Neurotransmitter and Depolarization-Stimulated Accumulation of Inositol 1,3,4,5-Tetrakisphosphate Mass in Rat Cerebral Cortex Slices

[32P]Inositol 1,3,4,5-tetrakisphosphate ([32P]Ins(1,3,4,5)P4) binds to a rat cerebellar membrane site with high affinity (KD = 2.8 +/- 0.6 nM) and low capacity (Bmax = 176 +/- 34 fmol/mg of protein). Evidence for a low-affinity site (KD = 164 +/- 48 nM) was also apparent. The high-affinity site displayed marked specificity for the Ins(1,3,4,5)P4 isomer, compared with several other inositol polyphosphates, and has been used as the basis of a radioreceptor assay for Ins(1,3,4,5)P4 in extracts of rat cerebral cortex slices. The resting Ins(1,3,4,5)P4 concentration (1.89 +/- 0.11 pmol/mg of protein) in the slices was rapidly and dramatically increased by carbachol and quisqualate. K+ depolarization of cerebral cortex slices also stimulated Ins(1,3,4,5)P4 accumulation, with at least 50% of the response being sensitive to atropine, a result indicating that muscarinic receptor stimulation by released acetylcholine contributes significantly to the K+ effect.     

Lithium Selectively Inhibits Muscarinic Receptor-Stimulated Inositol Tetrakisphosphate Accumulation in Mouse Cerebral Cortex Slices

The in vitro effects of Li on agonist- and depolarization-stimulated accumulation of inositol phosphates were determined in mouse cerebral cortex slices. Of the agents examined, only the cholinergic agonist carbachol produced a significant accumulation of inositol tetrakisphosphate (InsP4) in the absence of Li. Lithium at 5 mM enhanced the accumulation of inositol monophosphate (InsP1) and inositol bisphosphate (InsP2) due to all the stimuli used and potentiated inositol trisphosphate (InsP3) accumulation due to histamine and noradrenaline, although at lower Li concentrations, carbachol-stimulated InsP3 accumulation was reduced. Li also enhanced InsP4 accumulation in the presence of noradrenaline, histamine, and elevated KCl level but, in marked contrast, reduced carbachol-stimulated InsP4 accumulation with an IC50 of 100 microM. There was a significant time delay between the initiation of carbachol stimulation and the beginning of the InsP4 inhibition due to Li. The phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate did not mimic the effects of Li. The results suggest that muscarinic receptor-mediated InsP4 production might be one of the targets for the therapeutic action of Li.     

Differential Effects of Lithium on Muscarinic Receptor Stimulation of Inositol Phosphates in Rat Cerebral Cortex Slices

The accumulation of labelled inositol mono-, bis-, and trisphosphate in rat cerebral cortex slices was examined following preincubation with [3H]inositol. The muscarinic receptor agonist carbachol produced a rapid and sustained increased accumulation of each labelled inositol phosphate both in the presence and absence of 5 mM lithium. Lithium potentiated carbachol-stimulated accumulation of inositol monophosphate (EC50 0.5 mM) and inositol bisphosphate (EC50 4 mM) in a concentration-dependent manner. However, exposure to lithium in the presence of the muscarinic agonist produced a concentration- and time-dependent inhibition of inositol trisphosphate accumulation that was not related to receptor desensitisation. Although the present data do suggest that polyphosphoinositides are substrates for agonist-stimulated phospholipase C in brain, these results may not be entirely consistent with the production of inositol mono- and bisphosphate through inositol trisphosphate dephosphorylation. Furthermore, these data suggest site(s) additional to inositol monophosphatase that are affected by lithium.     

