神经生长因子对小鼠突触体内Ca^2＋水平的调节作用

观察了多次海马内微注射ＮＧＦ对小鼠突触体内游离钙水平的影响，并在离体情况下观察ＮＧＦ对ＥＧＴＡ和ＣａＣｌ２分别造成突触体内低钙和高钙状态的调节作用。结果如下：（１）在体实验表明，一定剂量的ＮＧＦ可显著降低老年小鼠海马突触体内游离钙水平（Ｐ＜００５）；（２）离体实验表明，当突触体游离钙水平降低时，适当剂量的ＮＧＦ具有升高游离钙水平的作用；而突触体内游离钙水平升高时，则ＮＧＦ有降低游离钙水平的作用。提示ＮＧＦ对游离钙水平的双向调节作用可能是ＮＧＦ改善老年性记忆衰退的作用机制。     

腺苷对突触体^45Ca摄取的抑制作用

用同位素标记~(15)Ca法测定大鼠突触体内游离钙的浓度,探讨了腺苷对高钾、N-甲基-D-天冬氨酸(NMDA)谷氨酸(Glu)等刺激所致的Ca~(2 )内流增加的影响。结果发现腺苷在10nmol/L～0.1μmol/L范围内对高钾、NMDA、Glu等刺激所致的Ca~(2 )内流有抑制作用,并呈剂量依赖关系,最大抑制率分别为41.68±7.68％、31.32±6.17％、37.52±2.29％。这可能是腺苷对缺血性脑损伤保护作用的机理。     

Subsynaptosomal distribution of calcium during aging and 3,4-diaminopyridine treatment

Since previous studies showed that calcium uptake by synaptosomes from rodents declines with aging [30], the subsynaptosomal distribution of calcium was determined with the disruption method of Scott et al. [37]. Calcium uptake by the mitochondrial (digitonin-resistant) and non-mitochondrial (digitonin-labile) compartments, as well as total uptake, were determined at 2, 5 and 10 min. After a 10 min incubation under resting conditions (5 mM-KCl), total calcium uptake decreased at 10 months (−14.6%) and 30 months (−33.0%) of age; mitochondrial calcium uptake increased by 10 months (+11.2%) but declined by 30 months (−17.5%); the nonmitochondrial calcium compartment declined at 10 (−34.7%) and 30 (−43.4%) months when compared to the 3 month old control. With potassium depolarization (31 mM-KCl), total calcium uptake declined from 100% (3 months) to 73.8% (10 months) or 53.0% (30 months); mitochondrial calcium uptake declined from 100% (3 months) to 85.6% (10 months) or 68.4% (30 months); non-mitochondrial calcium uptake decreased at 10 (−34.3%) and 30 (−57.7%) months of age when compared to 3 months (100%). The deficits in calcium homeostasis are not due to changes in synaptosomal volumes or to diminished membrane potentials, as assessed by tetraphenylphosphonium ion accumulation. 3,4-Diaminopyridine partially reversed the alterations in total, mitochondrial and non-mitochondrial calcium uptake by synaptosomes from aged mice.     

Permanent brain ischemia induces marked increments in hsp72 expression and local protein synthesis in synapses of the ischemic hemisphere

Transient focal ischemia induced in rat brain by occlusion of the middle cerebral artery (MCAo) elicits a generalized induction of the 72 kDa heat-shock protein (hsp72) heralding functional recovery. As this effect implies activation of protein synthesis, and local systems of protein synthesis are present in brain synapses, and may be analyzed in preparations of brain synaptosomes, we evaluated hsp72 expression and protein synthesis in synaptosomal fractions of spontaneously hypertensive rats (SHRs) subjected to permanent MCAo. SHRs were randomly divided in ischemics and sham controls, anaesthesia controls and passive controls. Focal ischemia was induced under chloral hydrate anaesthesia by unilateral permanent MCAo. Protein synthesis was determined by [³⁵S]methionine incorporation into synaptosomal proteins from ischemic and contralateral cortex/striatum, and from cerebellum. Hsp72 expression was measured in the same fractions by immunoblotting. Our data demonstrate that under these conditions synaptic hsp72 markedly increases in the ischemic hemisphere 1 and 2 days after MCAo, progressively declining in the following 2 days, while no significant change occurs in control rats. In addition, in the ischemic hemisphere the rate of synaptic protein synthesis increases more than two-fold between 1 and 4 days after MCAo, without showing signs of an impending decline. The present data provide the first demonstration that synaptic protein synthesis is massively involved in brain plastic events elicited by permanent focal ischemia.     

