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An optimized method for measuring hypocretin-1 peptide in the mouse brain reveals differential circadian regulation of hypocretin-1 levels rostral and caudal to the hypothalamus
Neuroscience 310 (2015) 354–361
AN OPTIMIZED METHOD FOR MEASURING HYPOCRETIN-1 PEPTIDE IN THE MOUSE BRAIN REVEALS DIFFERENTIAL CIRCADIAN REGULATION OF HYPOCRETIN-1 LEVELS ROSTRAL AND CAUDAL TO THE HYPOTHALAMUS J. L. JUSTINUSSEN, a A. HOLM a AND B. R. KORNUM a,b*
dian, metabolic and limbic structures. Lack of hypocretin signaling is implicated in type 1 narcolepsy, a sleep disorder characterized by excessive daytime sleepiness, cataplexy (loss of muscle tone induced by positive emotions), and rapid-eye-movement sleep disturbances (Partinen et al., 2014). The complex symptomatology of narcolepsy demonstrates the complexity of hypocretin signaling. Hypocretin projections are found throughout the brain (Peyron et al., 1998; Cutler et al., 1999; Nambu et al., 1999), innervating areas important for wakefulness (locus coeruleus), reward seeking and motivation (ventral tegmental area), mostly positive emotions and social interaction (amygdala), and central motor control (Harris et al., 2005; Carter et al., 2010; Blouin et al., 2013; Hu et al., 2015). The hypocretin system is modulated by homeostatic states (Yoshida et al., 2001; Zeitzer et al., 2007; Carter et al., 2009), but circadian regulation has also been suggested. Further, it is known that the circadian clock is sensitive to feedback from arousal stimuli, and it has been suggested that the hypocretin system plays an important role in this feedback via the neuropeptide Y-containing neurons of the intergeniculate leaflet (Nixon and Smale, 2005; Pekala et al., 2011; Belle et al., 2014). Circadian oscillation in prepro-hypocretin messenger ribonucleic acid (mRNA) or hypocretin-1 peptide (hypocretin-2 is difficult to measure reliably) levels have been observed in rat, non-human primates, and humans with a peak during the waking state (Estabrooke et al., 2001; Fujiki et al., 2001; Yoshida et al., 2001; Salomon et al., 2003; Zeitzer et al., 2003). The only study of 24-h fluctuations of hypocretin-1 peptide levels in the mouse brain so far did not reveal circadian variations, likely due to methodological limitations (Lin et al., 2002). Circadian fluctuation in hypocretin-1 peptide levels in the rat brain can be ablated by suprachiasmic nucleus lesioning (Deboer et al., 2004; Zhang et al., 2004). However, there is no evidence of a direct synaptic connection between the suprachiasmic nucleus and hypocretin neurons. Instead, the circadian clock has been suggested to drive the hypocretin-1 system through suprachiasmic nucleus output circuits (Deurveilher and Semba, 2005). Taheri et al. (2000) measured hypocretin-1 peptide in rat brain homogenate from different areas and found diurnal variation in the hypothalamus and pons but not in other regions suggesting local regulatory mechanisms. It has also been
a
Molecular Sleep Lab, Department of Clinical Biochemistry, Rigshospitalet, Glostrup, Denmark b Danish Center for Sleep Medicine, Department of Clinical Neurophysiology, Rigshospitalet, Glostrup, Denmark
Abstract—The hypocretin/orexin system regulates, among other things, sleep and energy homeostasis. The system is likely regulated by both homeostatic and circadian mechanisms. Little is known about local differences in the regulation of hypocretin activity. The aim of this study was to establish an optimized peptide quantification method for hypocretin-1 extracted from different mouse brain areas and use this method for investigating circadian fluctuations of hypocretin-1 levels in these areas. The results show that hypocretin-1 peptide can be extracted from small pieces of intact tissue, with sufficient yield for measurements in a standard radioimmunoassay. Utilizing the optimized method, it was found that prepro-hypocretin mRNA and peptide show circadian fluctuations in the mouse brain. This study further demonstrates that the hypocretin-1 peptide level in the frontal brain peaks during dark as does prepro-hypocretin mRNA in the hypothalamus. However, in midbrain and brainstem tissue caudal to the hypothalamus, there was less circadian fluctuation and a tendency for higher levels during the light phase. These data suggest that regulation of the hypocretin system differs between brain areas. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: hypocretin, orexin, circadian rhythm, peptides, hypothalamus.
INTRODUCTION Hypocretin-1 and -2 (also known as orexin A and B) are neuropeptides exclusively produced in the lateral hypothalamus (de Lecea et al., 1998; Sakurai et al., 1998). Hypocretin signaling regulates homeostatic functions through integration of multiple inputs from circa*Correspondence to: B. R. Kornum, Molecular Sleep Laboratory, Rigshospitalet, Glostrup, Nordre Ringvej 69, 2600 Glostrup, Denmark. Abbreviations: CPM, counts per minute; CV, coefficient of variation; DPBS, dulbecco’s phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; KO, knock out; mRNA, messenger ribonucleic acid; RIA, radioimmunoassay; qPCR, quantitative real-time polymerase chain reaction; DCT, change in the threshold count. http://dx.doi.org/10.1016/j.neuroscience.2015.09.050 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 354
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demonstrated that hypocretin neurons in the perifornical region exhibit more diurnal variation in their activity than hypocretin neurons in the lateral hypothalamic area (Estabrooke et al., 2001). This could indicate a functional heterogeneity across the hypocretin nucleus. This paper presents an optimized method for extracting and measuring prepro-hypocretin mRNA and hypocretin-1 peptide in different brain areas from the same mouse. This method was next used in an investigation of circadian fluctuation of hypocretin-1.
