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Distribution and daily variations of PACAP in the chicken brain
Peptides 22 (2001) 1371–1377
Distribution and daily variations of PACAP in the chicken brain R. Jo´zsaa, A. Somogyva´ri-Vighb,c,*, D. Reglo¨dia, T. Hollo´sya, A. Arimurab,c a
Department of Anatomy, University of Pe´cs, Pe´cs 7624 Hungary Department of Medicine, Tulane University, New Orleans, LA 70112, USA c US -Japan Biomedical Research Laboratories, Tulane University Hebert Center, Belle Chasse, LA 70037, USA b
Abstract Levels of PACAP38 were measured in different areas of the chicken brain under various lighting conditions by radioimmunoassay (RIA). Selected groups of animals were maintained under light for 14 h alternating with 10 h of darkness (LD), reversed lighting conditions (DL) and constant light (LL) or constant dark (DD). Daily variations of PACAP levels were observed in the brainstem, diencephalon, telencephalon and retina. In the brainstem and diencephalon, levels of PACAP increased during subjective nighttime, except in the DL group where levels were elevated between 15–21 h. In the telencephalon, the lowest level of PACAP was measured between 12–21 h except in the DL group where two peaks occurred at 18 and 03 h. In the retina, all 4 groups showed a similar level and pattern, with lowest levels during midday hours. No daily variation was observed in the pineal gland. According to the present observations, it is suggested that PACAP levels differ in several areas of the chicken brain under various lighting conditions and photic stimuli do not appear to be the main regulators of the circadian variations of PACAP. © 2001 Elsevier Science Inc. All rights reserved. Keywords: PACAP; chicken; daily variation; radioimmunoassay
1. Introduction Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalami on the basis of its adenylate cyclase activating activity. It belongs to the VIP/secretin/glucagon peptide family, and shows closest homology to VIP. The primary structure of PACAP is highly conserved during phylogeny: in vertebrate species it shows only 1– 4 amino acid differences from mammalian PACAP [2]. PACAP has been shown also in the chicken nervous system. Chicken PACAP has 1 amino acid substitute compared to human PACAP. The genes encoding chicken PACAP and its receptors are also similar to human PACAP genes [23,29,38]. PACAP and PACAP receptor immunoreactivity and their mRNAs are widely distributed in different regions of the chicken brain and peripheral organs [29,30]. It has also been shown that PACAP stimulates cAMP formation in various areas of the chicken brain [27]. PACAP is a pleiotropic peptide, having numerous actions in the central nervous system (CNS) and peripheral organs [2]. One of its recently shown functions is the mod* Corresponding author. Tel.: ⫹1-504-394-7199; fax: ⫹1-504-3947169. E-mail address: [email protected] (A. Somogyvari-Vigh).
ulation of circadian activity. Circadian rhythms are coordinated by endogenous mechanisms, that can be entrained by environmental stimuli, most notably by the daily changes in light intensity. In mammals, the suprachiasmatic nucleus (SCN) is a major circadian pacemaker. Photic cues are transmitted to the SCN via the retinohypothalamic tract [11]. In this way, light entrains the SCN and its driven output rhythms to the 24-h day. PACAP is abundant in the rat SCN, in the retinohypothalamic pathway and retinal ganglionic cells [11,14]. Daily variations of PACAP receptor mRNA have been demonstrated in the SCN and supraoptic nuclei of the rat hypothalamus, suggesting that PACAP receptors are differentially expressed across the 24-h cycle [5]. PACAP has also been shown to reset the circadian clock in a manner similar to light [6,16]. These and other results suggest that PACAP is involved in the circadian pacemaker clock in mammals [5,15,21,37]. Avian circadian rhythms are coordinated by oscillators consisting of the retina, pineal gland and SCN, and the relative importance of each structure and their connections varies among avian species [22,39]. Interactions among these structures serve to generate coordinated circadian outputs. These outputs control a vast array of circadian rhythms of biological processes. No data are presently available on the circadian variations of PACAP in the chicken brain. In the present study, we investigated the distribution of
0196-9781/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 1 ) 0 0 4 7 7 - 6
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PACAP-like immunoreactivity (PACAP-LI) by means of radioimmunoassay (RIA) in different regions of the chicken brain, and we examined the daily variation of PACAP-LI under different lighting conditions. 2. Methods
The detailed RIA procedure and HPLC verification of chicken PACAP have been described previously [3,38]. The PACAP38 antiserum [88111–3] was generated against synthetic PACAP [24 –38] and recognizes PACAP38 as well as peptides containing the epitope of PACAP [24 –38], but not PACAP27.