Interaction between 5-HT uptake inhibition and activation of 5-HT autoreceptors by exogenous agonists in rat cerebral cortex slices and synaptosomes

Summary Rat cerebral cortex slices or synaptosomes were labelled with ³H-5-hydroxytryptamine (³H-5-HT) and subsequently superfused. They were depolarized by electrical stimulation (slices) or with high K⁺ (slices and synaptosomes). Continuous electrical stimulation (2 Hz, 24 mA, 2 ms) and continuous or discontinuous K⁺ depolarization (15–25 mM) were used. 1. Continuous electrical stimulation or continuous K⁺-depolarization of slices evoked a steady overflow of tritium that slowly decayed with time. 2. Exposure to increasing concentrations of 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole succinate (RU 24969) (0.001–0.1 M) during continuous electrical stimulation produced a concentration-dependent decrease in tritium overflow. Citalopram (1 M) counteracted the effect of RU 24969. 3. RU 24969 inhibited the evoked ³H-overflow and citalopram reduced the effect of RU 24969 also during continuous depolarization of slices with 20 mM K⁺. Similar results were obtained by using 5-methoxytryptamine or LSD. 4. In slices 1 M citalopram increased significantly the tritium overflow evoked by electrical stimulation or by 20 mM K⁺-depolarization. 5. Increasing the K⁺ concentration from 20 mM to 25 mM mimicked the effects of 1 M citalopram both on the RU 24969 activity and on the evoked tritium overflow. 6. RU 24969 (0.001–0.1 M) decreased in a concentration-dependent way the release of tritium from cortical synaptosomes depolarized with K⁺ (15–20 mM). The presence of 1 M citalopram did not modify significantly the effect of the agonist. Citalopram was ineffective also when the serotonin uptake carrier in superfused synaptosomes was activated by tryptamine. In conclusion, in slices of rat cerebral cortex, the action of exogenous 5-HT autoreceptor agonists is inhibited by 5-HT uptake blockers independently of the depolarizing agent (electrical stimulation or high-K⁺) used to elicit ³H-5-HT release. Increasing K⁺-concentration, which probably increases serotonin in the biophase, mimics the presence of the reuptake inhibitor. These data together with the finding that, in superfused synaptosomes, 5-HT uptake inhibition did not affect the potency of autoreceptor agonists, favours the idea that, in cerebral cortex slices, inhibitors of 5-HT reuptake prevent activation of autoreceptors by exogenous agonists by increasing the concentration of 5-HT in the autoreceptor biophase. Send offprint requests to M. Raiteri at the above address     

Choline inhibits acetylcholine release via presynaptic muscarine receptors

Summary The intraperitoneal administration of gabaculine, aminooxyacetic acid (s.c.), -acetylenic GABA, -vinyl GABA, ethanolamine-O-sulphate, sodium valproate and GABA caused significant increases in the GABA content of both the whole brain and brain nerve endings (synaptosomes) in mice, a linear relationship being observed between the two parameters. When the relative effects on GABA concentrations were compared with the potency of the different drugs to elevate the thresholds for electroconvulsions and clonic pentetrazole seizures, only the increase in the pentetrazole seizure threshold was significantly correlated with the elevation of brain and synaptosomal GABA content. The present results indicate that the threshold for pentetrazole seizures is an appropriate test-system to study the relationship between alterations of overall GABA levels in the brain and nerve terminals and anticonvulsant activity of GABA-elevating agents.     