EXPERIMENTAL PROCEDURES Animals All mice were wild type C57BL/6 (from Nordic Taconic) or hypocretin knock out (KO) of the same background. The hypocretin KO mouse strain was kindly provided by Associate Professor Giovanna Zoccoli and Associate Professor Alessandro Silvani, PRISM lab, Bologna University. Eight-week-old female mice were used with the exception of two control experiments (non-specific binding and comparison of homogenized and intact brains) where ten-week-old male mice were used (animal approval 2014-15-2934-01025). During the course of the optimization experiments, it was found that some cages of mice were fighting, so it was decided to use females to reduce the chance of this type of behavior. No data are included from the fighting mice, but the optimization in male brain tissue was not repeated in females. All mice were sacrificed by cervical dislocation without anesthesia (the effects of anesthesia on peptide hypocretin extraction and quantification are not known) and all dissections were done between 13:00 and 15:00 with the exception of the 24-h study. Mice had ad libitum access to food and water and were kept on a 12:12 h light:dark cycle (lights on at 7:00 am). Mice were generally housed four to a cage, except in the 24-h study when they were housed six to a cage. During this experiment, one cage was removed from the housing area for sacrifice every three hours starting at Z = 2 (Z = 0 is lights on) until the following day at the same time (Z = 22). For the optimization experiments, 47 animals were used. Fifty-four animals were used for the 24-h study. These experiments were carried out in accordance with the Council Directive 2010/63EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes. Dissections For optimization of peptide extraction from the brain, different brain dissections were tested. (A) Halves: the brain tissue was divided in two equal halves following the corpus callosum and extending this cut through the cerebellum and brainstem. (B) Three parts: The brain tissue was divided by a coronal cut, immediately rostral to the optic chiasm and another cut perpendicular to the corpus callosum, immediately rostral to the start of the cerebellum (Fig. 1a). All cortex was removed from the section between the two cuts. This section includes
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hypothalamus, thalamus and other midbrain regions, but is referred to as ‘‘hypothalamus”. The tissue section rostral to this is referred to as ‘‘rostral” and the section caudal to this is referred to as ‘‘caudal”. All brain tissue samples were snap-frozen on dry ice. Tissue preparation We tested three different preparations of tissue for peptide extraction. (A) The tissue was kept intact (referred to as ‘‘intact”). (B) The tissue thawed just enough so that a scalpel could cut the tissue into smaller pieces and immediately refrozen on dry ice (referred to as ‘‘cut”). In optimization experiments, the tissue was cut into smaller pieces than in the 24-h study. In the 24-h study the tissue was cut twice, approximately in half and then each half in half again (four pieces). This kept the dissection more simple and reproducible and reduced the amount of time that the tissue was thawed. (C) The tissue was homogenized by mixing with lysing matrix D ceramic beads (MP biomedicals, Santa Ana, California, USA) and 600 lL of cold homogenizing buffer. The homogenizing buffer consisted of one protease inhibitor tablet (complete protease inhibitor cocktail tablet from Roche Diagnostics GmbH, Mannheim, Germany), 500 lL of EDTA and 49.5 mL of dulbecco’s phosphate-buffered saline (DPBS) to make 50 mL of solution. The sample in buffer was placed in a FastPrep FP120 (speed: 4.0 for 30 s) and then incubated on ice for five minutes. Three hundred micro liter was transferred to a new tube for peptide quantification and RNA purification (see below). Peptide extraction Peptide was extracted from the tissue by adding 0.5 M cold acetic acid to the tube containing the intact or cut tissue (10 lL acetic acid solution for 1 mg tissue). For the homogenized samples, 1.2 mL of acetic acid solution was added to each sample. The tubes containing tissue with the acetic acid solution were placed in boiling water for ten minutes and cooled on ice for five minutes. Samples were centrifuged at 1700g for five minutes and the supernatant was moved to a new tube kept on ice and then placed in a Heto SpeedVac to evaporate overnight at room temperature. Radioimmunoassay Hypocretin-1 peptide was quantified using a radioimmunoassay (RIA) kit from Phoenix Pharmaceuticals, Inc, Karlsruhe, Germany (Catalog No: RK-003-30). Samples were resuspended in the provided radioimmunoassay buffer with deionized water containing a dissolved complete protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany) and run in duplicates following the manufacturer’s instructions. Final results were calculated in GraphPad Prism 5.0 using the ‘‘log(inhibitor) vs. response-variable slope (four parameters)” function to fit the standard curve. An experiment was conducted in eight-week-old male and female hypocretin KO mice from the same litter with a littermate control to test for nonspecific binding in tissue and there was no evidence of non-specific binding
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Fig. 1. Hypocretin-1 peptide can be measured reliably in smaller mouse brain areas. (A) Schematic representation of the dissection method. The cortex was removed from the middle section before processing. Arrows represent direction of cut. (B) Non-specific binding controls. Standard curve from the RIA along with the counts per minute (CPM) of a typical wild-type littermate (gray point). The lower dashed line represents the mean CPM of the hypocretin KO mice (n = 5). The top dashed line is a control included in the RIA kit that does not contain any tissue. (C) Hypocretin-1 peptide concentration in right brain, left brain, and tissue rostral to the optic chiasm (n = 8). Caudal sections were not included in this experiment. (D) Hypocretin-1 peptide concentration in the caudal section and the weight of the tissue (n = 54). Data from the rostral section also show no correlation with weight (not shown). (E) Comparison of tissue processing methods. Hypocretin-1 peptide levels from rostral or caudal sections either intact, cut into smaller pieces, or homogenized before peptide extraction (n = 10, 14, and 3) respectively. The data are combined from two different experiments.
(Fig. 1b). For the 24-h study, all samples were measured in one assay. The optimization experiments were run on different assays for each optimization step.
and glyceraldehyde 3-phosphate dehydrogenase (Mm99999915_g1) genes as controls. All samples were run in technical triplicates.
RNA quantification
Statistics
For the 24-h study, the middle section described in the dissections part of the methods sections was used for messenger RNA quantification by real-time polymerase chain reaction. This tissue was homogenized in 300 lL buffer (provided with the NucleospinÒ miRNA isolation kit from Macherey–Nagel) using lysing matrix D ceramic beads (from MP biomedicals, Santa Ana, California, USA) and the same FastPrep settings as described above. Total RNA containing miRNAs were isolated from the tissue using the NucleospinÒ miRNA isolation kit from Macherey–Nagel according to the manufacturer’s guidelines. RNA was eluted in a total volume of 30 lL and concentration determined using NanoDrop2000C (Thermo Scientific, Waltham, Massachusetts, United States). Quantitech Reverse Transcription kit (Qiagen, Venlo, Netherlands) was used for cDNA synthesis. TaqMan Gene Expression Master Mix and prepro-hypocretin (Mm01964030_s1) probe were used for quantitative real-time polymerase chain reaction (qPCR) with beta-actin (Mm00607939_s1)
All statistical analyses were done in Prism 5.0 (GraphPad). In all experiments, data were checked for points three standard deviations from the mean. No points were excluded as outliers in any experiment. For the dissection experiments, groups were compared using an unpaired t-test and the halves were compared using a paired t-test. For the linear regressions, time points were grouped and the linear regressions plotted by the individual time points. For the tissue processing experiments and non-specific binding control experiments, all groups are compared using an unpaired t-test. For the 24-h experiment, results were divided into mRNA results and peptide results and then combined in a final analysis. For the mRNA results, the mean of the change in the threshold count (DCT) values for the light phase samples was used as the reference in the fold change calculation. The rostral and caudal comparisons of light and dark phase time points are compared using unpaired t-tests. In the final figure comparing peptide and mRNA results, the same fold change and peptide
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concentrations are calculated as before and a linear regression was fit to the data.