2.1. Animals
2.4. Protein assay
One-day old chicken were purchased from a local supplier (Mohacs, Hungary), and kept under different lighting conditions starting from the day of purchase for 4 weeks. Experiments were carried out in summer (June–July), with room temperatures ranging between 22–25°C. The animals (n ⫽ 128) were distributed into 4 groups: LD group was maintained under 14L:10D light/dark cycle (lights on at 06 h, lights off at 20 h); DL group was kept under reversed conditions: 10D:14L dark/light cycle (lights off at 06 h, lights on at 16 h). Light intensity was about 300 lux at bird level provided by ceiling-mounted fluorescent tubes. The animals in the LL group were illuminated continuously in a room with no natural light. The DD group was kept in complete darkness. Animals belonging to one group were kept in one room, distributed among environmental chambers that were identical, each comfortably housed up to 5 birds. The rooms were designated to permit animal management without admitting any extraneous light and to provide continuous ventilation and temperature control (only in DD group, infrared viewer was used). To prevent synchronization via treatment, animals were fed at different times of the day. Water and food were accessible ad libitum.
The protein content of each fraction was assayed using the Bradford method (Bio-Rad protein assay, Bio-Rad, Hercules, CA) with BSA as the standard.
2.2. Tissue extraction for RIA The animals (n ⫽ 4 in each group) were decapitated every 3 h, starting at 12 h under brief halothane anesthesia, and brains and retinas were removed. For the DD experiment, birds were sacrificed by cervical dislocation in darkness using an infrared viewer. Samples from different brain areas were dissected under a dissecting microscope immediately after sacrifice. The following tissues were collected: whole diencephalon, brainstem (pons and medulla oblongata), tectum, pineal gland and hypophysis; anterior part of telencephalon; and parts of cerebellum and retina. The tissues were transferred into an ice-chilled tube containing 1 ml of 5% trifluoroacetic acid (Sigma-Aldrich, Hungary). Each specimen was homogenized with a glass homogenizer, and an aliquot was transferred into another tube for protein assay. The tissue homogenate was centrifuged at 12,000 ⫻ g at room temperature for 30 min. The supernatant was transferred into a test tube and lyophilized twice. 2.3. PACAP radioimmunoassay The dried residues were dissolved in RIA buffers and assayed for PACAP38 using a highly specific procedure.
2.5. Data analysis Four separate extractions and RIAs were performed on each tissue examined. Tissue content of PACAP-LI was expressed as ng per mg protein. The mean of four determinations for each tissue was subjected to statistical analysis, using a one-way analysis of variance followed by Duncan’s multiple range test when appropriate. Comparison between multiple groups at different time points was assessed by one-way analysis of variance (ANOVA); post hoc comparisons were performed using the Tukey Multiple Comparison test. Statistical significance was given when P ⬍ 0.05. In addition, cosinor analysis was used to assess circadian variation of PACAP levels in the different brain areas [13,18, 19]. The terms “subjective day and night” were used to define periods, when the animals (LD and DL group) were kept under light or darkness, respectively. For animals kept under constant lighting conditions, the same time periods were considered as “subjective day and night” as for the animals in the LD group.
3. Results The amount of endogenous PACAP-LI showed marked topographical variations in the various brain regions of the chicken (Fig. 1). All areas of the chicken brain contained high levels of PACAP38. Highest average daily levels were found in the diencephalon and in the brainstem (7–19 ng/mg protein). The telencephalon, tectum and cerebellum contained levels of 2.5–7 ng PACAP/mg protein. Lowest values were measured in the hypophysis, pineal body and retina (0.5–3 ng/mg protein). In addition, daily variations of PACAP38 were observed under different lighting conditions in the brainstem, diencephalon, telencephalon and retina. 3.1. Brainstem Most pronounced patterns were observed in the brainstem (Fig. 2a). The daily variation of PACAP values was similar in the LD, LL and DD groups. During subjective day hours, no significant difference was found between the dif-
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Fig. 1. Averages of PACAP levels of all subjective day (06 –20) and subjective night (20 – 06) hours in (1) diencephalon; (2) brainstem; (3) telencephalon; (4) tectum; (5) cerebellum; (6) pituitary gland; (7) pineal gland and (8) retina. Pairs of bars represent the four groups under different lighting conditions (LD, DL, LL, DD, respectively). Stars indicate significant difference between averages of subjective day and night hours (P ⬍ 0.05). Values are expressed as ng PACAP/mg protein ⫾ SEM.
ferent time points, values varied between 4 –10 ng/mg protein. Only the lowest levels at 15 and 18 h in the DD group were significantly different from all previous day-time hours. During subjective night hours, PACAP levels increased to 12–19 ng/mg protein. Average PACAP levels of all subjective night hours were significantly higher in all 3 groups than those of subjective day hours (Fig. 1). In the LD group, a single peak was observed at 24 h, which was significant compared to all other time points. In the LL
group, the peak was observed at 21 h, with levels significantly different from all previous time points. In the DD group, the peak occurred also at 21 h, being significantly different from the lowest levels at 15, 18 and 03 h. A different pattern was observed in the DL group: levels of PACAP were highest between 15 and 21 h, which were significantly different from the preceding and succeeding time points. Cosinor analysis revealed significant daily rhythms for LD and DL groups. The observed elevations
Fig. 2. Daily rhythms of PACAP levels in the (a) brainstem; (b) diencephalon; (c) telencephalon; and (d) retina. Values are expressed as ng PACAP/mg protein ⫾ SEM.
