Integrative coordination of circadian mammalian diversity: neuronal networks and peripheral clocks.

Diverse circadian rhythms are generated, maintained and/or coordinated by brain structures constituting the circadian timing system. However, the mechanisms underlying the variety in activity types and circadian rhythm phases and amplitudes are currently unknown. We address this problem by comparing rhythms in diurnal and nocturnal mammals, while focusing on alterations not involving the central circadian oscillator. The circadian rhythms are divided into two groups: activity-independent and activity-related. The rhythms in the first group have similar acrophases in all mammals and are anticipated to function as an internal zeitgeber (time giver). Analysis of activity-related circadian rhythms in behavior, blood pressure (BP) and renal excretion suggests separate mechanisms in their regulation in addition to the central suprachiasmatic nuclei-located circadian oscillator. We propose that: (a) a passive hypothalamic oscillator coordinates the phases and underlies the high amplitude of behavioral circadian rhythms; (b) a separate rostral hypothalamic network participates in the regulation of the low-amplitude circadian BP rhythm; and (c) a circadian oscillator in the kidney generates electrolyte excretion rhythms. A model is offered where the overt activity is determined by the phase-relationship between the circadian and the passive hypothalamic oscillator. Specific brain structures or peripheral circadian oscillators integrate circadian and other signals for different activity-related circadian rhythms. The hypothalamic structures implicated in regulation of behavioral and blood pressure rhythms belong to the circadian timing system since they underlie circadian rhythms diversity. The same hypothalamic areas selectively modulate circadian rhythms in response to homeostatic stimuli or stress without engaging the circadian oscillator.

Gene expression in suprachiasmatic nucleus and circadian rhythms.

The suprachiasmatic nuclei (SCN) contain a circadian system consisting of circadian oscillator (clock) that is normally synchronized by the light/dark cycle (input) and drives circadian rhythms (output) that are intrinsic to the SCN. Gene expression of immediate-early genes, such as c-fos and jun-B, in the ventrolateral SCN is associated with circadian synchronization by light pulses and subjected to circadian control. Vasopressin and somatostatin gene expression shown distinct circadian rhythms intrinsic to the dorsomedial SCN with higher peptide levels occurring during the day. In addition, embryonic SCN grafted into the brain of an SCN-lesioned arrhythmic host define the period of the restored circadian locomotor rhythm. Taken together, these and other findings support the notion that the expression of genes underlying circadian synchronization, oscillation and output takes place within individual SCN neurons. However, no information regarding the nature and number of those neurons as well as the molecular mechanisms of the single cell-circadian oscillator and output is currently available. Therefore, we propose a simple two-neuron model as a framework for critically discussing the molecular genetic strategies to analyze the circadian system in SCN.

Molecular mechanisms underlying mood stabilization in manic-depressive illness: the phenotype challenge.

OBJECTIVE: The authors critically examine the evidence supporting the hypothesis that lithium's therapeutic effects in bipolar affective disorder are mediated by alterations in the expression of specific genes in critical neuronal circuits. METHOD: Using the heuristic "initiation and adaptation paradigm," the authors appraise the biological effects and underlying molecular and cellular mechanisms of lithium's action. The evidence is critically reviewed, with special attention to the reductive and integrative approaches necessary for identifying lithium's clinically relevant cellular and molecular targets. RESULTS: Lithium's acute effects are mediated through inhibition of specific enzymes involved in two distinct but interacting signaling pathways--the protein kinase C and glycogen synthase kinase 3 beta signaling cascades--that converge at the level of gene transcriptional regulation. The expression of different genes, including transcription factors, is markedly altered by chronic lithium administration. Chronic lithium treatment also robustly increases the expression of the neuroprotective protein Bcl-2, raising the intriguing possibility that some of lithium's effects are mediated through underappreciated neurotrophic/neuroprotective effects. The importance of lithium's effect on circadian rhythms and the related methodological problems in validating the role of specific genes in lithium's therapeutic effects are discussed. CONCLUSIONS: Despite the plethora of lithium effects at the genomic level, direct evidence that the genes identified thus far are responsible for phenotypic changes associated with chronic lithium treatment is still lacking. The combination of sensitive molecular technologies, appropriately designed paradigms, better behavioral analysis, and a chronobiologic approach seems necessary for the future identification of one or more clinically relevant lithium-target genes.