Cholesterol and phospholipid composition of cerebellar fractions enriched in unmyelinated axons and synaptosomes

Summary Three distinct phospholipid patterns occurred in the mitochondrial subcellular fractions: a) myelin, b) axonal-synaptosomal and c) mitochondrial. Axonal and synaptosomal fractions were found to be very similar in phospholipid and cholesterol composition. In all submitochondrial fractions cholesterol, phosphatidyl serine and sphingomyelin occurred in the relatively constant molar ratio of 1.00.180.11. The phospholipid patterns reported in this paper correspond well to others published for synaptosomes and synaptic membranes from mammalian sources as well as axonal membranes from invertebrate sources. However, morphological analysis has indicated that axonal as well as synaptosomal fractions contained significant amounts of microsomes and abundant membranes of undetermined origin which must also be considered when attempting morphological-biochemical correlations. Attention was given to the possible functional and chemical similarity of axonal and synaptosomal membranes other than that portion presumably specialized for chemical transmission, and to the difficulty of establishing the lipid pattern of the true synaptic membrane in presently available preparations.     

The effects produced by prostaglandin D2 on serotonin turnover and release and tryptophan uptake

In earlier studies, it was proposed that there was a serotonergic involvement in the ability of prostaglandin D2 (PGD2) to potentiate pentobarbital sleeping time. The actions of PGD2 on neuronal turnover and release of serotonin and uptake of tryptophan were examined in mice. The effect of PGD2 administration on serum tryptophan levels was also investigated. PGD2 (1 and 4 mg/kg) increased the concentrations in whole brain of endogenous tryptophan (TRYP) and of 3H-tryptophan (3H-TRYP) following an intravenous (IV) injection of 3H-tryptophan. Formation of 3H-5-hydroxyindoleacetic acid (3H-5HIAA) was doubled after PGD2 administration (1 and 4 mg/kg). Whole brain concentrations of endogenous serotonin (5HT) and 3H-serotonin (3H-5HT) were unchanged after the administration of the prostaglandin. PGD2 (10(-4) to 10(-10)M) in vitro had no effect on spontaneous or K+-evoked release of 3H-5HT from whole brain synaptosomes. Uptake of 3H-tryptophan in synaptosomes was neither stimulated nor depressed by (10(-4) to 10(-12)M) PGD2. There was also no change in serum tryptophan levels after administration of this prostaglandin. Thus, PGD2 administration does affect the serotonergic system but no direct neurochemical correlate of sedation can be shown.     

Evaluation of the effects of aluminium,ethanol and their combination on rat brain synaptosomal integral proteins in vitro and after 90-day oral exposure

The effects of aluminium lactate (Al-lactate) on the rat cerebral synaptosome integral proteins adenosinetriphosphatase (ATPase) and acetylcholinesterase (AChE) were studied in vitro and in vivo. Coexposure with ethanol (EtOH) was studied in both situations. Isolation of synaptosomes was carried out using isoosmotic Percoll gradients. In in vitro experiments, the synaptosomes were exposed to different concentrations of Al-lactate in the incubation mixture. Al-lactate caused decreases in total ATPase and AChE activities concentration dependently. The decrease in ATP activity started at 0.2 mM concentration, and concentration for the 50% decrease of the enzyme activity (EC₅₀ ) was 1.1 mM. The decrease in AChE activity started at 5–10 mM concentration, and the EC₅₀ value was 15.8 mM. Coexposure with ethanol (2 mM) increased the EC₅₀ values similarly in both cases. After 90-day oral exposure of rats to Al-lactate (91.8 mg/kg/day), the serum aluminium level was 0.9–1.3 M/l. Coexposure with EtOH (3.0 g/kg/day) did not significantly increase the blood Al (0.7–2.2 M/l). Aluminium exposure caused a decrease in the blood EtOH concentration (0.6 mM/l) compared with blood EtOH (12.3 mM/l) in the rats exposed to ethanol only. In the rats studied 2 weeks after the Al exposure, the activities of ATPase and AChE were significantly lower than in the rats studied immediately after the exposure. Correspondingly, a significant decrease in AChE activity was found in Al- and EtOH-exposed rats, but in the control rats there were no differences between the study groups. Immediately after the 90-day dosing, the exposed rats did not differ significantly from the control rats. Based on the in vitro results, the neural membrane integral proteins ATPase and AChE may be considered as targets for the effects of aluminium and ethanol. Ninety-day in vivo exposure of rats to aluminium caused decrease in ATPase and AChE activities, detectable 2 weeks after the exposure.     

