Proteomic and gene expression analysis of zebrafish brain undergoing continuous light/dark stress

Several organisms irrespective of their complexity in structure and function have an inbuilt circadian rhythm. Zebrafish could be used as an alternate model animal in sleep research as it exhibits similar sleep–wake dynamics as mammals and Drosophila. In this study, we have analysed the adult zebrafish brain for its differential proteome and gene expression during perturbed light/dark cycle. A total of 53 and 25 proteins including sncb, peroxiredoxins and TCR alpha were identified based on two‐dimensional gel electrophoresis Fourier transform mass spectrometer/ion trap tandem mass spectrometer and differential in‐gel electrophoresis MALDI TOF MS/MS analysis, respectively, with at least 1.5‐fold changes between the control and experimental brains. Real time‐polymerase chain reaction revealed that many circadian pathway‐associated genes, such as per1b, bmal1b, cry1b, bmal2 and nr1d2, were differentially regulated during continuous light/dark exposures. It is hypothesized that the differential regulation of these genes might lead to the discovery of potential diagnostic markers for gaining insight into the light/dark‐associated stress in humans.     

Heritable natural variation of light/dark preference in an outbred zebrafish population

Abstract

Anxiety is a fear-like response to stimuli perceived to be threatening. Excessive or uncontrollable anxiety is a debilitating psychiatric disorder which affects many people throughout their lifetime. In unravelling the complex genetic and environmental regulations of anxiety-like phenotypes, models measuring the natural dark avoidance of larval zebrafish have shed light on the individual variation and heritability of this anxiety-related trait. Using the light/dark choice paradigm and selective breeding, this study aims to validate previous findings of the variable (VDA) and strong dark aversion (SDA) heritability in AB-WT larval zebrafish using the outbred zebrafish strain EK, which offers more genetic diversity to aid in future molecular mapping efforts. 190 larvae (6?days post fertilization [dpf] and 7 dpf) were tested across four trials and divided into variable (VDA), medium (MDA) and strong (SDA) dark aversion for further in-crosses. VDA and MDA larvae became more explorative with time, whereas SDA larvae rarely left the preferred light zone. The SDA and VDA in-crosses significantly increased the respective phenotypes in the second generation of larvae, whereas VDA?×?MDA inter-crosses did not. For the second-generation SDA cohort, dark aversion correlated with increased thigmotaxis, which reinforces SDA as an anxiety-like phenotype. Our finding that the dark aversion trait and SDA and VDA phenotypes are heritable in an outbred zebrafish population lays an important foundation for future studies of genetic underpinnings using whole-genome mapping methods. This conserved fear/anxiety-like response in a highly accessible model organism also allows for further pharmacological and behavioral studies to elucidate the etiology of anxiety and the search for novel therapeutics for anxiety disorders.     

Stimuli affecting zebrafish (Danio rerio) behavior in the light/dark preference test

Ethanol has been suggested to have an anxiolytic effect on zebrafish, primarily based on its disruption of the novel tank diving response and of some social behaviors. The light/dark preference test offers a complementary measure of anxiety-like behavior in fish, and the purpose of the current study was to determine the effects of acute ethanol exposure on behavior in the light/dark task. In Experiment 1, the stimuli used to induce light/dark preference in zebrafish were varied in order to determine how best to measure the behavior. Subjects exhibited phototaxis (preference for light) when illumination was manipulated, but scototaxis (preference for dark) when wall and substrate color were manipulated. There was a clear interaction between locomotor activity and color preference, with animals preferentially freezing in darker locations. Because of ambiguity in interpreting behavior in the open/covered version of the test, the black/white version was used in Experiment 2. In Experiment 2, zebrafish were exposed to ethanol (0.25%, 0.5%, or 1.0%) or water for 30 minutes, and then placed in a black/white preference tank containing either ethanol (same doses) or water for a 30-minute test. Ethanol exposure increased locomotor activity and reduced freezing. Additionally, there was a significant interaction between ethanol treatment and locomotor activity on side preference. Low doses of ethanol increased white avoidance in normally swimming fish, while high doses did not.     