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Table 1 Cosinor analysis of PACAP circadian rhythms
Brainstem
Hypothalamus
Telencephalon
Retina
LD DL LL DD LD DL LL DD LD DL LL DD LD DL LL DD
MESOR ng
Amplitude ng
Acrophase degree
F
p
8.11 8.52 10.92 8.58 9.8 8.47 9.95 8.63 4.68 8.28 10.11 8.56 0.7 0.79 0.62 0.55
3.02 4.27 2.66 2.41 2.13 3.57 2.41 2.12 1.3 3.27 2.22 2.18 0.3 0.23 0.19 0.31
⫺5.22 ⫺283.84 ⫺339.97 ⫺21.48 ⫺32.9 ⫺274.07 ⫺34.11 ⫺24.32 ⫺95.02 ⫺276.24 ⫺29 ⫺16.96 ⫺14.14 ⫺16.46 ⫺35.67 ⫺77.24
7.28 3.98 1.45 2.73 3.25 6.06 4.53 3.5 3.83 10.89 2.4 4.38 13.21 3.57 8.08 15.56
0.022 0.039 0.253 0.087 0.049 0.011 0.021 0.047 0.031 0.577 0.004 ⬍0.001 ⬍0.001 0.049 0.002 0.007
during subjective night in the LL and DD groups did not fit the circadian curve determined by the cosinor analysis (Table 1).
of the light hours, respectively), representing significantly higher values compared to all other time points. Using cosinor analysis, significant rhythm was obtained in all groups except in the DL group (Table 1).
3.2. Diencephalon 3.4. Retina In the diencephalon, the daily pattern of PACAP was found to be similar to that in the brainstem (Fig. 2b). In the LD, LL and DD groups, PACAP levels varied in the range of 6 –10 ng/mg protein during the subjective day. In the subjective nighttime, PACAP levels increased in all 3 groups (11–14 ng/mg protein), with highest levels at 21 h in the animals kept under constant lighting conditions (LL, DD), and at 24 h in the LD group (Fig. 2b). Although no significant difference could be shown comparing values at different time points using t test, averages of PACAP levels of all subjective night hours were significantly higher in all 3 groups than those of all subjective day hours, regardless of the lighting conditions (Fig. 1). The pattern observed in the DL group was similar to that of the brainstem: values were highest at the end of dark and beginning of light hours (15–21 h). Cosinor analysis revealed significant circadian variations in all four groups (Table 1). 3.3. Telencephalon In the telencephalon, three groups, the LD, LL and DD groups showed a nearly similar daily variation of PACAP (Fig. 2c). PACAP levels showed a decreasing pattern during subjective daytime, reaching low levels between 12–21 h, with lowest values at 21, 12–15 or at 18 h in the LD, LL and DD groups, respectively. Values gradually returned to the original levels after these time points. The difference between the low and high levels was significant in all 3 groups. Under reverse lighting conditions (DL), two peaks were observed, at 18 and 03 h (at the beginning and the end
In the retina, all 4 groups showed a similar pattern: values started decreasing after 06 h, with lowest levels reached at 12–15 h (Fig. 2d). Levels gradually returned to the original levels during the subjective night hours independent of the lighting conditions. The difference between the lowest and highest levels was significant in all 4 groups. In the LD group, high values were measured at 03, 06, 21 and 24 h, low levels at 9, 12 and 15 h. Similar pattern could be observed in the LL group, although all values of PACAP were lower than in the LD group. In the DL group, high levels were measured at 06 and 24 h, which were significantly different from all values of all other time points. In the DD group, highest levels were observed at 03 and 06 h, which were significantly higher compared to values at all other time points. Significant difference was found in the LD and LL groups between average values measured at subjective day time points compared to those at subjective night hours, with higher levels during the night hours (Fig. 1). Cosinor test also revealed significant daily variation in all groups (Table 1). 3.5. Tectum, cerebellum, hypophysis, pineal body No circadian rhythmicity of PACAP levels was observed in the tectum, cerebellum, hypophysis and pineal glands using either statistical tests. Levels of PACAP varied in the tectum and cerebellum in the range of 4 –5.5 and 2–3.3 ng/mg protein, respectively. However, a slight but not significant increase in average PACAP levels could be seen
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under reversed lighting conditions (DL group) during subjective day hours in the tectum, and during subjective night in the cerebellum (Fig. 1). In the pineal gland, significant increase was observed only in the DL group during subjective night hours (Fig. 1). The hypophysis showed a slight but not significant increase only in the LD and DD groups during subjective night hours (Fig. 1).
