Altered responsiveness of adenosine 3′,5′-monophosphate-generating systems in brain slices from adult rats after neonatal treatment with 6-hydroxydopamine

Sprague-Dawley rat pups were administered 6-hydroxydopamine (100 mg/kg) subcutaneously on each of the first 4 days after birth. At 4–5 mo of age the rats and untreated littermates were killed, and the accumulation of [¹⁴C]cyclic AMP elicited by various agents in [¹⁴C]adenine-labeled brain slices was investigated. The accumulation of cyclic AMP elicited by norepinephrine was significantly greater in slices from neocortex and midbrain of 6-hydroxydopamine-treated rats than in corresponding slices from littermate controls. Accumulations in slices from medulla-pons and cerebellum were not significantly altered by the neonatal treatment with 6-hydroxydopamine. Thus, an enhanced response to norepinephrine pertains both in a region, the neocortex, where norepinephrine-levels were permanently reduced by neonatal treatment with 6-hydroxydopamine and in a region, the midbrain, where adult levels of norepinephrine were nearly doubled by the neonatal treatment with 6-hydroxydopamine. Enhanced responses of cyclic AMP-generating systems in neocortical slices from 6-hydroxydopamine-treated rats also pertained with isoproterenol, prostaglandin E₁ and an adenosine-norepinephrine combination, but not with adenosine alone. Depletion of norepinephrine throughout the brain by administration of either reserpine or intraventricular 6-hydroxydopamine to adult rats enhanced responses to norepinephrine in slices from neocortex, but not midbrain. Reserpine had no effect on responses in either neocortex or midbrain of rats treated neonatally with 6-hydroxydopamine.     

α2-Adrenergic receptors mediate inhibition of cyclic AMP production in neurons in primary culture

The actions of adrenergic agents on the intracellular production of cyclic adenosine monophosphate (AMP) was examined in intact cortical and striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to the β-adrenergic agonist isoproterenol (10 μM) resulted in a 5-fold increase in intraneuronal cyclic AMP; norepinephrine (100 μM), alone or in combination with isoproterenol, produced only a 3-fold increase in cyclic AMP levels. However, in the presence of yohimbine (10 μM), cyclic AMP productions due to norepinephrine or isoproterenol plus norepinephrine were identical to isoproterenol alone. When striatal or cortical neurons were exposed to pertussis toxin (100 ng/ml) overnight, there was no detectable difference between isoproterenol- and norepinephrine-stimulated cyclic AMP production. These data suggest thatα₂-adrenergic receptors mediate the attenuation of cyclic AMP production in neurons and do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.     

Comparison of the effects of biogenic amines on cyclic GMP and cycle AMP levels in mouse cerebellum in vitro.

Norepinephrine (NE) elevates levels of both 3',5'-guanosine monophosphate (cyclic GMP) and 3',5'-adenosine monophosphate (cyclic AMP) in incubated slices of mouse cerebellum. As little as 1 muM NE is capable of increasing the level of either cyclic nucleotide. Maximal elevations of cyclic AMP and cyclic GMP levels produced by NE are 15- to 40-fold and 2- to 4-fold, respectively. Dopamine, serotonin and histamine, other biogenic amines considered to be neurotransmitters in CNS, have no effect on mouse cerebellum cyclic nucleotide levels except at relatively high (1 mM) concentrations. NE-induced accumulation of cyclic GMP, but not cyclic AMP, is blocked by omission of Ca2+ from the incubation media. Theophylline does not alter the effect of this catecholamine on either cyclic nucleotide. In tissue slices incubated in buffered sucrose or in choline-Krebs buffer, NE is still capable of increasing both cyclic GMP and cyclic AMP levels; however, these elevations are less than those observed in brain slices incubated in Krebs-Ringer buffer. NE, in combination with glutamate, produces supra-additive elevations of both cyclic GMP and cyclic AMP levels. However, NE in combination with adenosine or high levels of K+ only has a synergistic effect on cyclic AMP and not on cyclic AMP and not on cyclic GMP combination. The elevation of cyclic AMP levels produced by NE appears to be mediated via both alpha- and beta-adrenergic receptor sites. In contrast, the receptor site(s) mediating cyclic AMP accumulation do not appear to be either typical alpha- or beta-adrenergic receptors.     

