Effects of barbiturates on energy and intermediary metabolism in cultured astrocytes

Effects of barbiturates on utilization of the two substrates, glucose and glutamate, were studied in astrocytes in primary cultures. Carbon dioxide formation from glucose was under ordinary conditions not affected by barbiturates but in the presence of 10 μM malate there, was a potassium-induced stimulation (20–25%) which was significantly (P < 0.001) inhibited (30–35%) by pentobarbital (0.5 mM). Glutamate oxidation was not enhanced by excess potassium but there was a distinct dose-dependent reduction in the presence of pentobarbital. In contrast, pentobarbital or phenobarbital had no effect on the formation of glutamine from glutamate.     

Deletion of astrocytic BMAL1 results in metabolic imbalance and shorter lifespan in mice

Disruption of the circadian cycle is strongly associated with metabolic imbalance and reduced longevity in humans. Also, rodent models of circadian arrhythmia, such as the constitutive knockout of the clock gene Bmal1, leads to metabolic disturbances and early death. Although astrocyte clock regulates molecular and behavioral circadian rhythms, its involvement in the regulation of energy balance and lifespan is unknown. Here, we show that astrocyte-specific deletion of Bmal1 is sufficient to alter energy balance, glucose homeostasis, and reduce lifespan. Mutant animals displayed impaired hypothalamic molecular clock, age-dependent astrogliosis, apoptosis of hypothalamic astrocytes, and increased glutamate and GABA levels. Importantly, modulation of GABAA-receptor signaling completely restored glutamate levels, delayed the reactive gliosis as well as the metabolic phenotypes and expanded the lifespan of the mutants. Our results demonstrate that the astrocytic clock can influence many aspects of brain function and neurological disease and suggest astrocytes and GABAA receptor as pharmacological targets to prevent the metabolic dysfunctions and shortened lifespan associated with alterations of circadian rhythms.     

Glutamate decreases pyruvate carboxylase activity and spares glucose as energy substrate in cultured cerebellar astrocytes.

The effects of glutamate on [U-(13)C]glucose metabolism were studied in cerebellar astrocytes using (13)C magnetic resonance spectroscopy. Labeled glutamate, glutamine, aspartate, lactate, and alanine were observed both in the cell extracts and in media, and, additionally, labeled glycogen was detected in the cell extracts. However, only labeled lactate and alanine were quantifiable in the medium in addition to [U-(13)C]glucose. In the presence of unlabeled glutamate, the amount of [U-(13)C]glucose removed from the medium was decreased, indicating that glutamate might spare glucose as an energy substrate and thus decrease the uptake of glucose. Labeled glycogen, [4,5-(13)C]glutamate, [3,4,5-(13)C]glutamate, [3,4-(13)C]aspartate, and [U-(13)C]alanine were increased in the presence of glutamate. However, the increase in the amount of [3,4,5-(13)C]glutamate from the second turn in the tricarboxylic acid (TCA) cycle was less pronounced than that of [4,5-(13)C]glutamate from the first turn in the TCA cycle. This indicates the dilution of label, probably resulting from the synthesis of unlabeled oxaloacetate from glutamate in the TCA cycle. Furthermore, exogenous glutamate had an inhibiting effect on pyruvate carboxylation, presumably by formation of oxaloacetate from 2-oxoglutarate derived from glutamate. It could be shown that glucose is a better substrate for energy production than glutamate; it is, however, less efficient in labeling amino acids than glutamate in cerebellar astrocytes.     

Circadian rhythm of plasma melatonin in endogenous depression

1. The circadian rhythm of plasma melatonin was investigated in normal men 18–30 years (N=5), normal men 50–70 years (N=5) and in six patients with endogenous depression.

