CEREBRAL METABOLIC CHANGES IN PROFOUND, INSULIN-INDUCED HYPOGLYCEMIA, AND IN THE RECOVERY PERIOD FOLLOWING GLUCOSE ADMINISTRATION

Severe hypoglycemia was induced by insulin in lightly anaesthetized (70°_o N₂O) and artificially ventilated rats. Brain tissue was frozen in situ after spontaneous EEG potentials had disappeared for 5. 10. 15 or 30 min and cerebral cortex concentrations of labile organic phosphates, glycolytic metabolites, ammonia and amino acids were determined. In other experiments, recovery was induced by glucose injection at the end of the period of EEG silence. All animals with an isoelectric EEG showed extensive deterioration of the cerebral energy state. and gross perturbation of amino acid concentrations. The latter included a 4-fold rise in aspartate concentration and reductions in glutamate and glutamine concentrations to 20 and 5^o_o of control levels respectively. There was an associated rise in ammonia concentration to about 3μmol-g^-1. Administration of glucose brought about extensive recovery of cerebral energy metabolism. For example, after an isoelectric period of 30 min tissue concentrations of phosphocreatine returned to or above normal, the accumulation of ADP and AMP was reversed, there was extensive resynthesis of glycogen and glutamine and full normalisation of tissue concentrations of pyruvate. α-ketoglutarate. GABA and ammonia. However, even after 3 h of recovery there was a reduction in the ATP concentration and thereby in adenine nucleotide pool, moderate elevations of lactate content and the lactate pyruvate ratio, and less than complete restoration of the amino acid pool. It is concluded that some cells may have been irreversibly damaged by the hypoglycemia.     

OXIDATIVE METABOLISM OF THE CEREBRAL CORTEX OF THE RAT IN SEVERE INSULIN-INDUCED HYPOGLYCAEMIA

—Measurements were made of organic phosphates, carbohydrate substrates, amino acids and ammonia in the cerebral cortex, as well as of cerebral blood flow and of cerebral metabolic rate for oxygen and glucose in rats that developed an isoelectric EEG pattern (‘coma’) during insulin-induced hypoglycaemia. The results were compared to those obtained in control animals, as well as in hypoglycaemic animals with an EEG pattern of slow waves and polyspikes. In animals with slow waves and polyspikes, there was a decrease in all citric acid cycle intermediates except succinate and oxaloacetate, and a decrease in the pool size of intermediates. In animals that had an isoelectric EEG for 5–15 min, there were further decreases in citrate, isocitrate, α-ketoglutarate, malate and fumarate, but since the concentration of succinate (and oxaloacetate) increased, the pool size remained the same. In isoelectric animals, the results revealed extensive utilization of amino acids by both transamination and deamination reactions. However, since glycogen had disappeared and the amino acid pattern was constant after the first 5 min of isoelectric EEG, further oxidation must have occurred at the expense of non-carbohydrate, non-amino acid substrates. There were two- to three-fold increases in cerebral blood flow in animals with slow waves and polyspikes and in animals with isoelectric EEG, and no decrease in the cerebral metabolic rate for oxygen. Since less than half of the oxygen consumption could be accounted for in terms of glucose extraction, the data indicate that severe hypoglycaemia is associated with extensive oxidation of endogenous substrates other than carbohydrates and free acids.     

CEREBRAL ENERGY STATE IN INSULIN-INDUCED HYPOGLYCEMIA, RELATED TO BLOOD GLUCOSE AND TO EEG

—Concentrations of phosphocreatine, creatine, ATP, ADP and AMP were measured in the cerebral cortex of rats during insulin-induced hypoglycemia. Blood glucose concentrations were related to clinical symptoms in unanaesthetized animals and to the EEG pattern in paralysed and lightly anaesthetized animals. There was an excellent correlation between blood glucose concentration and EEG pattern. In animals showing a pronounced slowing of the EEG or convulsive polyspike activity for up to 20 min, there were no changes in any of the phosphates. However, after prolonged convulsive activity some animals showed clear signs of energy failure, and in all animals with an isoelectric EEG there was a major derangement of the energy state. Since the majority of those animals did not show signs of cerebral hypoxia or ischemia it is concluded that hypoglycemic coma is accompanied by substrate deficiency of a degree sufficient to induce energy depletion of brain tissue.     