Norepinephrine depletion and responsiveness of norepinephrine-sensitive cyclic AMP generating systems in guinea pig brain.

The accumulation of radioactive cyclic AMP was examined in [¹⁴C]adeninelabeled slices of guinea pig neocortex following in vivo treatments designed to lower the levels of putative neurotransmitters such as norepinephrine, serotonin, and histamine. Lesions of the medial forebrain bundle and treatment with 6-hydroxydopamine or reserpine decreased cortical concentrations of norepinephrine in guinea pig. Based on studies in rats, enhanced responses of norepinephrine-sensitive cortical cyclic AMP-generating systems were to be expected after such treatments. Instead, no evidence for enhanced responses of cyclic AMP-generating systems to norepinephrine, serotonin, or histamine was obtained with cortical slices from the treated guinea pigs. Adenosine-sensitive cyclic AMP-generating systems in guinea pig cortical slices did become hyperresponsive after reserpine.     

The accumulation of adenosine 3',5'-monophosphate in cerebral cortical slices of the quaking mouse, a neurologic mutant

Norepinephrine, adenosine, veratridine, and adenosine-biogenic amine combinations elicit an accumulation of cyclic AMP in cerebral cortical slices from C57B1/6J control (+/+), quaking (qk/qk) and heterozygous (qk/+) mice. The percent conversion of radioactive adenine nucleotides to cyclic AMP in slices previously incubated with radioactive adenine or adenosine is markedly lower in slices from quaking and heterozygous mice compared to controls with all stimulatory agents except an adenosine-norepinephrine combination. Total incorporation of radioactive adenine or adenosine into adenine nucleotides is, however, significantly higher with slices of quaking and heterozygous mice. The absolute amount of radioactive cyclic AMP and the levels of endogenous cyclic AMP are nearly identical in the 3 groups of mice following incubation with all stimulatory agents except adenosine. The absolute accumulation of both radioactive and endogenous cyclic AMP was significantly lower in quaking and heterozygous mice after incubation with adenosine. The ratio of [¹⁴C]- to [³H]cyclic AMP in slices previously incubated with [¹⁴C]adenine and [³H]adenosine is dependent upon the stimulatory agent. The ratio with each agent is consistently lower in quaking mice compared to controls. The data provide evidence for biochemical alterations in nucleotide metabolism in cortical slices ofboth quaking and heterozygous mice. These effects would not appear to be directly associated with hypomyelination and tremor of the quaking mouse, since the heterozygous mouse, with one quaking gene does not show the latter gross abnormalities.     

Altered responsiveness of adenosine 3′,5′-monophosphate-generating systems in rat cortical slices after lesions of the medial forebrain bundle

The magnitude of the accumulation of radioactive cyclic AMP elicited by norepinephrine in [¹⁴C]adenine-labeled slices of rat cortical slices was enhanced 1.4-fold in ipsilateral slices from animals with unilateral lesions of the medial forebrain bundle as compared to slices from the contralateral hemisphere. This enhanced responsiveness to norepinephrine was first manifested 2 days after the lesion, and persisted for at least 3 weeks. The level of norepinephrine in cortical tissue from the hemisphere with lesion underwent a concomitant decline to less than 20% of the level in tissue from the contralateral hemisphere. Responses of cyclic AMP-generating systems to isoproterenol and to a combination of histamine and a phosphodiesterase inhibitor were also significantly enhanced in ipsilateral cortical slices after lesions of the medial forebrain bundle, while responses to a combination of norepinephrine and the β-adrenergic antagonist, propranolol, to adenosine, veratridine, prostaglandin E₁, and a combination of serotonin and a phosphodiesterase inhibitor were not significantly altered. The response to histamine, in the presence of a phosphodiesterase inhibitor (isobutylmethylxanthine), was blocked effectively only by an H₂-antagonist, metiamide. Responses to norepinephrine after lesions of the medial forebrain bundle were enhanced in ipsilateral hippocampal slices, but not in ipsilateral slices from midbrain or cerebellum.     

