Circadian Rhythms: Methods and Protocols

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Tagged studies were separately screened afterwards for inclusion based on the following criterium besides being included in our mapping : studies measuring one or more of the compounds of interest during an undisturbed baseline period of minimally 6 hours including a light-dark transition. Most of the excluded tagged studies measured for shorter periods after pharmacological interventions, or after retrodialysis of various compounds.

During full-text screening, labels were added to all included references by one of the screeners CL or KJ to reflect for which of the compound s of interest histamine and the amino acids asparagine, aspartate, GABA, glutamate, glutamine, glycine, proline and taurine baseline concentrations were provided.

The EROS-file was exported to Excel, where references were sorted by these labels to create the tables provided in the appendices. An individual paper is included in multiple tables if it describes baseline dialysate concentrations of more than one compound of interest. Likewise, when references described measurements in several brain regions e.

In our protocol, we explained that the available risk-of-bias tools are hardly applicable to baseline measurements. We thus planned to provide an indication of study quality, internal validity and risk of bias by tabulating the extracted study characteristics. For the mapping review, only limited data were extracted, and all references are provided. We did not perform a formal risk of bias assessment for these included studies.

For the studies included in our systematised review, the risk of bias was estimated by one experimenter. We added the following elements to the analysis: blinding of sample analysis for detection bias and incomplete outcome data for attrition bias, [ 25 ], and for other sources of bias: verification of probe placement, reporting of an a priori power analysis, approval of an ethical board, and conflicts of interest. Our search in PubMed retrieved references, that in Embase After removal of duplicates, references remained for title-abstract screening.

Of these, were screened full-text, and were included in the mapping review. An overview of the flow of papers is provided in Figure 1. We tagged 47 references during full-text screening as relevant for circadian rhythms, sleep and sleep deprivation. Of these, 7 met the inclusion criterium for our systematised review on circadian rhythms. Our search for relevant title words added 4 references, resulting in 11 references included in our SR on circadian rhythms.

The number of references retrieved by compound is provided in Table 1. The number of references by compound varies from only 6 references for proline to references for glutamate. Concordance of labelling in the subset of studies ranged from Concordance percentages are also provided in Table 1.

JNCASR Library catalog › Results of search for '(su:{Circadian rhythms})'

Only one study described the baseline microdialysate levels for all our compounds of interest [ 26 ], and 4 more described 8 out of 9 [ 4 , 27 , 28 , 29 ]. Most of the studies reported only one studies or two studies of these compounds. References for the included studies are provided by compound in appendix 1—9. The titles in a specific table can be screened one by one, or a search for title words of interest can be performed.

When searching for title words, we highly recommend searching for all known synonyms of a term of interest, and searching for the first letters of the term to retrieve both the singular and the plural use and different spellings. One reference did not mention the sex of the rats, the others all used male animals only. The light schedule was adequately described by 10 out of 11 references, one only mentioned a 12 h light-dark cycle without providing actual times, but expressed the data in Zeitgeber time [ 30 ]. For the 10 papers describing the light-dark cycle, the cycle was normal , lights on at — for 7, reversed for 2 , lights on at or , or partially reversed , lights on at h.

Probe lengths 0. Membrane type e. None of the references mentioned probe reuse. All references described HPLC for sample analysis, 9 with fluorescence detection, 1 with electrochemical detection, one with both detection types. Either histamine only 6 references , or several amino acids 5 references were measured.

Circadian Rhythms: Methods and Protocols

Table 2 lists the 2 studies on aspartate circadian rhythms. In rats, Honma et al. Baseline dialysis commenced at different points of the light-dark cycle, and lasted 24 h. Group averages were calculated after smoothing of the individual animal data with a three-point running average. Circadian effects were tested with a two-way analysis of variance ANOVA which showed a significant effect.

According to the authors, aspartate levels started to decrease before light onset, and increase before dark onset. In hamsters kept on a light-dark cycle lights on at and measured for 24 h, Glass et al. Aspartate peaked during the dark phase; — and — h. To conclude, in the suprachiasmatic nucleus, aspartate is higher during the dark than during the light phase in both rats and hamsters, and the rhythm is also present under urethane anaesthesia [ 24 , 31 ].

Table 3 lists the 3 studies on GABA circadian rhythms. Castaneda et al. Values were expressed as a percentage of the average of the first 8 samples and somehow corrected for slow continuous changes in dialysate concentrations. Circadian rhythms were tested with the Cosinor method to fit the data to a sinusoidal model. If the fit was not significant, but differences between dark and light were apparent, samples 1 and 24 and samples 25 and 48 were compared with linear regression.

GABA did not show circadian variation. Marquez de Prado et al. Rats were on a light-dark cycle; lights ON at Meng et al. Samples were collected at 2 h intervals over a 24 h period. Data were analyzed with paired-samples t-test and independent-samples t-test.

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The authors observed a circadian pattern with relatively high levels during the dark period and low levels during the light period. To conclude, results for GABA are too inconsistent to generalise [ 23 , 32 , 33 ].