Cerebral energy metabolism in guinea pig fetuses during development.

During development fetal arterial oxygen tension falls, whereas cerebral oxygen consumption rises due to an increase in cerebral metabolism. To compensate for this increase in oxygen consumption, blood flow and therefore oxygen delivery to the cerebrum rises. To determine whether during development oxygen delivery to the cerebrum meets cerebral oxygen consumption, we measured the concentrations of high-energy phosphates and glycolytic intermediates in the cerebral cortex of fetal guinea pigs at different gestational ages. During development there was no change in the concentrations of adenosine triphosphate, creatine phosphate, adenosine monophosphate, and lactate. However, cerebral concentrations of adenosine diphosphate increased and those of glucose decreased. Our results suggest that the increase in fetal cerebral oxygen delivery during development meets cerebral oxygen consumption with increasing gestational age. We speculate that the measured rise in the concentrations of adenosine diphosphate may accelerate glycolysis during development and therefore may cause a rise in both cerebral blood flow to maintain oxygen delivery.     

METABOLITES AND ENZYMES OF THE PENTOSE PHOSPHATE PATHWAY IN ISOLATED NERVE ENDINGS1

Abstract— The activities of each enzyme associated with the pentose phosphate pathway as well as the non-enzymatic intermediates in this pathway were measured in synaptosomes isolated from rat cerebral cortex. The specific activities of transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) were significantly lower in synaptosomes than cerebral cortex; however, the specific activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribosephosphate isomerase (EC 5.3.1.6) and ribulosephosphate epimerase (EC 5.1.3.1.) were comparable in homogenates of synaptosomal fractions and cerebral cortex. Concentrations of most intermediates of the pentose pathway were also similar in extracts of synaptosomes and brain homogenates. Six hours after treatment of rats with the nicotinamide analog, 6-aminonicotinamide (6-AN), 6-phosphogluconate levels in synaptosomes were increased 5-fold; however, glucose-6-phosphate levels remained unchanged. During a 30 min in uitro incubation 6-phosphogluconate levels increased approx 2-fold in synaptosomes obtained from 6-AN treated rats but did not change in synaptosomes from untreated rats. During the same period glucose-6-phosphate levels decreased in synaptosomes from both control and 6-AN treated rats. The conversion of both [1-¹⁴C]glucose and [6-¹⁴C]glucose to ¹⁴CO₂ was depressed in synaptosomes from 6-AN treated rats; however, the ratio of the two isotopes converted to ¹⁴CO₂ was essentially the same. It is concluded that the pentose phosphate pathway is active in nerve endings both in vivo and in vitro.     

Cerebral Oxidative Metabolism and Blood Flow During Acute Hypoglycemia and Recovery in Unanesthetized Rats

