fat emulsion to the transient cerebral ischemic injury in rats is associated with the inhibition to the increase of [Ca2+]i to some extent.     

石杉碱甲对血管性痴呆小鼠模型海马神经细胞静息态[Ca~(2+)]i的影响

目的　观察血管性痴呆 (VD)小鼠海马神经细胞内静息态游离 Ca2 +浓度 ([Ca2 + ]i)变化 ,以及石杉碱甲 (哈伯因 )对 VD的治疗效果和 [Ca2 + ]i的影响 ,进而探讨 [Ca2 + ]i的改变在 VD发病机制中的作用。方法　采用双侧颈总动脉线结、反复缺血 -再灌注法 ,制作小鼠血管性痴呆动物模型 ,并设假手术组作为对照 ,服用石杉碱甲者为治疗组。分别于术后第 2 9天、第 30天进行学习、记忆和行为学测试 ;然后快速制取海马活细胞 ,以 Fluo- 3/ AM为荧光探针 ,在激光扫描共聚焦显微镜下观察各组海马神经细胞静息态 [Ca2 + ]i变化。结果　 (1)模型组的学习和记忆成绩低于假手术组及治疗组 (P<0 .0 5 ) ,治疗组及假手术组间无显著性差别 ;(2 )模型组神经细胞静息态 [Ca2 + ]i显著高于假手术组、治疗组 (P<0 .0 5 ) ,治疗组及假手术组间无显著性差别。结论　石杉碱甲作为中枢神经系统特异性胆碱酯酶抑制剂和 NMDA受体拮抗剂 ,可降低血管性痴呆小鼠海马静息态 [Ca2 + ]i,并改善其临床症状 ;因此提示 :海马神经细胞静息态 [Ca2 + ]i过高参与了血管性痴呆的发病过程。     

Effects of anisodamine on altered [Ca~(2+)]i and cerebral cortex ultrastructure following acute cerebral ischemia/reperfusion injury in rabbits

BACKGROUND： Calcium ion （Ca^2＋） overload plays an important role in cerebral ischemia/reperfusion injury. Anisodamine, a type of alkaloid, can protect the myocardium from ischemia and reperfusion injury by inhibiting intracellular calcium [Ca^2＋]i overload. OBJECTIVE： To investigate effects of anisodamine on [Ca^2＋]i concentration and cortex ultrastructure following acute cerebral ischemia/reperfusion in rabbits. DESIGN, TIME AND SETTING： Randomized and controlled trial was performed at the Department of Emergency, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from September to December 2006. MATERIALS： Forty healthy rabbits were used to establish models of acute cerebral ischemia/reperfusion. Anisodamine was provided by Lianyungang Dongfeng Pharmaceutical Factory; Fura-2 was purchased from Nanjing Jiancheng Bioengineering Institute; dual-wave length fluorescent spectrophotometry system and DM-300 software were provided by Bio-Rad, USA; OPTON-EM10C transmission electron microscope was product of Siemens, Germany. METHODS： Forty rabbits were randomly divided into the following groups： sham operation, ischemia, ischemia/reperfusion, and anisodamine, with ten rabbits in each group. Models of complete cerebral ischemia injury were established. In addition, blood was collected from the femoral artery of rats in the ischemia/reperfusion and anisodamine groups to induce hypotension and establish repeffusion injury models. The bilateral common carotid artery clamp was removed from the anisodamine group 20 minutes after ischemia, and anisodamine （10 mg/kg body mass） was injected via the femoral vein. Rabbits in the sham operation group underwent only venous cannulation. MAIN OUTCOME MEASURES： [Ca^2＋]i concentration was determined using a dual-wave length fluorescent spectrophotometry system, and cortical ultrastructure was observed following uranyl-lead citrate staining. RESULTS： The levels of [Ca^2＋]i in the ischemia and ischemia/reperfusion gro     

