Cerebral Cortical Slices Research Articles

6β-Acetoxy nortropane regulated processing of amyloid precursor protein in CHOm 1 cells and rat brain

The effects of the muscarinic receptor agonist 6β-acetoxy nortropane on amyloid precursor protein (APP) processing were studied in both transfected Chinese hamster ovary cells stably expressing muscarinic M 1 receptors (denoted as CHOm 1 cell line) and in cerebral cortical and hippocampal slices. Exposure of CHOm 1 cells to 6β-acetoxy nortropane for 1 h significantly increased the secretion of secretory amyloid precursor protein (derived from α-secretase cleavage) in a concentration-dependent manner. In the same system, 6β-acetoxy nortropane reduced the β-amyloid peptide production. Similar results were obtained in hippocampal and cerebral cortical slices, with 6β-acetoxy nortropane administration resulting in an increase in secretory amyloid precursor protein and a decrease in β-amyloid peptide release. The increase of secretory amyloid precursor protein secretion was abolished by preincubation with selective muscarinic M 1 receptor antagonist pirenzepine, but not by preincubation with selective muscarinic M 2 receptor antagonist methoctramine, suggesting that 6β-acetoxy nortropane promotes secretory amyloid precursor protein release in the brain via muscarinic M 1 receptor activation. These results suggest that 6β-acetoxy nortropane could exert a beneficial effect on the progress of Alzheimer's disease by promoting amyloid precursor protein processing through α-secretase.

Carbachol-induced network oscillations in the intact cerebral cortex of the newborn rat.

In mature cortex, activation of the cholinergic system induces oscillatory network activity and facilitates synaptic plasticity. We used an in vitro preparation of the intact cerebral cortex and cortical slices of the neonatal rat to study carbachol (CCh, >or=30 micro M)-induced network oscillations during the early postnatal period. Multi-site extracellular recordings revealed CCh-induced transient beta oscillations with an average duration of 4.6 +/- 0.2 s, amplitude of 123 +/- 7.4 microV and frequency of 17.7 +/- 0.5 Hz. These oscillations propagated uniformly at 0.5-1.5 mm/s over the cortex and were reversibly blocked by tetrodotoxin (TTX) and atropine, indicating that they depended on action potentials and activation of muscarinic receptors. The activity was not blocked by bicuculline methiodide or gabazine, but was reversibly abolished by kynurenic acid, indicating that activation of glutamate receptors, but not GABA-A receptors, was required. CNQX caused a significant decrease in the power of the Fourier frequency spectrum of the CCh-induced oscillations and CPP or MK-801 completely blocked the activity, indicating a contribution of AMPA/kainate receptors and an essential role of NMDA receptors. Oscillations were synchronized between sites separated horizontally by approximately 1 mm and for delays of 2-8 ms. Synchronized activity between neighboring recording sites was very stable over repeated applications of CCh. Whole-cell recordings from morphologically identified pyramidal neurons in the intact cortex revealed a close temporal correlation between CCh-induced membrane oscillations and local field potential recordings. In contrast, CCh-induced oscillations recorded in coronal neocortical slices were smaller in amplitude and frequency, suggesting that a widespread network of intracortical axonal connections is required for their generation.

Excitotoxic mechanism of cell swelling in rat cerebral cortical slices treated acutely with ammonia

Swelling of CNS cells due to endogenous ammonia is a major cause of cerebral oedema in hyperammonaemic encephalopathies. In the present study, incubation in the presence of 5mM ammonium acetate (“ammonia”) decreased steady-state distribution of [14C]inulin within incubated rat cerebrocortical minislices, indicating cell swelling. NMDA receptor antagonists, MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cycloheptene-5,10-imine maleate,10μM) and dl-AP-5 (dl-2-amino-5-phosphonovaleric acid, 250μM), a nitric oxide synthase inhibitor, l-nitroarginine (l-NNA, 500μM), and an antioxidant, taurine (Tau, 10mM), markedly attenuated the cell volume-increasing effect of ammonia. The effect of Tau (10mM) was abolished by the GABA(A) receptor antagonist bicuculline (100μM), but was unaffected by the Tau transport inhibitor guanidynoethyl-sulfonate (GES, 500μM). Ammonia increased the slice content of Gln, an amino acid whose excess accumulation has been implicated in hyperammonemic oedema. However, treatments that reduced the cell volume did not affect Gln content. These results indicate that ammonia-induced cell swelling is in a large degree mediated by overactivation of NMDA receptors and the ensuing generation of NO and free radicals.