Abstract: Progressive neurological depression leading to coma was produced in unanesthetized rats at a constant level of hypoglycemia induced by insulin. High-energy phosphate concentrations in brain remained normal during hypoglycemic lethargy, but ATP declined by 6% during stupor and by 40% during coma that was characterized by an isoelectric EEG. Cerebral blood flow (CBF) remained normal during hypoglycemia whereas the cerebral metabolic rates for oxygen (CMRo₂) and glucose (CMR_glucose) decreased by 45 and 73%, respectively, indicating oxidation of nonglucose fuels. A plot of CMRo₂ and CMR_glucose versus plasma glucose indicated increasing oxidation of alternate substrates (elevated CMRo²/CMR_glucose) at plasma glucose concentrations below 2.5 mm . The cerebral uptake of β-hydroxybutyrate increased during hypoglycemic stupor and its complete oxidation could account for the CMRo₂ in excess of glucose utilization. Brain ammonia, a byproduct of amino acid metabolism, reached a level during hypoglycemic coma sufficient to produce coma in normoglycemic animals. The rate and degree of recovery after glucose administration depended on the duration of hypoglycemia and the pretreatment neurological state of the animal. Following 10 min of glucose infusion, ATP levels that were modestly depressed in stuporous rats recovered fully, paralleling the animals' apparently full neurological recovery. Rats that had been in hypoglycemic coma for 1 min or less fully recovered high-energy phosphate concentrations in brain. However, when normalization of plasma glucose was delayed for more than 1 min of coma, the CMRo₂ remained depressed, CBF decreased to 40% of control, and high-energy substrates failed to normalize. In keeping with the depression of oxidative metabolism and blood flow, neurological function and the EEG remained abnormal even after 1 h of glucose infusion. The findings suggest that irreversible brain injury may develop within the first minutes of hypoglycemic coma.     

Failing atrial myocardium: energetic deficits accompany structural remodeling and electrical instability

The failing ventricular myocardium is characterized by reduction of high-energy phosphates and reduced activity of the phosphotransfer enzymes creatine kinase (CK) and adenylate kinase (AK), which are responsible for transfer of high-energy phosphoryls from sites of production to sites of utilization, thereby compromising excitation-contraction coupling. In humans with chronic atrial fibrillation (AF) unassociated with congestive heart failure (CHF), impairment of atrial myofibrillar energetics linked to oxidative modification of myofibrillar CK has been observed. However, the bioenergetic status of the failing atrial myocardium and its potential contribution to atrial electrical instability in CHF have not been determined. Dogs with (n = 6) and without (n = 6) rapid pacing-induced CHF underwent echocardiography (conscious) and electrophysiological (under anesthesia) studies. CHF dogs had more pronounced mitral regurgitation, higher atrial pressure, larger atrial area, and increased atrial fibrosis. An enhanced propensity to sustain AF was observed in CHF, despite significant increases in atrial effective refractory period and wavelength. Profound deficits in atrial bioenergetics were present with reduced activities of the phosphotransfer enzymes CK and AK, depletion of high-energy phosphates (ATP and creatine phosphate), and reduction of cellular energetic potential (ATP-to-ADP and creatine phosphate-to-Cr ratios). AF duration correlated with left atrial area (r = 0.73, P = 0.01) and inversely with atrial ATP concentration (r = -0.75, P = 0.005), CK activity (r = -0.57, P = 0.054), and AK activity (r = -0.64, P = 0.02). Atrial levels of malondialdehyde, a marker of oxidative stress, were significantly increased in CHF. Myocardial bioenergetic deficits are a conserved feature of dysfunctional atrial and ventricular myocardium in CHF and may constitute a component of the substrate for AF in CHF.     

Cerebral energy metabolism in immature and mature guinea pig fetuses during acute asphyxia.

In immature fetuses circulatory centralization caused by acute asphyxia is less effective than that in mature fetuses (Jensen & Berger, 1991). This suggests that cerebral oxygenation may be poor in immature fetuses during asphyxia. On the other hand cerebral oxygen consumption is lower in immature than that in mature fetuses. To determine, whether or not there is an imbalance between oxygen supply and demand in one or the other group, we compared the time course of the changes of cerebral concentrations of both high-energy phosphates and glycolytic intermediates between immature and mature guinea pig fetuses during acute asphyxia caused by arrest of uterine blood flow. The fall in the cerebral concentrations of adenosine triphosphate and glucose, and the rise in those of adenosine monophosphate and lactate were slower in immature than in mature fetuses. There were no differences between the levels of cerebral adenosine diphosphate and creatine phosphate of the two groups. From these results we conclude that during acute asphyxia the imbalance between cerebral oxygen supply and demand is less marked in immature than in mature fetuses.     