Lithium Enhances Accumulation of [3H]Inositol Radioactivity and Mass of Second Messenger Inositol 1,4,5-Trisphosphate in Monkey Cerebral Cortex Slices

We previously reported that lithium, in the presence of acetylcholine, increased accumulations of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in brain cortex slices from the guinea pig, rabbit, rat, and mouse. In the mouse and rat, the Li(+)-induced increases required supplementation of the medium with inositol. This probably relates to the following facts: (a) Brain cortices of the mouse and rat contain in vivo concentrations of inositol half of that of the guinea pig. (b) Incubated rat brain cortex slices are depleted of inositol by 80%. (c) The slices require 10 mM inositol supplementation to restore in vivo concentrations. We now show that in monkey brain cortex slices, therapeutic concentrations of Li+ increase accumulation of inositol 1,4,5-trisphosphate. The inositol 1,3,4,5-tetrakisphosphate level is not increased. Neither inositol nor an agonist is required. The same effects are seen whether inositol 1,4,5-trisphosphate is quantified by the [3H]inositol prelabeling technique or by mass assay, although mass includes a pool of inositol 1,4,5-trisphosphate that is metabolically inactive. Thus, in a therapeutically relevant model for humans, Li+ increases inositol 1,4,5-trisphosphate levels in brain cortex slices, as was previously seen in lower mammals at non-rate-limiting concentrations of inositol.     

Depolarization and Agonist-Stimulated Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulation in Rat Cerebral Cortex

Muscarinic receptor stimulation or depolarization with elevated extracellular K+ induced rapid and sustained increases in mass accumulations of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] in cerebral cortex slices. Synergistic but transient responses of both inositol polyphosphate second messengers were observed when slices were stimulated with carbachol under depolarizing conditions; this synergy was observed as an increase in the maximal responsiveness, with no significant change in EC50 values for carbachol. Omission of buffer Ca2+ ([Ca2+]e 10-20 microM) reduced basal Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentrations; the relative stimulatory effects of muscarinic receptor stimulation were maintained, but the effects of depolarization were markedly attenuated under these conditions. A component of the response to depolarization appeared to be indirectly mediated by the release of acetylcholine, because the K(+)-evoked increase in Ins(1,3,4,5)P4 was enhanced by the cholinesterase inhibitor physostigmine, and was partially attenuated by atropine. An additive suppression by nitrendipine suggests that entry of Ca2+ through L-type Ca2+ channels may serve to accelerate phosphorylation of Ins(1,4,5)P3 by 3-kinase. Norepinephrine did not significantly increase Ins(1,4,5)P3 or Ins(1,3,4,5)P4 accumulation; however, in the presence of depolarizing K+, norepinephrine caused a dramatic increase in Ins(1,3,4,5)P4 mass accumulation. In contrast, the excitatory amino acid quisqualate caused significant increases in the mass accumulations of both inositol polyphosphates measured, with no further increase being observed under depolarizing conditions. The results are discussed with respect to the interactive effects of agonist and depolarization stimuli on inositol polyphosphate accumulation which might more accurately reflect the conditions pertaining in vivo.     

Dual Effects of K+ Depoiarisation on Inositol Polyphosphate Production in Rat Cerebral Cortex

Depolarisation of [3H]inositol-prelabelled slices of rat cerebral cortex with elevated extracellular K+ induced a rapid and marked increase in inositol polyphosphate accumulation. Addition of the muscarinic antagonist atropine (10 microM) markedly inhibited the K+-induced accumulation of inositol tetrakisphosphate (InsP4), with only a slight reduction in stimulated inositol bis- and trisphosphate levels. Inhibitory effects on InsP4 were noted at the earliest time period measured (30 s) and suggested the involvement of released endogenous acetylcholine in part of the response. The atropine-insensitive component of depolarisation did not appear to be secondary to release of noradrenaline, histamine, or 5-hydroxytryptamine, because addition of prazosin, mepyramine, or ketanserin was without effect on the K+ response. Furthermore, secretion of a neuropeptide that could stimulate phosphoinositide hydrolysis was unlikely, because the peptidase inhibitor bacitracin was also without effect. The results suggest that endogenous acetylcholine can stimulate phosphoinositide metabolism by interacting with muscarinic receptors and that this is particularly evident on InsP4 accumulation. Atropine-insensitive responses may be secondary to Ca2+ entry via voltage-sensitive channels.     

