From Work Well-Being to Burnout: A Hypothetical Phase Model.

Upon exposure to chronic stressors, how do individuals move from being in a healthy state to a burnout? Strikingly in literature, this has prevailed a categorical view rather than a dimensional one, thus the underlying process that explains the transition from one state to another remains unclear. The aims of the present study are (a) to examine intermediate states between work engagement and burnout using cluster analysis and (b) to examine cortisol differences across these states. Two-hundred and eighty-one Argentine workers completed self-report measures of work engagement and burnout. Salivary cortisol was measured at three time-points: immediately after awakening and 30 and 40min thereafter. Results showed four different states based on the scores in cynicism, exhaustion, vigor, and dedication: engaged, strained, cynical, and burned-out. Cortisol levels were found to be moderate in the engaged state, increased in the strained and cynical states, and decreased in the burned-out state. The increase/decrease in cortisol across the four stages reconciles apparent contradictory findings regarding hypercortisolism and hypocortisolism, and suggests that they may represent different phases in the transition from engagement to burnout. A phase model from engagement to burnout is proposed and future research aimed at evaluating this model is suggested.

Light pollution: the possible consequences of excessive illumination on retina.

Light is the visible part of the electromagnetic radiation within a range of 380-780 nm; (400-700 on primates retina). In vertebrates, the retina is adapted to capturing light photons and transmitting this information to other structures in the central nervous system. In mammals, light acts directly on the retina to fulfill two important roles: (1) the visual function through rod and cone photoreceptor cells and (2) non-image forming tasks, such as the synchronization of circadian rhythms to a 24 h solar cycle, pineal melatonin suppression and pupil light reflexes. However, the excess of illumination may cause retinal degeneration or accelerate genetic retinal diseases. In the last century human society has increased its exposure to artificial illumination, producing changes in the Light/Dark cycle, as well as in light wavelengths and intensities. Although, the consequences of unnatural illumination or light pollution have been underestimated by modern society in its way of life, light pollution may have a strong impact on people's health. The effects of artificial light sources could have direct consequences on retinal health. Constant exposure to different wavelengths and intensities of light promoted by light pollution may produce retinal degeneration as a consequence of photoreceptor or retinal pigment epithelium cells death. In this review we summarize the different mechanisms of retinal damage related to the light exposure, which generates light pollution.

Synthesis of retinal ganglion cell phospholipids is under control of an endogenous circadian clock: daily variations in phospholipid-synthesizing enzyme activities.

Retinal ganglion cells (RGCs) are major components of the vertebrate circadian system. They send information to the brain, synchronizing the entire organism to the light-dark cycles. We recently reported that chicken RGCs display daily variations in the biosynthesis of glycerophospholipids in constant darkness (DD). It was unclear whether this rhythmicity was driven by this population itself or by other retinal cells. Here we show that RGCs present circadian oscillations in the labeling of [32P]phospholipids both in vivo in constant light (LL) and in cultures of immunopurified embryonic cells. In vivo, there was greater [32P]orthophosphate incorporation into total phospholipids during the subjective day. Phosphatidylinositol (PI) was the most 32P-labeled lipid at all times examined, displaying maximal levels during the subjective day and dusk. In addition, a significant daily variation was found in the activity of distinct enzymes of the pathway of phospholipid biosynthesis and degradation, such as lysophospholipid acyltransferases (AT II), phosphatidate phosphohydrolase (PAP), and diacylglycerol lipase (DGL) in cell preparations obtained in DD, exhibiting differential but coordinated temporal profiles. Furthermore, cultures of immunopurified RGCs synchronized by medium exchange displayed a circadian fluctuation in the phospholipid labeling. The results demonstrate that chicken RGCs contain circadian oscillators capable of generating metabolic oscillations in the biosynthesis of phospholipids autonomously.

c-Fos associates with the endoplasmic reticulum and activates phospholipid metabolism.