2. The environmental photoperiod was 11 hours.

3. The subjects and patients were indoors with lights on from 07:00 until 23:00 hours.

4. Blood samples were obtained every 4 hours over a 24 hour period, with additional sampling at 22:00 and 02:00 hours.

5. Plasma melatonin was estimated by radioimmunoassay compared to both groups of controls.

6. In the depressed patients, the levels of melatonin were low throughout the 24 hour period.

7. The depressives had a delayed onset of the dark phase of the rhythm.

8. The patients also showed peak melatonin levels occurring earlier than in the controls.

9. Circadian rhythm of melatonin and therefore of its pacemaker may be altered in endogenous depression.     

Plasma melatonin is reduced in Huntington's disease

This study was undertaken to determine whether the production of melatonin, a hormone regulating sleep in relation to the light/dark cycle, is altered in Huntington's disease. We analyzed the circadian rhythm of melatonin in a 24‐hour study of cohorts of control, premanifest, and stage II/III Huntington's disease subjects. The mean and acrophase melatonin concentrations were significantly reduced in stage II/III Huntington's disease subjects compared with controls. We also observed a nonsignificant trend toward reduced mean and acrophase melatonin in premanifest Huntington's disease subjects. Onset of melatonin rise was significantly more temporally spread in both premanifest and stage II/III Huntington's disease subjects compared with controls. A nonsignificant trend also was seen for reduced pulsatile secretion of melatonin. Melatonin concentrations are reduced in Huntington's disease. Altered melatonin patterns may provide an explanation for disrupted sleep and circadian behavior in Huntington's disease, and represent a biomarker for disease state. Melatonin therapy may help the sleep disorders seen in Huntington's disease. © 2014 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.     

Photic and circadian regulation of retinal melatonin in mammals

Several studies have established that melatonin synthesis occurs in the retina of vertebrates, including mammals. In mammals, a subpopulation of photoreceptors (probably the cones) synthesize melatonin. Melatonin synthesis in the retina is elevated at night and reduced during the day in a fashion similar to events in the pineal gland. Both the MT1 and MT2 melatonin receptors are present in the retina and retinal melatonin does not contribute to circulating levels, suggesting that retinal melatonin acts locally as a neurohormone and/or neuromodulator. Melatonin synthesis in the retina of mammals is under the control of a circadian oscillator, and circadian rhythms in melatonin synthesis and/or release have been described for several species of mammals. These rhythms are present in vivo, persist in vitro, are entrained by light and are temperature compensated. The cloning of the gene responsible for the synthesis of the enzyme arylalkylamine N-acetyltransferase (the key enzyme in the melatonin biosynthetic pathway) has allowed studies of the molecular mechanisms responsible for the generation of retinal melatonin rhythmicity. The present review focuses on the cellular and molecular mechanisms that regulate melatonin synthesis. In particular, we discuss how the photic environment and the circadian clock interact in determining melatonin levels, in addition to the role that melatonin plays in retinal physiology.     

PKCepsilon upregulates voltage-dependent calcium channels in cultured astrocytes

Astrocytes express voltage-gated calcium channels (VGCCs) that are upregulated in the context of the reactive astrogliosis occurring in several CNS pathologies. Moreover, the ability of selective calcium channel blockers to inhibit reactive astrogliosis has been revealed in a variety of experimental models. However, the functions and regulation of VGCC in astrocytes are still poorly understood. Interestingly, protein kinase C epsilon (PKCepsilon), one of the known regulators of VGCC in several cell types, induces in astrocytes a stellated morphology similar to that associated to gliosis. Thereby, here we explored the possible regulation of VGCC by adenovirally expressed PKCepsilon in astrocytes. We found that PKCepsilon potently increases the mRNA levels of two different calcium channel alpha(1) subunits, Ca(V)1.2 (L-type channel) and Ca(V)2.1 (P/Q-type channel). The mRNA upregulation was followed by a robust increase in the corresponding peptides. Moreover, the new calcium channels formed as a consequence of PKCepsilon activation are functional, since overexpression of constitutively-active PKCepsilon increased significantly the calcium current density in astrocytes. PKCepsilon raised currents carried by both L- and P/Q-type channels. However, the effect on the P/Q-type channel was more prominent since an increase of the relative contribution of this channel to the whole cell calcium current was observed. Finally, we found that PKCepsilon-induced stellation was significantly reduced by the specific L-type channel blocker nifedipine, indicating that calcium influx through VGCC mediates the change in astrocyte morphology induced by PKCepsilon. Therefore, here we describe a novel regulatory pathway involving VGCC that participates in PKCepsilon-dependent astrocyte activation.     

