Vasopressin immunoreactivity and release in the suprachiasmatic nucleus of wild-type and tau mutant Syrian hamsters

Brain Research 936 (2002) 38–46 www.elsevier.com / locate / bres

Research report

Vasopressin immunoreactivity and release in the suprachiasmatic nucleus of wild-type and tau mutant Syrian hamsters Eddy A. Van der Zee*, Malgorzata Oklejewicz, Koen Jansen, Serge Daan, Menno P. Gerkema Zoological Laboratory, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands Accepted 20 December 2001

Abstract Despite the prominent role of the Syrian hamster (Mesocricetus auratus) in studies of circadian rhythms, there are no data available on the temporal dynamics of the neuropeptide vasopressin (AVP), a major output system of the suprachiasmatic nucleus (SCN). We studied the hamster SCN-AVP system in vivo across the light period and in vitro using long-term organotypic SCN cultures. Additionally, we compared wild-type and tau mutant hamsters with an endogenous circadian period of |24 h and |20 h, respectively. The in vivo study revealed no differences in the number of SCN-AVP neurons between the two genotypes of hamsters studied at three time points across the light period of the circadian cycle. A significantly higher level of AVP-immunoreactivity, however, was found in the SCN of wild-type compared to tau mutant hamsters at the beginning and in the middle of the light period, but not at the end of the light period. SCN-AVP cell number and immunostaining decreased significantly across the light period in wild-type hamsters, but not in tau mutants. The in vitro study revealed a significantly higher rate of AVP release per 24 h from the tau mutant SCN compared to the wild-type SCN. Robust circadian oscillations in AVP release were not found in either type of hamster. These results may suggest that the SCN-AVP system of hamsters, irrespective of genotype, is relatively weak compared to other species. Moreover, the tau mutation seems to influence the SCN-AVP system by enhancing the rate of AVP release and by reducing AVP content and its daily fluctuation.  2002 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Circadian; Organotypic culture; Suprachiasmatic nucleus; Vasopressin; Syrian hamster, Tau mutation

1. Introduction The suprachiasmatic nucleus (SCN) of the laboratory rat and the Syrian hamster have been model systems in the field of chronobiology [16,33] since the discovery in the early 1970s of this nucleus as the main circadian oscillator [26,42]. Final, unequivocal proof for this role of the SCN was provided by transplantation experiments using tau mutant hamsters [32]. Most detailed anatomical and notably physiological data of the SCN are derived from the rat, but most behavioral observations are obtained from the hamster because of its high cycle-to-cycle circadian ac*Corresponding author. Tel.: 131-50-363-2062; fax: 131-50-3632148. E-mail address: [email protected] (E.A. Van der Zee).

curacy. Although the cytoarchitecture and retinal afferents of the SCN are identical in a wide variety of mammals including rat and hamster [5], minor differences in neuropeptidergic organization were observed between these species [27]. The neuropeptide vasopressin (AVP), a major output system of the SCN, is abundantly expressed in both species [3,4,10,22,40,43,44,51]. These studies show that SCN-AVP neurons are more densely packed in rat than in hamster, and a separate group of AVP neurons in the ventro-lateral subdivision of the SCN such as particularly present in mouse SCN [2] is more pronounced in hamster [3,4] compared to rat. AVP-immunoreactivity (AVPir) fluctuates in the rat SCN across the light–dark cycle, with high levels at the beginning of the light period (Zeitgeber Time—ZT 0) and lower levels at the beginning of the dark period (ZT 12) [29]. Hamsters kept under a natural LD

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cycle in April / May expressed more SCN-AVPir in the middle of the light period compared to the middle of the dark period [36]. To our knowledge, however, changes in AVPir in hamster SCN across the light period have not been reported in the literature. Most studies describe detailed anatomical characteristics of AVPir with no information of time of day [3,10,22,25,40,43], or restricted the analysis to a small part of the LD cycle [1]. AVP is released from the SCN during the light period, and protein levels are restored during the dark period (reviewed in Ref. [48]). AVP derived from the SCN fluctuates in a circadian fashion, which has been measured in the cerebral spinal fluid of rat, guinea pig, rabbit, sheep, goat, cat, and rhesus monkey (see for review Ref. [25]). AVP release from rat SCN has been thoroughly studied using a variety of techniques, such as acute SCN slices [12,13], microdialysis [23] or long-term organotypic SCN cultures [21,39]. Micropunches of rat SCN revealed essentially the same AVP rhythm as the other methods [19]. Surprisingly, however, no AVP release data are available from hamster SCN. Here, we studied the temporal dynamics of the SCNAVP system in Syrian hamsters (Mesocricetus auratus) using brain sections containing the SCN to examine AVPir at three time points across the light period, and by employing long-term organotypic SCN cultures to examine AVP release profiles across a 50-h sampling period. Additionally, we compared normal, wild-type (tau 1 / 1) hamsters with a free-running circadian period of |24 h with tau mutant (tau 2 / 2) hamsters with a free-running circadian period of |20 h [31]. Tau is a single, semidominant mutation in the gene encoding for casein kinase I´ [24], a critical component of the mammalian circadian clock. Levels of AVP mRNA are significantly reduced in the SCN of mutant hamsters compared to wild-types [35]. This difference may hint at a major impact of this mutation on SCN-AVP regulation at the protein level.