Oxidation of 4-hydroxynonenal in rat brain slices

4-Hydroxy-2-nonenal (HNE) is implicated as a neurotoxic ‘second messenger’ of oxidative damage in Alzheimer's disease (AD). The mechanism of HNE toxicity is due to alkylation of cellular nucleophilic groups. The C1 aldehyde is key to the alkylation ability of HNE. Oxidation of the C1 aldehyde to 4-hydroxy-2-nonenoic acid is catalyzed by aldehyde dehydrogenases. In this work, we tested the hypothesis that HNE oxidation to HNEAcid occurs in rat cerebral cortex utilizing rat cerebral cortical slices exposed extracellularly to HNE. HNEAcid formation occurs in a dose dependent manner with approximately 18–25% of the HNE consumed accounted for by HNEAcid formation. HNEAcid was found exclusively in the incubation media, suggesting that HNEAcid is exported from the cells of the slice. These data demonstrate that HNE detoxification through the oxidation pathway occur in the cerebral cortex.

Antagonism of VIP-stimulated cyclic AMP formation in chick brain.

Of eight peptides tested (0.01-5 microM), only two, that is, pituitary adenylate cyclase-activating polypeptide (PACAP27) and chicken vasoactive intestinal peptide (cVIP), potently stimulated cyclic AMP (cAMP) production in cerebral cortical slices of the chick. Mammalian VIP (mVIP) showed some activity only at the highest dose tested, whereas truncated forms of PACAP or VIP, that is, PACAP6-27, cVIP6-28, and mVIP6-28, or hybrid compounds, that is, neurotensin6-11-cVIP7-28 (NT-cVIP) and neurotensin6-11-mVIP7-28 (NT-mVIP), were inactive. Thirty-minute preincubation of chick cortical slices with 5 microM PACAP6-27, NT-cVIP, or NT-mVIP competitively antagonized the cAMP effects of cVIP (0.03-1 microM), with the truncated form of PACAP being the best antagonist. Preincubation of slices with 5 microM mVIP6-28 also produced a significant inhibition of the cVIP (0.1-1 microM)-induced increase in cAMP production; however its action was independent of the concentration of cVIP. In contrast to mVIP6-28, cVIP6-28 showed no antagonistic activity against the full-length peptide. In parallel experiments, 30-min pretreatment of cortical slices with 5 microM PACAP6-27 significantly antagonized the PACAP38-evoked increase in cAMP formation, whereas mVIP6-28 or the NT-mVIP hybrid was ineffective. It has been concluded that in the chick brain, PACAP and cVIP stimulate cAMP biosynthesis via PAC1 and VPAC-type receptors, respectively, and PACAP6-27 seems to be the most potent, yet PACAP/VIP receptor-nonselective antagonist. Unlike truncated PACAP, the NT-VIP hybrid peptides tested may represent VPACtype receptor-selective blocking activity.

Effects of glutamate on dehydroascorbate uptake and its enhanced vulnerability to the peroxidation in cerebral cortical slices.

Pro-oxidant properties of ascorbate have been studied with uses of brain tissues and neuronal cells. Here we address potential mechanism of ascorbate coupling with glutamate to generate oxidative stress, and the role which oxidized ascorbate (dehydroascorbate) transport plays in oxidative neuronal injury. Ascorbate in neurones can be depleted by adding glutamate in culture medium since endogenous ascorbate can be exchanged with glutamate, which enhances ascorbate/dehydroascorbate transport by depleting ascorbate in the neurons with the glutamate- heteroexchange. However, ascorbate is known readily being oxidized to dehydroascorbate in the medium. Glutamate enhanced the dehydroascorbate uptake by cells via a glucose transporter (GLUT) from extracellular region, and cytosolic dehydroascorbate enhanced lipid peroxide production and reduced glutathione (GSH) concentrations. Iso-ascorbate, the epimer of ascorbate was ineffective in generating the oxidative stress. These observations support the current concept that the high rates of dehydroascorbate transport via a GLUT after the release of ascorbate by glutamate leads to peroxidation, the role of glutamate on ascorbate/dehydroascorbate recycling being critical to induce neuronal death via an oxidative stress in the brain injury.