Functional interaction between neuropeptide Y receptors and modulation of calcium channels in the rat hippocampus

We investigated the functional interaction between neuropeptide Y (NPY) receptors using nerve terminals and cultured rat hippocampal neurons, and we evaluated the involvement of voltage-gated Ca(2+) channels (VGCCs) in NPY receptors-induced inhibition of Ca(2+) influx and glutamate release. The KCl-evoked release of glutamate from hippocampal synaptosomes was inhibited by 1 microM NPY and this effect was insensitive to either BIBP3226 (Y1 receptor antagonist) or L-152,804 (Y5 receptor antagonist), but was sensitive to BIIE0246 (Y2 receptor antagonist). We could also pharmacologically dissect the NPY receptors activity by using Y1, Y2 and Y5 receptor agonists ([Leu(31),Pro(34)]NPY, NPY13-36, NPY (19-23)-(Gly(1),Ser(3),Gln(4),Thr(6),Ala(31),Aib(32),Gln(34))-pancreatic polypeptide (PP), respectively), and in all the cases we observed that these agonists could inhibited the KCl-induced release of glutamate. However, the selective and specific co-activation of both Y1 and Y2 or Y2 and Y5 receptors resulted in non-additive inhibition, and this effect was prevented in the presence of the Y2 antagonist, but was insensitive to the Y1 or Y5 receptor antagonist. Moreover, as we previously showed for Y1 receptors, we also observed that the activation of Y5 receptors inhibited the glutamate release in the dentate gyrus and CA3 subregion, without significant effect in the CA1 subregion of the hippocampus. The same qualitative results were obtained when we investigated the role of NPY Y1 and Y2 receptors in modulating the changes in [Ca(2+)](i) due to KCl depolarisation in cultured hippocampal neurons. The inhibitory effect of nitrendipine (L-type VGCC blocker) or omega-conotoxin GVIA (omega-CgTx; N-type VGCC blocker) was not potentiated by the simultaneous activation of Y1 or Y2 receptors. Moreover, the exocytotic release of glutamate was inhibited by omega-agatoxin IVA (omega-Aga; P-/Q-type VGCC blocker), and this VGCC blocker did not potentiate Y1, Y2 or Y5 receptor-mediated inhibition of glutamate release. Also, the effect of ionomycin in inducing the exocytotic release of glutamate from hippocampal synaptosomes was insensitive to the activation of NPY receptors. In the present paper, we identified a role for NPY Y1, Y2 and Y5 receptors in modulating the exocytotic release of glutamate and the [Ca(2+)](i) changes in the rat hippocampus. In conditions of co-activation, there appears to exist a physiological cross-talk between Y1 and Y2 and also between Y2 and Y5 receptors, in which Y2 receptors play a predominant role. Moreover, we also show that Y1 and Y2 receptors exert their inhibitory action by directly modulating L-, N-, and P-/Q-type VGCCs, whereas the inhibition of glutamate release mediated by the Y5 receptors seems to involve P-/Q-type VGCCs.