Adenosine A(2A) receptors modulate glutamate uptake in cultured astrocytes and gliosomes

Glutamate is the primary excitatory neurotransmitter in the central nervous system, where its toxic build-up leads to synaptic dysfunction and excitotoxic cell death that underlies many neurodegenerative diseases. Therefore, efforts have been made to understand the regulation of glutamate transporters, which are responsible for the clearance of extracellular glutamate. We now report that adenosine A(2A) receptors (A(2A) R) control the uptake of D-aspartate in primary cultured astrocytes as well as in an ex vivo preparation enriched in glial plasmalemmal vesicles (gliosomes) from adult rats, whereas A(1) R and A(3) R were devoid of effects. Thus, the acute exposure to the A(2A) R agonist, CGS 21680, inhibited glutamate uptake, an effect prevented by the A(2A) R antagonist, SCH 58261, and abbrogated in cultured astrocytes from A(2A) R knockout mice. Furthermore, the prolonged activation of A(2A) R lead to a cAMP/protein kinase A-dependent reduction of GLT-I and GLAST mRNA and protein levels, which leads to a sustained decrease of glutamate uptake. This dual mechanism of inhibition of glutamate transporters by astrocytic A(2A) R provides a novel candidate mechanism to understand the ability of A(2) (A) R to control synaptic plasticity and neurodegeneration, two conditions tightly associated with the control of extracellular glutamate levels by glutamate transporters.     

Changes in circadian rhythm for mRNA expression of melatonin 1A and 1B receptors in the hypothalamus under a neuropathic pain‐like state

Several clinical reports on neuropathic pain of various etiologies have shown that it significantly interferes with sleep. Inadequate sleep due to neuropathic pain may contribute to the stressful negative consequences of living with pain. It is generally recognized that melatonin (MT) system in the hypothalmus is crusial for circadian rhythm and sleep‐wake transition. However, little, if any, is known about whether neuropathic pain could affect the MT system associated with sleep disturbance. In this study, we investigated the possible changes in circadian rhythm for the expression of MT receptors, especially MT1A and MT1B receptors, in the hypothalamus of mice with sciatic nerve ligation. The samples for real‐time RT‐PCR assay were prepared at 8:00, 14:00, 20:00, and 2:00 on day 7 after sciatic nerve ligation or sham operation. The mRNA expression of MT1A and MT1B receptors at 2:00 in sciatic nerve‐ligated mice, which exhibited thermal hyperalgesia along with an increase in wakefulness and a decrease in nonrapid eye movement sleep, was significantly greater than those in sham‐operated mice, whereas the levels of both MT1A and MT1B receptors at 8:00 in sciatic nerve‐ligated mice were significantly lower than those in sham‐operated mice. These findings suggest that neuropathic pain‐like stimuli lead to sleep disturbance in parallel with changes in circadian rhythm for mRNA expression of MT 1A and 1B receptors in the hypothalamus of mice. Synapse 68:153–158, 2014. © 2014 Wiley Periodicals, Inc.     

Endocannabinoid signaling in astrocytes

The study of the astrocytic contribution to brain functions has been growing in popularity in the neuroscience field. In the last years, and especially since the demonstration of the involvement of astrocytes in synaptic functions, the astrocyte field has revealed multiple functions of these cells that seemed inconceivable not long ago. In parallel, cannabinoid investigation has also identified different ways by which cannabinoids are able to interact with these cells, modify their functions, alter their communication with neurons and impact behavior. In this review, we will describe the expression of different endocannabinoid system members in astrocytes. Moreover, we will relate the latest findings regarding cannabinoid modulation of some of the most relevant astroglial functions, namely calcium (Ca²⁺) dynamics, gliotransmission, metabolism, and inflammation.     