c-Fos, a transcription factor that constitutes DNA-binding AP-1 complexes, regulates gene expression that promotes long-lasting cellular changes. We show that, in addition to its transcription factor activity, c-Fos regulates the metabolism of phospholipids cytoplasmically by an AP-1-independent activity. Two waves of c-Fos expression that promote subsequent waves of stimulation of 32P-orthophosphate incorporation into phospholipids are evidenced in quiescent cultured fibroblasts induced to re-enter the cell cycle. The first wave of c-Fos expression peaks at 7.5 min and returns to control levels by 15 min. The second wave starts by 30 min and remains elevated at 120 min. In the first wave, the lipids that incorporate 32P are predominantly second-messenger polyphosphoinositides (PIP, PIP2, PIP3); whereas in the second wave, membrane-biogenesis-related lipids (PI, PE, PA), become radioactive. Both waves of phospholipid activation depend on c-Fos expression. It is interesting that a peptide that blocks AP-1 nuclear import does not affect phospholipid activation. Immunocytochemical examination showed c-Fos immunoreactivity associated to the endoplasmic reticulum. We conclude that c-Fos, rapidly induced upon cell stimulation, associates to the endoplasmic reticulum where it first regulates the synthesis/ replenishment of phospholipids required for signal transduction pathways and subsequently regulates enzymes involved in the genesis of new membrane necessary for cell growth.

Circadian regulation of phospholipid metabolism in retinal photoreceptors and ganglion cells.

The neural retina is a key component of the vertebrate circadian system that is responsible for synchronizing the central circadian pacemaker to external light-dark (LD) cycles. The retina is itself rhythmic, showing circadian cycles in melatonin levels and gene expression. We assessed the in vivo incorporation of 32P-phosphate and 3H-glycerol into phospholipids of photoreceptor cells (PRCs) and retina ganglion cells (GCs) from chicks in constant illumination conditions (dark: DD or light: LL) over a 24-h period. Our findings showed that in DD there was a daily oscillation in 32P-labeling of total phospholipids synthesized in GCs and axonally transported to the brain. This metabolic fluctuation peaked during the subjective night (zeitgeber time [ZT] 20), persisted for several hours well into the subjective day and declined at subjective dusk (ZT 10-12). PRCs also exhibited an in vivo rhythm of 32P-phospholipid synthesis in DD. This rhythm peaked around ZT 22, continued a few hours into the day and declined by the end of subjective dusk. The major individual species labeled 1 h after 32P administration was phosphatidylinositol (PI) in both PRCs and GCs. Rhythmic phospholipid biosynthesis was also observed in DD after 3H-glycerol administration, with levels in GCs elevated from midday to early night. PRCs exhibited a similar rhythmic profile with the lowest levels of labeling during midnight. Phosphatidylcholine (PC) accounted for the individual species with the highest ratio of 3H-glycerol incorporation in both cell populations at all phases examined. By contrast, in LL the rhythm of 3H-glycerol labeling of phospholipids damped out in both cell layers. Our findings support the idea that, in constant darkness, the metabolism of retinal phospholipids, including their de novo biosynthesis, is regulated by an endogenous circadian clock.

Selective regional blockade of junB gene expression in the hamster suprachiasmatic nucleus by a tyrosine kinase inhibitor.

The hypothalamic suprachiasmatic nucleus (SCN) functions as a circadian pacemaker regulating a variety of physiological and behavioral rhythms in mammals. Retinal illumination evokes expression of several immediate-early genes, including junB, in the ventral SCN early in the subjective night and throughout the SCN later in the subjective night. junB mRNA and protein are also expressed spontaneously around subjective dawn in nocturnal rodents, but only in the dorsal SCN. We examined the biochemical signaling mechanisms underlying both spontaneous and light-evoked expression of junB mRNA in the SCN of Syrian hamsters. Hamsters were injected (i.p.) before subjective dawn with vehicle or with either tyrphostin or genistein, inhibitors of protein tyrosine kinase, and maintained in the dark for 30 min. They were then exposed to a light pulse or kept in darkness for another 30 min. In situ hybridization studies demonstrated that tyrphostin pretreatment (12 or 24 mg/kg) reduced both spontaneous and light-evoked expression of junB mRNA only in the dorsal, and not the ventral, portion of the SCN. Conversely, genistein had little effect on either spontaneous or light-evoked expression of junB mRNA in any part of the SCN. These results indicate that a protein tyrosine kinase sensitive to tyrphostin but not to genistein is involved in the transduction pathways leading to expression of junB mRNA selectively in the dorsal SCN, independently of circadian phase and independently of the involvement of light.