Increased Intracellular Calcium Stimulates 3H-Inositol Polyphosphate Accumulation in Rat Cerebral Cortical Slices

Agents that increase the intracellular Ca2+ concentration have been examined for their ability to stimulate 3H-inositol polyphosphate accumulation in rat cerebral cortex slices. Elevated extracellular K+ levels, the alkaloid sodium channel activator veratrine, the calcium ionophore ionomycin, and the marine toxin maitotoxin were all able to stimulate phosphoinositide metabolism. Certain features appear common to the agents studied. Thus, although [3H]inositol monophosphate, [3H]inositol bisphosphate ([3H]InsP2), and [3H]inositol trisphosphate were all stimulated, a proportionally greater effect was observed on [3H]InsP2 in comparison to stimulation by the muscarinic receptor agonist carbachol. However, only an elevated K+ level stimulated [3H]inositol tetrakisphosphate ([3H]InsP4) accumulation alone or produced marked synergy with carbachol on the formation of this polyphosphate. The results suggest that agents that elevate the cytoplasmic Ca2+ concentration in cerebral cells can increase the hydrolysis of membrane polyphosphoinositides. The pattern of the response differs from that produced by muscarinic receptor agonists and indicate that Ca2(+)-dependent hydrolysis may involve different pools of lipids, phosphoinositidase C enzymes, or both. However, clear differences in the ability of these agents to stimulate InsP4, alone or in the presence of muscarinic agonist, suggest that factors other than a simple elevated intracellular Ca2+ concentration are implicated.     

Subacute and Chronic In Vivo Lithium Treatment Inhibits Agonist- and Sodium Fluoride-Stimulated Inositol Phosphate Production in Rat Cortex

We have investigated the effects of in vivo lithium treatment on cerebral inositol phospholipid metabolism. Twice-daily treatment of rats with LiCl (3 mEq/kg) for 3 or 16 days resulted in a 25-40% reduction in agonist-stimulated inositol phosphate production, compared with NaCl-treated controls, in cortical slices prelabelled with [3H]inositol. A small effect was also seen with 5-hydroxytryptamine (5-HT) 24 h after a single dose of LiCl (10 mEq/kg). Dose-response curves to carbachol and 5-HT showed that lithium treatment reduced the maximal agonist response without altering the EC50 value. This inhibition was not affected by the concentration of LiCl in the assay buffer. Stimulation of inositol phosphate formation by 10 mM NaF in membranes prepared from cortex of 3-day lithium-treated rats was also inhibited, by 35% compared with NaCl-treated controls. Lithium treatment did not alter the kinetic profile of inositol polyphosphate formation in cortical slices stimulated with carbachol. Muscarinic cholinergic and 5-HT2 bindings were unaltered by lithium, as was cortical phospholipase C activity and isoproterenol-stimulated cyclic AMP formation. [3H]Inositol labelling of phosphatidylinositol 4,5-bisphosphate was significantly enhanced by 3-day lithium treatment. The results, therefore, indicate that subacute or chronic in vivo lithium treatment reduces agonist-stimulated inositol phospholipid metabolism in cerebral cortex; this persistent inhibition appears to be at the level of G-protein-phospholipase C coupling.     