4. Discussion In the present study, we showed distribution and daily variations of PACAP38 in different regions in the chicken brain. Our result shows that highest levels of PACAP38 were found in the diencephalon and brainstem followed by the cerebral cortex, tectum, and cerebellum. Lowest levels were found in the hypophysis, pineal gland and retina. Nowak et al. have demonstrated that PACAP stimulates the cAMP formation in different areas of the chicken brain [27]. They found highest stimulation in the hypothalamus followed by the cerebral cortex, optic lobes and the retina (other brain areas were not examined). Thus, the distributional pattern of PACAP observed in our study is in accordance with the regional differences of cAMP stimulation by PACAP. Our results are also in accord with those of Peeters et al. [29,30], who found high expression of PACAP mRNA and PAC1 receptor mRNA in the chicken telencephalon, hypothalamic, thalamic and brainstem nuclei, tectum and cerebellum. We found that the changes in levels of PACAP showed similar daily pattern in the diencephalon and brainstem. In the LD, DD and LL groups, the levels of PACAP increased during subjective night. Elevated levels of PACAP observed in LD conditions are in good accord with other studies demonstrating higher levels of PACAP, VIP and other peptides in rat hypothalamic nuclei during night hours [9,26]. In our present findings, the constant lighting conditions do not affect the normal circadian variation of PACAP in the brainstem and diencephalon. Cagampang et al. found similar daily variations of both VIP and PACAP receptor mRNA expression in rat hypothalamic nuclei during lightdark cycle and constant darkness [5]. Conflicting data also exist however; no daily variation of PACAP and VIP was found in the rat SCN under constant dark conditions [9,33], and highest VIP density was observed during light hours in the hamster SCN under diurnal lighting conditions [32]. Several studies describe reversed or disturbed hormonal patterns under reversed lighting conditions [11]. We found that under reversed lighting condition, the circadian variation did not follow the pattern observed in the LD,LL,DD groups; nor did it follow a reversed pattern. We observed a pronounced shift in elevation of PACAP values under this condition: values increased 6 h in advance compared to the other groups. Circadian rhythms have been reported in the brainstem, daily variations of melatonin-binding have been demonstrated in the pigeon brainstem and hypothalamus
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[36,40]. No circadian pattern has yet been described for PACAP in the brainstem of other species, although some studies show that the peptide is involved in circadian activities coordinated partly by the brainstem [1]. It is also possible that more detailed examination of PACAP content in different nuclei in the diencephalon and brainstem would reveal other patterns, since differential distribution of PACAP has been described in diencephalic and brainstem nuclei both in mammals and in chicken [2,29,30,37]. In the telencephalon, daily patterns were the same in LD, DD and LL groups. In contrast, rat telencephalon did not exhibit daily variations of PACAP and PAC1 receptor mRNA [5,9]. The average daily levels of PACAP in the retina were higher under changing lighting conditions than under constant conditions, which is in accordance with the findings of Herbst et al. [17], who found a suppression of VIP immunoreactivity in rat retina under constant darkness. Interestingly, the daily pattern of PACAP was similar in all 4 groups in the retina. Evidences suggest that the mammalian retina has a clock separate from the SCN [25]. Retinal rhythms have been also described in the chicken, and results indicate that several components coordinate these rhythms including melatonin, the photoreceptors themselves through light and other neuronal inputs [22]. Our observations imply that the retinal PACAP rhythm might be mainly influenced by other factors rather than light. In the four areas discussed (diencephalon, brainstem, telencephalon and retina), constant lighting conditions did not disturb the basic daily pattern of PACAP observed under LD. These observations correspond to studies showing that circadian rhythms are innate characteristics of organisms and do not simply reflect changes of environmental conditions. For many characteristics, a stable circadian rhythm can be seen even in the absence of measurable environmental signals such as lighting, while shifting the time of lights-on and lights-off can cause reestablishment of an appropriate relationship to the changed cycle [4,7,11]. In the above mentioned areas except the retina, the daily pattern was disturbed, but not exactly inverted in reversed (DL) conditions. Longer periods of adaptation and/or measuring more parameters of the animals (body temperature, activity), could reveal a more precise determination of this phenomenon. Considering these limitations in our study, under the used experimental conditions it seems that the variations of PACAP in the chicken brain are mainly influenced by factors other than light. Nonphotic stimuli, for instance, have been also shown to alter the diurnal oscillation of VIP in mammalian brain [12,31,32]. PACAP has been found to stimulate melatonin production in pineal cells similarly to VIP [34,37]. We measured low levels of PACAP in the chicken pineal gland and no significant daily variation could be detected. Fukuhara et al. [10] found elevated PACAP levels at night in rat pineal gland, similar to night-time elevations of NPY and VIP [20,33]. In contrast, Moller et al. [24] found no daily varia-
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ton of PACAP in the rat pineal gland measured by RIA. Fibers but no cell bodies immunoreactive for PACAP were reported suggesting that the origin of PACAP in the pineal gland arises from the superior cervical ganglion or other ganglia [24]. This apparent absence of PACAP synthesis in the pinealocytes may explain the low levels and the lack of daily pattern observed also in the chicken pineal gland. Few data have been published on factors influencing rhythms in tectum and cerebellum [8,28,35]. In our study, PACAP values did not show significant daily variations in the cerebellum, tectum and hypophysis. In summary, our results provide the first evidence that PACAP levels oscillate in a circadian manner in the avian brain and retina. Our data suggest that daily variation in PACAP levels in different brain areas and retina might be influenced by different regulatory mechanisms. The persistence of PACAP’s circadian variations in constant lighting schedules suggests that it is driven by endogenous circadian time structures rather than environmental lighting.