Nocturnal melatonin profiles before and one year after beginning shift-work

Abstract Nocturnal serum melatonin profiles were determined twice for seven single women, during their time of employment as nurses (baseline), and after one year (follow up), in order to investigate the effects of shift-work on nocturnal melatonin secretion. All subjects were working in the same hospital under an irregularly rotating three-shift system. Five (5) mL blood samples were drawn six times at 2 h intervals between 20:00–06:00 hours under dim light conditions (< 50 lux). The same sampling procedures were repeated the following year. The results showed pronounced inter-individual differences in melatonin concentrations. There was a trend towards increasing maximum melatonin concentration (MAX melatonin) at follow up, with a similar tendency seen in summed melatonin (the sum of six measured melatonin concentrations). A trend was also seen towards increasing melatonin ratio at 06:00 hours (the percentage of melatonin concentration at 06:00 hours by summed melatonin) at follow up. Melatonin concentration at 06:00 hours was significantly higher at follow up, and a significant correlation between Morningness-Eveningness score (M-E score) at baseline and increased summed melatonin at follow up was also seen. These results suggest that: nocturnal melatonin secretion does not significantly increase after beginning shift-work; and that greater increases in melatonin secretion at follow up are found in subjects with higher M-E scones (increased morning type). With more subjects, however, there may be significant increase in MAX melatonin and/or summed melatonin in the follow-up study.     

Influence of maternal melatonin on melatonin receptors in rat offspring

Summary Using quantitative autoradiography, we have studied the influence of maternal plasma melatonin on the expression and density of melatonin receptors in the brain and pituitary of rat offspring. At birth, the same structures displayed melatonin receptors whether the rats were born to and reared by intact or pinealectomized dams. The receptor density was, however, about 20% lower in the group born to pinealectomized dams. At postnatal day 9, when the pups of both groups synthetize rhythmically their own melatonin, this difference was suppressed. These results indicate that melatonin does not appear to be a requirement for the expression of its receptors, but seems to play a stimulatory role in their synthesis.     

pH-sensitive inwardly rectifying chloride current in cultured rat cortical astrocytes

The effect of pH(o) on plasma membrane chloride current of cultured rat cortical astrocytes was investigated using the whole-cell patch-clamp technique. In the presence of intra- and extracellular solutions with symmetrical high Cl(-) content and K(+) channel inhibitors, the cells exhibited an inwardly rectifying current. The current activated slowly at potentials negative to -40 mV and did not display time-dependent inactivation. The current was inhibited by 0.1 mM Cd(2+), 0.1 mM Zn(2+), 1 mM 9-anthracene-carboxylic acid, and 0.2 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not by 10 mM Ba(2+) or 3 mM Cs(+). Reversal potential of the current followed the chloride equilibrium potential and was not influenced by changes in K(+) or Na(+) concentration. The inwardly rectifying chloride current was augmented by extracellular acidosis and reduced by alkalosis. The pH sensitivity was most pronounced in the physiologically relevant pH(o) range of 6.9--7.9. Lowering pH to 6.4 induced no additional increase in steady-state current amplitude compared with pH(o) 6.9, but it substantially slowed the activation kinetics. According to its kinetic and pharmacological properties this chloride current is similar to that found in cultured rat astrocytes after long-term treatment with dibutyryl-cAMP, however, in our cultures it was consistently expressed without any treatment with the drug. Considering that astrocytes possess carbonic anhydrase and Cl(-)/HCO3(-) antiporter, this current may participate in the regulation of the interstitial and astrocyte pH.     

Optimization of light and melatonin to phase-shift human circadian rhythms

Both light and melatonin, appropriately timed, have been shown to phase-shift human circadian rhythms. In addition, both light and melatonin have acute physiological and behavioural effects. Depending on the dose, melatonin can reduce core body temperature and induce sleepiness. Conversely, light at night increases body temperature and enhances alertness and performance. The acute and phase-shifting effects of light and melatonin have justified their investigation and use in the treatment of circadian rhythm sleep disorders. Melatonin is the treatment of choice for blind people with non-24 h sleep/wake disorder. Current research is directed towards optimizing these therapies with respect to time of administration, dose and formulation of melatonin, intensity, duration and spectral composition of light. Our studies in totally blind people with non-24 h sleep/wake disorder have shown that, in addition to improving sleep, daily administration of melatonin can entrain their free-running circadian rhythms. The ability of melatonin to entrain free-running rhythms depends, in part, on the time of melatonin administration relative to the subject's circadian phase. Subjects who were entrained by melatonin began their treatment in the phase advance portion (CT 6-18) of the published melatonin phase-response curves (PRCs), whereas those who failed to entrain began their melatonin treatment in the delay portion of the PRC. Whether the effect of light on the human circadian axis can be optimized by altering its spectral composition has been investigated. Recently, it was demonstrated that light-induced melatonin suppression in humans is sensitive to short wavelength light (420-480 nm; lambda(max) approximately 460 nm), a response very different to the classical scotopic and photopic visual systems. Whether other nonvisual light responses (e.g. circadian phase resetting) show a similar spectral sensitivity is currently being studied.     