The nicotinic allosteric potentiating ligand galantamine facilitates synaptic transmission in the mammalian central nervous system.

In this study, the patch-clamp technique was used to determine the effects of galantamine, a cholinesterase inhibitor and a nicotinic allosteric potentiating ligand (APL) used for treatment of Alzheimer's disease, on synaptic transmission in brain slices. In rat hippocampal and human cerebral cortical slices, 1 microM galantamine, acting as a nicotinic APL, increased gamma-aminobutyric acid (GABA) release triggered by 10 microM acetylcholine (ACh). Likewise, 1 microM galantamine, acting as an APL on presynaptically located nicotinic receptors (nAChRs) that are tonically active, potentiated glutamatergic or GABA-ergic transmission between Schaffer collaterals and CA1 neurons in rat hippocampal slices. The cholinesterase inhibitors rivastigmine, donepezil, and metrifonate, which are devoid of nicotinic APL action, did not affect synaptic transmission. Exogenous application of ACh indicated that high and low levels of nAChR activation in the Schaffer collaterals inhibit and facilitate, respectively, glutamate release onto CA1 neurons. The finding then that the nAChR antagonists methyllycaconitine and dihydro-beta-erythroidine facilitated glutamatergic transmission between Schaffer collaterals and CA1 neurons indicated that in a single hippocampal slice, the inhibitory action of strongly, tonically activated nAChRs in some glutamatergic fibers prevails over the facilitatory action of weakly, tonically activated nAChRs in other glutamatergic fibers synapsing onto a given neuron. Galantamine is known to sensitize nAChRs to activation by low, but not high agonist concentrations. Therefore, at 1 microM, galantamine is likely to increase facilitation of synaptic transmission by weakly, tonically activated nAChRs just enough to override inhibition by strongly, tonically activated nAChRs. In conclusion, the nicotinic APL action can be an important determinant of the therapeutic effectiveness of galantamine.

Stimulatory effects of pituitary adenylate cyclase-activating polypeptide on inositol phosphates accumulation in avian cerebral cortex and hypothalamus

This study has demonstrated that the short and long form of the pituitary adenylate cyclase-activating polypeptide (PACAP), i.e. PACAP 27 and PACAP 38, moderately but significantly, and in a concentration (0.5–5 μM)-dependent manner, stimulated inositol phosphates (IPs) accumulation in myo-[ 3H]inositol-prelabeled cerebral cortical and hypothalamal slices of chick and duck, and in slices of rat cerebral cortex; both peptides had no effect on IPs formation in rat hypothalamus. Vasoactive intestinal peptide (VIP; 0.5–5 μM) weakly enhanced IPs accumulation in chick hypothalamus, had no significant action in chick cerebral cortex (in fact there was a tendency to attenuate the IPs response in this tissue), and slightly, but significantly, inhibited the IPs accumulation in rat cerebral cortex. VIP showed no activity in rat hypothalamus. It is concluded that the stimulatory action of PACAP on phosphoinositide metabolism in avian cerebral cortex, similar to rat cerebral cortex, is mediated via phospholipase C-linked PAC 1 type receptors. In chick hypothalamus, however, there may be a component of VPAC type receptors stimulating IPs formation.

Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide-stimulated cyclic AMP synthesis in rat cerebral cortical slices: interaction with noradrenaline, adrenaline, and forskolin.

Pituitary adenylate cyclase-activating polypeptide (PACAP; 0.001-1 microM) and vasoactive intestinal peptide (VIP; 0.01-1 microM) produced a concentration-dependent stimulation of cyclic AMP (cAMP) formation in rat cerebral cortical slices prelabeled with [3H]adenine. The effects of PACAP38 and PACAP27 were similar, and more efficacious (at 0.1 and 1 microM) than those of VIP. Adrenaline and noradrenaline (each at 100 microM) also stimulated cAMP formation, with the latter compound being more effective. Combination of PACAP38, PACAP27 (each at 0.1 microM) and VIP (1 microM) with adrenaline or noradrenaline resulted in most cases in additive effects, with some supraadditive (PACAP27 plus adrenaline) or subadditive (PACAP38 or VIP plus noradrenaline) fluctuations. In contrast, combination of each of the three peptides with 3 microM forskolin resulted in synergistic effects. These results indicate that in rat cerebral cortex there is no synergism between PACAP or VIP with noradrenaline or adrenaline; however, based on the forskolin data, it seems likely that synergistic effects may take place with VIP or PACAP and other cAMP-stimulating neuroregulators.