Acknowledgment This work was supported by the National Scientific Research Fund (OTKA No T032523).
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Distribution and daily variations of PACAP in the chicken brain R. Jo´zsaa, A. Somogyva´ri-Vighb,c,*, D. Reglo¨dia, T. Hollo´sya, A. Arimurab,c a
Department of Anatomy, University of Pe´cs, Pe´cs 7624 Hungary Department of Medicine, Tulane University, New Orleans, LA 70112, USA c US -Japan Biomedical Research Laboratories, Tulane University Hebert Center, Belle Chasse, LA 70037, USA b
Abstract Levels of PACAP38 were measured in different areas of the chicken brain under various lighting conditions by radioimmunoassay (RIA). Selected groups of animals were maintained under light for 14 h alternating with 10 h of darkness (LD), reversed lighting conditions (DL) and constant light (LL) or constant dark (DD). Daily variations of PACAP levels were observed in the brainstem, diencephalon, telencephalon and retina. In the brainstem and diencephalon, levels of PACAP increased during subjective nighttime, except in the DL group where levels were elevated between 15–21 h. In the telencephalon, the lowest level of PACAP was measured between 12–21 h except in the DL group where two peaks occurred at 18 and 03 h. In the retina, all 4 groups showed a similar level and pattern, with lowest levels during midday hours. No daily variation was observed in the pineal gland. According to the present observations, it is suggested that PACAP levels differ in several areas of the chicken brain under various lighting conditions and photic stimuli do not appear to be the main regulators of the circadian variations of PACAP. © 2001 Elsevier Science Inc. All rights reserved. Keywords: PACAP; chicken; daily variation; radioimmunoassay
1. Introduction Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalami on the basis of its adenylate cyclase activating activity. It belongs to the VIP/secretin/glucagon peptide family, and shows closest homology to VIP. The primary structure of PACAP is highly conserved during phylogeny: in vertebrate species it shows only 1– 4 amino acid differences from mammalian PACAP [2]. PACAP has been shown also in the chicken nervous system. Chicken PACAP has 1 amino acid substitute compared to human PACAP. The genes encoding chicken PACAP and its receptors are also similar to human PACAP genes [23,29,38]. PACAP and PACAP receptor immunoreactivity and their mRNAs are widely distributed in different regions of the chicken brain and peripheral organs [29,30]. It has also been shown that PACAP stimulates cAMP formation in various areas of the chicken brain [27]. PACAP is a pleiotropic peptide, having numerous actions in the central nervous system (CNS) and peripheral organs [2]. One of its recently shown functions is the mod* Corresponding author. Tel.: ⫹1-504-394-7199; fax: ⫹1-504-3947169. E-mail address: [email protected] (A. Somogyvari-Vigh).
ulation of circadian activity. Circadian rhythms are coordinated by endogenous mechanisms, that can be entrained by environmental stimuli, most notably by the daily changes in light intensity. In mammals, the suprachiasmatic nucleus (SCN) is a major circadian pacemaker. Photic cues are transmitted to the SCN via the retinohypothalamic tract [11]. In this way, light entrains the SCN and its driven output rhythms to the 24-h day. PACAP is abundant in the rat SCN, in the retinohypothalamic pathway and retinal ganglionic cells [11,14]. Daily variations of PACAP receptor mRNA have been demonstrated in the SCN and supraoptic nuclei of the rat hypothalamus, suggesting that PACAP receptors are differentially expressed across the 24-h cycle [5]. PACAP has also been shown to reset the circadian clock in a manner similar to light [6,16]. These and other results suggest that PACAP is involved in the circadian pacemaker clock in mammals [5,15,21,37]. Avian circadian rhythms are coordinated by oscillators consisting of the retina, pineal gland and SCN, and the relative importance of each structure and their connections varies among avian species [22,39]. Interactions among these structures serve to generate coordinated circadian outputs. These outputs control a vast array of circadian rhythms of biological processes. No data are presently available on the circadian variations of PACAP in the chicken brain. In the present study, we investigated the distribution of
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PACAP-like immunoreactivity (PACAP-LI) by means of radioimmunoassay (RIA) in different regions of the chicken brain, and we examined the daily variation of PACAP-LI under different lighting conditions. 2. Methods
The detailed RIA procedure and HPLC verification of chicken PACAP have been described previously [3,38]. The PACAP38 antiserum [88111–3] was generated against synthetic PACAP [24 –38] and recognizes PACAP38 as well as peptides containing the epitope of PACAP [24 –38], but not PACAP27.
2.1. Animals
2.4. Protein assay
One-day old chicken were purchased from a local supplier (Mohacs, Hungary), and kept under different lighting conditions starting from the day of purchase for 4 weeks. Experiments were carried out in summer (June–July), with room temperatures ranging between 22–25°C. The animals (n ⫽ 128) were distributed into 4 groups: LD group was maintained under 14L:10D light/dark cycle (lights on at 06 h, lights off at 20 h); DL group was kept under reversed conditions: 10D:14L dark/light cycle (lights off at 06 h, lights on at 16 h). Light intensity was about 300 lux at bird level provided by ceiling-mounted fluorescent tubes. The animals in the LL group were illuminated continuously in a room with no natural light. The DD group was kept in complete darkness. Animals belonging to one group were kept in one room, distributed among environmental chambers that were identical, each comfortably housed up to 5 birds. The rooms were designated to permit animal management without admitting any extraneous light and to provide continuous ventilation and temperature control (only in DD group, infrared viewer was used). To prevent synchronization via treatment, animals were fed at different times of the day. Water and food were accessible ad libitum.