Influence of melatonin on fatigue severity in patients with chronic fatigue syndrome and late melatonin secretion

The effect of melatonin, a chronobiotic drug, was explored in 29 patients with chronic fatigue syndrome (CFS) and Dim Light Melatonin onset (DLMO) later than 21.30 hours, reflective of delayed circadian rhythmicity. The patients took 5 mg of melatonin orally, 5 h before DLMO during 3 months. Their responses to the checklist individual strength (CIS), a reliable questionnaire measuring the severity of personally experienced fatigue, were assessed twice with a 6-week interval immediately before the treatment and once after 3 months treatment. In the pre-treatment period the fatigue sub-score improved significantly. After treatment, the total CIS score and the sub-scores for fatigue, concentration, motivation and activity improved significantly. The sub-score fatigue normalized in two of the 29 patients in the pre-treatment period and in eight of 27 patients during treatment. This change was significant. In the patients with DLMO later than 22.00 hours (n=21) the total CIS score and the sub-scores for fatigue, concentration and activity improved significantly more than in the patients (n=8) with DLMO earlier than 22.00 hours. Melatonin may be an effective treatment for patients with CFS and late DLMO, especially in those with DLMO later than 22.00 hours.     

Features of melatonin catabolism in chicks

Melatonin is a neurohormone that is involved in biorhythm synchronization that is produced by the pineal gland and retina. We have studied the production of the kynurenine products, N-acetyl-N-formyl-5-methoxykynurenamine (AFMK) and N-acetyl-5-methoxykynurenamine (AMK), during catabolism of melatonin in chicks (Gallus gallus). Analyses of blood serum samples, retinas, and pineal glands were performed using the HPLC method with fluorometric and electrochemical detection. Considerable amounts of melatonin were found in the pineal gland, retina, and blood serum, although endogenous AFMK and AMK were not found in these tissues. Nevertheless, after intraperitoneal injection of melatonin at a dose of 10 mg/kg, these compounds were found in the blood serum, and AMK was found in the retina. Analysis of AFMK and AMK pharmacodynamics in the blood serum showed that their peak concentrations were 4.8 ng/ml and 2 ng/ml, respectively; the peaks were observed within less than 5 minutes after melatonin injection for AFMK and in 20 minutes after melatonin injection for AMK. The amount of AMK in the retina also reached its peak (60 pg/retina) in 20 minutes. The concentration of both metabolites decreased to the detection limit within one hour. Thus, melatonin may be oxidized via the kynurenine pathway, and its oxidation products, AFMK and AMK, have short half lives. The total amount of both metabolites after melatonin injection was approximately 0.04% of its amount in the blood serum and retina, thus, this pathway of melatonin oxidation is not the major one.     

Circadian rhythms of melatonin,cortisol and prolactin in patients with obsessive-compulsive disorder

Plasma melatonin, cortisol and prolactin (PRL) levels were measured over a 24-h period in 13 drug-free patients with obsessive-compulsive disorder and in matched healthy subjects. The circadian profiles of melatonin and PRL were altered in patients; the circadian rhythm of cortisol was preserved, although at a higher level compared with normal controls. These changes were significantly related to the severity of the obsessive-compulsive symptoms. Further studies need to clarify the state- or trait-dependent character of these abnormalities.     

Sodium-dependent high-affinity uptake of taurine in cultured cerebellar granule cells and astrocytes

Taurine uptake in cultured cerebellar granule cells and astrocytes consisted of a saturable high-affinity component and nonsaturable diffusion. The transport constant (Km) was significantly lower and the maximal velocity (V) higher in granule cells than in astrocytes. The uptakes were strictly sodium dependent and also moderately decreased in potassium-free medium. The specificity profile of taurine uptake was similar in both cell types, hypotaurine, beta-alanine, and guanidinoethanesulphonic acid being the most potent inhibitors, followed by GABA and homotaurine. Glutamate inhibited taurine uptake more in astrocytes than in granule cells. In principle, the uptake systems were similar in granule cells and astrocytes, exhibiting features characteristic of uptake of a neurotransmitter or -modulator.