Brief constant light accelerates serotonergic re-entrainment to large shifts of the daily light/dark cycle

Brief (∼2 day) constant light exposure (LL_b) in hamsters dramatically enhances circadian phase-resetting induced by the 5-HT receptor agonist, (±)-2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT) and other nonphotic stimuli. The present study was undertaken to determine if LL_b can also amplify phase-resetting responses to endogenous 5-HT and accelerate re-entrainment to large-magnitude advance and delay shifts of the light/dark (LD) cycle. First, central serotonergic activity was increased by i.p. injection of l-tryptophan±the 5-HT reuptake inhibitor fluoxetine. Hamsters under LD or exposed to LL_b received vehicle or drugs during the early morning, and phase-shifts of the locomotor activity rhythm were measured after release to constant darkness. Neither drug phase-shifted animals not exposed to LL_b (P>0.5 vs. vehicle); however in animals receiving LL_b,l-tryptophan with and without fluoxetine produced large phase-advance shifts (means=2.5±0.4 h and 2.6±0.2 h, respectively; both P<0.035 vs. vehicle). Next, the effects of LL_b combined with 8-OH-DPAT or l-tryptophan+fluoxetine on serotonergic re-entrainment to 10 h phase-advance and phase-delay shifts of the LD cycle were assessed. In groups not exposed to LL_b, vehicle controls re-entrained slowly to the advance and delay shifts (means=16±1 and 24±4 days, respectively), but those treated with 8-OH-DPAT re-entrained faster (means=11±2 and 9±2 days, respectively; both P<0.05 vs. vehicle). In groups exposed to LL_b, vehicle controls re-entrained slowly to the advance and delay shifts (means=15±2 and 25±3 days, respectively); however those receiving 8-OH-DPAT rapidly re-entrained to the delay and advance shifts, with the majority (75%) requiring only 1–2 days (means=2±1 and 4±2 days, respectively; both P<0.05 vs. vehicle). Animals exposed to LL_b and treated with l-tryptophan+fluoxetine also exhibited accelerated re-entrainment to a 10 h advance shift (mean=5±2 days; P<0.05 vs. vehicle). Thus through enhancing serotonergic phase-resetting, LL_b facilitates rapid re-entrainment to large shifts of the LD cycle which offers a potential approach for treating circadian-related desynchronies.     

Resetting of the rat circadian clock after a shift in the light/dark cycle depends on the photoperiod.

Adjustment of the circadian clock to shifts in the light/dark (LD) cycle was assessed from the rat pineal N-acetyltransferase (NAT) rhythm which is controlled by a pacemaker in the suprachiasmatic nucleus of the hypothalamus. Re-entrainment to an 8-h delay in the LD cycle took more than 3 days in rats maintained under a regime with 18 h of light and 6 h of darkness per day (LD 18:6) whereas it was completed within 3 days in those maintained under LD 12:12. Re-entrainment to an advance in the LD cycle proceeded through a transient diminution or almost disappearance of the NAT rhythm amplitude following a 5-h, 3-h and even a mere 2-h advance shift under LD 18:6, whereas no such diminution occurred under LD 12:12 even after a 5-h advance shift. Altogether, the data indicate that resetting of the circadian clock after shifts in the LD cycle depends on the photoperiod.     

