Analysis of synaptic bodies in the Sprague–Dawley rat pineal gland under extreme photoperiods

Micron 38 (2007) 237–251 www.elsevier.com/locate/micron

Analysis of synaptic bodies in the Sprague–Dawley rat pineal gland under extreme photoperiods Holger Jastrow *, Jo¨rg Racke Department of Anatomy and Cell Biology, Histology, Johannes Gutenberg-University, Becherweg 13, D-55128 Mainz, Germany Received 4 May 2006; received in revised form 4 June 2006; accepted 5 June 2006

Abstract Synaptic bodies (SBs) are small, prominent organelles in pinealocytes, most probably involved in signal transduction processes. To check the influence of the photoperiod on their shape plasticity and number we chose two extreme lighting conditions, i.e. 20 h of illumination followed by 4 h of darkness (LD 20:4) versus (LD 4:20). Pineal glands were assessed at 0, 4 and 13 h after dark onset. Under both conditions reconstructed SBs were plates or ribbons but never spheres and there were no obvious differences in morphology. Photoperiodic changes in SB profile size and number were investigated: application of the established method for SB quantification based on single section profile counts (SSPC) of areas showed a significant increase of SB profiles under LD 20:4. However, it has to be noted that SSPC depend on both, number and size of the structures. In contrast to this, modification of the disector counting method, also applied for unbiased quantification of whole SBs, revealed that rat pinealocytes show insignificantly more SBs under LD 20:4 than under 4:20 conditions. The lengths of the SB profiles, which were first measured under different conditions in this study, depend on SB size. They increased significantly under LD 20:4. In conclusion, we detected only an increase in SB size but not in their number. We further prove that, at least for SBs, it is of no value to calculate disector levels from SSPCs. # 2006 Elsevier Ltd. All rights reserved. Keywords: Bidirectional disector counts; Synaptic ribbons; Pinealocytes; Three-dimensional reconstruction; Electron microscopy

1. Introduction Synaptic bodies (SBs) are presynaptic, electron-dense, proteinaceous structures surrounded by vesicles. They are present in pinealocytes, retinal photoreceptor and bipolar cells (Sjo¨strand, 1958, 1974) as well as in other sensory cells, e.g., hair cells in the organ of Corti (Sobkowicz et al., 1982; MerchanPerez and Liberman, 1996) or vestibular organs (Ross, 2000). While in sensory organs SBs are involved in signal transmission processes, the functional significance of pineal SBs remains to be elucidated. To analyze their detailed morphology we applied transmission electron microscopy (TEM) to serial sections and performed three-dimensional (3D) reconstructions of SBs for the first time in Sprague–Dawley (SD) rat pinealocytes. SB profiles are usually rod-like (200 nm  35 nm; Figs. 1, 2, 4 and 6b) whereas 3D reconstructions demonstrated that intact structures

* Correspondence to: Raiffeisenstr. 11, 55239 Gau-Odernheim, Germany. Tel.: +49 6733 949540; fax: +49 6733 949540. E-mail address: [email protected] (H. Jastrow). URL: http://www.drjastrow.de/HJ.html 0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2006.06.013

are plate- or ribbon-like in the pineal gland (Jastrow et al., 1997a, 2004, Figs. 5 and 6a). SBs have a constant thickness of 35 nm and two major parallel flat surfaces. When width to length ratio of these main surfaces is between 1:1 and 1:3 we define SBs as plate-like, whereas we term them ribbon-like when the ratio is 1:>3. The usually smaller plate-like structures were more frequent than ribbons and follow-up study of serial sections also indicated a predominance of plates. Due to these facts, we use the more general term ‘‘synaptic bodies’’ instead of ‘‘synaptic ribbons’’ the more frequently used term in the literature. In rat pinealocytes SBs undergo changes in number and size during a 24 h period (Kurumado and Mori, 1977, 1980; McNulty et al., 1985, 1987; Riemann et al., 1990; Jastrow et al., 1997a) and further show changes in different photoluminous ‘‘seasons’’ (Karasek et al., 1988a; Martı´nez-Soriano et al., 1992). Light mediates functional and ultrastructural changes in pinealocytes (Karasek, 1981; Martı´nez-Soriano et al., 2002) via the retina-hypothalamic-reticulo-superior cervical ganglia pathway, providing adrenergic innervation of the pineal gland (Wurtman et al., 1967). Constant light substantially decreases SB profile numbers, whereas constant darkness increases them in the rat pineal (King and Dougherty, 1982). Short light pulses

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Fig. 2. A field of rod-like SB profiles from almost parallel oriented ribbon- to plate-like SBs. Fields with more than five directly adjacent profiles were rare. Note the lamination: two central less electron-dense stripes are visible in five SR profiles, indicating that these profiles were cut at exactly 908 to the section plane and run parallel. Also note that the central vesicles of the field are attached to both neighbouring SBa and that they have considerably smaller diameters than the SER in vicinity to the field.