Cyclic GMP Formation and Inositol Phosphate Accumulation Do Not Share Common Origins in Rat Brain Slices

Cyclic GMP formation and inositol phospholipid hydrolysis were studied in rat brain slices to determine if the two processes have common origins. Muscarinic cholinergic stimulation enhanced [3H]inositol phosphate ([ 3H]IP) accumulation from slices prelabelled with [3H]inositol but did not affect cyclic GMP formation in the cortex, striatum, or cerebellum. An elevated level of extracellular K+ stimulated accumulation of both cyclic GMP and [3H]IP in cortex slices. The former, but not the latter, was reduced by lipoxygenase and phospholipase A2 inhibition. Calcium channel activation enhanced and blockade reduced K+-stimulated [3H]IP formation without affecting the cyclic GMP level, and there were differences in the Ca2+ requirements for the two responses. Thus, there is no support for the concept that guanylate cyclase activation inevitably accompanies inositol phospholipid breakdown, and the evidence presented demonstrates that K+ stimulation promotes cyclic GMP and [3H]IP accumulation by different transducing pathways.     

In Vitro and In Vivo Inhibition of Inositol Monophosphatase by the Bisphosphonate L-690,330

Abstract: We have previously described the synthesis of bis-phosphonate-containing inhibitors of inositol monophosphatase. In the present study, a more detailed examination of the in vitro and in vivo properties of one of these compounds, L-690,330, is described. L-690,330 is a competitive inhibitor of inositol monophosphatase with a K ₁, depending on the source of IMPase, of between 0.2 and 2 μM. Although ∼1,000-fold more potent in vitro than lithium, in muscarinic m1 receptor-transfected Chinese hamster ovary cells prelabelled with [³H]inositol, L-690,330 only produced 40% of the accumulation of [³H]inositol monophosphates achieved by lithium at the same concentration (10 m M ), suggesting that the ability of L-690,330 to cross the cell membrane is limited. Nevertheless, under conditions of cholinergic stimulation (100 mg/kg of pilocarpine s.c.), high doses of L-690,330 were able to increase brain inositol(1)phosphate levels in vivo to three- to fourfold control levels. This effect was dose dependent (ED₅₀= 0.3 mmol/kg s.c.) and was maximal after 1 h. In peripheral tissues, the effects of L-690,330 on inositol(1)phosphate levels mimicked those of lithium both qualitatively and quantitatively. However, in the brain, the effects of L-690,330 were much less than seen with lithium, consistent with the blood-brain barrier restricting access of the polar L-690,330 into the CNS, thereby further limiting entry of compound into cells in the brain. In the future, it may be possible to develop prodrugs of this compound, which circumvent many of the cell permeability problems inherent in bisphosphonate compounds.     

Stimulus-Induced Accumulation of Inositol Tetrakis-, Pentakis-, and Hexakisphosphates in N1E-115 Neuroblastoma Cells

When [3H]inositol-prelabelled N1E-115 cells were stimulated with carbamylcholine (CCh) (100 microM), high K+ (60 mM), and prostaglandin E1 (PGE1) (10 microM), a transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached its maximum level at 15 s and had declined to the basal level at 2 min. CCh, high K+, and PGE1 also caused accumulations of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], [3H]inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and [3H]inositol hexakisphosphate (InsP6). Muscarine and CCh induced accumulations of [3H]Ins(1,4,5)P3, [3H]-Ins(1,3,4,6)P4, [3H]InsP5, and [3H]InsP6 with a similar potency and exerted these maximal effects at 100 microM, whereas nicotine failed to do so at 1 mM. With a slower time course, CCh, high K+, and PGE1 caused accumulations of [3H]-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In an N1E-115 cell homogenate, [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3 were converted to [3H]InsP5 through [3H]-Ins(1,3,4,6)P4. The above results indicate that Ins(1,3,4,6)P4, InsP5, and InsP6 are rapidly formed by several kinds of stimulants in N1E-115 cells.     