Developmental changes in glutathione S-transferase isoforms expression and activity in intrasplenic fetal liver tissue transplants in rats

The aim of the present study was to characterise developmental changes in glutathione S-transferase (GST) isoforms expression and in glutathione conjugation capacity in intrasplenic liver tissue transplants. For this purpose, syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fischer 344 rats. Three days, 1, 2, 4 weeks, 2, 4, 6 months and 1 year later, transplant-recipients and control animals were sacrificed and class alpha, mu and pi GST isoforms expression and GST activities using the substrates o-dinitrobenzene and 1-chloro-2,4-dinitrobenzene were assessed in livers and spleens. In the hepatocytes of the adult livers no class pi, but a distinct class alpha and mu GST expression was seen. The bile duct epithelia were class pi GST positive. Fetal livers displayed almost no class alpha and mu, but a slight class pi GST expression. The same pattern was seen in 3-day-old intrasplenic liver tissue transplants. Up to 2 weeks after surgery the class alpha and mu GST expression increased in the hepatocytes of the transplants, whereas the immunostaining for class pi GST disappeared. No remarkable changes were seen thereafter. Normal conjugation capacities were observed with the livers of both groups of rats. Control spleens displayed only low GST activities. From 2 months after transplantation on activities were significantly higher in transplant-containing spleens than in respective control organs with a further increase up to one year after grafting. These results show that intrasplenically transplanted fetal liver cells proliferate and differentiate into mature cells displaying a GST expression pattern with respective enzyme activities similar to adult liver.     

Homeostatic response to sleep/rest deprivation by constant water flow in larval zebrafish in both dark and light conditions

Sleep—or sleep‐like states—have been reported in adult and larval zebrafish using behavioural criteria. These reversible quiescent periods, displaying circadian rhythmicity, have been used in pharmacological, genetic and neuroanatomical studies of sleep–wake regulation. However, one of the important criteria for sleep, namely sleep homeostasis, has not been demonstrated unequivocally. To study rest homeostasis in zebrafish larvae, we rest‐deprived 1‐week‐old larvae with a novel, ecologically relevant method: flow of water. Stereotyped startle responses to sensory stimuli were recorded after the rest deprivation to study arousal threshold using a high‐speed camera, providing an appropriate time resolution to detect species‐specific behavioural responses occurring in a millisecond time‐scale. Rest‐deprived larvae exhibited fewer startle responses than control larvae during the remaining dark phase and the beginning of the light phase, which can be interpreted as a sign of rest homeostasis—often used as equivalent of sleep homeostasis. To address sleep homeostasis further, we probed the adenosinergic system, which in mammals regulates sleep homeostasis. The adenosine A1 receptor agonist, cyclohexyladenosine, administered during the light period, decreased startle responses and increased immobility bouts, while the adenosine antagonist, caffeine, administered during the dark period, decreased immobility bouts. These results suggest that the regulation of sleep homeostasis in zebrafish larvae consists of the same elements as that of other species.     

Fluid intake of rats in constant light and during feeding restricted to the light or dark portion of the illumination cycle

Rats restricted to eating and drinking during the light phase of a 12:12 light-dark (L-D) cycle (L-drinkers) consumed much smaller volumes of saccharin and NaCl than did rats whose feeding and drinking was restricted to the dark phase (D-drinkers). The patterns of fluid intake within the respective 12-hr feeding periods were markedly different for L- and D-drinkers and the food/fluid ratios were significantly lower for D- than for L-drinkers. It was concluded that illumination affects fluid intake independently of changes in food intake and that light limits the rat's capacity for fluid ingestion. L- and D-drinkers did not differ in their preference for saccharin over water in two bottle preference tests; this suggests that light does not interfere with the rat's ability to detect and determine the quality of the taste stimulus. Differences in ingestive behavior between L- and D-drinkers may in part reflect constraints imposed by changes in the sleep-wakefulness cycle. Intake of a wide concentration range of saccharin solutions was decreased in ad lib fed rats housed in constant light (L-L); differences in fluid intake between the L-L and L-D groups varied directly with the hedonic value of the saccharin solutions. Only 44% of rats housed in constant light as compared to 100% of rats maintained in a L-D cycle decreased their intake of a saccharin solution whose ingestion was followed by injection of lithium chloride. This finding is relevant to studies of illness-induced aversions and was related to circadian rhythms in drug susceptibility and their alteration in constant light. The necessity for controlling and specifying environmental illumination in studies of ingestive behavior was emphasized and various methodological considerations related to illumination cycles were discussed.     