Fig. 1. Micrographs showing typical profiles of synaptic bodies (SBs) of the rat pineal gland. Singly lying profiles are attached to small plaques on the cell membranes of adjacent pinealocytes (a). Such pairs of directly opposite SBs were rare, whereas isolated singly lying profiles were most common, followed by pairs of roughly parallel oriented profiles (b).

at night also result in a reduction of SB profiles (Karasek et al., 1988b). Light information is transmitted to the pineal gland in different ways mainly via the retinohypothalamic tracts, suprachiasmatic nuclei paraventricular hypothalamic nuclei, spinal cord and superior cervical ganglia providing adrenergic fibres to the gland, further there is central innervation of the gland (reviews: Reuss, 1996, 2003). The most important neuronal input to the pineal gland is its adrenergic innervation, which is established about 8–10 days after birth (Machado et al., 1968; Kurumado and Mori, 1980) and has to be seen in the context of the maturation of the ocular photoreceptor system which exhibits morphological and physiological characteristics of adult retina at postnatal day 12 in rats (Weidmann and Kuwabara, 1968). The established method for quantification of SBs is a single section profile count (SSPC), from areas of a single section, to

avoid double counting. The size of the evaluated tissue ranges from 1470 mm2 (Karasek et al., 1982) to 65,630 mm2 (Jastrow et al., 1997b), but most investigations were performed on areas of 20,000–22,000 mm2. The quantity of SBs profiles in SD rat pineal glands of all available studies so far published is summarized in Table 1. Comparison of data, calculated to a standard area of 20,000 mm2, reveals considerable variations for similar conditions in many cases. Reviews providing quantitative data of pineal SB profiles of other species (McNulty and Fox, 1992; Bhatnagar, 1994) also show such variations. One reason for this may be that all the data collected so far (with one exception: Jastrow et al., 1997b) are based on SSPC in defined areas. Unfortunately, this procedure is biased (particle size influences the number of profiles). Some investigators simply counted ‘‘ribbon fields’’, whereby they defined them as single or groups of closely lying SB profiles. This should avoid double counting of profiles from inherently folded SBs. To obtain a more meaningful number of whole SBs in a defined volume of the tissue under scrutiny, further effort and an appropriate method is required. Our study was carried out to give reliable numbers of whole synaptic bodies per volume for the SD rat pineal gland under different extreme light conditions. Therefore, we decided to use the disector, an unbiased stereological tool which is not influenced by alterations in shape and size of particles. In this way quantification is performed on two adjacent sections, resulting in the number of counted particles in a defined volume (Gundersen, 1986; Gundersen et al., 1988). We used the adaptation of the disector counting method for rare and small particles introduced by Jastrow et al. (1997b) to cope with problems due to size and rareness of SBs. Thus, two sections are alternately scanned directly on the TEM screen (Fig. 3).

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In order to diminish the counting time, we checked, whether a factor, as suggested by Jastrow et al. (1997b), allows the calculation of SB number per unit volume from SSPC data with sufficient accuracy, when both quantification methods are applied to one animal of an identically treated group. Further we tested, whether multiplication of an SSPC value in combination with a profile length measurement would be sufficient for reasonable quantification of SBs. 2. Materials and methods Adult male and female Sprague–Dawley rats (n = 36; separated into six groups of six rats each; body weight: 150– 180 g) were kept under a protocol according to the NIH guidelines for care and use of laboratory animals (illumination with Osram L 65/25 white universal fluorescent strip lights, 200 lux at cage level; room temperature 20  1 8C; relative humidity 50%; food and water ‘‘ad libitum’’). Two different extreme light/dark cycles were applied for two weeks in January