Emergence of excitotoxicity in cultured forebrain neurons coincides with larger glutamate-stimulated [Ca(2+)](i) increases and NMDA receptor mRNA levels

We examined several factors related to the increase in susceptibility to excitotoxicity that occurs in embryonic forebrain neurons over time in culture. Neuronal cultures were resistant to a 5-min exposure to 100 microM glutamate/10 microM glycine at 5 days in vitro (DIV), but became vulnerable to the same stimulus by 14 DIV. We used the fluorescent indicators, fura-2 and magfura-2, which have high and low affinity for Ca(2+), respectively, to measure changes in [Ca(2+)](i). Glutamate-stimulated increases in the fura-2 and magfura-2 ratio reached maximum values by 10 DIV. Fura-2 reported similar [Ca(2+)](i) changes with exposure to 3 or 100 microM glutamate for 5 min, whereas magfura-2 reported larger [Ca(2+)](i) increases with 5-min exposure to 100 microM glutamate than with exposure to 3 microM glutamate, 100 microM kainate or 50 mM K(+) from 10 DIV onward. This suggests that the magnitude of the [Ca(2+)](i) changes correlated with the excitotoxicity potential of a stimulus when magfura-2, but not fura-2, was used to measure Ca(2+). We also used RNase protection assays to measure NMDA receptor subunit mRNA levels. NR1 and NR2A mRNA increased continuously over time in culture, whereas NR2B mRNA increased dramatically during the first 10 days and subsequently remained stable. The time course of the increase in NR2B mRNA most closely followed the increase in glutamate-stimulated changes in the magfura-2 signal and neuronal injury. Therefore, the increases in the glutamate-stimulated [Ca(2+)](i) responses and NMDA receptor subunit mRNA levels (especially NR2B) are likely involved in the development of susceptibility to excitotoxicity in cultured rat forebrain neurons.     

Weak effect of neuromodulators on climbing fiber-activated [Ca(2+)](i) increases in rat cerebellar Purkinje neurons.

The effect of several neuromodulators (carbachol (CCh), serotonin (5-HT), noradrenaline (NE), and dopamine (DA)) on the climbing fiber (CF)-induced [Ca(2+)](i) increase in the dendrites of cerebellar Purkinje cells was examined in slices from the rat cerebellum. Purkinje cells were filled with the Ca(2+) indicator bis-fura-2 with patch electrodes on the soma. [Ca(2+)](i) changes were measured from regions of interest in the dendrites with a high speed camera. Changes evoked by one or three responses were measured in control conditions and with neuromodulators added to the bath. None of these four classic modulators caused a significant change in the CF-induced [Ca(2+)](i) amplitude. Buspirone, a partial 5-HT(1A) agonist and a weak DA receptor antagonist caused a small (10-15%) reduction in the response.     

GluR6-containing KA receptor mediates the activation of p38 MAP kinase in rat hippocampal CA1 region during brain ischemia injury

Our previous study showed that kainate (KA) receptor subunit GluR6 played an important role in ischemia-induced MLK3 and JNK activation and neuronal degeneration through the GluR6-PSD95-MLK3 signaling module. However, whether the KA receptors subunit GluR6 is involved in the activation of p38 MAP kinase during the transient brain ischemia/reperfusion (I/R) in the rat hippocampal CA1 subfield is still unknown. In this present study, we first evaluated the time-course of phospho-p38 MAP kinase at various time-points after 15 min of ischemia and then observed the effects of antagonist of KA receptor subunit GluR6, GluR6 antisence oligodeoxynucleotides on the phosphorylation of p38 MAP kinase induced by I/R. Results showed that inhibiting KA receptor GluR6 or suppressing the expression of KA receptor GluR6 could down-regulate the elevation of phospho-p38 MAP kinase induced by I/R. These drugs also reduced the phosphorylation of MLK3, MKK3/MKK6, MKK4, and MAPKAPK2. Additionally, our results indicated administration of three drugs, including p38 MAP kinase inhibitor before brain ischemia significantly decreased the number of TUNEL-positive cells detected at 3 days of reperfusion and increased the number of the surviving CA1 pyramidal cells at 5 days of reperfusion after 15 min of ischemia. Taken together, we suggest that GluR6-contained KA receptors can mediate p38 MAP kinase activation through a kinase cascade, including MLK3, MKK3/MKK6, and MKK4 and then induce increased phosphorylation of MAPKAPK-2 during ischemia injury and ultimately result in neuronal cell death in the rat hippocampal CA1 region.     