Fluctuations in cellular proliferation across the light/dark cycle in the subgranular zone of the dentate gyrus in the adult male Syrian hamster

A lifelong persistent neurogenesis occurs in the dentate gyrus of the mammalian hippocampus. Research in peripheral cell tissue has shown that the timing of cellular division of these cells coincide with the light/dark cycle, however it remains unclear as to whether there is an association between the time of day and cellular proliferation in the brain. The timing of cellular division can be studied through the use of a cellular proliferation marker, such as 5-bromo-2-deoxyuridine (BrdU), which is taken up by the DNA of dividing cells during replication. The goal of this study was to determine whether the time of day affects the number of BrdU labeled cells in the subgranular zone of the dentate gyrus of adult male Syrian hamsters. Adult males received a single systemic injection of BrdU (300 mg/kg) at either the end of the light (ZT-13) or dark phase (ZT-23) of a 14:10 LD cycle and were sacrificed 24 h or 3 days later. Sections through the hippocampus were immunolabeled for BrdU. Cellular proliferation fluctuated across the light/dark cycle during the expansion phase rather than during initial cellular proliferation. A twofold increase in number was expected between 24 and 72 h following a single BrdU injection, but this increase was only seen in the population of cells injected at the end of the light phase.     

Influence of age on behavioural response in the light/dark paradigm.

We have compared the performance of male Swiss mice at different ages (correlated with different body weight; 12-34 g) in the light/dark test of anxiety. Mice received saline only. The best age at which control values were optimum was that of 4 weeks old. Mice at this age spent 58% of the total test duration in the dark compartment. The oldest mice (i.e., 8 weeks old) exhibited an increase in total activity characterised by increase in movements in each compartment, together with an increase in the number of transitions. An age-related effect was found suggesting caution when interpreting the results of mice in the light/dark paradigm, the best period being that of 4 weeks.     

GAD isoforms exhibit distinct spatiotemporal expression patterns in the developing mouse lens: correlation with Dlx2 and Dlx5.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter of the adult nervous system and its biosynthetic enzyme glutamic acid decarboxylase (GAD) are abundantly expressed in the embryonic nervous system and are involved in the modulation of cell proliferation, migration, and differentiation. Here we describe for the first time the expression of GABA and embryonic and adult GAD isoforms in the developing mouse lens. We show that the GAD isoforms are sequentially induced with specific spatiotemporal profiles: GAD65 and embryonic GAD isoforms prevail in primary fibers, while GAD67 is the predominant GAD expressed in the postnatal secondary fibers. This pattern correlates well with the expression of Dlx2 and Dlx5, known as upstream regulators of GAD. GABA and GAD are most abundant at the tips of elongating fibers and are absent from organelle-free cells, suggesting their involvement is primarily in shaping of the cytoskeleton during fiber elongation stages.     

Cholecystokinin concentration in specific brain areas of rats fed during the light or dark phase of the circadian cycle

Measurement of peptide concentration in specific areas can be used as an initial investigative method for identifying brain sites in which the peptides may be acting. In this study cholecystokinin (CCK) concentration in specific hypothalamic and hindbrain areas of male Sprague-Dawley rats was measured in order to determine whether changes occurred as a result of feeding activity during different portions of the circadian cycle. Three groups of 40 rats each were studied: Group 1 were fasted 16 hr during the dark phase then sacrificed immediately or after a 20 min light phase meal. Group 2 were fasted 16 hr during the light phase then sacrificed immediately after lights out or after a 20 min dark-onset meal. Group 3 were fed ad lib and sacrificed immediately after light out or after a 20 min dark-onset meal. CCK was extracted from dissected areas and concentration was measured by RIA. There was no difference in CCK concentration of any of the 9 brain areas in rats fasted during the dark phase and fed during the light phase. In rats fasted during the light phase CCK concentration of the paraventricular nucleus (PVN) was greater in those that subsequently ate a meal at dark-onset than in those that did not eat (p less than 0.05). In ad lib fed rats CCK concentration was less in the anterior hypothalamus (AH) and greater in the supraoptic nucleus (SON) in rats that ate a dark-onset meal than in rats that did not (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification, expression and tissue distribution of the three rat lysyl hydroxylase isoforms