RESULTS Hypocretin-1 peptide can be reliably measured using the rostral part of the mouse brain The purpose of these experiments was to determine if dissecting the brain before extracting peptide produced an adequate yield of hypocretin-1 peptide for quantification. We compared left and right halves (from the same brain) with the smaller rostral cut of mouse brain tissue. Our data showed that higher concentration of hypocretin-1 peptide was detected in the rostral sections than in the brain halves (n = 8/group, P = <0.0001 and P = 0.0008 comparing rostral to right and left respectively) (Fig. 1c). The brain halves show no significant differences between the right and left. The coefficient of variation (CV) for the right, left and rostral samples are 10.96%, 19.64% and 17.20% respectively. We determined whether or not the weight of the tissue sample correlated with peptide concentrations. For this hypocretin-1 was measured in both rostral and caudal sections of the same mice (n = 54 from the 24 h study). We did not see a correlation between hypocretin-1 concentration and weight of the tissue within the same brain section (Fig. 1d). Tissue processing affects the amount of hypocretin-1 peptide measured We also studied the effect of tissue processing on peptide yields (Fig. 1e). Three different processing protocols were tested: mouse brain tissue was homogenized, cut, or left intact. Our data showed that concentration of hypocretin1 varied between the different processing methods. First we compared intact and homogenized brains (triangles in Fig. 1e). The highest concentration was obtained from the intact tissue, while completely homogenizing the tissue resulted in 53.9% lower levels measured. The CV of the intact tissue was 3.84% while the CV of the homogenized tissue was CV of 31.65%. Next we compared the two milder processing strategies (intact and cut, circles Fig. 1e). In the cut tissue the hypocretin-1 concentration was 35.3% lower compared to intact tissue. For rostral tissue, the coefficients of variation for intact and cut tissue were 46.44% and 15.98% respectively. The CVs for the caudal samples were 60.24% and 42.26% respectively. All processing methods resulted in sufficient peptide concentrations for radioimmunoassay. From these data, we observed that intact tissue yielded higher concentrations of peptide, but cutting the tissue appeared to reduce the variation in concentration within groups. For the final experiment, it was decided to cut the tissue, but use fewer cuts than were used in these optimization experiments. Different patterns of hypocretin-1 peptide measurement in different parts of the brain We next wanted to determine rostral and caudal hypocretin-1 peptide concentrations over the 24-h cycle
Fig. 2. Circadian variation in hypocretin-1 peptide concentrations in different brain areas. Hypocretin-1 peptide concentration in tissue from (A) the rostral section and (B) the caudal section. For both panels A and B the shaded region represents the dark phase, n = 6 for each time point. (C) Average hypocretin-1 peptide concentration in the light and dark phase in different tissue sections (P = 0.0013 and 0.1401, left to right). n = 24 for light and n = 30 for dark condition in each tissue dissection.
(Fig. 2). Our analysis revealed different 24-h patterns for rostral samples and caudal samples. The rostral data showed lower expression in the light period than in the dark (active) phase (P = 0.0013). The caudal data showed less fluctuation across the 24-h period and did not show a significant difference overall between light and dark periods, but a tendency for higher levels in the light phase was observed. Peak time of hypocretin-1 concentration in rostral samples was Z = 22 (mean = 232.02 pg/mL) followed by Z = 13 (mean = 210.96 pg/mL). In caudal samples the peak time of hypocretin-1
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Fig. 3. Circadian variation in prepro-hypocretin mRNA. (A) Fold change in gene expression throughout a 24-h period (n = 6 for each timepoint). Z = 0 is lights on. The shaded regions represent the dark phases of the day and the unshaded region represents the light phase. The mean DCT of the light phase was used as reference in the fold change calculation. (B) Average prepro-hypocretin mRNA expression during the light and dark phases (P = 0.0010). The samples numbers for the light and dark sections are n = 24 and n = 30 respectively.
concentration was Z = 4 (mean = 200.94 pg/mL). Of note, the first and last time points were taken at the same time on different days and thus we expected to show similar concentrations, but in the rostral samples they did not. From the same brains where peptide levels were measured in rostral and caudal sections, we also measured prepro-hypocretin mRNA expression from the hypothalamic section (Fig. 3). These mRNA data showed a similar trend as the rostral data; with significantly lower expression in the light phase than in the dark phase (P = 0.0010). Comparing the mRNA expression to the peptide data shown above, the correlation with mRNA also differed between the tissue dissections (Fig. 4). The rostral hypocretin-1 peptide concentrations increased (P = 0.0033) with prepro-hypocretin mRNA expression while the caudal peptide levels decreased (P = 0.0276).
DISCUSSION Peptide data from different parts of the brain show different fluctuation patterns over 24 h The purpose of the 24-h study was to apply the method optimizations discussed in this paper and to observe
Fig. 4. Correlations between hypocretin-1 peptide concentration and prepro-hypocretin mRNA expression. Peptide concentration compared to the mRNA fold change in the hypothalamus sections using the mean of the light phase as a reference value in (A) rostral samples (P = 0.0033, R2 = 0.15) and (B) caudal samples (P = 0.0276, R2 = 0.09). n = 54 in both figures.