Inositol Lipid Metabolism in Rat Hippocampal Formation Slices: Basal Metabolism and Effects of Cholinergic Agonists

Incubation of rat hippocampal formation slices under steady-state conditions with [3H]inositol leads to only three phospholipids becoming labelled: phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. All three lipids incorporate [32P]Pi into their phosphodiester phosphate group with the polyphosphoinositides also incorporating this tracer into their monoester phosphate groups. As the concentrations of these lipids remain constant during these labelling processes we conclude that the phosphodiester phosphate, the inositol moiety, and the monoester phosphate groups undergo metabolic turnover in hippocampal formation slices incubated in vitro. The rate of incorporation of [3H]inositol into all three inositol phospholipids was stimulated by the addition of methacholine to the medium. Moreover, following steady-state labelling of the inositol lipids with [3H]inositol, methacholine in the presence of 10 mM LiCl caused a transient fall of 13% in the radiochemical concentration of phosphatidylinositol 4,5-bisphosphate after only 30 s stimulation and a fall of 15% in the radiochemical concentration of phosphatidylinositol after 30 min. Concomitantly, there was an approximately stoichiometric rise in the radiochemical concentration of inositol phosphates. Thus, we suggest that methacholine stimulates an inositol phospholipid phosphoinositidase C in rat hippocampal formation slices.     

Comparative Effects of Lithium on the Phosphoinositide Cycle in Rat Cerebral Cortex, Hippocampus, and Striatum

Abstract: The effects of lithium on muscarinic cholinoceptor-stimulated phosphoinositide turnover have been investigated in rat hippocampal, striatal, and cerebral cortical slices using [³H]inositol or [³H]cytidine prelabelling and inositol 1,4,5-trisphosphate [lns(1,4,5)P₃] and inositol 1,3,4,5-tetrakisphosphate [lns(1,3,4,5)P₄] mass determination methods. Carbachol addition resulted in maintained increases in lns(1,4,5)P₃ and lns(1,3,4,5)P₄ mass levels in hippocampus and cerebral cortex, whereas in striatal slices these responses declined significantly over a 30-min incubation period. Carbachol-stimulated lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations were inhibited by lithium in all brain regions studied in a time-and concentration-dependent manner. For example, in hippocampal slices significant inhibitory effects of LiCl were observed at times > 10 min after agonist challenge; IC₅₀ values for inhibition of agonist-stimulated lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations by lithium were 0.22 ± 0.09 and 0.33 ± 0.13 mM, respectively. [³H]CMP-phosphatidate accumulation increased in all brain regions when slices were stimulated by agonist and lithium. The ability of myo-inositol to reverse these effects, as well as lithium-suppressed lns(1,4,5)P₃ accumulation, implicates myo-inositol depletion in the action of lithium in the hippocampus and cortex at least. The results of this study suggest that although significant differences in the magnitude and time courses of changes in inositol (poly)phosphate metabolites occur in different brain regions, lithium evokes qualitatively similar enhancements of [³H]inositol monophosphate and [³H]CMP-phosphatidate levels and inhibitions of lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations. However, the inability of striatal slices to sustain carbachol-stimulated inositol polyphosphate accumulation in the absence of lithium and the inability to reverse effects with myo-inositol may indicate differences in phosphoinositide signalling in this brain region.     

Effect of Electroconvulsive Shock on Muscarinic Cholinergic Receptors in Rat Cerebral Cortex and Hippocampus

Abstract: Single electroconvulsive shock (ECS) induced no change in [³H]quinuclidinyl benzilate ([³H]QNB) binding to muscarinic cholinergic receptors in rat cortex and hippocampus. ECS administered once daily for 7 days induced a significant reduction in [³H]QNB binding in both brain areas. Concurrent ECS reversed the significant increase in cortical [³H]QNB binding induced by chronic atropine administration. These findings may have relevance to the antidepressant or amnestic effects of electroconvulsive therapy.     