Immediate early gene expression within the visual system: light and circadian regulation in the retina and the suprachiasmatic nucleus.

Immediate early genes are a family of genes that share the characteristic of having their expression rapidly and transiently induced upon stimulation of neuronal and non-neuronal cells. In this review, first a short description of the IEGs is given, then it is discussed the stimulus-induced and circadian-induced variations in the expression of IEGs in the visual system, mainly in the retina and the suprachiasmatic nucleus. The possible physiological consequences of these variations in IEG expression are also considered. Finally, we refer to two aspects of our recent studies and those of other laboratories involving light-driven IEG expression. The first is the finding that in the chick retina, the expression of c-fos is differentially modulated in the different cell types and that c-fos regulates the synthesis of the quantitatively most important lipids of all cells, the phospholipids, by a non-genomic mechanism. The second is the occurrence of differential waves of IEG expression in the mammalian suprachiasmatic nucleus regarding light induction or spontaneous oscillations.

A simple method to obtain retinal cell preparations highly enriched in specific cell types. Suitability for lipid metabolism studies.

The neural retina is a highly complex tissue composed of excitatory and inhibitory neurons and of glial cells. The biosynthesis of lipids that occurs in the retina may be distinctly regulated in one neuronal type of cells with respect to another. To study the cell-type-specific aspects of lipid metabolism, a method for the separation of different retinal cell populations is needed. Herein, we describe a very simple procedure to isolate preparations highly enriched in specific retinal cell types that are suitable for in vitro biochemical assays. The method consists of selectively obtaining photoreceptors (PRC) and retina ganglion cells (RGC) from lyophilized chicken retinas using Scotch tape to assess, then, the in vitro incorporation of labeled precursors into phospholipid moieties. When their metabolic capability was assayed, it was found that these cell preparations maintain their enzyme activities intact to incorporate (32)P-phosphate into phospholipids in vitro at a similar rate as observed in fresh tissue after 1 h incubation. The highest proportion of labeling was observed in phosphatidylethanolamine (PE), followed by phosphoinositides (PIPs), phosphatidylcholine (PC) and phosphatidic acid (PA). Phosphatidate-phosphohydrolase (PAPase), a key enzyme of glycerolipid metabolism, exhibits similar levels of activity when assessed in fresh or frozen cell preparations, indicating that the lyophilization procedure does not significantly affect this activity. It is concluded that different cell populations obtained by the experimental procedure described herein, are useful to study the cellular metabolism and its regulation.

Daily rhythm of spontaneous immediate-early gene expression in the rat suprachiasmatic nucleus.

Nocturnal light induces the expression of various immediate-early genes (IEGs) in the suprachiasmatic nucleus (SCN), the primary pacemaker of the circadian system of mammals, and causes phase shifts of behavioral rhythms. In the hamster SCN, some IEGs show both sensitivity to light induction at night and a daily peak of spontaneous expression near dawn in different regions of the nucleus. To investigate whether both patterns of IEG expression are observed in the rat SCN, the authors studied the expression of NGFI-A, junB, c-fos, and fosB near the time of subjective dawn in rats entrained to a light-dark 12:12 cycle and then maintained in constant total darkness for approximately 48 h. They found that there were two independent rhythms of expression for junB and c-fos mRNAs in the SCN: (1) a rhythm of photic sensitivity expressed throughout the night and (2) a spontaneous rhythm of expression triggered around dawn and persisting for at least 2 h into the day. By contrast, fosB and NGFI-A transcripts were expressed only after light exposure at night and did not exhibit significant levels of spontaneous expression in the absence of photic input. These observations demonstrate that the circadian clock gates expression of two independent rhythms related to IEG expression in the rat SCN. The rhythm of sensitivity to nocturnal light exposure is expressed more strongly in the ventral SCN and may be related to photic entrainment. The second rhythm is triggered spontaneously in darkness around subjective dawn and is expressed in more dorsal parts of the SCN.