hypocretin-1 peptide concentrations rostral and caudal to the hypothalamus over the 24-h cycle. This study found that different regions of the brain had different patterns over 24 h. The rostral tissue showed diurnal tendencies with higher hypocretin-1 peptide concentrations during the dark phase and lower concentrations in the light phase. The peak of the hypocretin-1 concentration occurred early in the lightsoff period in the rostral section while the nadir occurred early in the dark phase. The shape of this fluctuation (sinoidal or otherwise) is difficult to determine from this experiment without repeating the experiment for a higher/larger sample size and/or more time points. The peak and nadir of the caudal tissue shows an opposite trend compared to the rostral concentration with the peak being in the light phase and the nadir being in the dark phase. Overall, the caudal samples showed less diurnal variation in hypocretin-1 peptide concentration. When looking at gene expression, our data show a diurnal pattern in prepro-hypocretin expression with decreased concentrations toward the end of the light phase and increased concentrations in the dark phase. Taheri et al. (2000) also demonstrated a diurnal variation in prepro-hypocretin mRNA in rats with the peak expression in whole hypothalamus being at the beginning of
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lights on (ZT = 0) and the lowest level of expression being at lights off in rats. In our experiment peak mRNA expression was at the end of the dark face, with no difference between ZT = 19 and ZT = 22. We did not measure at ZT = 0, but taken together the two results support the current understanding of hypocretin activity increasing toward the end of the wake period, to counteract the increasing sleep pressure. Our results show that diurnal variation of hypocretin-1 peptide levels differs between regions. Previous research in rats has also shown that hypocretin-1 peptide concentrations fluctuate over the 24-h cycle. Yoshida et al. (2001) demonstrated using microdialysis that hypocretin-1 peptide levels in the hypothalamus and medial thalamus show diurnal variation in rats with a pattern similar to the one we found in the rostral part of the brain. No other brain areas were included in that study. Taheri et al. (2000) studied diurnal variation in hypocretin-1 peptide levels in 16 different rat brain areas and found diurnal fluctuation in the preoptic/anterior hypothalamus and pons, but not in other brain regions. Interestingly, their data show the same pattern as we observe in mice with peptide levels in the preoptic/anterior hypothalamus reflecting mRNA fluctuations peaking at the end of the dark phase, while pons showed much less fluctuation and almost the opposite pattern with an increase in hypocretin-1 levels during the light phase. This paper presents data supporting a differential regulation of hypocretin-1 peptide levels in different brain areas. This may be related to the function of hypocretin-1 signaling in these areas. For example, frontal areas where hypocretin signaling is related to wake functions have higher levels of hypocretin-1 during the dark phase and show more diurnal variation, while areas involved in functions such as motor control during sleep show different patterns of hypocretin-1 levels. Our data could suggest regional differences in time delay between changes in gene expression and peptide processing. There may also be regional differences in prepro-hypocretin translation and processing at the level of the hypocretin synapses. Another explanation is that there may be regional differences in release of peptide since, by not homogenizing the tissue, these peptide measurements do likely not include intracellular hypocretin-1. Finally, our data might also reflect the possible existence of subgroups of hypocretin neurons, as have been suggested before (Estabrooke et al., 2001). Future studies should focus on this. Further, a simple and fast method like the one described here will allow further studies exploring the basis of the differential regulation we observe. It would be interesting to see how changes in homeostatic sleep pressure or circadian rhythm would be reflected in the hypocretin-1 levels of different brain areas. Using mice, this regulation can further be explored in various transgene models. Methodological considerations Dissections. We demonstrate that dissecting the brain revealed information about regional differences in peptide concentrations. These experiments also revealed that
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higher concentrations of peptide were measured in the rostral and caudal sections than in the brain halves. Some previous experiments by other research groups utilized whole or half brain for peptide quantification (e.g. Lin et al., 2002). Our experiment shows that using brain halves leads to lower concentrations of hypocretin-1 peptide being extracted. One consideration for these experiments is that all of the dissections were done by the same person, even in the 24-h study, in order to have more consistent dissections. During the 24-h study the dissection speed increased and plateaued across the time points. This may be evidence that the person who performed the dissection became more comfortable with the protocol through the course of the experiment (although the researcher was familiar with the dissection) but may also be evidence of the researcher becoming more tired throughout the experiment. Both are certain to introduce some variation into the experiment. The coefficient of variation percentage (CV%) between time points did not increase throughout the experiment or vary drastically from one time point to another so no time points were excluded.
Tissue processing. Our results demonstrate that homogenizing the tissue is not necessarily the best way to prepare the tissue because there is more variation and the concentrations are lower compared to using intact tissue or cutting the tissue into smaller pieces. The release of peptidases from the homogenized tissue could be a possible explanation for this observation. Further experiments are needed to see if extracting from the intact tissue results in more extracellular peptide being extracted than intracellular, which could also explain some of the variation seen in cutting or homogenizing the tissue. Based on the results in this paper, it is difficult to conclude if intact tissue or cutting into smaller pieces is the superior method, although it is advisable to be consistent in how the tissue is cut. Too many cuts results in concentrations similar to those of the homogenized tissue; low concentrations and more variation within the group.
Experimental execution. In this experiment, time points were set-up with the intention that the first and last time points would be at the same time of day and it was expected that they would have similar concentrations of hypocretin-1. For rostral samples and gene expression data, it can be seen that there is a large difference between the first and last time points, which are done at the same time on different days. This is not the case in the caudal samples. It is possible that the cycle is not necessarily exactly 24 h, as many ‘‘circadian cycles” are not exactly 24 h, but are additionally regulated by the suprachiasmic nucleus or light input. It is also possible that the nature of the experiment (collecting tissue at many time points with two people in 30 h) contributed to this discrepancy. No changes to the external environment in the animal facility were noticed between days, but there could have
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been changes in smell or sound undetectable to the experimenters.
CONCLUSION The purpose of this paper was to optimize peptide quantification by exploring modifications in dissections and peptide extraction. We conclude that homogenizing the tissue may not be the best way to prepare tissue for peptide extraction because it leads to higher variation and lower concentrations of peptide and that dissecting the tissue before peptide extraction can allow for the collection of gene expression data from the same animals and allow for more insights on the regional regulation of hypocretin-1. These experiments further demonstrated that hypocretin-1 peptide concentrations measured over a 24-h period show different fluctuations when tissue rostral to the hypothalamus and tissue caudal to the hypothalamus are measured independently. This experiment also compared peptide quantification to gene expression over 24-h and found a stronger correlation with peptide levels rostral to the hypothalamus compared to caudal. Further studies are needed to detangle the functional consequence of this. This study demonstrates, for the first time, clear differences in circadian variation of hypocretin-1 peptide levels in frontal and caudal mouse brain areas. This observation of regional differences in the regulation of hypocretin-1 is of importance for understanding hypocretin signaling and function.
CONTRIBUTION JLT: performed experiments and data analysis, contributed to the design of the study, and wrote the manuscript. AH: Contributed to the 24-h study, performed qPCR experiments and analysis, and revised the manuscript along with responsible for submitting the manuscript. BRK: Designed the study, data analysis, wrote the manuscript, and provided financial support. Acknowledgments—The authors would like to thank Associate Professors Giovanna Zoccoli and Alessandro Silvani, PRISM lab, Bologna University for providing the hcrt-KO mice. This study was funded by the Lundbeck Foundation – Denmark. The authors have no conflict of interest.