2. Materials and methods

2.1. Animals Male Syrian hamsters (Mesocricetus auratus) were obtained from a breeding stock at the Zoological Laboratory, Haren, The Netherlands. Wild-type (tau 1 / 1) and homozygote tau (tau 2 / 2) mutant hamsters (F2 generation) were obtained from crossings between heterozygous parents (F1 generation; [28]). All hamsters (age 5–6 months) were housed individually in cages (l3w3h5403 24315 cm) in a climate-controlled room at 2360.5 8C. Food (standard Hope Farms  pellets) and water were available ad libitum. Tau 1 / 1 hamsters were entrained to a light–dark cycle of 12 h light and 12 h dark (LD 12:12) and tau 2 / 2 were kept in LD 10:10 for at least 14 days prior to the experiment. Principles of Laboratory Animal

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Care (NIH publication No. 86-23, revised 1985) were followed, and experiments were approved by the Animal Experimentation Committee of the University of Groningen (Dec. No. 2091 and 2093).

2.2. Experimental protocol Thirty-six adult hamsters were used at three time points across the light period to establish whether the level of AVPir changes during the light period. Hamsters (18 tau 1 / 1 and 18 tau 2 / 2) were sacrificed at the beginning of the light period (ZT 0; seven tau 1 / 1 and six tau 2 / 2), halfway through the light period (ZT 6; six tau 1 / 1 and six tau 2 / 2) and at the end of the light period (ZT 12; five tau 1 / 1 and six tau 2 / 2). It should be noted that tau 2 / 2 hamsters were entrained to a 10:10 LD cycle. Therefore, ZT 6 is the middle of the light period (5 h after lights on), and ZT 12 is the end of the light period (10 h after lights on). Hence, one ZT-hour is 60 min for tau 1 / 1 hamsters, and 50 min for tau 2 / 2 subjects.

2.3. AVP immunocytochemistry Animals were deeply anesthetized with an overdose of sodium pentobarbital (900 mg / kg, i.p.), followed by a quick dissection of the brain. Thereafter, brains were immersion fixed for 24 h in 4% paraformaldehyde at 4 8C. Subsequently, brains were stored in phosphate buffer with 0.1% sodium azide. Before sectioning on a cryostat, brains were cryoprotected in 30% sucrose phosphate buffer for 24 h. Coronal brain sections were cut at a thickness of 25 mm, and collected in phosphate buffered saline (PBS, pH 7.4) and stored in this medium with 0.1% NaN 3 at 4 8C until further processing. Brain sections were rinsed in PBS and pre-incubated with 0.1% H 2 O 2 for 20 min, followed by three rinses with PBS. Thereafter, brain sections were pre-incubated with 5% normal goat serum and subsequently incubated overnight with the primary rabbit anti-vasopressin antibody (1:100; ICN) at room temperature. Sections were preincubated again with normal goat serum (5%), and incubated with biotinylated goat anti-rabbit (1:200; Zymed) for 2 h at room temperature. After rinsing with PBS, the sections were exposed to HRP-conjugated Streptavidin (1:200; Zymed) for 2 h at room temperature. Triton X-100 (0.5%) was added during all incubation steps. Finally, the sections were processed with diaminobenzidine (DAB)–H 2 O 2 (30 mg DAB / 100 ml PBS) under visual guidance.