Ontogeny of rolipram-sensitive,low-Km,cyclic AMP-specific phosphodiesterase in rat brain

The postnatal development of rolipram-sensitive, low-K_m, cyclic AMP-specific phosphodiesterase (PDE4) was investigated in discrete regions of rat brain using a PDE4 activity assay and immunoblot analyses with K116, a PDE4 antibody. The V_max for cyclic AMP hydrolysis by PDE4 was lower at birth when compared to adult levels in cerebral cortex, cerebellum, and neostriatum. K_m values for cyclic AMP hydrolysis by PDE4, in contrast, did not change throughout the observed period in any brain region tested. The developmental patterns for PDE4 were significantly different among the examined brain regions. PDE4 activity in olfactory bulb and hippocampus also was found to be lower at birth in comparison to adult levels. Immunoblot analyses showed that developmental patterns of PDE4 were significantly different for the various subtypes, and also varied substantially across brain regions. The results suggest that PDE4 might be differentially regulated by different ontogenetic events.     

Prostacyclin biosynthesis in vascular endothelium is not inhibited by cyclic AMP. Studies with 3-isobutyl-1-methylxanthine and forskolin

We have previously reported ($\text{Proc. Natl. Acad. Sci.}$ 79, 495–499, 1982) that the cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (IBMX), stimulates cyclic AMP accumulation and inhibits prostacyclin (PGI₂) production in primary monolayer cultures of human umbilical vein endothelium. The present study was carried out to determine whether these effects are causally related. Incubation of endothelial monolayers with the diterpene, forskolin, increased the intracellular concentration of cyclic AMP by 10-fold. Despite this marked increase in cyclic AMP, neither baseline production of PGI₂ nor release in response to stimulation by thrombin or the divalent cation ionophore, A23187, was affected. Both forskolin and isoproterenol were found to potentiate the effect of IBMX on cyclic AMP accumulation without causing further inhibition of PGI₂ biosynthesis. Inhibition of cyclic nucleotide phosphodiesterase activity with 2,6-bis-(diethanolamino)-4-piperidinopyrimido-[5,4-d]pyrimidine increased cyclic AMP levels to the same extent as IBMX; however, this agent had no effect on PGI₂ biosynthesis. These findings demonstrate that increases in the intracellular concentration of cyclic AMP have no short-term effects on PGI₂ biosynthesis in vascular endothelium and suggest that inhibition of PGI₂ production by IBMX is the result of some other, cyclic AMP-independent action of the drug.     

Receptor-linked cyclic amp systems in rat neostriatum: Differential localization revealed by kainic acid injection

Various receptor-linked cyclic AMP systems were measured in rat neostriatum 2--14 days after selective destruction of neuronal cell bodies and dendrites by micro-injection of 3 microgram of kainic acid. Basal adenylate cyclase activity was reduced by up to 56% in the injected side and the sensitivity to dopamine was abolished. Up to 84% of cyclic nucleotide phosphodiesterase activity, hydrolyzing either cyclic AMP or cyclic GMP, was destroyed by kainic acid injection. Specific binding of [3H]etorphine and [3H]spiroperidol was reduced by up to 62% in the injected side, while non-specific binding was unchanged. All of these changes were time-dependent, and were greatest 7--14 days after kainic acid treatment. On the other hand, intrastriatal kainic acid injection caused no change in the steady-state concentration of cyclic AMP in striatal slices, or in the in vivo cyclic AMP content in the striatum of rats killed by microwave irradiation. Receptor-mediated increases in cyclic AMP accumulation in striatal slices were either unchanged or markedly potentiated by kainic acid treatment. The maximum response to adenosine was unchanged, while the response to isoprenaline was increased up to 3.7-fold, the response to dopamine increased up to 6.7-fold, and the response to PGE1 increased up to 30-fold. The effect of dopamine in kainic acid-treated striatal slices was no longer blocked by fluphenazine, but was blocked by propranolol, suggesting an interaction of dopamine with a beta-adrenoceptor in kainic acid-treated slices. The results suggest differential cellular localizations of the various receptor-linked cyclic AMP systems in rat neostriatum. Some dopamine and opiate receptors, as well as most of the phosphodiesterase activity, are associated with local neuronal elements, while beta-adrenoceptor, adenosine and PGE1 alterations in cyclic AMP are not. The potentiation of the beta-adrenoceptor and PGE1 responses suggests that they may occur in glial cells. In addition, the pool of adenylate cyclase destroyed by kainic acid appears to make little contribution to normal levels of cyclic AMP in the tissue.     

