Differential modulation of GABAA and NMDA receptors by α7-nicotinic receptor desensitization in cultured rat hippocampal neurons.

AIM: To explore the modulatory effect of desensitized α7-containing nicotinic receptors (α7-nAChRs) on excitatory and inhibitory amino acid receptors in cultured hippocampal neurons and to identify the mechanism underlying this effect. METHODS: Whole-cell patch-clamp recordings were performed on cultured rat hippocampal neurons to measure α7-nAChR currents and to determine the role of desensitized α7-nAChRs on brain amino acid receptor activity. RESULTS: Pulse and perfusion applications of the α7-nAChR agonist choline were applied to induce different types of α7-nAChR desensitization in cultured hippocampal neurons. After a brief choline pulse, α7-nAChR was desensitized as a result of receptor activation, which reduced the response of the A type γ-aminobutyric acid (GABAA) receptor to its agonist, muscimol, and enhanced the response of the NMDA receptor to its agonist NMDA. By contrast, the responses of glycine or AMPA receptors to their agonists, glycine or AMPA, respectively, were not affected. Pretreatment with the α7-nAChR antagonist methyllycaconitine (MLA, 10 nmol/L) blocked the choline-induced negative modulation of the GABAA receptor and the positive modulation of the NMDA receptor. The regulation of the GABAA and NMDA receptors was confirmed using another type of α7-nAChR desensitization, which was produced by a low concentration of choline perfusion. The negative modulation of the GABAA receptor was characterized by choline-duration dependency and intracellular calcium dependency, but the positive modulation of the NMDA receptor was not associated with cytoplasmic calcium. CONCLUSION: Brain GABAA and NMDA receptors are modulated negatively and positively, respectively, by desensitized α7-nAChR as a result of choline pretreatment in cultured hippocampal neurons.

[Change of memory function and decrease of nitric oxide level of whole brain in the transgenic mice expressing human tau 40 with P301L mutation].

OBJECTIVE: To study the mechanism of learning and memory dysfuction in the transgenic mouse expressing human tau 40 isoform with P301L mutation (F10). METHODS: The human tau protein expression and phosphor-tau protein levels were detected with Western blot method. The neurofibrillary tangles were observed with Bielshowsky silver stain. The behavior changes of learning and memory were observed by open field test and passive avoidance test. Acetyleholine level, activities of acetycholinesterase and choline acetyltransferase of whole brain was detected by colorimetry method. The nitric oxide level of whole brain was detected by nitrate enzyme reduction method. RESULTS: Exogenous human tau gene was expressed and an elevation of phosphor-tau protein level in 7 and 3-month transgenic mice's hippocampus andcerebrocortex was observed. The neurofibrillary tangles were observed in cerebrocortex of 7-month transgenic mice; the 7-month transgenic mice also presented an evident reduction of learning and memory ability and nitric oxide level of the whole brain, but not changes in acetylcholine level, acetycholinesterase activity, choline acetyltransferase activity and expression in whole brain. CONCLUSION: Tau transgenic mice (F10) can still inherit their parents' biologiccal characters, and develop learning and memory dysfunction awnodh san obvious decrease in nitric oxide level of whole brain in the 7-month old mice, suggesting a decrease of nitric oxide level of whole brain would be involved in the mechanism of learning and memory dysfunction in these transgenic mice.

[Generation of Tau/App/PS1 triple-transgenic mouse model and the study of its biological characteristics].

OBJECTIVE: To establish the triple-transgenic mouse model and study their biological characteristics by molecular biology, behavior and pathology. METHODS: Hybrid the Tau and amyloid precursor protein (APP)/presenilins (PS1) transgenic mouse, the genotype of offspring mice were identified by PCR. Transcribed target genes were detected by RT-PCR. The protein expression of exogenous genes was detected by Western-blot. The pathological change of neurofibrillary tangles and senile plaque were observed by Bielschowsky silver staining and ABC immunohistochemical method. The changes time of learning and memory were observed by Morris water maze. RESULTS: APP, PS1 and Tau genes were transcript in Tau/APP/PS1 mice. In 6 to 8 months old Tau/APP/PS1 mice, the neurofibrillary tangles and senile plaque could be found in cortex and hippocampus. In 6 months old Tau/APP/PS1 mice, the learning and memory abilities were worse. CONCLUSION: With the behavior change and pathological changes in Tau and beta-amyloid protein (AP), the Tau/APP/PS1 triple-transgenic mice can be used as a further study animal model of AD's pathogenesis and the target of drug treatment.

Similar potency of catechin and its enantiomers in alleviating 1-methyl-4-phenylpyridinium ion cytotoxicity in SH-SY5Y cells.