2.4. Quantitative analyses of AVP immunoreactivity AVPir was quantified in two complementary ways by counting AVP cells and by image analysis. Quantitative analyses of either way described below were performed ‘blind’ to the genotype of hamster. Cell counts in brain sections were performed as described earlier [2,14,45]. In

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short, AVP-positive SCN cells were counted (left and right hemisphere were summed) in two sections containing the medial SCN in the rostro-caudal axis. Optical densities (ODs) of AVPir were determined in the SCN and in the paraventricular nucleus (PVN). The PVN served as a control region expressing high levels of AVP. The OD in the SCN and PVN was measured in the same two brain sections used for cell counting. The OD is expressed in arbitrary units corresponding to gray levels using a Quantimet 600 image analysis system. The value of background labeling was measured in a hypothalamic area devoid of AVP cell bodies, fibers or punctae. The OD of the area of interest was related to the background value by the formula [(OD area 2OD background ) / OD background ], thus eliminating the variability in background staining among sections.

2.5. Preparation of organotypic cultures Pup SCN explants were cultured according to the organotypic slice culture technique using the roller-drum method [46]. In short, pups of 7 days old were deeply anesthetized with an overdose of sodium pentobarbital (900 mg / kg, i.p.) before decapitation. The brains were quickly removed and a block of hypothalamic tissue containing the bilateral SCN was cut coronally in 400-mmthick slices using a tissue chopper. Slices containing the SCN were trimmed and placed in Gey’s balanced salt solution and cooled for 2 h at 5 8C. The slice was embedded in a plasma clot (10 ml chicken plasma and 10 ml thrombin) on a coverslip and placed in a culture tube with 700 ml of culture medium (25% horse serum, 45% Eagle’s basal medium with 62 mM D-glucose and 4.16 mM NaHCO 3 , and 30% Hanks’ balanced salt solution with 4.16 mM NaHCO 3 ). The SCN slice was cultured at 36 8C with rotation (12 revolutions / h). In all cases, the medium was replaced once or twice a week. The morphological development of the cultures was followed daily by light microscopic inspection. Drawings as well as photomicrographs were made by phase-contrast microscopy. After 10 days of culturing, samples were collected from each culture by changing the sample medium (700 ml, consisting of 10% horse, 60% Eagle’s basal medium, and 30% Hanks’ balanced salt solution) every 2 h for 2 days (25 time points). The collected medium was immediately frozen in liquid nitrogen and stored at 220 8C until analysis.

2.6. Radioimmunoassay for AVP In total, 240 ml of each sample was analyzed with a highly sensitive radioimmunoassay (RIA) for AVP according to Watanabe et al. [49], with minor modifications. In short, we used rabbit anti-AVP (‘Peter’, 3-10-1980; The Netherlands Institute for Brain Research, Amsterdam) in a

final dilution of 1:16,000. For each tube, 50 ml of Sac-cel solid phase second antibody coated cellulose suspension (IDS, UK) was used as precipitation reagents. Standard curves ranged from 0.25 to 64 pg AVP per 50 ml and the assay had a detection limit of 0.25 pg / ml. Cross-reactivity of ‘Peter’ besides AVP was 0.04% with vasotocin, and ,0.01% with oxytocin. In total, 240 ml of each sample was analyzed in triplicate (80 ml per tube). The interspecific assay coefficient of variation was 12%. To be able to compare the total amount of AVP release between cultures analyzed in different RIAs, a ‘calibration RIA’ was performed. In this RIA, four samples from each culture (with values close to the average AVP release of that culture) were taken and analyzed in one RIA. The outcome of the AVP concentration in the calibration RIA was then used to calibrate all cultures. RIA data were further analyzed with different approaches to determine whether peak values in AVP release, circadian periods and rhythmicity were present. The number of peaks was determined by calculating the standard deviation of the average of the relative values within an individual assay. When a relative value exceeded the average plus .2 S.D., it was considered to be a significant peak value.

2.7. Statistical analyses Differences in AVP release between the genotypes were tested for statistical significance by a Mann–Whitney Utest. Differences in AVP OD measures were tested by two-way analysis of variance followed by a post-hoc Tukey test [41]. A probability level of P,0.05 was used as an index of statistical significance. All tests were twotailed, and all data are presented as means6S.E. unless mentioned otherwise.