Differential display protocol with selected primers that preferentially isolates mRNAs of moderate- to low-abundance in a microscopic system.

A modified reverse transcription polymerase chain reaction (RT-PCR)-based differential display procedure with selected primers (SPR) was developed to increase the bias toward isolating moderate- to low-abundance transcripts that are differentially expressed during synapse formation in a microscopic neuronal system, the embryonic chicken ciliary ganglion. Major modifications, in comparison with available arbitrarily primed RT-PCR protocols, include the use of (i) experimentally selected primer pairs (50% GC-rich 15-21-mers) that avoid the amplification of highly abundant ribosomal and mitochondrial transcripts; (ii) a higher PCR annealing temperature (50 degrees C instead of 40 degrees C); (iii) selection of sequencing gel bands that are dependent on the two primers for amplification; (iv) tests for reproducibility by SPR amplification of independent sets of RNA extractions and Southern blot analysis of the products with an isolated radiolabeled clone; and (v) quantitative RT-PCR, instead of Northern blot analysis, to confirm the differential expression of individual cDNAs. Thirty-six cDNAs were isolated and sequenced using SPR. None showed significant homology to highly abundant transcripts. In contrast, when no criterion for primer or band selection was applied, 22% of 55 cDNAs were identical to ribosomal and mitochondrial transcripts. Reproducible amplification of 9 out of 10 SPR-isolated cDNAs was established by Southern blot analysis. Differential expression was then confirmed for 4 selected sequences by quantitative RT-PCR. Thus, SPR is a reproducible and efficient procedure for identifying differentially regulated transcripts of moderate- to low-abundance in microscopic biological systems.

Lithium treatment in ovo: effects on embryonic heart rate, natural death of ciliary ganglion neurons, and brain expression of a highly conserved chicken homolog of human MTG8/ETO.

Understanding the action of the mood stabilizer lithium is dependent on availability of experimental models where lithium treatment at clinically relevant concentrations induces marked phenotypic and genotypic changes. Here we report on such changes in the chicken embryo. Lithium chloride (0.6 mM), applied in ovo 60 h after incubation, markedly delayed the heart rate increase observed from ED2.5 to ED5, and induced the brain expression of a new chicken gene cETO from ED7 to ED15. At the same time the overall developmental dynamics and embryo survival, or the expression of chicken gephyrin were not significantly affected. Furthermore, lithium treatment (0.3 mM, 48 h after incubation) abolished the difference in neuronal number between ED12 ciliary ganglia developing in the presence or absence of postganglionic target muscles. We show that cETO is a close homologue of the human transcription factor MTG8/ETO; named after its location on chromosome 8, and participation in chromosomal translocation 8;21 in myeloid leukemia. The mRNA and protein levels of ETO and gephyrin had a parallel course in chicken brain development suggesting that the expression of both genes is regulated mainly at the level of gene transcription. However, the patterns of expression were markedly different. ETO peaked at ED7 and decreased five-fold at ED15. In contrast, gephyrin levels increased five-fold from ED7 to ED15. We propose that the induction of ETO expression, in concert with lithium-induced upregulation of other genes, such as PEBP2beta and bcl-2, is participating in the neuroprotective effect of chronic lithium treatment.

Feeding pattern and light-dark variations in water intake and renal excretion after suprachiasmatic nuclei lesions in rats.

Bilateral destruction of the suprachiasmatic nuclei (SCN) eliminated light-dark (L/D) variations in water intake and urine output in albino rats. The lesions abolished also the circadian rhythm of food intake, without changing significantly the 24 hour number of meals, total meal duration and 24 hour food intake. Only the L/D distribution of the number of meals was changed from 5.6/16.9 in control period to 12.7/12.9 after lesions. In contrast, the L/D distribution of sodium, potassium and chlorides excretions demonstrated attenuated but persistent nocturnal type. These data imply that SCN play a role of driving oscillator for the circadian rhythm of food intake, but probably are not the main synchronizer for the rhythms of electrolyte excretions.