The action of valproate on spontaneous epileptiform activity in the absence of synaptic transmission and on evoked changes in [Ca2+]o and [K+]o in the hippocampal slice

The effects of valproate (VPA) on neuronal excitability and on changes in extracellular potassium ([K⁺]₀) and calcium ([Ca²⁺]₀) were investigated with ion selective-reference electrode pairs in area CA₁ of rat hippocampal slices. Field potential responses to single ortho- and antidromic stimuli were unaltered by VPA (1–5 mM). The afferent volley evoked in the Schaffer-commissural fibers was also unaffected. In contrast, VPA (1 mM) depressed frequency potentiation and paired pulse facilitation markedly. Decreases in [Ca²⁺]₀ induced either by repetitive stimulation or by application of the excitatory amino acids N-methyl-d-aspartate and quisqualate were reduced, and the latter results suggest that VPA interferes with postsynaptic Ca²⁺ entry. When synaptic transmission was blocked by lowering [Ca²⁺]₀ (0.2 mM) and elevating [Mg²⁺]₀ (7 mM), prolonged afterdischarges elicited by antidromic stimulation were blocked by VPA. VPA also suppressed the spontaneous epileptiform activity seen when [Ca²⁺]₀ was lowered to 0.2 mM, without elevating [Mg²⁺]₀. The amplitudes of the rises in [K⁺]₀ induced by repetitive orthodromic stimulation were only slightly depressed and those elicited by antidromic stimulation were generally unaltered by VPA, as were laminar profiles of stimulus-evoked [K⁺]₀ signals. These results indicate that VPA has membrane actions in addition to known effects on excitatory and inhibitory transmitter pools.     

Vascular endothelial growth factor acutely reduces calcium influx via inhibition of the Ca2+ channels in rat hippocampal neurons

Vascular endothelial growth factor (VEGF) protects neurons against ischemic injury. An overload of intracellular calcium ions (Ca(2+)) caused by the excessive release of glutamate is widely considered to be one of the molecular mechanisms of ischemic neuronal death. In the present study, we investigated whether VEGF could modulate the activity of Ca(2+) channels on the neuronal membrane. We used the Fluo-3 image method assisted by confocal laser scan microscopy to detect any Ca(2+) influx in primary cultured hippocampal neurons. Whole-cell patch-clamp techniques were used to record the activity of the high-voltage-activated (HVA) Ca(2+) currents in the CA1 pyramidal neurons of hippocampal slices that were freshly prepared from neonatal brains of rats. The results obtained from the Fluo-3 image experiments showed that VEGF pretreatment of cultured neurons at a final concentration of 50, 100, or 200 ng/ml acutely and dose dependently attenuated the Ca(2+) influx induced by application of KCl (60 mM) or glutamate (50 microM). This effect was blocked by SU1498, an antagonist of Flk-1 VEGF receptor. The influx of Ca(2+) returned to basal levels after removal of VEGF. Furthermore, electrophysiological recording data showed that VEGF could acutely reduce the amplitudes of the HVA Ca(2+) currents in a dose- and voltage-dependent manner. The HVA Ca(2+) currents also returned to the levels of the control after removal of VEGF from the system. Taken together, the results obtained from the present study demonstrated that VEGF specifically reduced the influx of Ca(2+) via the inhibitory activity of the HVA Ca(2+) channels in hippocampal neurons.     