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2001: one with 4 h of light followed by 20 h of darkness (LD 4:20; groups 1–3), the other with 20 h of light followed by 4 h of darkness (LD 20:4; groups 4–6). Animals were killed by decapitation under open ether anaesthesia. Animals of group 1 were killed some minutes before light off, those of group 2 after 4 h of darkness and those of group 3 after 13 h of scotophase. Rats in group 4 were sacrificed a few minutes before onset of darkness, those of group 5 after 4 h of darkness and those of group 6 after 13 h of the onset of the dark phase lasting for 4 h (which means 9 h after light on; more details in Table 1). 2.1. Tissue preparation Pineal glands were quickly removed (during scotophase under dim red light) and immediately fixed in a modified Karnovsky (1965) solution of 2% (w/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 0.1 M PBS (phosphate-buffered saline, pH 7.4) at 4 8C for 24 h. After washing in 0.1 M PBS with 6.8% (w/v) sucrose, pineal glands were postosmicated for

Table 1 Number of all synaptic body profiles (irrespective of form) and/or fields expressed as means  S.E.M. per 20,000 mm2 of tissue of untreated (controls of most studies) Sprague–Dawley rats in vivo sorted according to LD, time of sacrifice, month and sex

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90 min (2%, w/v, OsO4 in 0.1 M PBS, pH 7.4), block-stained in 0.5% (w/v) uranyl acetate plus 1% (w/v) phosphotungstic acid in 70% (v/v) acetone overnight, dehydrated in acetone and embedded randomly oriented in Epon1. Tissue blocks were trimmed to a wedge shape and notches were cut into margins to enable recognition of corresponding areas more easily. Serial sections were cut with diamond knives on a Reichert Ultratome III set to a section thickness of 50 nm. This thickness was chosen in agreement with the recommendation that for disector counts the height of the investigated volume should be a third to a quarter of the particle size to be investigated (Austin et al., 1995; Mayhew and Gundersen, 1996), since the average SB size is 100–300 nm (Jastrow et al., 1997a,b). The requirement that the separation of paired section planes of physical disectors should be less than the smallest linear dimension of the particles being counted was fulfilled (Austin et al., 1995). Sections were mounted on one-hole copper grids coated with a Formvar1 film, and were stained in 8% (w/v) uranyl acetate in distilled water for 10 min followed by 0.7% (w/v) lead nitrate + 0.9% (w/v) sodium citrate for 5 min, according to Reynolds (1963). Table 1 (Continued )

2.2. Examination and image acquisition Thirty-six series of up to 21 sections were examined using a LEO (Zeiss, Oberkochen, Germany) TEM 906E. Disector counts (DC) and SSPCs were performed simultaneously on two adjacent sections taken from the centre of a series (examined areas detailed in Table 1; magnification: 21,560). Due to the rareness of straight undamaged tissue edges in animal no. 9 two pairs of sections lying at a distance of some micrometers (to avoid double counting of large SBs) were analyzed. Thereby evaluated areas and counting results were summed. A modification of the disector method (Jastrow et al., 1997b) was applied to cope with problems due to size and rareness of SBs. The disector was applied bidirectionally to improve efficiency during investigation of the reference volume. Counting results were expressed per standard area of 20,000 mm2 and for a volume of 1000 mm3 (standard area  50 nm section thickness). All counted profiles as well as the corresponding areas in the adjacent disector-counted sections were digitized using a 14 bit Slow Scan CCD Camera (Proscan, Scheuring,

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Table 1 (Continued )

SBP: number of synaptic body profiles; SBF: synaptic body fields; g: grams; d: days; w: weeks; m: male; f: female; f18: female on day 18 of pregnancy; L/D: light/ dark hours; L on: Light on at; : time of sacrifice; area: area of count per animal (mm2); [n]: number of animals; month (1–12); LL: constant light; *mean, no normal distribution of results; #mean of six different counted areas; dark shading for all animals killed during the dark phase.