Blood pressure and heart rate rhythmicity: differential effects of late pregnancy.

Arterial blood pressure (BP) and heart rate (HR) of 31 hospitalized pregnant women at low risk of hypertension were automatically monitored for 48 h at 15-min intervals. Each of the recorded 56 data series for systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and HR was chronobiologically assessed by linear-nonlinear rhythmometry. The rhythm-adjusted mean (MESOR), circadian amplitude, circadian acrophase, and best-fitting period were grouped by pregnancy trimester and further subjected to analysis of variance. BP MESOR remained unaltered, whereas HR MESOR increased significantly in middle and late pregnancy. Ultradian rhythms, with an amplitude higher than that of the circadian rhythm, were found in 25% of the SAP records in the second and third trimester. Such ultradian rhythms were not detected in the simultaneously recorded HR. Finally, the group BP and HR circadian acrophases coincided in the first trimester, but were significantly apart in mid and late pregnancy. These observations support the notion that the coordination of BP and HR rhythmicity involves different physiological mechanisms. Analysis of the individual variability in the chronobiological end points (based on the records of nine women monitored in each pregnancy trimester) revealed that only the BP MESOR was well reproducible in the course of pregnancy and may be useful in early diagnosis of gestational hypertension.

Suprachiasmatic nuclei lesions do not eliminate the circadian rhythms of electrolyte excretion in the rat.

The 24-hour variations in 2-hour diuretic and saluretic action of furosemide (4 mg/kg) given at 8, 12, 16, 20, 24 or 4 hr and the circadian rhythms of food and water intake, urine and electrolyte excretion followed for two consecutive days at 4-hour intervals were investigated in suprachiasmatic nuclei (SCN)-lesioned or sham-operated male Wistar rats. The results showed that: a distinct 24-hour rhythm in furosemide-induced urine, sodium and chloride excretion persisted after SCN lesions, and the lesions abolished the circadian rhythms of food and water intake but only desynchronized the individual 24-hour variations in electrolyte excretion. We concluded that SCN play a role as a central synchronizer but not as a major oscillator of the circadian rhythms of electrolyte excretion in the rat.

Peripheral p-chloroamphetamine is an unsuitable probe for investigation of central serotoninergic control on renal renin secretion in the rat.

Intraperitoneal application of p-chloroamphetamine (PCA) is considered a suitable probe for investigation of central serotoninergic control on renin release in the rat, although it causes several behavioral and autonomic changes including negative water balance (increased urination and loss of body weight). The possibility that PCA-induced renin release is secondary to the alterations in water balance was investigated 1 hour after intraperitoneal PCA in male Wistar (Wi) (Experiment I). Long-Evans (LE) and diabetes insipidus (DI) (Experiment II), DI rats pretreated by the inhibitor of angiotensin I-converting enzyme captopril (Experiment III), and water-loaded or propranolol-pretreated Wi rats (Experiment IV). PCA treatment induced significant body weight loss, increase in hematocrit, stimulation of renin-aldosterone system (RAS) and elevation of plasma creatinine level. A toxic damage of the kidney and liver was documented histologically 72 h after 5 mg/kg PCA in Wi rats. The blockade of PCA-induced stimulation of RAS (by captopril or propranolol) markedly potentiated the attendant negative water balance, whereas positive water balance (oral water load) abolished PCA-induced renin secretion. In conclusion, intraperitoneal PCA is an unsuitable probe for investigation of central serotoninergic control on renin release in the rat since PCA-induced renin release is caused by the attendant negative water balance.

Suprachiasmatic nuclei lesions eliminate light/dark variations in short-term feeding responses to deprivation or insulin treatment in rats.