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(Accepted 19 September 2015) (Available online 28 September 2015)
AN OPTIMIZED METHOD FOR MEASURING HYPOCRETIN-1 PEPTIDE IN THE MOUSE BRAIN REVEALS DIFFERENTIAL CIRCADIAN REGULATION OF HYPOCRETIN-1 LEVELS ROSTRAL AND CAUDAL TO THE HYPOTHALAMUS J. L. JUSTINUSSEN, a A. HOLM a AND B. R. KORNUM a,b*
dian, metabolic and limbic structures. Lack of hypocretin signaling is implicated in type 1 narcolepsy, a sleep disorder characterized by excessive daytime sleepiness, cataplexy (loss of muscle tone induced by positive emotions), and rapid-eye-movement sleep disturbances (Partinen et al., 2014). The complex symptomatology of narcolepsy demonstrates the complexity of hypocretin signaling. Hypocretin projections are found throughout the brain (Peyron et al., 1998; Cutler et al., 1999; Nambu et al., 1999), innervating areas important for wakefulness (locus coeruleus), reward seeking and motivation (ventral tegmental area), mostly positive emotions and social interaction (amygdala), and central motor control (Harris et al., 2005; Carter et al., 2010; Blouin et al., 2013; Hu et al., 2015). The hypocretin system is modulated by homeostatic states (Yoshida et al., 2001; Zeitzer et al., 2007; Carter et al., 2009), but circadian regulation has also been suggested. Further, it is known that the circadian clock is sensitive to feedback from arousal stimuli, and it has been suggested that the hypocretin system plays an important role in this feedback via the neuropeptide Y-containing neurons of the intergeniculate leaflet (Nixon and Smale, 2005; Pekala et al., 2011; Belle et al., 2014). Circadian oscillation in prepro-hypocretin messenger ribonucleic acid (mRNA) or hypocretin-1 peptide (hypocretin-2 is difficult to measure reliably) levels have been observed in rat, non-human primates, and humans with a peak during the waking state (Estabrooke et al., 2001; Fujiki et al., 2001; Yoshida et al., 2001; Salomon et al., 2003; Zeitzer et al., 2003). The only study of 24-h fluctuations of hypocretin-1 peptide levels in the mouse brain so far did not reveal circadian variations, likely due to methodological limitations (Lin et al., 2002). Circadian fluctuation in hypocretin-1 peptide levels in the rat brain can be ablated by suprachiasmic nucleus lesioning (Deboer et al., 2004; Zhang et al., 2004). However, there is no evidence of a direct synaptic connection between the suprachiasmic nucleus and hypocretin neurons. Instead, the circadian clock has been suggested to drive the hypocretin-1 system through suprachiasmic nucleus output circuits (Deurveilher and Semba, 2005). Taheri et al. (2000) measured hypocretin-1 peptide in rat brain homogenate from different areas and found diurnal variation in the hypothalamus and pons but not in other regions suggesting local regulatory mechanisms. It has also been
a
Molecular Sleep Lab, Department of Clinical Biochemistry, Rigshospitalet, Glostrup, Denmark b Danish Center for Sleep Medicine, Department of Clinical Neurophysiology, Rigshospitalet, Glostrup, Denmark
Abstract—The hypocretin/orexin system regulates, among other things, sleep and energy homeostasis. The system is likely regulated by both homeostatic and circadian mechanisms. Little is known about local differences in the regulation of hypocretin activity. The aim of this study was to establish an optimized peptide quantification method for hypocretin-1 extracted from different mouse brain areas and use this method for investigating circadian fluctuations of hypocretin-1 levels in these areas. The results show that hypocretin-1 peptide can be extracted from small pieces of intact tissue, with sufficient yield for measurements in a standard radioimmunoassay. Utilizing the optimized method, it was found that prepro-hypocretin mRNA and peptide show circadian fluctuations in the mouse brain. This study further demonstrates that the hypocretin-1 peptide level in the frontal brain peaks during dark as does prepro-hypocretin mRNA in the hypothalamus. However, in midbrain and brainstem tissue caudal to the hypothalamus, there was less circadian fluctuation and a tendency for higher levels during the light phase. These data suggest that regulation of the hypocretin system differs between brain areas. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: hypocretin, orexin, circadian rhythm, peptides, hypothalamus.
INTRODUCTION Hypocretin-1 and -2 (also known as orexin A and B) are neuropeptides exclusively produced in the lateral hypothalamus (de Lecea et al., 1998; Sakurai et al., 1998). Hypocretin signaling regulates homeostatic functions through integration of multiple inputs from circa*Correspondence to: B. R. Kornum, Molecular Sleep Laboratory, Rigshospitalet, Glostrup, Nordre Ringvej 69, 2600 Glostrup, Denmark. Abbreviations: CPM, counts per minute; CV, coefficient of variation; DPBS, dulbecco’s phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; KO, knock out; mRNA, messenger ribonucleic acid; RIA, radioimmunoassay; qPCR, quantitative real-time polymerase chain reaction; DCT, change in the threshold count. http://dx.doi.org/10.1016/j.neuroscience.2015.09.050 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 354
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demonstrated that hypocretin neurons in the perifornical region exhibit more diurnal variation in their activity than hypocretin neurons in the lateral hypothalamic area (Estabrooke et al., 2001). This could indicate a functional heterogeneity across the hypocretin nucleus. This paper presents an optimized method for extracting and measuring prepro-hypocretin mRNA and hypocretin-1 peptide in different brain areas from the same mouse. This method was next used in an investigation of circadian fluctuation of hypocretin-1.