Characterization of antidepressant-like effects of p-synephrine stereoisomers.

We previously reported that p-synephrine has antidepressant-like activity in the murine models of forced swimming and tail suspension. In the present study, we characterized antidepressant-like effects of p-synephrine stereoisomers in both in vivo and in vitro systems. In the tail suspension test, S-(+)-p-synephrine (3 mg/kg, p.o.) reduced the duration of immobility, while R-(-)-p-synephrine (0.3-3 mg/kg, p.o.) had no effect. S-(+)-p-synephrine (0.3, 1 and 3 mg/kg, p.o.) and R-(-)-p-synephrine (1 mg/ kg and 3 mg/kg, p.o.) significantly reversed the reserpine (2.5 mg/kg, i.p.)-induced hypothermia. S-(+)-p-synephrine was more effective than R-(-)-p-synephrine in inhibition of both [3H]noradrenaline uptake in rat cerebral cortical slices (maximal inhibition 85.7 +/- 7.8% vs. 59.8 +/- 4.3%; EC50 5.8 +/- 0.7 microM vs. 13.5 +/- 1.2 microM) and [3H]nisoxetine binding (Ki 4.5 +/- 0.5 microM vs. 8.2 +/- 0.7 microM). In contrast, R-(-)-p-synephrine was more effective than S-(+)-p-synephrine in stimulation of [3H]noradrenaline release from rat cerebral cortical slices (maximal stimulation 23.9 +/- 1.8% vs. 20.1 +/- 1.7%; EC50 8.2 +/- 0.6 microM vs. EC50 12.3 +/- 0.9 microM). The stimulatory effect of R-(-)-p-synephrine on [3H]noradrenaline release was inhibited by nisoxetine (100 nM), but tetrodotoxin (1 microM) and elimination of extracellular calcium had no effect. It is suggested that S-(+)-p-synephrine has more effective antidepressant-like activity than R-(-)-p-synephrine.

Activation of CB1 cannabinoid receptors in rat hippocampal slices inhibits potassium-evoked cholecystokinin release, a possible mechanism contributing to the spatial memory defects produced by cannabinoids

Cannabinoid use is known to disrupt learning and memory in a number of species. cholecystokinin (CCK) release and CCK receptors have been implicated in spatial memory processes in rodents. Rat hippocampal CCK interneurons express cannabinoid 1 receptors (CB1). The CB1 agonist R(+)WIN 55,212–2 (WIN+), at 1 and 10 μmol, strongly inhibited potassium-evoked CCK release from rat hippocampal slices, while the inactive isomer S(−)WIN,55,212–3 (WIN−) had no effect. CCK release from cerebral cortical slices was not altered by WIN+.

Effects of metabotropic glutamate receptor agonists and antagonists on D-aspartate release from mouse cerebral cortical and striatal slices.

The cytosolic release of L-glutamate has been held to be responsible for the increase in extracellular glutamate to toxic levels in the brain. The mechanism and regulation of this release was now studied in cerebral cortical and striatal slices with D-[3H]aspartate, a non-metabolized analogue of L-glutamate and a poor substrate for vesicular uptake. L-Glutamate and D-aspartate strongly stimulated the release in a concentration-dependent manner. Of the ionotropic glutamate receptor agonists, only kainate enhanced the basal release in the striatum. Of the metabotropic glutamate receptor ligands, the group I agonist (S)-3,5-dihydroxyphenylglycine (S-DHPG) failed to affect the basal release but inhibited the D-aspartate-evoked release in the striatum. The group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) had no effect on the basal release in either preparation but enhanced the L-glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum, not however in the cerebral cortex. The group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) and the group II antagonist (2S)-2-ethylglutamate (EGLU) were without effect on the basal, D-aspartate- and L-glutamate-evoked releases of D-[3H]aspartate in either preparation. The group III agonist L-serine-O-phosphate (L-SOP) failed to affect the basal release but reduced the D-aspartate-evoked release in the striatum. The group III antagonist (RS)alpha-methylserine-O-phosphate (MSOP) failed to affect the basal release but increased the glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum. Both L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) and (2S,1'S,2'R)-2-carboxycyclopropyl)glycine (L-CCG-III), transportable inhibitors of the high-affinity glutamate uptake, enhanced the basal release, more strongly in the striatum than in the cerebral cortex. L-CCG-III also increased the L-glutamate-evoked release in the striatum. Nontransportable dihydrokainate enhanced the basal release much less and failed to affect the glutamate-evoked release. The results indicate that the release of glutamate from cytosolic pools is carrier-mediated via homoexchange. This process is regulated in the striatum by metabotropic group I and group III receptors in a manner different from the regulation of the vesicular release of glutamate from presynaptic terminals.