Short-term (1-h, 4-h and 12-h) and long-term (24-h) feeding responses to 24-h food deprivation (FD) or insulin treatment (IT) (8 mU/kg IP) were studied in male rats under a 12/12-h light/dark (L/D) cycle. The 24-h FD or the IT began either at onset (Dawn) or offset (Dusk) of the lights. In sham-operated rats (Shams) both protocols elicited greater short-term feeding responses at Dusk (p less than 0.05 or less). In suprachiasmatic nuclei (SCN)-lesioned rats the L/D variations in short-term responses were absent. In both SCN and Shams the 24-h feeding responses did not depend on stimuli time-schedule. We conclude that the regulation of short-term (circadian), but not long-term, feeding responses to metabolic stimuli is dependent on SCN integrity in the rat.

A method for continuous infusion and blood sampling in unanaesthetized rats.

We describe a technique for long-term cardiac catheterization and blood sampling in unanaesthetized rats. The use of metabolism cage and swivel joint-equipped infusion system allows also continuous infusion of fluids in freely-moving animals.

Circadian regulation of feeding in rats: suprachiasmatic versus ventromedial hypothalamic nuclei.

The role of the suprachiasmatic nuclei as a major component of a specific circadian system controlling feeding periodicity is reviewed. Evidence is presented supporting the assumption that the ventromedial hypothalamus and the suprachiasmatic nucleus may act as a constant (tonic) regulator and a circadian modulator respectively of feeding in rats. It is concluded that a specific circadian system differing from the metabolic control mechanism superimposes the circadian periodicity of feeding. A model is put forward for the possible functional relationships between circadian and metabolic (homeostatic) control mechanisms of feeding in rats.

Effect of constant light and darkness on the circadian rhythms in rats: I. Food and water intake, urine output and electrolyte excretion.

The light/dark (L/D) rhythms of food and water intake, urine output and Na+, Cl- and K+ excretion were studied in male Wistar albino rats subjected either to constant light (LL) or darkness (DD). The LL regimen had a suppressive effect on the daily food and water intake, urine output and sodium excretion, combined with an elimination of L/D variations in all parameters studied. The DD regimen caused a decrease of the daily food and water intake, and sodium and chloride excretion. The L/D rhythms of water and electrolyte excretion were eliminated, while the diurnal rhythms of food and water intake were attenuated but still persisted after 16 days of DD. Our results suggest that the L/D cycle is the major external synchronizer of the circadian rhythms of water and electrolyte excretion in the rat. In the absence of L/D cyclicity, the persisting periodicity of food intake is not a potent enough synchronizer of the circadian rhythms of water and electrolyte excretion. Probably, the circadian rhythms of food and water intake from one hand, and the rhythms of renal excretion from the other hand, are regulated via different circadian mechanisms in the rat.

A reliable technique for midline stereotaxic cannulation of the third ventricle in the rat.

A simple technique is described for chronic or acute cannulation of the third ventricle in the rat slightly displacing the superior sagittal sinus to allow midline implantation of a vertically oriented cannula. The results of an experiment successfully employing the technique for transplantation of embryonic hypothalamic tissue in rats are briefly described.

Effect of bevelling on C1-selective microelectrodes: experimental and model study.

Bevelling of the ultra-fine tips of C1-selective microelectrodes (ME) based on liquid ion-exchanger Corning 477913 caused significant decrease in ME resistivity, increase in sensitivity dependent on its initial value and decrease in selectivity constant for bicarbonates (KC1,HCO3) from 10:1 to approximately 8:1. Bevelling did not affect KC1,NO3 which had value below 1 due to higher selectivity of the ion-exchanger for nitrates. The observed experimental alterations in ME behavior after bevelling were in agreement with those predicted by a relatively simple electrical model comprising of two conductive pathways in parallel having opposite in sign sensitivities.

Effect of morphine withdrawal on food and water intake, urine output and electrolyte excretion in the rat: participation of the renin-aldosterone-system in renal excretory changes.