OBJECTIVES: Previously, the flavonoid (±)-catechin was shown to exert potent neuroprotective action in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease model. The purpose of this study was to investigate whether the different enantiomers of catechin ((+)-catechin, (-)-catechin and (±)-catechin, a 50:50 mixture of (+)-catechin and (-)-catechin) could protect SH-SY5Y cells against 1-methyl-4-phenylpyridinium ion (MPP(+) ) toxicity by decreasing the generation of oxygen free radicals. The inhibitive effect of (±)-catechin on JNK/c-Jun activation was investigated. METHODS: The effects of (+)-catechin, (-)-catechin or (±)-catechin in protecting against MPP(+) toxicity were evaluated and compared in SH-SY5Y cells by testing the release of lactate dehydrogenase. The generation of reactive oxygen species (ROS) was measured by immunochemistry and the phosphorylation level of JNK/c-Jun was determined by Western blotting. KEY FINDINGS: In SH-SY5Y cells, (+)-catechin, (-)-catechin or (±)-catechin reduced apoptosis induced by MPP(+) and decreased ROS generation caused by MPP(+) . Different enantiomers of catechin showed protective effects at similar potency. Moreover (±)-catechin decreased JNK/c-Jun phosphorylation which was increased by MPP(+). CONCLUSIONS: Catechin and its two enantiomers could protect SH-SY5Y cells against MPP(+) cytotoxicity at a similar potency. Antioxidative stress and inhibition of the JNK/c-Jun signalling pathway might have been involved in the neuroprotective mechanisms of catechin against MPP(+) cytotoxicity in SH-SY5Y cells.

Down-regulation of peroxisome proliferator-activated receptor γ coactivator-1α expression in fatty acid-induced pancreatic beta-cell apoptosis involves nuclear factor-κB pathway.

BACKGROUND: Pancreatic beta-cell apoptosis induced by lipotoxicity, to a large extent, contributes to the progression of type 2 diabetes. To investigate the mechanism of free fatty acid induced apoptosis, we aimed to study the effects of palmitic acid (PA) on the apoptosis and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression in ßTC3 cells as well as the possible role of nuclear factor-κB (NF-κB) in this process. METHODS: Hoechst 33258 was used to detect ßTC3 cell apoptosis, which was induced by PA stimulation for 12 hours. PGC-1α expression was analyzed by reverse transcription polymerase chain reaction, IκB kinase ß (IKKß), IκBα, NF-κB-inducing kinase (NIK) and Rel-B expressions were analyzed by Western blotting. MG132 was employed to block the endogenous IκBα degradation before PA administration, and then its effect on PA-inducing cell apoptosis and PGC-1α mRNA expression was analyzed. RESULTS: Significant increased cell apoptosis was found at the concentration of 0.5 mmol/L and 1.0 mmol/L PA administration. PA (0.5 mmol/L) could extensively reduced the expression of PGC-1α mRNA. After exposing ßTC3 cells to 0.5 mmol/L PA for different time periods (0, 4, 6, 8, 10 and 12 hours), IKKß protein expression increased while IκBα, NIK and Rel-B protein expression declined in a time-dependent manner. Pretreatment with MG132 to inhibit the degradation of IκBα, partially prevented the down-regulation of PGC-1α mRNA expression after 12-hour PA treatment in accordance with the decrease of PA induced apoptosis. CONCLUSIONS: NF-κB canonical pathway was activated in PA-mediated ßTC3 cell apoptosis, whereas non-canonical pathway was inhibited. Reduced PGC-1α expression by PA in ßTC3 cells could involve the activation of canonical NF-κB pathway, so as to deteriorate the PA induced apoptosis.

Effects of carvedilol on delayed rectifier and transient inactivating potassium currents in rat hippocampal CA1 neurons.

1. The aims of the present study were to investigate the mechanism(s) underlying the protective effect of carvedilol against neural damage. 2. The transient inactivating potassium current (I(A) ) and the delayed rectifier potassium current (I(K) ) in rat hippocampal CA1 pyramidal neurons were recorded using whole-cell patch-clamp techniques. 3. Carvedilol (0.1-3 µmol/L) significantly inhibited I(K) with an IC(50) of 1.3 µmol/L and the inhibition was voltage independent. Over the same concentration range, carvedilol had no effect on the amplitude of I(A). At 1 µmol/L, carvedilol did not significantly change the steady state activation curves of I(A) and I(K), but did negatively shift their steady state inactivation curves. Recovery from inactivation was slowed for both I(A) and I(K). The inhibitory effect of carvedilol on I(K) was not affected by the adrenoceptor agonists phenylephrine and prazosin or the adrenoceptor antagonist isoproterenol, but propranolol was able to shift the dose-response curve of carvedilol for I(K) to the right. 4. Because I(K) is the main pathway for loss of intracellular potassium from depolarized neurons, selective obstruction of I(K) by carvedilol could be useful for neuroprotection.