Involvement of noradrenaline transporters in S-nitrosocysteine-stimulated noradrenaline release from rat brain slices: existence of functional Na +-independent transporter activity

Noradrenaline (NA) can be released by both exocytosis and by the membrane transporter responsible for transmitter uptake. Previously, we reported that S-nitrosocysteine (SNC), an S-nitrosothiol, stimulated [ 3H]NA release from the rat hippocampus. In this study, we investigated the involvement of the NA transport system in SNC-stimulated NA release from rat brain (cerebral cortex and hippocampus) slices. [ 3H]NA release by SNC in normal Na + (148 mM)-containing buffer from both slices was slightly, but significantly, inhibited by 1 μM desipramine, an NA transporter inhibitor. [ 3H]NA release in low Na + (under 14 mM)-containing buffer was inhibited by over 50% by desipramine. [ 3H]NA release by tyramine from both slices in normal and low Na + buffer was almost completely inhibited by desipramine. [ 3H]NA uptake into cerebral cortical slices was observed in low Na + buffer at 20–30% of normal Na + buffer levels. [ 3H]NA uptake in both normal and low Na + buffers was inhibited by desipramine and by SNC. Although [ 3H]NA uptake in normal Na + buffer was almost completely inhibited by 500 μM ouabain, the uptake in low Na + buffer was resistant to ouabain. These findings suggest the existence of a functional Na +-independent NA transport system and that SNC stimulates NA release at least partially via this system in brain slices.

Neuronal nicotinic receptors in synaptic functions in humans and rats: physiological and clinical relevance

The present report describes the participation of nicotinic receptors (nAChRs) in controlling the excitability of local neuronal circuitries in the rat hippocampus and in the human cerebral cortex. The patch-clamp technique was used to record responses triggered by the non-selective agonist ACh and the α7-nAChR-selective agonist choline in interneurons of human cerebral cortical and rat hippocampal slices. Evidence is provided that functional α7- and α4β2-like nAChRs are present on somatodendritic and/or preterminal/terminal regions of interneurons in the CA1 field of the rat hippocampus and in the human cerebral cortex and that activation of the different nAChR subtypes present in the preterminal/terminal areas of the interneurons triggers the tetrodotoxin-sensitive release of GABA. Modulation by nAChRs of GABAergic transmission, which can result either in inhibition or disinhibition of pyramidal neurons, depends both on the receptor subtype present in the interneurons and on the agonist acting upon these receptors. Not only do α7 nAChRs desensitize faster than α4β2 nAChRs, but also α7 nAChR desensitization induced by ACh lasts longer than that induced by choline. These mechanisms, which appear to be retained across species, might explain the involvement of nAChRs in cognitive functions and in such neurological disorders as Alzheimer’s disease and schizophrenia.

Effect of acute hepatic encephalopathy on 3H-dopamine release from rat cerebral cortex and striatum in vitro: role of Ca2+

Hepatic encephalopathy (HE) is characterized by motor symptoms associated with disturbed functions of the dopaminergic systems, but the underlying mechanisms are not clear. A previous study from our laboratories revealed that HE, induced in rats by repeated treatment with thioacetamide, enhanced the 50 mM potassium (KCI)-stimulated release of newly loaded 3H-dopamine in both striatal and frontal cerebral cortical slices in the presence of Ca2+. In the present study we compared the effects of HE on dopamine release in striatal and frontal cerebral cortical slices and synaptosomes in the presence and absence of Ca2+. HE enhanced the KCl-stimulated 3H-dopamine release from striatal and frontal cortical synaptosomes in the presence of Ca2+ to the same extent as in slices prepared from the respective brain regions. In the absence of Ca2+ a slight reduction in dopamine release was observed in frontal cortical synaptosomes from HE rats when compared to control rats, while no effect of HE on the release was discernible in frontal cortical and striatal slices and striatal synaptosomes. We conclude that in both brain regions studied HE stimulates dopamine exocytosis triggered by Ca2+ influx without affecting the release mediated by means of plasma membrane transporters or exocytosis involving intraterminal Ca2+.