The protein content of each fraction was assayed using the Bradford method (Bio-Rad protein assay, Bio-Rad, Hercules, CA) with BSA as the standard.
2.2. Tissue extraction for RIA The animals (n ⫽ 4 in each group) were decapitated every 3 h, starting at 12 h under brief halothane anesthesia, and brains and retinas were removed. For the DD experiment, birds were sacrificed by cervical dislocation in darkness using an infrared viewer. Samples from different brain areas were dissected under a dissecting microscope immediately after sacrifice. The following tissues were collected: whole diencephalon, brainstem (pons and medulla oblongata), tectum, pineal gland and hypophysis; anterior part of telencephalon; and parts of cerebellum and retina. The tissues were transferred into an ice-chilled tube containing 1 ml of 5% trifluoroacetic acid (Sigma-Aldrich, Hungary). Each specimen was homogenized with a glass homogenizer, and an aliquot was transferred into another tube for protein assay. The tissue homogenate was centrifuged at 12,000 ⫻ g at room temperature for 30 min. The supernatant was transferred into a test tube and lyophilized twice. 2.3. PACAP radioimmunoassay The dried residues were dissolved in RIA buffers and assayed for PACAP38 using a highly specific procedure.
2.5. Data analysis Four separate extractions and RIAs were performed on each tissue examined. Tissue content of PACAP-LI was expressed as ng per mg protein. The mean of four determinations for each tissue was subjected to statistical analysis, using a one-way analysis of variance followed by Duncan’s multiple range test when appropriate. Comparison between multiple groups at different time points was assessed by one-way analysis of variance (ANOVA); post hoc comparisons were performed using the Tukey Multiple Comparison test. Statistical significance was given when P ⬍ 0.05. In addition, cosinor analysis was used to assess circadian variation of PACAP levels in the different brain areas [13,18, 19]. The terms “subjective day and night” were used to define periods, when the animals (LD and DL group) were kept under light or darkness, respectively. For animals kept under constant lighting conditions, the same time periods were considered as “subjective day and night” as for the animals in the LD group.
3. Results The amount of endogenous PACAP-LI showed marked topographical variations in the various brain regions of the chicken (Fig. 1). All areas of the chicken brain contained high levels of PACAP38. Highest average daily levels were found in the diencephalon and in the brainstem (7–19 ng/mg protein). The telencephalon, tectum and cerebellum contained levels of 2.5–7 ng PACAP/mg protein. Lowest values were measured in the hypophysis, pineal body and retina (0.5–3 ng/mg protein). In addition, daily variations of PACAP38 were observed under different lighting conditions in the brainstem, diencephalon, telencephalon and retina. 3.1. Brainstem Most pronounced patterns were observed in the brainstem (Fig. 2a). The daily variation of PACAP values was similar in the LD, LL and DD groups. During subjective day hours, no significant difference was found between the dif-
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Fig. 1. Averages of PACAP levels of all subjective day (06 –20) and subjective night (20 – 06) hours in (1) diencephalon; (2) brainstem; (3) telencephalon; (4) tectum; (5) cerebellum; (6) pituitary gland; (7) pineal gland and (8) retina. Pairs of bars represent the four groups under different lighting conditions (LD, DL, LL, DD, respectively). Stars indicate significant difference between averages of subjective day and night hours (P ⬍ 0.05). Values are expressed as ng PACAP/mg protein ⫾ SEM.
ferent time points, values varied between 4 –10 ng/mg protein. Only the lowest levels at 15 and 18 h in the DD group were significantly different from all previous day-time hours. During subjective night hours, PACAP levels increased to 12–19 ng/mg protein. Average PACAP levels of all subjective night hours were significantly higher in all 3 groups than those of subjective day hours (Fig. 1). In the LD group, a single peak was observed at 24 h, which was significant compared to all other time points. In the LL
group, the peak was observed at 21 h, with levels significantly different from all previous time points. In the DD group, the peak occurred also at 21 h, being significantly different from the lowest levels at 15, 18 and 03 h. A different pattern was observed in the DL group: levels of PACAP were highest between 15 and 21 h, which were significantly different from the preceding and succeeding time points. Cosinor analysis revealed significant daily rhythms for LD and DL groups. The observed elevations
Fig. 2. Daily rhythms of PACAP levels in the (a) brainstem; (b) diencephalon; (c) telencephalon; and (d) retina. Values are expressed as ng PACAP/mg protein ⫾ SEM.