Table 4 lists the 5 studies on glutamate circadian rhythms. These studies were all described above. According to Castaneda et al. Honma et al. Glass et al. To conclude, glutamate seems consistently higher during the dark than during the light phase in the suprachiasmatic nucleus in hamsters and in several brain regions in freely behaving and anaesthetised rats [ 23 , 24 , 31 , 32 , 33 ]. Table 5 lists the 3 studies on glutamine circadian rhythms. The study by Marquez de Prado et al. To conclude, no rhythmicity is observed in glutamine in different brain regions and different species [ 23 , 31 , 32 ].

Table 6 lists the 6 studies on histamine circadian rhythms. Fell et al. Rats were kept on a light-dark cycle; lights ON at The circadian rhythm was not specifically analysed, but from the figures it is clear that baseline histamine increases during the dark compared to the light period. Chu et al. The circadian rhythm was not specifically analysed, but the figure shows that baseline histamine increases during the dark compared to the light period.

Hong et al. Dialysates were collected for h starting at The circadian rhythm was again not specifically analysed, but the figure shows that histamine is higher at the onset of the dark phase compared to the light phase. Zant et al. Individual animal data also show higher histamine during dark than during light.

Mochizuki et al. They collected dialysates over variable periods of time over the circadian cycle. Average histamine concentrations were higher during dark than during light. Rozov et al. The authors found h and overlaid 8-h periodicities in histamine release, with an orthophase at To conclude, histamine seems consistently higher during the dark than during the light phase in the hypothalamus in mice and in several brain regions in rats [ 34 , 35 , 36 , 37 , 38 , 39 ].

Only one included reference measured taurine; in the neostriatum of Wistar rats. This study by Marquez de Prado et al. Taurine showed no circadian rhythm. Reporting of study characteristics was analysed by reference. None of the included references described blinding of sample analysis, missing samples or power analyses. Only 1 out of 11 references contained a statement on conflicts of interest. Approval of the protocol by an ethical board was reported in 6 out of 11 references. Methods for verification of probe placement were reported in 8 out of 11 references, but only 3 showed the results.

Our systematic map provides an overview of the papers describing baseline microdialysis measurements of histamine and the amino acids asparagine, aspartate, GABA, glutamate, glutamine, glycine, proline and taurine. In our results section, we describe how scientists can use this systematic map, to save the time involved in performing a systematic search and selection for one or more of the analysed compounds themselves.

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In our systematized review, we provide an example. The map shows that many more microdialysis studies have measured glutamate and GABA than proline and histamine. This partially echoes researchers interests in compounds as a neurotransmitter but is probably also reflecting the relative ease of measuring glutamate in dialysates. Included studies describe analytical methods [ 40 , 41 ], functional neuroanatomy [ 42 , 43 ], basic pharmacology [ 44 , 45 ], the neurochemistry of behaviour [ 46 , 47 , 48 ], and the neurochemistry of several disorders described in the introduction.

The 11 references on circadian rhythms show a relative abundance of studies on histamine, but none of them addressed asparagine, glycine or proline, and only one was on taurine. For asparagine, GABA, glycine, proline and taurine, further primary studies on circadian rhythms are warranted. Several authors have published SRs on the microdialysis technique, but we are aware of only one SR on amino acids [ 49 ]. Fliegel et al. We do not know if they followed a prespecified review protocol. Their search was more focussed than ours, they used key search terms and searched one database, which resulted in a selection of the available literature; studies reporting baseline glutamate and or GABA levels. While methodology of this SR was not described to the extent that we can evaluate its quality, it provides extremely useful overviews of the baseline concentrations of glutamate and GABA for several brain regions.

Our comprehensive search strategy makes this the most complete review of amino acids in microdialysates up to date. Because the size of the review was larger than anticipated, we had to adapt our protocol. By our labelling approach we managed to create a map of the literature by amino acid of interest.

We only labelled studies for measurements of asparagine, aspartate, GABA, glutamate, glutamine, glycine, histamine, proline and taurine. In the included papers, we regularly came across microdialysis measurements of other amino acids, e. Future systematic reviews should summarise the findings for these compounds.

RNA Extraction from mammalian tissues. Stuart N. Peirson and Jason N. Butler Northern analysis of sense and antisense frequency RNA in Neurospora crassa. RNase protection assay. Quantitative PCR. Butler V. Gene Expression: Proteins. Protein extraction, fractionation and purification from cyanobacteria. Natalia B. Ivleva and Susan S. Protein extraction from Drosophila heads. Plant protein extraction. Helen E. Conlon and Michael G. Salter Protein extraction from mammalian tissues. Choogon Lee Western blotting.

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    Foulkes Manipulation of mammalian cell lines for circadian studies. Filippo Tamanini Reporter assays. Use of firefly luciferase activity assays to monitor circadian molecular rhythms in vivo and in vitro.


    Wangjie Yu and Paul E. Hardin SCN cultures that maintain rhythmic properties in vitro. Tominaga-Yoshino, T. Ueyama and H.

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    Okamura VII. Microscopy analysis. RNA in situ hybridisations on Drosophila whole mounts. Corinna Wulbeck and Charlotte Helfrich-Forster In situ hybridisation of SCN slices. Horacio O. Immunohistochemistry in Drosophila, sections and whole mounts. Charlotte Helfrich-Forster Immunofluorescence analysis of circadian protein dynamics in cultured mammalian cells. Filippo Tamanini. Show more. Not what you expected?

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