Differential Uptake of Lithium Isotopes by Rat Cerebral Cortex and Its Effect on Inositol Phosphate Metabolism

Twenty hours following the subcutaneous administration of 5 mEq/kg doses of 6LiCl and 7LiCl to two groups of rats, the cerebral cortex molar ratio of 6Li+/7Li+ is 1.5. The effects of the lithium isotopes on cortex myo-inositol and myo-inositol-l-phosphate levels are the same as we have reported earlier: a Li+ concentration-dependent lowering of myo-inositol and increase in myo-inositol-1-phosphate. Thus 6LiCl, when administered at the same dose as 7LiCl, produces the larger effect on inositol metabolism. When the 6LiCl and 7LiCl doses were adjusted to 5 mEq/kg and 7 mEq/kg, respectively, the cortical lithium myo-inositol and myo-inositol-1-phosphate levels of each group of animals became approximately equal, suggesting that the isotope effect occurs at the level of tissue uptake, but not on inositol phosphate metabolism. The inhibition of myo-inositol-1-phosphatase by the two lithium isotopes in vitro showed no differential effect. The isotope effect on cerebral cortex uptake of lithium is in the same direction as that reported by others for erythrocytes and for the CSF/plasma ratio, but of larger magnitude.     

Effects of Lithium on Inositol Phospholipid Hydrolysis and Inhibition of Dopamine D1 Receptor-Mediated Cyclic AMP Formation by Carbachol in Rat Brain Slices

The in vitro and ex vivo effects of lithium on muscarinic cholinergic inositol phospholipid hydrolysis and muscarinic cholinergic inhibition of dopamine D1-receptor-stimulated cyclic AMP formation were examined in rat brain slices. Following chronic lithium feeding, carbachol-stimulated inositol phosphate accumulation was reduced ex vivo in slices of cerebral cortex but not in striatal slices. Lithium (1 mM) in vitro had no direct effect on dopamine D1-receptor-stimulated cyclic AMP formation, but enhanced the inhibitory effect of carbachol on the D1 response, in striatal slices, and this was not significantly altered by prior lithium feeding. Lithium therefore has effects on two discrete muscarinic responses in rat brain which are apparently maintained after chronic exposure to the ion and might be relevant to its antimanic actions.     

Adenosine Inhibition of Histamine-Stimulated Inositol Phospholipid Hydrolysis in Mouse Cerebral Cortex

The effects of adenosine on inositol phospholipid hydrolysis in mouse cerebrocortical slices were examined. Despite having no effect alone, adenosine and some structural analogues inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The responses to carbachol, noradrenaline, 5-hydroxytryptamine, and elevated KCl levels were unaffected. The effect of adenosine was on the maximal response to histamine rather than on its EC50. Several adenosine antagonists competitively blocked the inhibition due to adenosine. The results are discussed in relation to the previously reported enhancement of histamine-stimulated hydrolysis of inositol phospholipids in guinea pig brain.     

Potentiation by γ-Aminobutyric Acid of α1-Agonist-Induced Accumulation of Inositol Phosphates in Slices of Rat Cerebral Cortex

Noradrenaline-induced accumulation of 3H-labeled inositol mono-, bis-, and trisphosphate (IP1, IP2, and IP3, respectively) in lithium-treated slices of rat cerebral cortex preincubated with [3H]inositol was potentiated by gamma-aminobutyric acid (GABA). However, the effect on [3H]IP2 accumulation was much greater than that on [3H]IP1 or [3H]IP3 accumulation. The principal effect of GABA on noradrenaline concentration-response curves for both [3H]IP1 and [3H]IP2 was to cause an increase in the maximal response attainable. However, whereas the EC50 for GABA potentiation of [3H]IP1 formation was 0.5 mM, the curve for the potentiation of [3H]IP2 formation showed a marked upturn at GABA concentrations of greater than 1 mM. Prazosin (1 microM) blocked the noradrenaline-induced formation of all three inositol phosphates (IPs), in both the presence and the absence of 2 mM GABA. 3H-IP formation induced by phenylephrine and methoxamine was also potentiated by GABA, and again the greatest effect was on [3H]IP2 accumulation. The ratio of [3H]IP2/[3H]IP1 formed in response to 100 microM noradrenaline was increased by 2 mM GABA at all times from 10 to 60 min, whereas the ratio of [3H]IP3/[3H]IP1 was little altered. The effect of GABA was not mimicked by the GABAA agonists isoguvacine and 3-aminopropanesulphonic acid and was not blocked by bicuculline methiodide. (-)-Baclofen, a GABAB agonist, did produce some stimulation of the response to noradrenaline, but to a much lesser extent than GABA. Of the agents tested, nipecotic acid came nearest to reproducing the effect of GABA, in that the major effect was on [3H]IP2 accumulation. The effects of 2 mM GABA and 2 mM nipecotic acid were not additive. GABA potentiation of noradrenaline-induced 3H-IP formation was still apparent in the absence of Li+, but the increase of [3H]IP2 content was less than that of [3H]IP1 content.     