EXPERIMENTAL PROCEDURES Animals All mice were wild type C57BL/6 (from Nordic Taconic) or hypocretin knock out (KO) of the same background. The hypocretin KO mouse strain was kindly provided by Associate Professor Giovanna Zoccoli and Associate Professor Alessandro Silvani, PRISM lab, Bologna University. Eight-week-old female mice were used with the exception of two control experiments (non-specific binding and comparison of homogenized and intact brains) where ten-week-old male mice were used (animal approval 2014-15-2934-01025). During the course of the optimization experiments, it was found that some cages of mice were fighting, so it was decided to use females to reduce the chance of this type of behavior. No data are included from the fighting mice, but the optimization in male brain tissue was not repeated in females. All mice were sacrificed by cervical dislocation without anesthesia (the effects of anesthesia on peptide hypocretin extraction and quantification are not known) and all dissections were done between 13:00 and 15:00 with the exception of the 24-h study. Mice had ad libitum access to food and water and were kept on a 12:12 h light:dark cycle (lights on at 7:00 am). Mice were generally housed four to a cage, except in the 24-h study when they were housed six to a cage. During this experiment, one cage was removed from the housing area for sacrifice every three hours starting at Z = 2 (Z = 0 is lights on) until the following day at the same time (Z = 22). For the optimization experiments, 47 animals were used. Fifty-four animals were used for the 24-h study. These experiments were carried out in accordance with the Council Directive 2010/63EU of the European Parliament and the Council of 22 September 2010 on the protection of animals used for scientific purposes. Dissections For optimization of peptide extraction from the brain, different brain dissections were tested. (A) Halves: the brain tissue was divided in two equal halves following the corpus callosum and extending this cut through the cerebellum and brainstem. (B) Three parts: The brain tissue was divided by a coronal cut, immediately rostral to the optic chiasm and another cut perpendicular to the corpus callosum, immediately rostral to the start of the cerebellum (Fig. 1a). All cortex was removed from the section between the two cuts. This section includes
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hypothalamus, thalamus and other midbrain regions, but is referred to as ‘‘hypothalamus”. The tissue section rostral to this is referred to as ‘‘rostral” and the section caudal to this is referred to as ‘‘caudal”. All brain tissue samples were snap-frozen on dry ice. Tissue preparation We tested three different preparations of tissue for peptide extraction. (A) The tissue was kept intact (referred to as ‘‘intact”). (B) The tissue thawed just enough so that a scalpel could cut the tissue into smaller pieces and immediately refrozen on dry ice (referred to as ‘‘cut”). In optimization experiments, the tissue was cut into smaller pieces than in the 24-h study. In the 24-h study the tissue was cut twice, approximately in half and then each half in half again (four pieces). This kept the dissection more simple and reproducible and reduced the amount of time that the tissue was thawed. (C) The tissue was homogenized by mixing with lysing matrix D ceramic beads (MP biomedicals, Santa Ana, California, USA) and 600 lL of cold homogenizing buffer. The homogenizing buffer consisted of one protease inhibitor tablet (complete protease inhibitor cocktail tablet from Roche Diagnostics GmbH, Mannheim, Germany), 500 lL of EDTA and 49.5 mL of dulbecco’s phosphate-buffered saline (DPBS) to make 50 mL of solution. The sample in buffer was placed in a FastPrep FP120 (speed: 4.0 for 30 s) and then incubated on ice for five minutes. Three hundred micro liter was transferred to a new tube for peptide quantification and RNA purification (see below). Peptide extraction Peptide was extracted from the tissue by adding 0.5 M cold acetic acid to the tube containing the intact or cut tissue (10 lL acetic acid solution for 1 mg tissue). For the homogenized samples, 1.2 mL of acetic acid solution was added to each sample. The tubes containing tissue with the acetic acid solution were placed in boiling water for ten minutes and cooled on ice for five minutes. Samples were centrifuged at 1700g for five minutes and the supernatant was moved to a new tube kept on ice and then placed in a Heto SpeedVac to evaporate overnight at room temperature. Radioimmunoassay Hypocretin-1 peptide was quantified using a radioimmunoassay (RIA) kit from Phoenix Pharmaceuticals, Inc, Karlsruhe, Germany (Catalog No: RK-003-30). Samples were resuspended in the provided radioimmunoassay buffer with deionized water containing a dissolved complete protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany) and run in duplicates following the manufacturer’s instructions. Final results were calculated in GraphPad Prism 5.0 using the ‘‘log(inhibitor) vs. response-variable slope (four parameters)” function to fit the standard curve. An experiment was conducted in eight-week-old male and female hypocretin KO mice from the same litter with a littermate control to test for nonspecific binding in tissue and there was no evidence of non-specific binding
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Fig. 1. Hypocretin-1 peptide can be measured reliably in smaller mouse brain areas. (A) Schematic representation of the dissection method. The cortex was removed from the middle section before processing. Arrows represent direction of cut. (B) Non-specific binding controls. Standard curve from the RIA along with the counts per minute (CPM) of a typical wild-type littermate (gray point). The lower dashed line represents the mean CPM of the hypocretin KO mice (n = 5). The top dashed line is a control included in the RIA kit that does not contain any tissue. (C) Hypocretin-1 peptide concentration in right brain, left brain, and tissue rostral to the optic chiasm (n = 8). Caudal sections were not included in this experiment. (D) Hypocretin-1 peptide concentration in the caudal section and the weight of the tissue (n = 54). Data from the rostral section also show no correlation with weight (not shown). (E) Comparison of tissue processing methods. Hypocretin-1 peptide levels from rostral or caudal sections either intact, cut into smaller pieces, or homogenized before peptide extraction (n = 10, 14, and 3) respectively. The data are combined from two different experiments.
(Fig. 1b). For the 24-h study, all samples were measured in one assay. The optimization experiments were run on different assays for each optimization step.
and glyceraldehyde 3-phosphate dehydrogenase (Mm99999915_g1) genes as controls. All samples were run in technical triplicates.
RNA quantification
Statistics
For the 24-h study, the middle section described in the dissections part of the methods sections was used for messenger RNA quantification by real-time polymerase chain reaction. This tissue was homogenized in 300 lL buffer (provided with the NucleospinÒ miRNA isolation kit from Macherey–Nagel) using lysing matrix D ceramic beads (from MP biomedicals, Santa Ana, California, USA) and the same FastPrep settings as described above. Total RNA containing miRNAs were isolated from the tissue using the NucleospinÒ miRNA isolation kit from Macherey–Nagel according to the manufacturer’s guidelines. RNA was eluted in a total volume of 30 lL and concentration determined using NanoDrop2000C (Thermo Scientific, Waltham, Massachusetts, United States). Quantitech Reverse Transcription kit (Qiagen, Venlo, Netherlands) was used for cDNA synthesis. TaqMan Gene Expression Master Mix and prepro-hypocretin (Mm01964030_s1) probe were used for quantitative real-time polymerase chain reaction (qPCR) with beta-actin (Mm00607939_s1)
All statistical analyses were done in Prism 5.0 (GraphPad). In all experiments, data were checked for points three standard deviations from the mean. No points were excluded as outliers in any experiment. For the dissection experiments, groups were compared using an unpaired t-test and the halves were compared using a paired t-test. For the linear regressions, time points were grouped and the linear regressions plotted by the individual time points. For the tissue processing experiments and non-specific binding control experiments, all groups are compared using an unpaired t-test. For the 24-h experiment, results were divided into mRNA results and peptide results and then combined in a final analysis. For the mRNA results, the mean of the change in the threshold count (DCT) values for the light phase samples was used as the reference in the fold change calculation. The rostral and caudal comparisons of light and dark phase time points are compared using unpaired t-tests. In the final figure comparing peptide and mRNA results, the same fold change and peptide
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concentrations are calculated as before and a linear regression was fit to the data.