Introduction   Lysyl hydroxylases (LHs) (procollagen-lysine 2-oxoglutarate 5-dioxygenase; PLOD) catalyse the hydroxylation of lysine residues during the post-translational modification of collagenous proteins.
Results   In this poster, we describe the first identification and cloning of LH isoforms 2 and 3 from the rat, including both LH2-splice variants (LH2a and LH2b). The rat LHs are expressed in almost all tissue and cell types examined, indicating a probable lack of tissue specificity for LH function. All LH isoforms were stably transfected into CHO-K1 cells, and this represents the first example of recombinant LH production in a eukaryotic cell line. Expression and production of all LH isoforms led to an increase in total collagen synthesis. LH1 and LH2a expression and production led to an increase in total pyridinium cross-link production.
Discussion   Evidence that LH2a possesses telopeptide lysyl hydroxylase activity, previously thought to be a novel enzyme, is presented.     

GAD67-GFP精神分裂症小鼠行为学改变及海马齿状回颗粒细胞层GABA能神经元的表达

目的:探讨谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠制备精神分裂症模型后学习与记忆功能的改变及海马齿状回颗粒细胞层GABA能神经元的表达。方法:利用聚合酶链式反应(PCR)鉴定GAD67-GFP基因敲入小鼠,MK-801连续腹腔注射2周制备精神分裂症动物模型,通过悬尾实验、Morris水迷宫实验、免疫荧光标记技术等,观察GAD67-GFP基因敲入小鼠的学习与记忆功能的改变及GABA能神经元在海马齿状回颗粒细胞层的表达。结果:停药后实验组与对照组比较:(1)实验组体重增加明显低于对照组(P0.05);(2)行为学改变:1悬尾实验:实验组不动时间明显小于对照组(P0.05);2Morris水迷宫实验:定位航行实验中实验组逃避潜伏期,游泳总路程明显长于对照组(P0.05),而其平均游泳速度与对照组没有明显差异(P0.05);空间探查实验中实验组经过平台所在点的次数和在平台所在象限的时间明显小于对照组(P0.05);(3)在海马齿状回颗粒细胞层中实验组的GFP阳性细胞明显多于对照组(P0.05)。结论:通过对GAD67-GFP基因敲入小鼠进行腹腔注射MK-801制备精神分裂症模型后,其学习与记忆功能显著下降,且海马齿状回颗粒细胞层GABA能神经元明显增加。提示精神分裂症后学习记忆功能减退可能与GABA能神经元的表达有关。     

Changes in gravitational force cause changes in gene expression in the lens of developing zebrafish.