Measurement of Hippocampal Levels of Cellular Second Messengers Following In Situ Freezing

Abstract: The in situ freezing technique has been widely used to fix labile metabolites and cellular second messengers in cerebral cortex. In this study, we isolated specific brain regions at 0°C from coronal sections of frozen heads following in situ brain freezing and measured regional concentrations of labile metabolites and cellular messengers. These levels in the cortex were compared with those in cortical punches obtained at freezing temperature (less than −40°C) from the same in situ frozen brains and those of cortex dissected from decapitated animals. In both isoflurane- and pentobarbital-anesthetized animals, we observed that the levels of lactate, free fatty acids, inositol 1,4,5-trisphosphate, and diacylglycerol, as well as the proportion of protein kinase C associated with the membrane fraction, were similar in cortical punches taken at freezing temperature and those dissected at 0°C. However, with animals decapitated at room temperature, cortical and hippocampal levels of lactate, free fatty acids, and inositol 1,4,5-trisphosphate and the proportion of membrane protein kinase C were significantly higher than those of corresponding brain regions isolated at 0°C from in situ frozen brains ( p < 0.05). These results indicate that dissection of cortex and hippocampus at 0°C following in situ freezing will eliminate decapitation-induced production of artifacts and changes in the levels of cellular second messengers such as inositol 1,4,5-trisphosphate, diacylglycerol, and protein kinase C. The present technique, used in conjunction with in situ freezing, will fix cellular second messengers and labile metabolites in several regions of brain and may facilitate accurate characterization of molecular and cellular mechanisms underlying CNS function.     

Noradrenaline Stimulation Unbalances the Phosphoinositide Cycle in Rat Cerebral Cortical Slices

Abstract: Muscarinic cholinergic and α₁-adrenoceptor-mediated stimulation of phosphoinositide hydrolysis in rat cerebral cortex were compared by measuring carbachol- and noradrenaline-induced accumulation of various intermediates of the phosphoinositide cycle. Unlike carbachol, noradrenaline in the presence of guanosine 5'- O -(3-thiotriphosphate) did not stimulate phospholipase C activity in brain cortical membranes. In cortical slices, the efficacy of noradrenaline to stimulate accumulation of ³H-inositol phosphates and [³²P]phosphatidic acid was 2.5 to threefold that of carbachol. However, noradrenaline was less effective than carbachol in stimulating accumulation of [³H]CDP-diacylglycerol and resynthesis of phosphatidylinositol. This was not due to calcium inhibition of CTP:phosphatidate cytidyltransferase or to different lithium requirements for carbachol- and noradrenaline-stimulated accumulation of [³H]CDP-diacylglycerol. The noradrenaline-induced unbalance of the phosphoinositide cycle, which was most apparent at relatively high concentrations of calcium (2.5 m M ) in the incubation buffer, was qualitatively reproduced with ionomycin. The use of the α_1a-subtype-selective adrenoceptor antagonists WB4101 and 5-methylurapidil revealed a single α_1a-like component mediating the effects of noradrenaline. Our results suggest that the primary mechanism for phospholipase C activation by brain α₁ adrenoceptors involves an increase in intracellular calcium concentration.