3. Results

3.1. AVP immunoreactivity in vivo The expression of SCN-AVPir for both genotypes at three time points across the light period is shown in Fig. 1. AVP cells were predominantly found in the dorsomedial part of the SCN, whereas AVP-positive fibers and terminals were predominantly present in the dorsomedial and ventrolateral parts of the SCN. The parvocellular AVP cells of the SCN were moderately to weakly stained, compared to the densely stained hypothalamic magnocellular AVP cells of the PVN, supraoptic nucleus and nucleus circularis. AVP-positive fibers and notably terminals, however, were densely stained in the SCN as seen in other hypothalamic regions. Cell counts of AVPir neurons in the medial SCN revealed that the number of detectable AVPir neurons did

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Fig. 1. AVPir in the hamster SCN. AVPir in the SCN of wild-type (tau 1 / 1; left panels) and tau mutant (tau 2 / 2; right panels) hamsters at the beginning (ZT 0), halfway (ZT 6), and end (ZT 12) of the light period. It should be noted that the tau 2 / 2 hamsters are entrained to a 10:10 LD cycle, and that the middle of the light period, ZT6, means 5 h after lights on, and at the end of the light period, ZT12, means 10 h after lights on. Scale bar5255 mm.

not differ between the two genotypes (F(1,35) 50.24, P5 0.6) at any of the time points studied (Fig. 2A). However, the number of AVP neurons in tau 1 / 1 hamsters was significantly higher at ZT 0 compared to the other two time points (F( 2,17 ) 517.61, P,0.001). No such ZT-dependent difference was observed in tau mutant hamsters (F( 2,17 ) 5 0.28, P50.8). OD measures of AVPir in the same brain sections containing the medial SCN as studied for the number of cells revealed a significant difference between the genotypes (F( 1,35 ) 525.15, P,0.001) and between time points (F(2,35) 54.3, P50.02). The two genotypes differed in OD measures of AVPir at ZT 0 and 6 (P,0.05), but not at ZT 12 (P.0.05; Fig. 2B). Within tau 1 / 1 hamsters, AVPir differed significantly between ZT 0 and 12 and between ZT 6 and 12 (P,0.05; Fig. 2B), but not between the other time points. In contrast, no significant differences in AVPir across the light period were found in tau 2 / 2 hamsters. In contrast to the SCN, the OD measures of AVPir in the PVN revealed no differences between the genotypes at any of the three time points across the light period (P.0.05; Fig. 2C). Moreover, also no ZT-dependent difference in

PVN-AVPir was found in either genotype (P.0.05; Fig. 2C).

3.2. Development and AVP release from SCN organotypic cultures The finding of differences in AVPir across the light period between the two genotypes raised questions about the regulation of AVP release, which could be studied in detail in vitro. Following explantation of the SCN at postnatal day 7, the morphology of the cultures of tau 2 / 2 and 1 / 1 hamsters was studied by means of phase contrast microscopy on a daily basis until the beginning of sampling 12 days later. In all cultures, a clear morphological organization in ependymal zone, neuronal zone, and dispersed cell zone was seen as described in the literature for SCN organotypic cultures of other species (see Ref. [46], and references therein). No consistent differences in development were observed between the genotypes, and the development was similar to that described previously for wild-type hamsters with the exception that these cultures developed more towards 1–3 layers of cells in the

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Fig. 2. Quantification of AVPir. Quantification of the number of SCN-AVP neurons at three time points (A) in two sections containing the medial SCN, and (B) the optical density values measured in the same sections. The optical density of AVPir in the PVN (C) was measured as a AVP-positive control region. It should be noted that the tau 2 / 2 hamsters are entrained to a 10:10 LD cycle, and that the middle of the light period, ZT6, means 5 h after lights on, and at the end of the light period, ZT12, means 10 h after lights on.

neuronal zone compared to 3–5 observed previously [46]. In the majority of cultures (seven out of nine tau 1 / 1, and six out of nine tau 2 / 2 cultures), a circulation of fluid in the ependymal zone was present. AVP release was measured over a period of 50 h, with 2-h intervals. Two cultures of each genotype were discarded from analyses because they were (partly) lost

during the sampling procedure. Representative examples of 50-h AVP release profiles of both genotypes are presented in Fig. 3. The presence of significant peak values revealed no differences between tau 1 / 1 and 2 / 2 hamsters. Using statistical criteria for the presence of a peak in AVP release of .1, .1.5, or .2 S.D. above average levels of AVP release, all cultures but one tau 2 / 2 culture at .2

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Fig. 3. AVP release profiles. Representative examples of AVP release (in percentages of the mean level) of three wild-type (left panels; a, b, c) and three tau mutant (right panels; d, e, f) hamsters; dashed line represents the value of 1.5 times the S.D.