METABOLISM OF BRAIN DURING SLEEP AND WAKEFULNESS

Abstract— The levels in brain of lactate, pyruvate, creatine phosphate, ATP, ADP and AMP were examined in sleeping and waking adult rats. The animals were monitored electrophysiologically and the biochemical measurements were made after approx. 25 min of sleep or wakefulness. The previous treatment of the animals had a marked effect on the levels of brain metabolites during sleep. In animals not acclimatized to the observation chamber, brain levels of lactate and pyruvate rose during sleep above those in the waking state: creatine phosphate and ATP were depressed somewhat. When the animals were acclimatized by being placed in the observation chamber for at least 2 h on four or more consecutive days prior to the experiment, sleep was accompanied by a depression of brain levels of lactate and pyruvate and slight elevations of brain levels of creatine phosphate and ATP. No significant differences in the EEG recordings were noted between the sleeping rats of the acclimatized and non-acclimatized groups. These observations on the effect of acclimatization on brain metabolism during sleep may have clinical relevance in man.     

Regionally Selective Metabolic Effects of Hypoglycemia in Brain

Abstract: Regional CNS levels of glucose reserves, glycolytic intermediates, and high-energy phosphate reserves were measured in insulin-treated, hypoglycemic rats and correlated with EEG activity. Intravenous administration of insulin to paralyzed, ventilated animals causes concomitant reduction of blood glucose levels and progressive abnormality and eventual loss of EEG activity. In all regions of brain examined, glucose and glycogen levels decrease until they are essentially depleted, and glucose-6-phosphate and fructose-1,6-biphosphate fall approximately 80%. Pyruvate levels decrease 50% in cerebral cortex and brain stem and a lesser amount in striatum, hippocampus, thalamus, and cerebellum. Lactate levels fall 50–60% in all regions except cerebellum, where no change is observed. ATP and phosphocreatine levels remain normal until the EEG is isoelectric, and then decrease in all regions except cerebellum. These results demonstrate that hypoglycemia does not have a uniform effect on brain glucose and energy metabolism, and cerebellum seems to be relatively protected.     

Relation of ATP and creatine phosphate to fast axoplasmic transport in mammalian nerve

—ATP and creatine phosphate (CP) levels in cat sciatic nerve maintained in vitro were measured. Anoxia produced by N₂ or NaCN or the uncoupling of phosphorylation with DNP reduced the combined levels of ATP + CP to approximately one-half of control levels within 15 min. These agents also blocked fast axoplasmic transport in vitro within 15 min. A block of glycolysis with iodoacetic acid (IAA) reduced the combined levels of ATP + CP to approximately one half of control levels within 1.5–2 h and exposure of nerve in vitro to IAA caused a block of fast axoplasmic transport within the same interval. The correlation of the time at which block of transport occurred with the fall in the level of high-energy phosphates is consistent with the hypothesis that ATP supplies the energy required by the mechanism underlying fast exoplasmic transport.     

The effect of electroshock on regional CNS energy reserves in mice

ATP, phosphocreatine, glycogen, glucose and lactate levels were measured in the cerebral cortex, thalamus, cerebellum, brain stem and spinal cord of mice following supramaximal electroshock. During the initial 17 s after the onset of a 2 s electrical stimulus high energy phosphate expenditure exceeded formation in all regions but was slower in spinal cord than in the other regions. In cerebral cortex high energy phosphate utilization continued to exceed formation for 32 s which was twice as long as in any other region studied. Altered levels of metabolites recovered most rapidly in spinal cord and least rapidly in cerebral cortex. Pretreatment with a non-anaesthetic dose of phenobarbitone influenced the effect of electroshock. Most of the clinical seizure was prevented, and increased high energy phosphate utilization was sustained for a much shorter period. Only in cerebral cortex did high energy phosphate expenditure exceed formation for as long as 15 s after the electrical stimulus; but even in this region the excess of expenditure over formation was much less than in untreated animals.     