Evidence for a presynaptic adenylate cyclase system facilitating [3H]norepinephrine release from rat brain neocortex slices and synaptosomes

The effects of drugs known to enhance intracellular cyclic AMP levels on depolarization-induced [3H]norepinephrine release from superfused rat neocortical slices and synaptosomes were investigated. The adenylate cyclase activator forskolin, the membrane-permeating cyclic AMP analogues 8-bromo-cyclic AMP and dibutyryl cyclic AMP, as well as the phosphodiesterase inhibitors isobutylmethylxanthine and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrolidone (ZK 62771) enhanced the electrically evoked release of [3H]norepinephrine from superfused rat brain neocortex slices. 8-Bromo-cyclic GMP was without effect on the electrically evoked release. When [3H]norepinephrine release was enhanced by prolonging the electrical pulse duration from 2 msec to 10 msec, the relative inhibitory effect of the Ca2+ channel blocker Cd2+ and the relative facilitatory effect of the K+ channel blocker 4-aminopyridine remained unaffected. In striking contrast, the relative facilitatory effects of forskolin and 8-bromo-cyclic AMP were strongly reduced, whereas the effect of ZK 62771 was almost doubled. When veratrine-induced release of [3H]norepinephrine from cortex synaptosomes was examined, the facilitatory effects of forskolin, 8-bromo-cyclic AMP, and ZK 62771 were even more pronounced than in brain slices. The data strongly support the hypothesis that a presynaptic adenylate cyclase system plays a facilitatory role in the stimulus-secretion coupling process in central noradrenergic nerve terminals.     

Monoacylglycerol lipase

The presence of monoacylglycerol lipase was established in extracts of acetone-dried powders from rat and bovine brains using thioester substrate analogs. At pH 7.4, the apparentK _m andV _max values for 1-S-decanoyl-1-mercapto-2,3-propanediol were 56 μM and 227 nmol/h/mg protein in bovine gray matter. The divalent metal ions Ca²⁺ and Mg²⁺ had no effect on enzymic activity, but Zn²⁺ at 500 μM produced a 50% inhibition of this enzyme. Free fatty acids also caused a marked inhibition of monoacylglycerol lipase activity. Norepinephrine and 5-hydroxytryptamine slightly stimulated the enzymic activity. Hypoxic-hypoxia and 30-s postdecapitation ischemia resulted in a considerable increase in monoacylglycerol lipase activity of rat brain. However, the increased activity of monoacylglycerol lipase returned to normal after 5 min of ischemia. The increased activity of monoacylglycerol lipase during hypoxic-hypoxia and short-time ischemia may be partially responsible for increased levels of free fatty acids during these processes.     