S.D. had significant peak values. The number of peaks, depending on the criterion used, is shown in Table 1. No significant differences were found in the number of peaks between the genotypes. After a calibration procedure (see Materials and methods), the total amount of AVP release was obtained by summation of all AVP sample concentrations. Total AVP release was significantly higher in tau 2 / 2 than in tau 1 / 1 cultures (2.22-fold; P,0.01). Because the endogenous circadian rhythm of tau 2 / 2 hamsters is |4 h shorter than that of tau 1 / 1 hamsters, also in vitro [7], a correction was performed to see whether the total amount of AVP still differed when expressed per predicted circadian period. Also after this correction, tau 2 / 2 hamsters

release significantly more AVP per unit of time (1.85-fold; P,0.05).

4. Discussion

4.1. Conclusions Two parameters of the hamster SCN-AVP system were measured in two different experimental designs, i.e. AVPir in vivo and AVP release in vitro. The major findings reported here are: (1) the significantly higher level of AVPir in the SCN of wild-type compared to tau mutant hamsters at ZT 0 and 6 (measured in vivo), (2) the absence

Table 1 Analyses of AVP release patterns from organotypic SCN cultures

Averaged number of significant peaks per culture: Based on .1 S.D. Based on .1.5 S.D. Based on .2 S.D. Total amount of AVP released over 50 h ( pg) AVP released per predicted cycle ( pg) *P,0.05; **P,0.01.

tau 1 / 1 (n57)

tau 2 / 2 (n57)

2.8660.37 2.0060.33 1.1460.16 406.07676.57 194.92636.76

3.0060.47 2.1460.37 1.0060.24 902.146102.23** 360640.89*

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of a decrease in SCN-AVP cell number and SCN-AVPir across the light period in tau mutant hamsters in contrast to wild-types (measured in vivo), and (3) the significantly higher rate of AVP release per unit time from organotypic tau mutant SCN compared to organotypic wild-type SCN in the absence of robust circadian oscillations in AVP release in both genotypes (measured in vitro). The absence of significant differences in AVPir of the PVN between the genotypes and across the light period (measured in vivo) indicates that the findings for SCN-AVPir are specific to the SCN.

4.2. Features of the hamster SCN-AVP system The results of AVPir reveal some of the dynamic properties of the hamster SCN-AVP system across the light period. AVPir expressed as OD values can be interpreted as the content of AVP in the SCN, i.e. AVP present in the cellular compartments (cell body, dendrites, axons, varicosities) at the moment of fixation. AVP cell numbers in tau 1 / 1 SCN drop significantly in the middle (ZT 6) and the end (ZT 12) of the light period compared with the beginning (ZT 0) of the light period. In general, the number of immunocytochemically detectable peptidergic SCN neurons is a direct reflection of the total amount of protein (such as AVP) present in the somata [21,38,47]. Therefore, the higher number of cells found at ZT 0 in tau 1 / 1 hamsters is most likely due to accumulation of newly synthesized and not yet released AVP. While AVP cell numbers in tau 1 / 1 hamsters drop between ZT 0 and 6, AVP OD values (reflecting AVP content) decrease between ZT 6 and 12. This time lag of alterations in the two parameters of AVPir can be explained by transport of AVP from the soma to the axon and terminals, depleting the cell body of AVP (by which AVPir in already weakly stained somata declines below immunocytochemical detection) whereas total SCN-AVP content does not change clearly yet. Later in time, AVP is released by the terminals and varicosities, causing the drop in AVP content between ZT 6 and 12. A similar time lag between these two SCN-AVP parameters has been observed in mouse SCN [47]. The number of AVP cells did not differ significantly between tau 1 / 1 and tau 2 / 2 hamsters at any time point, although light microscopic examination revealed that AVP staining intensity was notably lower in cell bodies of tau mutant hamsters at ZT 0 and 6. This was confirmed by the OD measures, suggesting that the individual tau 2 / 2 AVP cells have a lower content of AVP. Combining the in vivo and in vitro data, one could speculate that this is a consequence of the twofold higher rate of AVP release. Shimomura and Menaker [37] suggested that the coupling between individual oscillators of the circadian clock might be weakened by the tau mutation. This higher quantity of AVP released by tau-mutant hamsters could be a compensatory action to enhance coupling and internal synchronization of individual SCN