Extracellular concentrations of taurine, glutamate, and aspartate in the cerebral cortex of rats at the asymptomatic stage of thioacetamide-induced hepatic failure: modulation by ketamine anesthesia.

Subclinical hepatic encephalopathy (SHE) was produced in rats by two intraperitoneal injections of TAA at 24 h intervals and the animals were examined 21 days later. Concentrations of the neuroactive amino acids taurine (Tau), glutamate (Glu) and aspartate (Asp), were measured in the cerebral cortical microdialysates of thioacetamide (TAA)-treated and untreated control rats. During microdialysis some animals were awake while others were anesthetized with ketamine plus xylazine. There was no difference in the water content of cerebral cortical slices isolated from control and SHE rats, indicating a recovery from cerebral cortical edema that accompanies the acute, clinical phase of hepatic encephalopathy in this model. When microdialysis was carried out in awake rats, dialysate concentrations of all the three amino acids were 30% to 50% higher in SHE rats than in control rats. Ketamine anesthesia caused a 2.2% increase of water content of cerebral cortical slices and increased Asp, Glu, and Tau concentration in microdialysates of control rats. In SHE rats, ketamine anesthesia produced a similar degree of cerebral edema, however, it did not alter Asp and Glu concentrations in the microdialysates. These data may reflect on one hand a neuropathological process of excitotoxic neuronal damage related to increased Glu and Asp, on the other hand neuroprotection from neuronal swelling indicated by Tau redistribution in the cerebral cortex. The reduction of the effects of SHE on Glu and Asp content in ketamine-anesthesized rats is likely to be due to interference of ketamine with the NMDA receptor-mediated component of the SHE-evoked excitatory neurotransmitter efflux and/or reuptake of the two amino acids. By contrast, the SHE-related increase of Tau content was not affected by ketamine anesthesia, indicating that the mechanism(s) underlying SHE-evoked accumulation of Tau must be different from the mechanism causing release of excitatory amino acids. The results with ketamine advocate caution when using this anesthetic in studies employing the cerebral microdialysis technique for measurement of extracellular amino acids.

Stimulation of Noradrenaline Release by T-588, a Cognitive Enhancer, in PC12 Cells

Previously, we reported that (R)-(-)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N- diethylamino)ethoxy] ethanol hydrochloride (T-588), a novel putative cognitive enhancer, stimulated noradrenaline (NA) release from rat cerebral cortical slices. In this study, we investigated the effects of T-588 compared to other secretagogues on NA release from PC12 cells. Addition of as little as 10 microM T-588 stimulated [3H]NA release in a dose-dependent and an extracellular Ca(2+)-independent manner from PC12 cells. Ten micromolar ionomycin-, 300 microM adenosine-5'-O-(gamma-thiotriphosphate)- and 10 microM forskolin-induced extracellular Ca(2+)-dependent [3H]-NA release was further enhanced by 30 microM T-588. Cytosolic synaptophysin and 25-kDa synaptosome-associated protein immunoreactivity was increased by addition of T-588 in a dose-dependent manner. Interestingly, increases in synaptic vesicle-related proteins triggered by T-588 had a 4-min lag time and were completely dependent on extracellular CaCl2. These findings suggest that T-588 stimulates NA release from PC12 cells in a Ca(2+)-independent manner. T-588 also induced the translocation of synaptic vesicles in a Ca(2+)-dependent manner.

Platelet-activating factor modulates brain sphingomyelin metabolism.