THE DISTRIBUTION OF LIPIDS IN THE HUMAN NERVOUS SYSTEM-V. GANGLIOSIDES AND ALLIED NEUTRAL GLYCOLIPIDS OF INFANT BRAIN

—Gangliosides and allied neutral glycosylceramides were isolated from human infant (2-24 months of age) cerebral cortex and white matter. The individual glycolipids were separated quantitatively by a combination of column and thin-layer chromatographic methods on silica gel, DEAE-cellulose and Sephadex G-25. In cerebral cortex G_D1a and G_M1 were the major fractions and constituted more than 70 per cent of the total gangliosides. The concentrations of neutral glycolipids, except for galactosylceramides, were very low: lactosylceramide and glucosylceramide comprised 30 and 5 nmol/g wet weight, respectively. In white matter their concentrations were 10 times higher. The ganglioside concentration was only 50 per cent of that in cerebral cortex: the difference was accounted for mainly by the much lower content of the major di- and trisialogangliosides. Stearic acid was the predominant fatty acid of all brain gangliosides. G_M3, and G_D3 had a considerable content of the very long-chain fatty acids, C₂₂-C₂₄, particularly in the white matter. Glucosylceramide and lactosylceramide had almost identical fatty acid patterns between each other in cerebral cortex and white matter. In the cerebral cortex stearic acid and in the white matter the very long-chain acids predominated. d20:1 Sphingosine comprised more than 20 per cent of total sphingosine in all the gangliosides of the G_l- and G₂-series. G_M3, and G_D3 like lactosylceramide contained significantly less of d20:1 sphingosine. The findings suggest the existence of separate compartments for the biosynthesis of the gangliosides. Glucosylceramides and lactosylceramides of white matter have the same ceramide composition as the galactosylceramides with normal fatty acids and are thus unlikely to be intermediates in the metabolism of the major brain gangliosides which have a completely different fatty acid composition.     

Recovery of Postischemic Brain Metabolism and Function Following Treatment with a Free Radical Scavenger and Platelet-Activating Factor Antagonists

We have studied the metabolic and functional effects of two new platelet-activating factor (PAF) antagonists (BN 50726 and BN 50739) and their diluent (dimethyl sulfoxide; DMSO) during reoxygenation of the 14-min ischemic isolated brain. Blood gases, EEG, auditory evoked potentials, cerebral metabolic rate for glucose (CMRglc), and cerebral metabolic rate for oxygen (CMRO2) were monitored throughout the study. Frozen brain samples were taken for measurement of brain tissue high-energy phosphates, carbohydrate content, and thiobarbituric acid-reactive material (TBAR, an indicator of lipid peroxidation) at the end of the study. Following 60 min of reoxygenation in the nontreated 14-min ischemic brains, lactate, AMP, creatine (Cr), intracellular hydrogen ion concentration [H+]i), and TBAR values were significantly higher and ATP, creatine phosphate (PCr), CMRglc, CMRO2, and energy charge (EC) values were significantly lower than the corresponding normoxic control values. PCr and CMRO2 values were significantly higher, and glycogen, AMP, and [H+]i values were significantly lower in the BN 50726-treated ischemic brains than in DMSO-treated ischemic brains. In brains treated with BN 50739, ATP, ADP, PCr, CMRO2, and EC values were significantly higher, and lactate, AMP, Cr, and [H+]i values were significantly lower than corresponding values in the DMSO-treated ischemic brains. TBAR values were near control levels in all brains exposed to DMSO. There was also marked recovery of EEG and auditory evoked potentials in brains treated with DMSO. Treatment with BN 50726 or BN 50739 in DMSO appeared to improve brain mitochondrial function and energy metabolism partly as the result of DMSO action as a free radical scavenger.(ABSTRACT TRUNCATED AT 250 WORDS)     

Are polyphosphates or phosphate esters prebiotic reagents?