[Isolation and identification of Suavissimoside R1 from roots of Rubus parvifollus used for protecting dopaminergic neurons against MPP+ toxicity].

OBJECTIVE: Suavissimoside R1 was isolated and identified as an active ingredient from Roots of Rubus parvifollus L, which exhibited protective effect on dopaminergic neurons against MPP+ toxicity. METHODS: The protective effects of crude extracts were investigated after mice were treated with 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). According to the protective effects of crude extracts, suavissimoside R1, one kind of triterpenoid saponin, was separated. It was investigated that whether Suavissimoside R1 can protect DA neurons from toxicity induced by MPP+ in rat mesencephalic cultures. RESULTS: Suavissimoside R1 was isolated from Roots of Rubus parvifollus L. Moreover, Suavissimoside R1, in dose of 100 micromol/L, alleviated the death of DA neurons induced by MPP+ obviously. CONCLUSION: These results suggest that suavissimoside R1 possesses potent neuroprotective activity and can be developed to be a potential anti-Parkinson's disease drug worthy for further study.

Noradrenaline release by activation of κ-bungarotoxin-sensitive nicotinic acetylcholine receptors participates in long-term potentiation-like response induced by nicotine.

Nicotine enhances the function of learning and memory, but the underlying mechanism still remains unclear. Hippocampal long-term potentiation (LTP) is assumed to be a cellular mechanism of learning and memory. Our previous experiments showed that with the single pulses evoking 80% of the maximal population spike (PS) amplitude, nicotine (10 µmol/L) induced LTP-like response in the hippocampal CA1 region. In the present study, the nicotinic acetylcholine receptor (nAChR) subtypes and relevant neurotransmitter releases involved in LTP-like response induced by nicotine were investigated by extracellularly recording the PS in the pyramidal cell layer in the hippocampal CA1 region in vitro. LTP-like response induced by nicotine was blocked by mecamylamine (1 µmol/L) or κ-bungarotoxin (0.1 µmol/L), but not by dihydro-ß-erythtroidine (DHßE, 10 µmol/L). Moreover, it was inhibited by propranolol (10 µmol/L), but not by phentolamine (10 µmol/L) or atropine (10 µmol/L). The results suggest that noradrenaline release secondary to the activation of κ-bungarotoxin-sensitive nAChRs participates in LTP-like response induced by nicotine in the hippocampal CA1 region.

Increase of beta1-adrenergic receptor gene expression induced by nicotine in hippocampal slice of rat.

AIM: To investigate the effect of nicotine on beta1-adrenergic receptor (beta1-AR) in the hippocampal slice of rat. METHODS: Hippocampal slices (400 microm thick) were incubated in artificial cerebrospinal fluid (ACSF) previously saturated with 95 % O2 and 5 % CO2 at 28 degree for 120 min, and then incubated with nicotine 10 micromol/L for 30, 60, 90, and 120 min. mRNA of the beta1-adrenergic receptor was examined with semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), and the protein level was measured by Western blot and RIA. RESULTS: The mRNA gene expression and the protein level of beta1-adrenergic receptor in hippocampal slices were increased after nicotine treatment. The peak of protein occurred later but higher than that of mRNA level. CONCLUSION: Both expression of beta1- adrenergic receptor gene transcription and post-transcriptional protein level in rat hippocampus were altered by nicotine.

Different synaptic mechanisms of long-term potentiation induced by nicotine and tetanic stimulation in hippocampal CA1 region of rats.

AIM: To investigate whether long-term potentiation (LTP) induced by nicotine and tetanic stimulation in the hippocampal CA1 region shares different mechanisms. METHODS: Extracellular population spikes of the pyramidal cell layer in the hippocampal CA1 region were recorded in vitro. RESULTS: LTP induced by the tetanic stimulation could be facilitated by nicotine 10 micromol/L, meanwhile, the tetanic stimulation did the same effect on LTP induced by nicotine 10 micromol/L. MK-801 10 micromol/L or NG-nitro-L-arginine methyl ester (L-NAME) 20 micromol/L blocked LTP induced by tetanic stimulation, but did not inhibit LTP induced by nicotine. CONCLUSION: LTP induced by nicotine shares different synaptic mechanisms with LTP induced by tetanic stimulation.