PKC and PKA, but not PKG mediate LPS-induced CGRP release and [Ca(2+)](i) elevation in DRG neurons of neonatal rats.

Calcitonin gene-related peptide (CGRP), is produced in dorsal root ganglia (DRG) neurons and released from primary afferent neurons to mediate hemodynamic effects and neurogenic inflammation. In this work, we determined whether lipopolysaccharide (LPS), an inflammatory stimulator, could trigger CGRP release from cultured DRG neurons and if so, which cellular signaling pathway was involved in this response. Cytoplasmic concentration of calcium ([Ca(2+)](i)) plays a key role in neurotransmitter release, therefore [Ca(2+)](i) was also determined in cultured DRG cells using fluo-3/AM. The results showed that LPS (0.1-10 microg/ml) evoked CGRP release in a time- and concentration-dependent manner from DRG neurons. LPS also increased [Ca(2+)](i) in a concentration-dependent manner. The protein kinase C (PKC) inhibitors, calphostin C 0.5 microM or RO-31-8220 0.1 microM, and the cAMP-dependent protein kinase (PKA) specific inhibitor RP-CAMPS 30 microM or nonspecific inhibitor H8 1 microM inhibited 1 microg/ml LPS-evoked CGRP release and [Ca(2+)](i) increase from DRG neurons. The cGMP-dependent protein kinase (PKG) inhibitor Rp-8-pCPT-cGMPS 30 microM did not block the LPS response. These data suggest that LPS may stimulate CGRP release and [Ca(2+)](i) elevation through PKC and PKA, but not PKG signaling pathway in DRG neurons of neonatal rats.     

Characterization of 5-HT2A receptor desensitization and the effect of cycloheximide on it in C6 cells

Summary. Effect of prolonged pretreatment with serotonin (5-HT) on 5-HT_2A receptor desensitization was examined by the measurement of intracellular calcium ([Ca²⁺]_i) mobilization in C6 cells. 5-HT-induced desensitization of [Ca²⁺]_i mobilization was in a time and dose dependent manner and reached a plateau after 3 hr. After 1 and 3 hr 5-HT pretreatment, 5-HT concentration in the medium little changed. 5-HT pretreatment with cycloheximide, a protein synthesis inhibitor, produced an enhancement of the desensitization for 3 and 6 hr pretreatment. However, 5-HT pretreatment for 3 and 6 hr caused no marked change in the 5-HT_2A receptor mRNA level or Gαq/11 protein in this study, suggesting that 5-HT may decrease 5-HT-induced [Ca²⁺]_i mobilization independent of 5-HT_2A receptor mRNA or G-proteins. Endothelin-1-induced [Ca²⁺]_i mobilization did not alter after 5-HT and/or cycloheximide pretreatment. These results showed that activation of the 5-HT_2A receptor induced homologous desensitization and pretreatment with 5-HT and/or cycloheximide did not change the efficacy of the second messenger pathway from Gq to a [Ca²⁺]_i rise. Received June 6, 2000; accepted October 17, 2000     

Catalase prevents elevation of [Ca(2+)](i) induced by alcohol in cultured canine cerebral vascular smooth muscle cells: Possible relationship to alcohol-induced stroke and brain pathology