Adenylate cyclase and the search for new compounds with the clinical profile of lithium

It is possible to evaluate the beta-adrenergic receptor-adenylate cyclase complex in the human periphery by measuring the plasma cyclic AMP rise after adrenergic agonists. A clinical trial of the beta 2 adrenergic agonist salbutamol in depression provided an opportunity to test whether adrenergic receptor subsensitivity does occur during clinical antidepressant treatment. After 1 and 3 weeks of oral salbutamol treatment, depression scores declined significantly in 11 depressed patients, while the plasma cyclic AMP response to i.v. salbutamol declined over 60%. The results support the concept that receptor sensitivity changes occur during human antidepressant therapy. Data are presented that Li, too, markedly reduces activity of beta-adrenergic adenylate cyclase in humans. The effect was evaluated by studying the effect of Li at therapeutic serum concentrations on the plasma cyclic AMP response to subcutaneous epinephrine. The Li effect is specific, since the plasma cyclic AMP response to glucagon is not inhibited. In rat cortical slices Li inhibition of noradrenaline-induced cyclic AMP accumulation is clearly demonstrable only at concentrations close to 2 mM Li. However, fresh human brain slices from edges of surgically-removed tumors show Li inhibition at 1 mM Li concentrations. These results imply that in brain as well as periphery, human noradrenergic adenylate cyclase is inhibited by therapeutic concentrations of Li. Demeclocyclin, a tetracycline-derived antibiotic, was found to inhibit noradrenaline-sensitive adenylate cyclase in rat cortical slices and to inhibit amphetamine-induced hyperactivity in rats in an open field. Clinical trials should search for new compounds with the clinical profile of Li.     

Metabolic studies on dysmyelinating mutant “pt” rabbit brain in vitro

“Paralytic tremor” (pt) rabbit mutant is characterized by a severe hypomyelination of the CNS, however it is not defined if the defect in myelinogenesis is an “assembly” or “synthesis” type. In this study, we have compared the general metabolic and biosynthetic properties of the myelinating mutant brain with unaffected controls of the same age. In the brain slices of 4 wk old “pt” rabbits the incorporation of U-[¹⁴C] glucose, 6-[³H] galactose, and U-[¹⁴C] leucine into macromolecules (total lipids and proteins, galactolipids, and myelin basic protein) was substantially elevated. In isolated myelin fraction, the total reduction of the radioactivity was followed by the increased specific activity of all examined macromolecules. The myelin to homogenate specific activity ratio was similar in control and “pt” rabbits. Distribution of the label and myelin marker, cyclic nucleotide 3′-phosphodiesterase (CNP-ase) among the membranous fractions suggests the partial inhibition of myelin formation in “pt” rabbits on the step of premyelin, unilamellar membranes.¹⁴CO₂ yields derived from differently labeled glucose were used for the evaluation of the basal oxidative metabolism in “pt” brain slices.¹⁴CO₂ production from U-[¹⁴C] glucose was normal. The depolarization of the slices by 50 mM K⁺ stimulated glucose oxidation to a higher extent in “pt” than in control. Hexose monophosphate pathway (HMP), the route providing much of NADPH required for lipid biosynthesis, did not change significantly by mutation. The activity of glucose 6-phosphate dehydrogenase (GIc-6-P DH), an oligodendroglia enriched, HMP connected enzyme, was slightly lower in “pt” homogenates by 13–17%, whereas CNP-ase was lowered more than 30% in the same samples. All this data suggest that the capacity for the synthesis of myelin constituents is well preserved in the mutant brain and the impairment of myelogenesis is probably caused by increased elimination of already synthesized, myelin-related components.     

N-phenylisopropyladenosine stimulates in normal and inhibits in adrenalectomized rats the low KM cyclic AMP phosphodiesterase in the brain

In crude rat brain membranes, apparent adenylate cyclase activity tested in the absence of phosphodiesterase inhibitors was dose-dependently inhibited by the adenosine ‘R-site’ agonist N⁶-phenylisopropyladenosine (N⁶-PIA). In membranes from adrenalectomized rats, however, N⁶-PIA induced, under the same conditions, an activation. However, in the presence of the phosphodiesterase inhibitor Ro-20-1724, brain adenylate cyclase responsiveness to N⁶-PIA resulted in a dose-dependent inhibition in both sham-operated and adrenalectomized rats. Thus, the low K_M cyclic AMP phosphodiesterase activity present in these brain membranes was investigated. Although this activity (tested in the presence of GTP) was unaltered by adrenalectomy, the dose-response curve of this enzyme to low concentrations of N⁶-PIA showed an activation in sham-operated and an inhibition in adrenalectomized rats, two effects which were suppressed by sodium (80 mM). These results showing that N⁶-PIA modulates both adenylate cyclase and cyclic AMP phosphodiesterase in the brain, provide an additional argument for a potential role of adenosine in the regulation of cyclic AMP metabolism in normal as well as in pathological brain.     