Male Wistar rats received two i.p. injections of morphine-HCl, 2.5 mg/kg at 8.00 a.m. and 2.00 p.m. on the 1st day: the dose was doubled every other day to reach a total daily dose of 40 mg/kg on the 4th day. This schedule was maintained for 12 days. On day 16 the animals received the last injection of morphine, 20 mg/kg. One hour later (9.00 a.m.) six rats were decapitated and PRA, PAC and ACTH were measured by radioimmunoassay. Groups of six rats were killed at 9.00 a.m. on the 1st, 2nd, 5th and the 8th day after morphine withdrawal. Control data for PRA, PAC and ACTH were obtained from eighteen saline-injected rats. Nine out of morphine-treated animals were kept in metabolism cages to investigate simultaneously food and water intake. and renal excretion. Morphine withdrawal after chronic morphine treatment in the rat resulted in antidiuresis and a reduction of electrolyte excretion which were not due to a reduction in water and food intake. The simultaneous increase of PRA and PAC associated with decreased electrolyte excretion indicates that, in addition to antidiuretic hormone, also the renin-aldosterone-system probably play a relevant role in the renal excretory changes after morphine withdrawal.

Plasma angiotensin-converting enzyme activity and blood pressure during the first year of life in normotensive and spontaneously hypertensive rat.

Plasma angiotensin-converting enzyme (ACE) activity and systolic blood pressure were studied every consecutive month during the first years of life in male spontaneously hypertensive (SHR) and in normotensive rats (NWR). During the first month after birth neither ACE activity nor systolic blood pressure showed significant difference between SHR and NWR. ACE activity in SHR was significantly reduced from 2nd till 12th month of age in comparison with age-matched NWR. In the 2nd month of age the systolic blood pressure was significantly higher in SHR than in NWR, it increased further until the 5th month and was maintained at this high level till the 12th month. No correlation between changes in the systolic blood pressure and the ACE activity was found in SHR after the 2nd month of age. During the investigation period no age-related dynamics of ACE activity was observed in SHR. The observed difference of ACE activity was not due to an increase of plasma his-leu hydrolyzing activity in SHR and was not abolished after a 24-hour dialysis of plasma. This difference could not be caused by the altered effect of chloride ion on the enzyme since similar pattern of Cl-dependent activation of plasma ACE in 4-month-old SHR and NWR was observed. Lineweaver-Burke plot analysis revealed that this difference appears to be due to a decrease of the enzyme maximal velocity in SHR but to a change of the Km value of ACE for the substrate hippuryl-1-his-1-leu. Our data provide evidence for a lower concentration of the available active enzyme molecules in SHR plasma in respect to NWR after the 1st month of life. Whether the reduced ACE activity in SHR is a consequence of the increased blood pressure remains to be elucidated.

PIKfyve lipid kinase is a protein kinase: downregulation of 5'-phosphoinositide product formation by autophosphorylation.

A subset of phosphoinositide 3-kinase family members are dual specificity enzymes; their protein kinase activity is thought to bring about an additional level to their intracellular regulation. Here we have examined whether the 5'-phosphoinositide kinase PIKfyve, reported previously to catalyze the formation of PtdIns 5-P and PtdIns 3,5-P(2) in vitro [Sbrissa et al. (1999) J. Biol. Chem. 274, 21589-21597], displays dual specificity. We now report that PIKfyve possesses an intrinsic protein kinase activity inseparable from its lipid kinase activity and, besides itself, can phosphorylate exogenous proteins in a substrate-specific manner. Both the autophosphorylation and transphosphorylation were demonstrated with PIKfyve immunopurified or affinity-purified from heterologously transfected COS cells, infected Sf9 cells, or native 3T3-L1 adipocytes. Conversely, no protein kinase activity was associated with immunopurified lipid kinase dead point (K1831E) or truncated (Delta1812-2052) PIKfyve mutants. PIKfyve autophosphorylation or transphosphorylation engaged Ser but not Thr or Tyr residues. PIKfyve autophosphorylation was largely abrogated upon pretreatment with PIKfyve lipid substrates or phosphatases. The impact of autophosphorylation on the PIKfyve lipid kinase activity was further examined with purified PIKfyve preparations. A decrease of 70% in the lipid product formation was associated with PIKfyve autophosphorylation, which was reversed upon treatment with phosphatases. In the cellular context, PIKfyve, or a fraction of it, was found in a phosphorylated form. Collectively, these results indicate that PIKfyve is a dual specificity kinase, which can generate and relay protein phosphorylation signals to regulate the formation of its lipid products, and possibly other events, in the context of living cells.