Light exposure activates retina ganglion cell lysophosphatidic acid acyl transferase and phosphatidic acid phosphatase by a c-fos-dependent mechanism.

We previously reported that the biosynthesis of phospholipids in the avian retina is altered by light stimulation, increasing significantly in ganglion cells in light and in photoreceptor cells in dark. In the present work, we have determined that light significantly increases the incorporation of [3H]glycerol into retina ganglion cell glycerophospholipids in vivo by a Fos-dependent mechanism because an oligonucleotide antisense to c-fos mRNA substantially blocked the light-dark differences. We also studied in vitro the enzyme activities of phosphatidate phosphohydrolase (PAPase), lysophosphatidate acyl transferase (AT II), and phosphatidylserine synthase from retinas of chickens exposed to light or dark. Higher PAPase I and AT II activities were found in incubations of retinal ganglion cells from animals exposed to light; no increase was observed in preparations obtained from light-exposed animals treated with the c-fos antisense oligonucleotide. No light-dark differences were found in phosphatidylserine synthase activity. These findings support the idea that a coordinated photic regulation of PAPase I and AT II is taking place in retina ganglion cells. This constitutes a reasonable mechanism to obtain an overall increased synthesis of glycerophospholipids in stimulated cells that is mediated by the expression of Fos-like proteins.

Circadian and photic regulation of immediate-early gene expression in the hamster suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus is the site of an endogenous circadian clock synchronized by daily light-dark cycles. At some daily phases, light exposure both shifts the clock and alters the expression of several immediate-early genes in cells of the suprachiasmatic nucleus. We have studied both spontaneous circadian and light-induced expression of several immediate-early gene messenger RNAs and proteins in hamsters in constant darkness or in response to brief light exposure. There was no detectable spontaneous expression of NGFI-A messenger RNA in suprachiasmatic nucleus cells at any circadian phase, but light pulses induced its expression selectively during the subjective night, with highest levels of expression 6 h into the night. We also found that there are two independent rhythms of expression of junB messenger RNA and JunB protein, as well as c-fos messenger RNA and c-Fos protein, in the suprachiasmatic nucleus of hamsters: a rhythm of photic sensitivity expressed throughout the night and a spontaneous rhythm of expression triggered around dawn. Induction of NGFI-A messenger RNA and c-fos messenger RNA and c-Fos protein in response to a light pulse were found throughout the suprachiasmatic nucleus, with the highest levels of expression in the ventrolateral subdivision; however, the spontaneous expression of JunB and c-Fos proteins was confined mainly to the dorsomedial suprachiasmatic nucleus. The temporal and anatomical differences in the expression of these immediate-early genes in the mammalian suprachiasmatic nucleus suggest that their protein products may be involved in different signaling mechanisms mediating either photic entrainment or endogenous oscillations within distinct subpopulations of suprachiasmatic nucleus cells.

Differential effects of glutamatergic blockade on circadian and photic regulation of gene expression in the hamster suprachiasmatic nucleus.