cells. This could increase the strength of circadian rhythmicity, since AVP appears to play an important role in determining the power (amplitude) of circadian rhythms [18], and the strength of circadian rhythms are reduced in tau-mutant hamsters (see below). AVP mRNA fluctuates in a circadian fashion in wildtype hamsters [11,35], and a significant reduction in the amount of AVP mRNA has been observed in tau-mutant hamsters comparing circadian times 4 and 16 [35]. The tau 2 / 2 hamsters, however, had lower mRNA levels at both these time points. It should be noted that Scarbrough and Turek [35] used Charles River’s hamsters where wildtypes and mutants were bred separately, whereas we used hamsters of the Wright’s strain with both genotypes bred from heterozygous crossings. Nevertheless, based on these mRNA data one could expect to find time-dependent changes at the protein level in both types of hamsters. Tau 2 / 2 hamsters, however, do not show changes in AVPir over time. Again, by combining the in vivo and in vitro data, this absence in tau-mutant hamsters may indicate that a twofold higher rate of AVP release from their SCN neurons is too high to fully replenish AVP levels at ZT 0 as seen in wild-type hamsters. Although it remains to be determined that AVP mRNA fluctuates in our tau 2 / 2 hamsters, it also raises the possibility that AVP mRNA translation (or AVP feedback on its own production) differs between the genotypes.

4.3. Species comparison The absence of AVPir fluctuation across the light period in tau-mutant hamsters suggests that this genotype has an SCN-AVP system with a low amplitude in circadian rhythmicity. If SCN-AVP contributes to the regulation of circadian rhythmicity of locomotor activity in hamsters, it would imply that tau-mutant hamsters have a weaker circadian organization of behavior. Indeed, tau-mutant hamsters do show a less precise rhythm with higher fragmentation of the rhythm [30]. Similar differences in precision of activity onset have been observed in our tau-mutant hamsters compared to wild-type hamsters [6], both with a randomized genetic background. So, within a species the positive correlation of the strength of circadian organization of behavior with the amplitude of the SCNAVP system holds, as previously shown for the common vole [14,21]. In this species, arrhythmic subjects release very low quantities of AVP in the absence of a circadian fluctuation, whereas rhythmic ones release relatively high quantities of AVP in a clear circadian pattern. However, inter-species comparison is less unambiguous. Factors other than AVP contributing to the strength of circadian organization of behavior may differ considerably between species, currently hampering a clear picture of the role of AVP in the SCN. The expression of circadian fluctuation in AVP release from hamster organotypic cultures is relatively weak,

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irrespective of type of hamster. No clear circadian fluctuation in AVP release was observed in either genotype of hamster, in contrast to vole, mouse, and rat long-term organotypic SCN cultures [21,47]. This may suggest that the AVP system in hamster SCN is less robust than that of the other rodents, despite the strong circadian organization of behavior of this species. SCN cultures obtained from (circadian rhythmic) voles [15,21], rats [21,39] or mice [47] explanted at similar postnatal ages under identical conditions in our laboratory generally reveal more robust circadian AVP release profiles with higher amplitudes (especially rat pups which were used to validate our organotypic culturing methodology and RIA sensitivity [21]). This difference cannot be attributed to a morphologically poorly developed SCN-AVP system of the hamster at the age of explanting (postnatal day 7), since the hamster SCN develops at least as early [8] or even earlier [34] than that of rat [9,20], mouse [17] or vole (K. Jansen, unpublished observations). It may, however, be related to the relatively lower numerical density of AVP neurons in hamster SCN (at least of those used in our laboratory where wild-type and tau mutant hamsters were bred from heterozygote tau mutant parents) compared to the other species. Analyzed in brain sections, mice, voles, and rats have approximately 8, 5, and 4 times more AVP neurons in the mid-SCN region compared to wild-type hamsters at ZT 0 [2,14,51]. In monolayered organotypic SCN cultures, mice, voles, and rats have approximately 4, 2, and 1.5 times more AVP neurons, respectively [47]. Alternatively (or additionally), poor AVP release rhythms of hamster SCN cultures may also be due to phase-angle differences in AVP release rhythms among individual AVP cells. It has been shown, for example, that circadian firing rhythms of individual SCN cells in culture can be out of phase [50]. The observation of the distinct nature of the SCN-AVP system of the tau mutant hamster may indicate that casein kinase I´ [24] influences temporal dynamics of AVP. From this point of view it is worthwhile to further scrutinize the impact of this mutation on peptide regulation at the protein level, for example for vasoactive intestinal polypeptide, somatostatin, or other SCN-derived neuropeptides.

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