Several studies have suggested that alcohol-induced brain injury is associated with generation of reactive oxygen species (ROS). The recent findings, that antioxidants (Vitamin E and pyrrolidine dithiocarbamate (PDTC)) prevent intracellular Ca(2+) ([Ca(2+)](i)) overload in cerebral vascular smooth muscle cells, induced by alcohol, demonstrate indirectly that ROS formation is related to cerebral vascular injury. The present experiments were designed to test the hypothesis that catalase, an hydrogen peroxide (H(2)O(2)) scavenging enzyme, can prevent or ameliorate alcohol-induced elevation of [Ca(2+)](i). Preincubation of cultured canine cerebral vascular smooth muscle cells with catalase (20-1000 units/ml) didn't produce any apparent changes from controls in resting levels of [Ca(2+)](i) after 1-3 days. Exposure of the cerebral vascular cells to culture media containing 10-100mM ethanol resulted in significant rises in [Ca(2+)](i) (p<0.01). Although exposure of these cells to a low concentration of catalase (20 units/ml) failed to prevent the increased level of [Ca(2+)](i) induced by ethanol, concomitant addition of higher concentrations of catalase (100-1000 units/ml) and ethanol (10-100mM) inhibited or ameliorated the rises of [Ca(2+)](i) induced by ethanol either at 24h or at 3 days, in a concentration-dependent manner. Catalase, in the range of 100-200 units/ml, inhibited approximately 50% of the [Ca(2+)](i) increases caused by ethanol in the first 24h. Catalase at a concentration of 1000 units/ml inhibited completely excessive [Ca(2+)](i) accumulation. The present results when viewed in light of other recently published data suggest that H(2)O(2) generation may be one of the earliest events triggered by alcohol in alcohol-induced brain-vascular damage, neurobehavioral actions and stroke.     

Activation of 5-HT2 receptors enhances the release of acetylcholine in the prefrontal cortex and hippocampus of the rat

The role of 5-HT2 receptors in the regulation of acetylcholine (ACh) release was examined in the medial prefrontal cortex and dorsal hippocampus using in vivo microdialysis. The 5-HT(2A/2C) agonist +/-1-(2,5-dimethoxy-4-iodophenyl) -2- aminopropane hydrochloride (DOI) (1 and 2 mg/kg, i.p.) significantly increased the extracellular concentration of ACh in both brain regions, and this response was attenuated in rats treated with the 5-HT(2A/2B/2C) antagonist LY-53,857 (3 mg/kg, i.p.). Treatment with LY-53,857 alone did not significantly alter ACh release in either brain region The 5-HT(2C) agonist 6-chloro-2-(1-piperazinyl)-pyrazine) (MK-212) (5 mg/kg, i.p.) significantly enhanced the release of ACh in both the prefrontal cortex and hippocampus, whereas the 5-HT2 agonist mescaline (10 mg/kg, i.p.) produced a 2-fold increase in ACh release only in the prefrontal cortex. Intracortical, but not intrahippocampal, infusion of DOI (100 microM) significantly enhanced the release of ACh, and intracortical infusion of LY-53,857 (100 microM) significantly attenuated this response. These results suggest that the release of ACh in the prefrontal cortex and hippocampus is influenced by 5-HT2 receptor mechanisms. The increase in release of ACh induced by DOI in the prefrontal cortex, but not in the hippocampus, appears to be due to 5-HT2 receptor mechanisms localized within this brain region. Furthermore, it appears that the prefrontal cortex is more sensitive than the dorsal hippocampus to the stimulatory effect of 5-HT2 agonists on ACh release.     

3,4-Methylenedioxymethamphetamine (MDMA) enhances the release of acetylcholine by 5-HT4 and D1 receptor mechanisms in the rat prefrontal cortex

3,4-Methylenedioxymethamphetamine (MDMA), an amphetamine analog, has been shown recently to increase the release of acetylcholine (ACh) in the prefrontal cortex (PFC). The present study further characterizes the stimulatory effect of MDMA on cortical ACh release and examines the role of serotonin (5-HT) and dopamine (DA) receptors in this response. The extracellular concentration of ACh was increased dose-dependently and similarly by the (+) and (-) enantiomers of MDMA (5 and 20 mg/kg, i.p.). The systemic administration of the 5-HT(4) antagonist SDZ 205,557 (1 mg/kg, i.p.), but not the 5-HT(2A/2B/2C) antagonist LY-53,857 (3 mg/kg, i.p.), significantly decreased cortical ACh release induced by MDMA. The MDMA-induced increase in the extracellular concentration of ACh also was significantly blunted in rats treated with the D(1) receptor antagonist SCH 23390 (0.5 mg/kg, i.p.). The extent to which the coadministration of SDZ 205,557 and SCH 23390 suppressed the MDMA-induced release of ACh in the PFC was no greater than that produced by either antagonist alone. These results suggest that the 5-HT(4) and D(1) receptor subtypes contribute to the mechanism by which MDMA increases ACh release in the PFC.     