In the present study the modulatory action of platelet-activating factor (PAF) on sphingolipid metabolism in cerebral cortical slices was studied. PAF did not alter the basal levels of either sphingomyelin (SM) or ceramide. However, the SMase-elicited reciprocal alterations in SM and ceramide levels were partially prevented by the PAF treatment. The PAF effect was dose-dependent, with 10-8 m being the lowest effective concentration, and receptor-mediated as it was abolished by WEB 2086, a PAF receptor antagonist. Neither N-oleoylethanolamine (OE, ceramidase inhibitor) or d,l-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP, an inhibitor of glucosylceramide synthase and the formation of 1-O-acyl ceramides) prevented the action of PAF. Therefore, the effect of PAF was unlikely to be dependent upon transformation of ceramides into glycosphingolipids, 1-O-acyl ceramides or sphingosine. Experiments with different labeled compounds ([14C]serine, [14C]arachidonate and phosphatidyl [N-methyl-3H]choline) were also performed to test whether PAF could affect the resynthesis of SM. Data obtained agree with the idea that selective pools of both choline and ethanolamine phospholipids were used as precursors for the resynthesis of SM elicited by SMase treatment. PAF itself did not evoke any variation in the lipids analyzed but always prevented the SMase-evoked alterations. Together the data suggest the interesting possibility that PAF increases the overall turnover of SM. In summary, the present data demonstrate that PAF is able to regulate the cellular ceramide levels in brain by accelerating the SM cycle.

Galanin inhibits acetylcholine release from rat cerebral cortex via a pertussis toxin-sensitive Giprotein

Galanin has been implicated in various physiological functions including memory, feeding and pain perception. Using rat cerebral cortical slices and synaptosome preparations incubated with [3H]choline in Kreb’s-Ringer solution, galanin was shown to inhibit both spontaneous and K+-stimulated [3H]ACh release in a concentration-related manner [EC50= 35 nM]. The galanin-mediated inhibition on spontaneous and K+-stimulated [3H]ACh release was respectively regulated by pertussis toxin-sensitive Gαi3and Gαi1. These suggest that galanin is a negative modulator of cortical cholinergic function and most probably acting on presynaptic cholinergic terminals. Although galantide blocked the galanin-mediated inhibitory effect on [3H]ACh release, it mimicked galanin in blocking K+-stimulated [3H]ACh release, indicating that galantide may have a more complicated pharmacology than being a galanin receptor antagonist. In addition, we demonstrate that galanin and β-amyloid peptide1–42synergistically attenuated K+-evoked [3H]ACh release from synaptosomes prepared from rat cerebral cortex. Since galanin is increased in Alzheimer’s disease brain, our results suggest that galanin may be involved in cholinergic dysfunctions that occur in Alzheimer’s disease.

Muscarinic receptor- and phorbol ester-stimulated phosphorylation of protein kinase C substrates in adult and neonatal cortical slices

The neuron-specific protein B-50 (GAP-43) is a major presynaptic substrate for protein kinase C (PKC). Phosphorylation of B-50 by PKC at serine-41 is functionally related to signal transduction in association with process outgrowth and neurotransmitter release. Thus, it is important to characterize the factors which modulate phosphorylation of B-50 by PKC. Phosphoinositide (PI)-coupled muscarinic acetylcholine receptor (mAchR) activation would be expected to increase PKC activity through production of the second messenger, diacylglycerol. To test the hypothesis that activation of mAchR also increases phosphorylation of B-50, protein phosphorylation has been examined in cerebral cortical slices in response to the cholinergic agonist, carbachol (Cch) in comparison to the phorbol ester, 4β-phorbol 12, 13-dibutyrate (PDB), a known activator of PKC. At short times of incubation with 1 mM Cch, a concentration which maximally activates PI metabolism, increased phosphorylation of a group of synaptosomal proteins, including B-50 and myristoylated, alanine-rich C kinase substrate (MARCKS), was observed. This increase was approximately half of that obtained in response to 1 μM PDB. Differing patterns of protein phosphorylation were observed in neonatal and adult slices: neonatal samples contained more MARCKS and a PKC substrate with a M r of 46 kDa. Phosphorylation of B-50 and MARCKS was sensitive to Cch in both cases. Immunoblotting demonstrated less m1 acetylcholine receptor (the predominant mAchR subtype coupled to PI metabolism in the cortex) in neonatal, as compared to adult, synaptosomal fractions. These results are consistent with a coupling between mAchR-stimulated PI metabolism and PKC-mediated protein phosphorylation that is developmentally regulated.