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Table 1 Cosinor analysis of PACAP circadian rhythms
Brainstem
Hypothalamus
Telencephalon
Retina
LD DL LL DD LD DL LL DD LD DL LL DD LD DL LL DD
MESOR ng
Amplitude ng
Acrophase degree
F
p
8.11 8.52 10.92 8.58 9.8 8.47 9.95 8.63 4.68 8.28 10.11 8.56 0.7 0.79 0.62 0.55
3.02 4.27 2.66 2.41 2.13 3.57 2.41 2.12 1.3 3.27 2.22 2.18 0.3 0.23 0.19 0.31
⫺5.22 ⫺283.84 ⫺339.97 ⫺21.48 ⫺32.9 ⫺274.07 ⫺34.11 ⫺24.32 ⫺95.02 ⫺276.24 ⫺29 ⫺16.96 ⫺14.14 ⫺16.46 ⫺35.67 ⫺77.24
7.28 3.98 1.45 2.73 3.25 6.06 4.53 3.5 3.83 10.89 2.4 4.38 13.21 3.57 8.08 15.56
0.022 0.039 0.253 0.087 0.049 0.011 0.021 0.047 0.031 0.577 0.004 ⬍0.001 ⬍0.001 0.049 0.002 0.007
during subjective night in the LL and DD groups did not fit the circadian curve determined by the cosinor analysis (Table 1).
of the light hours, respectively), representing significantly higher values compared to all other time points. Using cosinor analysis, significant rhythm was obtained in all groups except in the DL group (Table 1).
3.2. Diencephalon 3.4. Retina In the diencephalon, the daily pattern of PACAP was found to be similar to that in the brainstem (Fig. 2b). In the LD, LL and DD groups, PACAP levels varied in the range of 6 –10 ng/mg protein during the subjective day. In the subjective nighttime, PACAP levels increased in all 3 groups (11–14 ng/mg protein), with highest levels at 21 h in the animals kept under constant lighting conditions (LL, DD), and at 24 h in the LD group (Fig. 2b). Although no significant difference could be shown comparing values at different time points using t test, averages of PACAP levels of all subjective night hours were significantly higher in all 3 groups than those of all subjective day hours, regardless of the lighting conditions (Fig. 1). The pattern observed in the DL group was similar to that of the brainstem: values were highest at the end of dark and beginning of light hours (15–21 h). Cosinor analysis revealed significant circadian variations in all four groups (Table 1). 3.3. Telencephalon In the telencephalon, three groups, the LD, LL and DD groups showed a nearly similar daily variation of PACAP (Fig. 2c). PACAP levels showed a decreasing pattern during subjective daytime, reaching low levels between 12–21 h, with lowest values at 21, 12–15 or at 18 h in the LD, LL and DD groups, respectively. Values gradually returned to the original levels after these time points. The difference between the low and high levels was significant in all 3 groups. Under reverse lighting conditions (DL), two peaks were observed, at 18 and 03 h (at the beginning and the end
In the retina, all 4 groups showed a similar pattern: values started decreasing after 06 h, with lowest levels reached at 12–15 h (Fig. 2d). Levels gradually returned to the original levels during the subjective night hours independent of the lighting conditions. The difference between the lowest and highest levels was significant in all 4 groups. In the LD group, high values were measured at 03, 06, 21 and 24 h, low levels at 9, 12 and 15 h. Similar pattern could be observed in the LL group, although all values of PACAP were lower than in the LD group. In the DL group, high levels were measured at 06 and 24 h, which were significantly different from all values of all other time points. In the DD group, highest levels were observed at 03 and 06 h, which were significantly higher compared to values at all other time points. Significant difference was found in the LD and LL groups between average values measured at subjective day time points compared to those at subjective night hours, with higher levels during the night hours (Fig. 1). Cosinor test also revealed significant daily variation in all groups (Table 1). 3.5. Tectum, cerebellum, hypophysis, pineal body No circadian rhythmicity of PACAP levels was observed in the tectum, cerebellum, hypophysis and pineal glands using either statistical tests. Levels of PACAP varied in the tectum and cerebellum in the range of 4 –5.5 and 2–3.3 ng/mg protein, respectively. However, a slight but not significant increase in average PACAP levels could be seen
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under reversed lighting conditions (DL group) during subjective day hours in the tectum, and during subjective night in the cerebellum (Fig. 1). In the pineal gland, significant increase was observed only in the DL group during subjective night hours (Fig. 1). The hypophysis showed a slight but not significant increase only in the LD and DD groups during subjective night hours (Fig. 1).