AMPA/kainate receptor-mediated up-regulation of GABAA receptor delta subunit mRNA expression in cultured rat cerebellar granule cells is dependent on NMDA receptor activation

We have studied the effects of AMPA/kainate receptor agonists on GABA(A) receptor subunit mRNA expression in vitro in cultured rat cerebellar granule cells (CGCs). Kainate (KA) (100 microM) and high K(+) (25 mM) dramatically up-regulated delta subunit mRNA expression to 500-700% of that in control cells grown in low K(+) (5 mM). KA or high K(+) had no effect on the expression of the other major GABA(A) receptor subunits alpha1, alpha6, beta2, beta3 or gamma2. Up-regulation of delta mRNA was also detected with the AMPA receptor-selective agonist CPW-399 and to a lesser extent with the KA receptor-selective agonist ATPA. AMPA/kainate receptor-selective antagonist DNQX completely inhibited KA-, CPW-399- and ATPA-induced delta mRNA up-regulation indicating that the effects were mediated via AMPA and KA receptor activation. NMDA receptor-selective antagonist MK-801 inhibited 76% of the KA- and 57% of the CPW-399-induced delta up-regulation suggesting that KA and CPW-399 treatments may induce glutamate release resulting in NMDA receptor activation, and subsequently to delta mRNA up-regulation. In CGCs, delta subunit is a component of extrasynaptic alpha6betadelta receptors that mediate tonic inhibition. Up-regulation of delta during prolonged glutamate receptor activation or cell membrane depolarization may be a mechanism to increase tonic inhibition to counteract excessive excitation.     

Ryanodine receptor-mediated [Ca(2+)](i) release in glomus cells is independent of natural stimuli and does not participate in the chemosensory responses of the rat carotid body

Advanced glycation endproducts (AGEs), protein-bound oxidation products of sugars, have been shown to be involved in the pathophysiological processes of Alzheimer’s disease (AD). AGEs induce the expression of various pro-inflammatory cytokines and the inducible nitric oxide synthase (iNOS) leading to a state of oxidative stress. AGE modification and resulting crosslinking of protein deposits such as amyloid plaques may contribute to the oxidative stress occurring in AD. The aim of this study was to immunohistochemically compare the localization of AGEs and β-amyloid (Aβ) with iNOS in the temporal cortex (Area 22) of normal and AD brains. In aged normal individuals as well as early stage AD brains (i.e. no pathological findings in isocortical areas), a few astrocytes showed co-localization of AGE and iNOS in the upper neuronal layers, compared with no astrocytes detected in young controls. In late AD brains, there was a much denser accumulation of astrocytes co-localized with AGE and iNOS in the deeper and particularly upper neuronal layers. Also, numerous neurons with diffuse AGE but not iNOS reactivity and some AGE and iNOS-positive microglia were demonstrated, compared with only a few AGE-reactive neurons and no microglia in controls. Finally, astrocytes co-localized with AGE and iNOS as well as AGE and were found surrounding mature but not diffuse amyloid plaques in the AD brain. Our results show that AGE-positive astrocytes and microglia in the AD brain express iNOS and support the evidence of an AGE-induced oxidative stress occurring in the vicinity of the characteristic lesions of AD. Hence activation of microglia and astrocytes by AGEs with subsequent oxidative stress and cytokine release may be an important progression factor in AD.