Relative phosphocreatine and nucleoside triphosphate concentrations in cerebral gray and white matter measured in vivo by P nuclear magnetic resonance

Rates of ATP metabolism generally are higher in cerebral gray matter compared to white matter. In order to study the physiology of this regional difference in vivo, the 1-dimensional chemical shift imaging technique (1D-CSI) was used to acquire ³¹P nuclear magnetic resonance spectra from 2.5 mm slices of 4-week old piglet brains. Spectra from predominantly gray matter slices (estimated 76% gray matter, 7 mm below the scalp) were compared to predominantly white matter slices (56% estimated white matter, 13 mm below the scalp) as assessed by magnetic resonance images. The 1D-CSI technique introduced no systematic changes in the ratio of signals from a single chamber phantom containing a phosphocreatine (PCr) and ATP solution. Gray matter slices showed a PCr/NTP ratio of 0.93 ± 0.11 (mean ± S.D.) using a 2 s interpulse interval, a value very close to the ratio in surface coil localized spectra. The predominantly white matter slices showed a PCr/NTP ratio of 1.32 ± 0.18 (P < 0.02 for gray versus white matter). Using the estimated percentages of gray and white matter in the two slices and calculated concentrations from fully relaxed spectra, the gray matter PCr/NTP ratio is approximately 0.77, while the ratio in white matter is approximately 2.18. The difference in PCr/NTP measured in vivo suggests that either the total NTP concentration is higher or the steady state PCr concentration is lower in gray matter than in white matter in the piglet brain.     

Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from the cytosol of human platelets

A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase was purified to near homogeneity from the 150,000 g supernatant fraction of human platelets by a combination of DEAE-cellulose chromatography and cyclic GMP affinity chromatography. Overall purification was about 7400-fold with a 10% to 15% recovery of activity. On NaDodSO4-containing polyacrylamide gels, the purified enzyme migrates as a single band Mr = 105,000. Phosphodiesterase activity co-migrates with the protein band on native polyacrylamide gels. Both Mg2+ and Mn2+ support the activity of this phosphodiesterase. The enzyme hydrolyzes both cyclic AMP and cyclic GMP with similar maximal rates. The hydrolysis of both nucleotides exhibits positive homotropic cooperativity with S0.5 values of 50 +/- 12 microM for cyclic AMP and 35 +/- 15 microM for cyclic GMP and Hill coefficients of 1.2 to 1.5 for both nucleotides. Low levels of cyclic GMP stimulate the rate of cyclic AMP hydrolysis from 3- to 10-fold. The activity of this phosphodiesterase is not stimulated by the calcium binding protein, calmodulin. The cyclic GMP stimulation of cyclic AMP hydrolysis by this phosphodiesterase may provide a possible regulatory link between the metabolism of these two nucleotides in platelets.     

Lithium effects on noradrenergic-linked adenylate cyclase activity in intact rat brain: an in vivo microdialysis study.

The effects of chronic lithium treatment on adenylate cyclase activity in intact rat brain were examined using in vivo microdialysis. Basal extracellular cyclic adenosine monophosphate (AMP) increased in a dose-dependent manner after norepinephrine was added to the perfusate. Chronic lithium treatment increased basal brain extracellular fluid cyclic AMP levels, while decreasing the magnitude of the cyclic AMP response to stimulation with 100 microM norepinephrine.     