It is widely held that there was a phosphate compound in prebiotic chemistry that played the role of adenosine triphosphate and that the first living organisms had ribose-phosphate in the backbone of their genetic material. However, there are no known efficient prebiotic synthesis of high-energy phosphates or phosphate esters. We review the occurrence of phosphates in Nature, the efficiency of the volcanic synthesis of P₄O₁₀, the efficiency of polyphosphate synthesis by heating phosphate minerals under geological conditions, and the use of high-energy organic compounds such as cyanamide or hydrogen cyanide. These are shown to be inefficient processes especially when the hydrolysis of the polyphosphates is taken into account. For example, if a whole atmosphere of methane or carbon monoxide were converted to cyanide which somehow synthesized polyphosphates quantitatively, the polyphosphate concentration in the ocean would still have been insignificant. We also attempted to find more efficient high-energy polymerizing agents by spark discharge syntheses, but without success. There may still be undiscovered robust prebiotic syntheses of polyphosphates, or mechanisms for concentrating them, but we conclude that phosphate esters may not have been constituents of the first genetic material. Phosphoanhydrides are also unlikely as prebiotic energy sources. Correspondence to: S.L. Miller     

Studies on the Linkage of Energy Metabolism and Neuronal Activity in the Isolated Perfused Rat Brain

An isolated rat brain preparation was perfused using glucose-free (=aglycemic) media. The high-energy phosphates, substrates of the glycolytic pathway, free atnino acids, acetylcholine as well as the intracellular distribution of hexokinase activity were determined in brain tissues. The EEG was evaluated visually. The levels of glycolytic substrates, glutamate, and glutamine in cortical tissue decreased after aglycemic perfusion, whereas the aspartate level increased and the GABA level remained unchanged. The high-energy phosphate content seemed to be unaffected for about 15 min of aglycemic perfusion and fell significantly after 20 min. The EEG of the isolated brain changed rapidly after starting aglycemic perfusion and became isoelectric after 12–15 min. Hyperglycemic perfusion (35 mmol glucose per liter perfusion medium) did not alter the energy metabolism of the isolated brain. The breakdown of cerebral energy metabolism and of EEG activity was postponed when thiopental was added to the perfusion medium. The soluble hexokinase activity measured in cortical tissue was reduced after aglycemic perfusion and was enhanced after thiopental. Hyperglycemic perfusion did not influence the intracellular hexokinase distribution. The acetylcholine level in the striatum of the isolated rat brain was significantly decreased by aglycemia and was increased in hypothalamus by thiopental. It was suggested that hexokinase bound to the mitochondrial membrane may play an important role in the relationship of energy metabolism and neuronal activity.     

Determination of myocardial high-energy phosphates using bioluminescence

The measurement of myocardial high-energy phosphates (HEP) has become essential in the evaluation of current methods of myocardial protection both in the experimental and clinical setting. Assays for high-energy phosphates have required as much as 50 mg of myocardial tissue which prevents repeated biopsies in the clinical setting as well as in the experimental laboratory. Using the reaction of bioluminescence described by McElroy W. D. and B. L. Strehler (1949, Arch. Biol. Chem.22, 420), we have developed a technique to measure both adenosine triphosphate and creatine phosphate on samples of myocardial tissue weighing less than 10 mg. A liquid scintillation counter measures the light produced by ATP when added to a firefly extract containing luciferin/luciferase. The reaction is complete in seconds and is detected in the counter during the first 10-s count. Repeated samples have demonstrated a variation of less than 4% between samples. A 25-μl sample is diluted up to 40 μl of firefly extract for detection of adenosine triphosphate. Creatine phosphate is measured by the in vitro production of adenosine triphosphate which is maximum in 10 min when adenosine diphosphate and creatine kinase are added. Again reproducibility of repeated analyses demonstrates a 4% difference in creatine phosphate values. The rapidity, reproducibility, and ability to use ultramicrosamples allows investigators to analyze high-energy phosphates during various methods of myocardial protection currently used in clinical setting.     

Superoxide scavengers augment contractile but not energetic responses to hypoxia in rat diaphragm.