Gravity has been a constant physical factor during the evolution and development of life on Earth. We have been studying effects of simulated microgravity on gene expression in transgenic zebrafish embryos expressing gfp under the influence of gene-specific promoters. In this study, we assessed the effect of microgravity on the expression of the heat shock protein 70 (hsp70) gene in lens during development using transgenic zebrafish embryos expressing gfp under the control of hsp70 promoter/enhancer. Hsp70:gfp expression was up-regulated (45%) compared with controls during the developmental period that included the lens differentiation stage. This increase was lens specific, because the entire embryo showed only a 4% increase in gfp expression. Northern blot and in situ hybridization analysis indicated that the hsp70:gfp expression recapitulated endogenous hsp70 mRNA expression. Hypergravity exposure also increased hsp70 expression during the same period. In situ hybridization analysis for two lens-specific crystallin genes revealed that neither micro- nor hypergravity affected the expression level of betaB1-crystallin, a non-hsp gene used as a marker for lens differentiation. However, hypergravity changed the expression level of alphaA-crystallin, a member of the small hsp gene family. Terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) assay analysis showed that altered-gravity (Deltag) decreased apoptosis in lens during the same period and the decrease correlated with the up-regulation of hsp70 expression, suggesting that elimination of nuclei from differentiating lens fiber cells was suppressed probably through hsp70 up-regulation. These results support the idea that Deltag influences hsp70 expression and differentiation in lens-specific and developmental period specific manners and that hsp family genes play a specific role in the response to Deltag.     

Relationship between the expression of cyclins/cyclin-dependent kinases and sex-steroid receptors/Ki67 in normal human endometrial glands and stroma during the menstrual cycle

Cell cycle regulatory molecules were analysed in normal humanendometrial tissue using antibodies against cyclins D1, E, A,and B1 and cyclin-dependent kinases (CDKs) cdk4, cdk2, and cdc2.The expression of these regulatory molecules in gland cellsand stromal cells was compared with the expression of oestrogenreceptors (ER), progesterone receptors (PR), and Ki67 (a growth-relatedmolecule). In general, a substantially higher percentage ofthe gland cells stained positive for cyclins and CDKs duringthe proliferative phase of the menstrual cycle. Cyclin E, cdk2and/or cdk4 were especially apparent in the cytoplasm of mostof the gland cells as well as in the stromal cells. In contrast,most of the regulatory molecules were undetectable in the glandcells by the end of the secretory phase of the cycle, but theydid not decline in the stromal cells. The data also revealedthat ER, PR, and Ki67 in both gland cells and stromal cellsfollow the same basic pattern of expression as the cyclins andCDKs. These results suggest that cyclins and CDKs are functionallyinvolved in the rhythmic proliferation of normal human endometrialtissue, and the action of these agents may be related to theendometrial levels of sex steroids and Ki67. cyclin/cyclin-dependent kinase/endometrium/Ki67/steroid receptors     

GABAergic neurons in inferior colliculus of the GAD67-GFP knock-in mouse: electrophysiological and morphological properties

Utilizing slice preparations of GAD67-GFP knock-in mouse, in which GABAergic neurons are specifically labeled with GFP fluorescence, we studied electrophysiological characteristics of GABAergic neurons of IC by whole-cell patch clamp-recording combined with biocytin-intracellular-staining techniques. GABAergic neurons of IC fell into two distinct firing types; (1) tonic type neurons and (2) transient (phasic) type neurons. Tonic type neurons showed regularly repetitive discharge pattern in response to a long depolarizing current pulse (200 ms), and transient type neurons showed spike discharges just at the onset of current pulse. Most of neurons of both types showed depolarizing sag in response to hyperpolarizing current pulse, which were blocked by 0.1 mM ZD7288 (Ih blocker). All two types of tonic neurons showed an AHP, which was blocked by Cd2+ (0.1 mM) and high concentration of apamin (2 microM). One of tonic type neurons (BP) revealed a long delay in spike onset or a longer first spike interval when they were stimulated from hyperpolarized potentials. The remaining tonic neurons (RS) did not show this property. Tonic type neurons were distributed in all region of IC. Morphologically, they were not identical; heterogeneous in somatic diameter, dendritic field size and its orientation. One of transient type neurons (Th-) revealed an AHP after the spike. The other transient type neurons (Th+) showed a depolarization hump after the spike, which were blocked by 0.1-0.2 mM Ni2+. Th+ type neurons were found only in the dorsolateral region of IC, having small dendritic field. Th+ type neurons are likely to be a distinct, homogenous group of GABAergic neuron in IC.