Mammalian cell morphology and endocytic membrane homeostasis require enzymatically active phosphoinositide 5-kinase PIKfyve.

The dual specificity mammalian enzyme PIKfyve phosphorylates in vitro position d-5 in phosphatidylinositol (PtdIns) and PtdIns 3-P, itself or exogenous protein substrates. Here we have addressed the crucial questions for the identity of the lipid products and the role of PIKfyve enzymatic activity in mammalian cells. CHO, HEK293, and COS cells expressing PIKfyve(WT) at high levels and >90% efficiencies increased selectively the intracellular PtdIns 3,5-P(2) production by 30--55%. In these cell types PtdIns 5-P was undetectable. A kinase-deficient point mutant, PIKfyve(K1831E), transiently transfected into these or other cells elicited a dramatic dominant phenotype. Subsequent to a dilation of the PIKfyve-containing vesicles, PIKfyve(K1831E)-expressing cells progressively accumulated multiple swollen lucent vacuoles of endosomal origin, first in the perinuclear cytoplasm and then toward the cell periphery. Despite their drastically altered morphology, the PIKfyve(K1831E)-expressing cells were viable and functionally active, evidenced by several criteria. This phenotype was completely reversed by introducing PIKfyve(WT) into the PIKfyve(K1831E)-transfected cells. Disruptions of the localization signal in the PIKfyve kinase-deficient mutant yielded a PIKfyve(K1831E Delta fyve) protein, incompetent to vacuolate cells, implying that an active PIKfyve enzyme at distinct late endocytic membranes is crucial for normal cell morphology. This was further substantiated by examining the vacuolation-induced potency of several pharmacological stimuli in cells expressing high PIKfyve(WT) levels. Together, the results indicate that PIKfyve enzymatic activity, possibly through the generation of PtdIns 3,5-P(2), and/or yet to be identified endogenous phosphoproteins, is critical for cell morphology and endomembrane homeostasis.

PIKfyve, a mammalian ortholog of yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides. Effect of insulin.

One or more free hydroxyls of the phosphatidylinositol (PtdIns) head group undergo enzymatic phosphorylation, yielding phosphoinositides (PIs) with key functions in eukaryotic cellular regulation. Two such species, PtdIns 5-P and PtdIns 3,5-P(2), have now been identified in mammalian cells, but their biosynthesis remains unclear. We have isolated a novel mammalian PI kinase, p235, whose exact substrate specificity remained to be determined (Shisheva, A., Sbrissa, D., and Ikonomov, O. (1999) Mol. Cell. Biol. 19, 623-634). Here we report that recombinant p235 expressed in COS cells, like the authentic p235 in adipocytes, displays striking specificity for PtdIns over PI substrates and generates two products identified as PtdIns 5-P and PtdIns 3,5-P(2) by HPLC analyses. Synthetic PtdIns 3-P substrates were also converted to PtdIns 3,5-P(2) but to a substantially lesser extent than PtdIns isolated from natural sources. Important properties of the p235 PI 5-kinase include high sensitivity to nonionic detergents and relative resistance to wortmannin and adenosine. By analyzing deletion mutants in a heterologous cell system, we determined that in addition to the predicted catalytic domain other regions of the molecule are critical for the p235 enzymatic activity. HPLC resolution of monophosphoinositide products, generated by p235 immune complexes derived from lysates of 3T3-L1 adipocytes acutely stimulated with insulin, revealed essentially the same PtdIns 5-P levels as the corresponding p235 immune complexes of resting cells. However, the acute insulin action resulted in an increase of a wortmannin-sensitive PtdIns 3-P peak, suggestive of a plausible recruitment of wortmannin-sensitive PI 3-kinase(s) to p235. In conclusion, mouse p235 (renamed here PIKfyve) displays a strong in vitro activity for PtdIns 5-P and PtdIns 3,5-P(2) generation, implying PIKfyve has a key role in their biosynthesis.