Acute exposure to severe hypoxia depresses contractile function and induces adaptations in skeletal muscle that are only partially understood. Previous studies have demonstrated that antioxidants (AOXs) given during hypoxia partially protect contractile function, but this has not been a universal finding. This study confirms that specific AOXs, known to act primarily as superoxide scavengers, protect contractile function in severe hypoxia. Furthermore, the hypothesis is tested that the mechanism of protection involves preservation of high-energy phosphates (ATP, creatine phosphate) and reductions of P(i). Rat diaphragm muscle strips were treated with AOXs and subjected to 30 min of hypoxia. Contractile function was examined by using twitch and tetanic stimulations and the degree of elevation in passive force occurring during hypoxia (contracture). High-energy phosphates were measured at the end of 30-min hypoxia exposure. Treatment with the superoxide scavengers 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron, 10 mM) or Mn(III)tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride (50 microM) suppressed contracture during hypoxia and protected maximum tetanic force. N-acetylcysteine (10 or 18 mM) had no influence on tetanic force production. Contracture during hypoxia without AOXs was also shown to be dependent on the extracellular Ca(2+) concentration. Although hypoxia resulted in only small reductions in ATP concentration, creatine phosphate concentration was decreased to approximately 10% of control. There were no consistent influences of the AOX treatments on high-energy phosphates during hypoxia. The results demonstrate that superoxide scavengers can protect contractile function and reduce contracture in hypoxia through a mechanism that does not involve preservation of high-energy phosphates.     

CYCLIC AMP IN THE RAT CEREBRAL CORTEX AFTER ACTIVATION OF NORADRENALINE NEURONS OF THE LOCUS COERULEUS

The levels of cyclic AMP in the rat brain were studied in vivo following destruction or stimulation of the noradrenergic pathway originating in the locus coeruleus. After chronic lesion of the locus coeruleus no alterations in cyclic AMP content were found. Electrical stimulation of the locus coeruleus produced an elevation of cyclic AMP in the cerebral cortex of chloral hydrate anaesthetized rats of 30%. Maximal increases were found after 15–60 s stimulation at a frequency of 30–100 Hz. This maximal response was slightly inhibited by phenoxybenzamine, an α-adrenergic blocking agent, and by the β-blocker propranolol. When the α and β blockers were administered together a highly significant decrease in cyclic AMP response was observed. Pretreatment of the rats with reserpinc +α methyl-p-tyrosine prevented the cyclic AMP response. In addition to the effect in the cerebral cortex, cyclic AMP-levels were also enhanced in the hippocampus, in the striatum and in the hypothalamus. These results suggest that the locus coeruleus regulates a small fraction of cerebral cyclic AMP levels, by both α- and β-adrenergic receptors.     

PYRIDOXINE DEFICIENCY AND DEVELOPMENT OF THE CENTRAL NERVOUS SYSTEM IN THE RAT

Pyridoxine (vitamin B₆) deficiency was produced in rats during the period of development of the central nervous system. The levels of pyridoxal phosphate and y-amino-butyric acid in whole brains of these rats were determined, together with the activities of glutamate decarboxylase (EC 4.1.1.15) and γ-aminobutyrate aminotransferase (EC 2.6.1.19). The lowered contents of pyridoxal phosphate and γ-aminobutyrate in the brains confirmed the existence of pyridoxine deficiency. The activity of the glutamate decarboxylase holo-enzyme was decreased, whereas the activity of the apoenzyme was increased. However, there appeared to be no difference in the activity of γ-aminobutyrate aminotransferase. Concomitantly, some electrophysiological parameters, such as EEG and auditory evoked potentials, were analysed. The EEG of pyridoxine-deficient animals showed spike activity, presumably indicative of the existence of seizures in many of the deficient rats. Evoked potentials presented abnormalities in their latency, wave form and response to repetitive stimuli, but the extent to which they were affected depended upon the intensity of the deficiency.