Nocturnal light exposure induces immediate-early gene (IEG) expression in the hypothalamic suprachiasmatic nucleus (SCN) and causes phase shifts of activity rhythms in mammals. Some IEGs also show a circadian rhythm of expression in the SCN. While excitatory amino acids (EAAs) are known to be involved in mediating photic regulation of entrainment and gene expression, their involvement in spontaneous rhythms of gene expression has not been studied. We assessed the role of NMDA receptors in the expression of NGFI-A, junB and fosB mRNAs induced by light pulses of different intensities late in the night (Zeitgeber Time [ZT] 18). We also examined the spontaneous expression of junB mRNA near subjective dawn (ZT 0). Both dim (5 lx) and bright (100 lx) light pulses induced similar levels of expression of NGFI-A and junB in the SCN late in the night. fosB mRNA was strongly induced by bright light but was less sensitive to dim light. At ZT 18, dizocilpine (MK-801) (3 mg/kg, i.p. ), a non-competitive NMDA receptor antagonist, almost completely blocked light-evoked expression of IEG mRNAs in the ventral SCN but not in the dorsolateral region at a mid-caudal level using either light intensity. At ZT 0, MK-801 strongly reduced light-evoked expression of junB mRNA in both SCN subdivisions, but inhibited spontaneous expression significantly only in the dorsal region. NMDA receptors appear to play an important role in mediating photic input regulating IEG expression only in the ventral SCN at night. At dawn, however, NMDA receptors are involved in mediating photic effects in both parts of the SCN, as well as being involved in spontaneous activation of junB expression selectively in the dorsal SCN. These findings support the idea that the effects in the dorsolateral SCN of nocturnal light exposure are mediated by different mechanisms than those in other portions of the nucleus.

Light affects c-fos expression and phospholipid synthesis in both retinal ganglion cells and photoreceptor cells in an opposite way for each cell type.

Retina photoreceptor and ganglion cells isolated from chicks that in vivo were exposed to light have a different phospholipid labeling capacity than those from chicks in the dark. In the light exposed animals, the phospholipid labeling in the ganglion cells is higher (Delta% 45, p<0.005) than in those maintained in the dark, whereas in the photoreceptor cells, the opposite occurs, that is, the phospholipid labeling is higher in the dark than in light. The light-dark differences for phospholipid labeling correlate with the expression of c-fos: when c-fos expression increases (both in mRNA and in c-Fos protein content), phospholipid labeling increases concomitantly. That is, in ganglion cells, c-fos expression and the phospholipid synthesis is higher in light with respect to dark, whereas in photoreceptor cells, c-fos expression and phospholipid synthesis is higher in dark with respect to light. Moreover, when an oligonucleotide antisense to c-fos is administered intraocularly prior to separating the animals into light and dark, no differences in c-fos expression and, consequently, no differences in phospholipid synthesis are found between animals in light and dark. Taken together, these results point to a novel mechanism by which rapid genomic responses to cell stimulation are converted to longer lasting changes in the cell components.

Light exposure stimulates the activity of ganglioside glycosyltransferases of retina ganglion cells.

In chicks submitted to light stimulation, the synthesis of gangliosides of the retina ganglion cell increases with respect to chicks maintained in the dark. In an attempt to elucidate if the activation of glycosyltransferases participates in the establishment of these light-dark differences detected in vivo, we examined the activity of a key ganglioside glycosyltransferase, the GalNAc-T (N-acetylgalactosaminyltransferase) that converts GM3 to GM2, in the retina ganglion cells isolated from light and dark exposed chicks. We found that GalNAc-T and other glycosyltransferases are active in these ganglion cell preparations; the kinetic parameters for GalNAc-T were similar to those previously reported for chick retina. The other glycosyltransferase activities assayed were the galactosyltransferase (Gal-T2) that converts GM2 to GM1 and the N-acetylneuraminyltransferase (Sialyl-T1) that converts lactosylceramide to GM3. The three glycosyltransferase activities were higher in the ganglion cell preparations obtained from chicks exposed to light compared to those maintained in the dark. For the GalNAc-T activity, the differences disappear when the cell preparations are sonicated or if the assays are carried out in the presence of detergents or if the end product of the reaction is added to the incubates. The results indicate that the activation of the glycosyltransferases is part of the phenomenon required for cells to achieve the precise rate of synthesis of gangliosides needed in vivo.

Melatonin does not influence the expression of c-fos in the suprachiasmatic nucleus of rats and hamsters.