4. Discussion In the present study, we showed distribution and daily variations of PACAP38 in different regions in the chicken brain. Our result shows that highest levels of PACAP38 were found in the diencephalon and brainstem followed by the cerebral cortex, tectum, and cerebellum. Lowest levels were found in the hypophysis, pineal gland and retina. Nowak et al. have demonstrated that PACAP stimulates the cAMP formation in different areas of the chicken brain [27]. They found highest stimulation in the hypothalamus followed by the cerebral cortex, optic lobes and the retina (other brain areas were not examined). Thus, the distributional pattern of PACAP observed in our study is in accordance with the regional differences of cAMP stimulation by PACAP. Our results are also in accord with those of Peeters et al. [29,30], who found high expression of PACAP mRNA and PAC1 receptor mRNA in the chicken telencephalon, hypothalamic, thalamic and brainstem nuclei, tectum and cerebellum. We found that the changes in levels of PACAP showed similar daily pattern in the diencephalon and brainstem. In the LD, DD and LL groups, the levels of PACAP increased during subjective night. Elevated levels of PACAP observed in LD conditions are in good accord with other studies demonstrating higher levels of PACAP, VIP and other peptides in rat hypothalamic nuclei during night hours [9,26]. In our present findings, the constant lighting conditions do not affect the normal circadian variation of PACAP in the brainstem and diencephalon. Cagampang et al. found similar daily variations of both VIP and PACAP receptor mRNA expression in rat hypothalamic nuclei during lightdark cycle and constant darkness [5]. Conflicting data also exist however; no daily variation of PACAP and VIP was found in the rat SCN under constant dark conditions [9,33], and highest VIP density was observed during light hours in the hamster SCN under diurnal lighting conditions [32]. Several studies describe reversed or disturbed hormonal patterns under reversed lighting conditions [11]. We found that under reversed lighting condition, the circadian variation did not follow the pattern observed in the LD,LL,DD groups; nor did it follow a reversed pattern. We observed a pronounced shift in elevation of PACAP values under this condition: values increased 6 h in advance compared to the other groups. Circadian rhythms have been reported in the brainstem, daily variations of melatonin-binding have been demonstrated in the pigeon brainstem and hypothalamus
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[36,40]. No circadian pattern has yet been described for PACAP in the brainstem of other species, although some studies show that the peptide is involved in circadian activities coordinated partly by the brainstem [1]. It is also possible that more detailed examination of PACAP content in different nuclei in the diencephalon and brainstem would reveal other patterns, since differential distribution of PACAP has been described in diencephalic and brainstem nuclei both in mammals and in chicken [2,29,30,37]. In the telencephalon, daily patterns were the same in LD, DD and LL groups. In contrast, rat telencephalon did not exhibit daily variations of PACAP and PAC1 receptor mRNA [5,9]. The average daily levels of PACAP in the retina were higher under changing lighting conditions than under constant conditions, which is in accordance with the findings of Herbst et al. [17], who found a suppression of VIP immunoreactivity in rat retina under constant darkness. Interestingly, the daily pattern of PACAP was similar in all 4 groups in the retina. Evidences suggest that the mammalian retina has a clock separate from the SCN [25]. Retinal rhythms have been also described in the chicken, and results indicate that several components coordinate these rhythms including melatonin, the photoreceptors themselves through light and other neuronal inputs [22]. Our observations imply that the retinal PACAP rhythm might be mainly influenced by other factors rather than light. In the four areas discussed (diencephalon, brainstem, telencephalon and retina), constant lighting conditions did not disturb the basic daily pattern of PACAP observed under LD. These observations correspond to studies showing that circadian rhythms are innate characteristics of organisms and do not simply reflect changes of environmental conditions. For many characteristics, a stable circadian rhythm can be seen even in the absence of measurable environmental signals such as lighting, while shifting the time of lights-on and lights-off can cause reestablishment of an appropriate relationship to the changed cycle [4,7,11]. In the above mentioned areas except the retina, the daily pattern was disturbed, but not exactly inverted in reversed (DL) conditions. Longer periods of adaptation and/or measuring more parameters of the animals (body temperature, activity), could reveal a more precise determination of this phenomenon. Considering these limitations in our study, under the used experimental conditions it seems that the variations of PACAP in the chicken brain are mainly influenced by factors other than light. Nonphotic stimuli, for instance, have been also shown to alter the diurnal oscillation of VIP in mammalian brain [12,31,32]. PACAP has been found to stimulate melatonin production in pineal cells similarly to VIP [34,37]. We measured low levels of PACAP in the chicken pineal gland and no significant daily variation could be detected. Fukuhara et al. [10] found elevated PACAP levels at night in rat pineal gland, similar to night-time elevations of NPY and VIP [20,33]. In contrast, Moller et al. [24] found no daily varia-
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ton of PACAP in the rat pineal gland measured by RIA. Fibers but no cell bodies immunoreactive for PACAP were reported suggesting that the origin of PACAP in the pineal gland arises from the superior cervical ganglion or other ganglia [24]. This apparent absence of PACAP synthesis in the pinealocytes may explain the low levels and the lack of daily pattern observed also in the chicken pineal gland. Few data have been published on factors influencing rhythms in tectum and cerebellum [8,28,35]. In our study, PACAP values did not show significant daily variations in the cerebellum, tectum and hypophysis. In summary, our results provide the first evidence that PACAP levels oscillate in a circadian manner in the avian brain and retina. Our data suggest that daily variation in PACAP levels in different brain areas and retina might be influenced by different regulatory mechanisms. The persistence of PACAP’s circadian variations in constant lighting schedules suggests that it is driven by endogenous circadian time structures rather than environmental lighting.
Acknowledgment This work was supported by the National Scientific Research Fund (OTKA No T032523).
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