Cocaine andd-amphetamine induce changes in central ß-adrenoceptor sensitivity: Effects of acute and chronic drug treatment

The effects of acute and chronic treatment with psychomotor stimulants on specific binding of [³H]dihydroalprenolol to ß-adrenoceptors in rat brain were examined. At a dose of 10 mg/kg both acute and chronic treatment with cocaine and chronic treatment withd-amphetamine (10 mg/kg) caused increased binding of [³H]dihydroalprenolol. The molecular mechanism for this enhanced binding appears to be augmentation of the density of ß-adrenoceptors in rat brain. At a lower dose (5 mg/kg), however, chronic administration ofd-ampethamine caused a decrease in the density of ß-adrenoceptors in rat brain. Chronic treatment with eitherd-amphetamine (10 mg/kg) or cocaine induced a marked increase in the magnitude of cyclic AMP accumulation in rat brain slices elicited by norepinephrine. Acute as well as chronic administration ofd-amphetamine in vivo inhibited the temperature-dependent uptake of [³H]norepinephrine in rat brain synaptosomal homogenates, but no such inhibition was observed after chronic or acute treatment with cocaine. The results suggest that psychomotor stimulants induced ß-adrenoceptor supersensitivity which may be involved in the phenomenon of reverse tolerance and possibly psychosis in humans. The development of ß-adrenoceptor supersensitivity does not appear to be mediated through alterations in norepinephrine transport at the presynaptic sites.     

Biophysical and metabolic bases of cooling effects on cortical membrane potentials in the cat

Neuronal and silent cell membrane potentials were recorded in cat cortex during focal cooling by 5–6 C, either transiently for 1–2 min or for 10–15 min. Effects were compared between locally anesthetized, immobilized preparations and those under pentobarbital general anesthesia. The findings suggest actions at common membrane sites in effects of cooling and general anesthesia. In immobilized animals, transient cooling depolarized neurons by 1–2 mv/C. Silent cells responded similarly but with a slower onset. Intraneuronal wave activity and neuronal firing patterns showed related changes during the cooling cycle. Increased cell firing during transient cooling was not confined to depolarizing shifts but also occurred during hyperpolarizing shifts. Raising topical Ca²⁺ levels to 20 mM blocked depolarization by transient cooling, but 7.5 mM solutions were without effect. Repeated transient cooling or continuous cooling for 10–15 min produced almost complete depolarization of neurons and silent cells for periods extending considerably beyond recovery of brain temperature. Moreover, these cumulative effects beyond the cooling period were potentiated by the weaker 7.5 mM Ca²⁺ solutions. Pentobarbital anesthesia blocked the effects of sustained cooling on neuronal and silent cell membrane potentials and on unit firing activity. Also, topical 7.5-mM Ca²⁺ solutions failed to potentiate effects of cooling during barbiturate anesthesia. The effects of transient cooling are discussed in terms of membrane leakage currents that may be blocked by Ca²⁺. Cumulative effects with prolonged cooling suggest interference with membrane metabolism, and the possible role of cooperative binding and release of cations from membrane surface glycoproteins in these responses is considered.     

Lateral difference in responsiveness of norepinephrine-sensitive cyclic AMP-generating systems of rat cerebral cortex with iron-induced epileptic activity

Responsiveness of norepinephrine-sensitive cyclic AMP-generating systems was examined in slices of different cortical areas of rats showing electrographic spike and wave complexes after unilateral injection of ferrous chloride solution into the sensorimotor cortex. Accumulation of cyclic AMP elicited by norepinephrine was greater on the injection side of the cortex than on the other. Similar lateral differences were detected in cyclic AMP levels antagonized by phentolamine or propranolol, in which 8-phenyltheophylline almost completely inhibited the elicitation of cyclic AMP accumulation by a norepinephrine-propranolol combination but not by a norepinephrine-phentolamine combination. These results suggest that alterations in cyclic AMP generation through the beta-adrenoceptor-coupled adenylate cyclase system and through alpha-adrenergic activation of the adenosine receptor-coupled adenylate cyclase system are closely related to the electrographic activity of iron-induced epilepsy.