We have assessed whether melatonin can induce c-fos expression at various circadian phases, and whether melatonin can inhibit photically induced c-fos expression in the suprachiasmatic nucleus (SCN) in both rats and Syrian hamsters. Subcutaneous administration of melatonin at a dose of 100 microg/kg neither induced expression of Fos, the protein product of the c-fos proto-oncogene, nor inhibited the expression of Fos-like immunoreactivity (Fos-lir) induced by a light pulse in the SCN of rats and hamsters. In situ hybridization studies also demonstrated the absence of induction by acute melatonin treatments of c-fos mRNA in the SCN. Taken together, these results demonstrate that melatonin effects on SCN cells involve signal transduction pathways that do not include regulation of c-fos gene expression.

Expression of fosB mRNA in the hamster suprachiasmatic nucleus is induced at only selected circadian phases.

We have studied the expression of fosB mRNA in the suprachiasmatic nucleus (SCN) of hamsters by in situ hybridization using oligonucleotides with sequences complementary to the C-terminal of the fosB mRNA sequence. In animals exposed for 48 h to darkness, there was little or no background expression in SCN cells of fosB mRNA at any circadian phase. Light pulses (30 min) were able to induce fosB expression only during the subjective night. Transcripts of fosB increased rapidly to peak by the end of a 30-min light pulse. Light-induced increases gradually declined in darkness, but levels were still elevated for up to 150 min after the light pulse. Induction in response to a light pulse was largely restricted to the ventrolateral portion of the nucleus which receives the heaviest retinal projection. The temporal and anatomical pattern of fosB mRNA expression in the hamster SCN therefore resembles that reported previously for other immediate-early genes, such as c-fos.

Spontaneous and light-evoked expression of JunB-like protein in the hamster suprachiasmatic nucleus near subjective dawn.

Some cells in the hamster suprachiasmatic nucleus (SCN) show a circadian rhythm of expression of junB mRNA in constant darkness, while others show junB mRNA only in response to light at night. We found that both the light-induced and spontaneous expressions of junB mRNA are translated into protein in SCN cells. In constant darkness, JunB-like immunoreactivity (lir) appears spontaneously in cells in the dorsal SCN around subjective dawn and persists for at least 4 h into the subjective day. During the subjective night, there is no spontaneous expression, but a light pulse can induce JunB-lir in cells throughout the SCN, and especially in the ventrolateral portion. As a component of AP-1 proteins, JunB may play a role both in mediating circadian responses to photic stimuli and in spontaneous oscillation of elements of the SCN circadian pacemaker.

Spontaneous circadian and light-induced expression of junB mRNA in the hamster suprachiasmatic nucleus.

We examined both spontaneous and light-evoked expression of junB mRNA in the hamster suprachiasmatic nucleus (SCN), an endogenous circadian pacemaker. junB was expressed in the SCN in response to a light pulse during the subjective night and early subjective day as well as spontaneously during the transition from subjective night to subjective day. Light-evoked expression was strongest in the ventral SCN, while spontaneous expression was stronger in the dorsal SCN. Spontaneous expression began around subjective dawn and persisted for at least 4 h into the subjective day. The expression of junB mRNA may play a role in both phase-shifting responses to light and in spontaneous oscillation of the SCN pacemaker.

Immediate early gene c-fos regulates the synthesis of phospholipids but not of gangliosides.

Retinal ganglion cells isolated from chicks that in vivo were exposed to light have a higher phospholipid labeling capacity than those obtained from animals in the dark. Actinomycin D or a mixture of protein synthesis inhibitors or of antisense oligonucleotides to c-fos plus c-jun injected intraocularly 1 hr prior to the stimulation period, abolished the light-dark differences for phospholipids but not for gangliosides. Light stimulation induced the formation (and/or stabilization) of c-fos mRNA and of the protein c-Fos, indicating that immediate early gene induction, and consequently the synthesis of the protein(s) encoded, is essential to increase the synthesis of phospholipids but not of gangliosides. These results suggest a novel mechanism by which immediate early genes engram neural cells, modifying specifically the metabolism of cell constituents producing long-lasting changes in the cells.