RESULTS Hypocretin-1 peptide can be reliably measured using the rostral part of the mouse brain The purpose of these experiments was to determine if dissecting the brain before extracting peptide produced an adequate yield of hypocretin-1 peptide for quantification. We compared left and right halves (from the same brain) with the smaller rostral cut of mouse brain tissue. Our data showed that higher concentration of hypocretin-1 peptide was detected in the rostral sections than in the brain halves (n = 8/group, P = <0.0001 and P = 0.0008 comparing rostral to right and left respectively) (Fig. 1c). The brain halves show no significant differences between the right and left. The coefficient of variation (CV) for the right, left and rostral samples are 10.96%, 19.64% and 17.20% respectively. We determined whether or not the weight of the tissue sample correlated with peptide concentrations. For this hypocretin-1 was measured in both rostral and caudal sections of the same mice (n = 54 from the 24 h study). We did not see a correlation between hypocretin-1 concentration and weight of the tissue within the same brain section (Fig. 1d). Tissue processing affects the amount of hypocretin-1 peptide measured We also studied the effect of tissue processing on peptide yields (Fig. 1e). Three different processing protocols were tested: mouse brain tissue was homogenized, cut, or left intact. Our data showed that concentration of hypocretin1 varied between the different processing methods. First we compared intact and homogenized brains (triangles in Fig. 1e). The highest concentration was obtained from the intact tissue, while completely homogenizing the tissue resulted in 53.9% lower levels measured. The CV of the intact tissue was 3.84% while the CV of the homogenized tissue was CV of 31.65%. Next we compared the two milder processing strategies (intact and cut, circles Fig. 1e). In the cut tissue the hypocretin-1 concentration was 35.3% lower compared to intact tissue. For rostral tissue, the coefficients of variation for intact and cut tissue were 46.44% and 15.98% respectively. The CVs for the caudal samples were 60.24% and 42.26% respectively. All processing methods resulted in sufficient peptide concentrations for radioimmunoassay. From these data, we observed that intact tissue yielded higher concentrations of peptide, but cutting the tissue appeared to reduce the variation in concentration within groups. For the final experiment, it was decided to cut the tissue, but use fewer cuts than were used in these optimization experiments. Different patterns of hypocretin-1 peptide measurement in different parts of the brain We next wanted to determine rostral and caudal hypocretin-1 peptide concentrations over the 24-h cycle
Fig. 2. Circadian variation in hypocretin-1 peptide concentrations in different brain areas. Hypocretin-1 peptide concentration in tissue from (A) the rostral section and (B) the caudal section. For both panels A and B the shaded region represents the dark phase, n = 6 for each time point. (C) Average hypocretin-1 peptide concentration in the light and dark phase in different tissue sections (P = 0.0013 and 0.1401, left to right). n = 24 for light and n = 30 for dark condition in each tissue dissection.
(Fig. 2). Our analysis revealed different 24-h patterns for rostral samples and caudal samples. The rostral data showed lower expression in the light period than in the dark (active) phase (P = 0.0013). The caudal data showed less fluctuation across the 24-h period and did not show a significant difference overall between light and dark periods, but a tendency for higher levels in the light phase was observed. Peak time of hypocretin-1 concentration in rostral samples was Z = 22 (mean = 232.02 pg/mL) followed by Z = 13 (mean = 210.96 pg/mL). In caudal samples the peak time of hypocretin-1
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Fig. 3. Circadian variation in prepro-hypocretin mRNA. (A) Fold change in gene expression throughout a 24-h period (n = 6 for each timepoint). Z = 0 is lights on. The shaded regions represent the dark phases of the day and the unshaded region represents the light phase. The mean DCT of the light phase was used as reference in the fold change calculation. (B) Average prepro-hypocretin mRNA expression during the light and dark phases (P = 0.0010). The samples numbers for the light and dark sections are n = 24 and n = 30 respectively.
concentration was Z = 4 (mean = 200.94 pg/mL). Of note, the first and last time points were taken at the same time on different days and thus we expected to show similar concentrations, but in the rostral samples they did not. From the same brains where peptide levels were measured in rostral and caudal sections, we also measured prepro-hypocretin mRNA expression from the hypothalamic section (Fig. 3). These mRNA data showed a similar trend as the rostral data; with significantly lower expression in the light phase than in the dark phase (P = 0.0010). Comparing the mRNA expression to the peptide data shown above, the correlation with mRNA also differed between the tissue dissections (Fig. 4). The rostral hypocretin-1 peptide concentrations increased (P = 0.0033) with prepro-hypocretin mRNA expression while the caudal peptide levels decreased (P = 0.0276).
DISCUSSION Peptide data from different parts of the brain show different fluctuation patterns over 24 h The purpose of the 24-h study was to apply the method optimizations discussed in this paper and to observe
Fig. 4. Correlations between hypocretin-1 peptide concentration and prepro-hypocretin mRNA expression. Peptide concentration compared to the mRNA fold change in the hypothalamus sections using the mean of the light phase as a reference value in (A) rostral samples (P = 0.0033, R2 = 0.15) and (B) caudal samples (P = 0.0276, R2 = 0.09). n = 54 in both figures.
hypocretin-1 peptide concentrations rostral and caudal to the hypothalamus over the 24-h cycle. This study found that different regions of the brain had different patterns over 24 h. The rostral tissue showed diurnal tendencies with higher hypocretin-1 peptide concentrations during the dark phase and lower concentrations in the light phase. The peak of the hypocretin-1 concentration occurred early in the lightsoff period in the rostral section while the nadir occurred early in the dark phase. The shape of this fluctuation (sinoidal or otherwise) is difficult to determine from this experiment without repeating the experiment for a higher/larger sample size and/or more time points. The peak and nadir of the caudal tissue shows an opposite trend compared to the rostral concentration with the peak being in the light phase and the nadir being in the dark phase. Overall, the caudal samples showed less diurnal variation in hypocretin-1 peptide concentration. When looking at gene expression, our data show a diurnal pattern in prepro-hypocretin expression with decreased concentrations toward the end of the light phase and increased concentrations in the dark phase. Taheri et al. (2000) also demonstrated a diurnal variation in prepro-hypocretin mRNA in rats with the peak expression in whole hypothalamus being at the beginning of
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lights on (ZT = 0) and the lowest level of expression being at lights off in rats. In our experiment peak mRNA expression was at the end of the dark face, with no difference between ZT = 19 and ZT = 22. We did not measure at ZT = 0, but taken together the two results support the current understanding of hypocretin activity increasing toward the end of the wake period, to counteract the increasing sleep pressure. Our results show that diurnal variation of hypocretin-1 peptide levels differs between regions. Previous research in rats has also shown that hypocretin-1 peptide concentrations fluctuate over the 24-h cycle. Yoshida et al. (2001) demonstrated using microdialysis that hypocretin-1 peptide levels in the hypothalamus and medial thalamus show diurnal variation in rats with a pattern similar to the one we found in the rostral part of the brain. No other brain areas were included in that study. Taheri et al. (2000) studied diurnal variation in hypocretin-1 peptide levels in 16 different rat brain areas and found diurnal fluctuation in the preoptic/anterior hypothalamus and pons, but not in other brain regions. Interestingly, their data show the same pattern as we observe in mice with peptide levels in the preoptic/anterior hypothalamus reflecting mRNA fluctuations peaking at the end of the dark phase, while pons showed much less fluctuation and almost the opposite pattern with an increase in hypocretin-1 levels during the light phase. This paper presents data supporting a differential regulation of hypocretin-1 peptide levels in different brain areas. This may be related to the function of hypocretin-1 signaling in these areas. For example, frontal areas where hypocretin signaling is related to wake functions have higher levels of hypocretin-1 during the dark phase and show more diurnal variation, while areas involved in functions such as motor control during sleep show different patterns of hypocretin-1 levels. Our data could suggest regional differences in time delay between changes in gene expression and peptide processing. There may also be regional differences in prepro-hypocretin translation and processing at the level of the hypocretin synapses. Another explanation is that there may be regional differences in release of peptide since, by not homogenizing the tissue, these peptide measurements do likely not include intracellular hypocretin-1. Finally, our data might also reflect the possible existence of subgroups of hypocretin neurons, as have been suggested before (Estabrooke et al., 2001). Future studies should focus on this. Further, a simple and fast method like the one described here will allow further studies exploring the basis of the differential regulation we observe. It would be interesting to see how changes in homeostatic sleep pressure or circadian rhythm would be reflected in the hypocretin-1 levels of different brain areas. Using mice, this regulation can further be explored in various transgene models. Methodological considerations Dissections. We demonstrate that dissecting the brain revealed information about regional differences in peptide concentrations. These experiments also revealed that
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higher concentrations of peptide were measured in the rostral and caudal sections than in the brain halves. Some previous experiments by other research groups utilized whole or half brain for peptide quantification (e.g. Lin et al., 2002). Our experiment shows that using brain halves leads to lower concentrations of hypocretin-1 peptide being extracted. One consideration for these experiments is that all of the dissections were done by the same person, even in the 24-h study, in order to have more consistent dissections. During the 24-h study the dissection speed increased and plateaued across the time points. This may be evidence that the person who performed the dissection became more comfortable with the protocol through the course of the experiment (although the researcher was familiar with the dissection) but may also be evidence of the researcher becoming more tired throughout the experiment. Both are certain to introduce some variation into the experiment. The coefficient of variation percentage (CV%) between time points did not increase throughout the experiment or vary drastically from one time point to another so no time points were excluded.
Tissue processing. Our results demonstrate that homogenizing the tissue is not necessarily the best way to prepare the tissue because there is more variation and the concentrations are lower compared to using intact tissue or cutting the tissue into smaller pieces. The release of peptidases from the homogenized tissue could be a possible explanation for this observation. Further experiments are needed to see if extracting from the intact tissue results in more extracellular peptide being extracted than intracellular, which could also explain some of the variation seen in cutting or homogenizing the tissue. Based on the results in this paper, it is difficult to conclude if intact tissue or cutting into smaller pieces is the superior method, although it is advisable to be consistent in how the tissue is cut. Too many cuts results in concentrations similar to those of the homogenized tissue; low concentrations and more variation within the group.
Experimental execution. In this experiment, time points were set-up with the intention that the first and last time points would be at the same time of day and it was expected that they would have similar concentrations of hypocretin-1. For rostral samples and gene expression data, it can be seen that there is a large difference between the first and last time points, which are done at the same time on different days. This is not the case in the caudal samples. It is possible that the cycle is not necessarily exactly 24 h, as many ‘‘circadian cycles” are not exactly 24 h, but are additionally regulated by the suprachiasmic nucleus or light input. It is also possible that the nature of the experiment (collecting tissue at many time points with two people in 30 h) contributed to this discrepancy. No changes to the external environment in the animal facility were noticed between days, but there could have
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been changes in smell or sound undetectable to the experimenters.
CONCLUSION The purpose of this paper was to optimize peptide quantification by exploring modifications in dissections and peptide extraction. We conclude that homogenizing the tissue may not be the best way to prepare tissue for peptide extraction because it leads to higher variation and lower concentrations of peptide and that dissecting the tissue before peptide extraction can allow for the collection of gene expression data from the same animals and allow for more insights on the regional regulation of hypocretin-1. These experiments further demonstrated that hypocretin-1 peptide concentrations measured over a 24-h period show different fluctuations when tissue rostral to the hypothalamus and tissue caudal to the hypothalamus are measured independently. This experiment also compared peptide quantification to gene expression over 24-h and found a stronger correlation with peptide levels rostral to the hypothalamus compared to caudal. Further studies are needed to detangle the functional consequence of this. This study demonstrates, for the first time, clear differences in circadian variation of hypocretin-1 peptide levels in frontal and caudal mouse brain areas. This observation of regional differences in the regulation of hypocretin-1 is of importance for understanding hypocretin signaling and function.
CONTRIBUTION JLT: performed experiments and data analysis, contributed to the design of the study, and wrote the manuscript. AH: Contributed to the 24-h study, performed qPCR experiments and analysis, and revised the manuscript along with responsible for submitting the manuscript. BRK: Designed the study, data analysis, wrote the manuscript, and provided financial support. Acknowledgments—The authors would like to thank Associate Professors Giovanna Zoccoli and Alessandro Silvani, PRISM lab, Bologna University for providing the hcrt-KO mice. This study was funded by the Lundbeck Foundation – Denmark. The authors have no conflict of interest.
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(Accepted 19 September 2015) (Available online 28 September 2015)