Germany) and a PC equipped with a frame grabber card, running Analysis 3.2 software (Soft Imaging Systems, Mu¨nster, Germany) for image acquisition. 2.3. Statistical analysis The lengths of the counted areas (at 400–775) and of SB profiles (at 21,560) were measured using Analysis 3.2. SB-, SB profile counts and length measurement results were expressed as means  standard error of the mean (S.E.M.). SigmaStat 2.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. One-way Anova comparing individual groups, all pairwise multiple comparison procedures (Fisher LSD method, Tukey test) or unpaired t-tests were performed for SSPC and DC analysis. Kruskal-Wallis one-way analysis of variance on ranks or unpaired t-tests were used for evaluation of data of SB profile length. P-values equal to or less than 0.05 were regarded as significant. 2.4. Three-dimensional reconstruction

Fig. 3. Demonstration of the modified disector counting method. The areas in which disector counts were performed (red) are well preserved in sections a and b and show straight undamaged edges. They reach 7 mm (diameter of the visible screen at 20,000) in the sections. The counting volume was the sum of the lengths of the areas (1–6a,b)  7 mm  0.05 mm (section thickness)  2 (both sections).

To demonstrate the true three-dimensional (3D) shape of SBs five SB-fields and of six singly lying SBs were chosen and followed up in all adjacent sections, as long as SB profiles were seen or the end of the section series was reached. Digital sections of the SB fields were overlaid for three-dimensional reconstruction using Adobe1 Photoshop1 7.01 (Adobe Systems Inc., San Jose, CA, USA), and imported as oriented image stacks into different 3D reconstruction software: either

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VoxelCruncher 5.1 (ConVis GmbH & Co KG, Mainz, Germany) or Amira 4.0 (Mercury Computer Systems, Chelmsford, MA, USA). Profiles of identical SB were joined to 3D objects and visualized in different ways. For better comprehension of the 3D arrangement of SBs animations were generated from different series of rendered views to simulate 3608 rotation of reconstructed objects. Some of these animations are published as supplementary material on the Internet at: http://www.drjastrow.de/SBrat.html. 3. Results 3.1. General observations The general ultrastructure of the investigated pineal glands of rats does not show any obvious differences to the literature

(Wolfe, 1965; Karasek, 1981; Vollrath, 1981; Calvo and Boya, 1984; Nowicki et al., 2002). Over 70% of all synaptic bodies (SBs) are located in a distance of less than 100 nm to the cell surface membrane. In many cases they are directly attached to it by fine electron-dense stalks, anchored in a small electron-dense plaque on the inner surface of the plasmalemma (Figs. 1a,b and 4). As mentioned in Section 1, the three-dimensional form of SBs is ribbon or plate-like (Figs. 5–7). The reconstructed SBs, which can be seen as colour stereo animations at http://www.drjastrow.de/SBrat.html, are long, bent and more-or-less twisted ribbons. In one case two ribbon-like SBs join at an acute-angle to form one larger bizarre SB (Fig. 5). Most singly lying profiles appear on only a few consecutive sections and are thus small plates when reconstructed (data not shown). Over 90% of the altogether 2828 SB profiles digitized and analyzed are rod-like. Virtually all remaining profiles are derived from tangential to

Fig. 4. Demonstration of the disector counting method by two examples of adjacent sections (a and b) with areas showing unusual high concentrations of SBs. Only those profiles were disector counted that were present in just one section (red circles). The criterion was that no trace of an electron-dense profile had to be present. The arrow marks a partial profile caused by the edge of an SB with a clear profile in the neighbouring section that was not disector counted. Note that further adjacent sections (not shown) need to be checked to be sure that two profiles deriving from one bent or crescent shaped SB were not counted twice.

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Fig. 5. Five stereo views of a three-dimensionally reconstructed field of SBs showing bent ribbon-like SB structures. Corresponding stereo motion pictures are published on: http://www.drjastrow.de/SBrat.html.

sagittal cuts of plates or ribbons. When other profile forms are followed up in consecutive sections it becomes evident that small round profiles or triangles or irregular shapes are near-edge sections of plates, or derived from twisted or strongly bent ribbons. Thus in our material, i.e. under the extreme light conditions applied in this study, no true spheres (spherules) and only four small irregular (i.e. non-plate- or ribbon-like) SBs are present. SBs consist of five laminae, the central of which and the two outer ones are extremely electron-dense; they enclose two less electron-dense intermediate laminae. This lamination is only visible when SBs extend straight throughout the section thickness and lie at exactly 908 to the plane of vision (Figs. 2 and 6b). This is demonstrated by the ‘‘true colour’’, i.e. original grey tone 3D animation on http://www.drjastrow.de/SBrat.html. The lamination of SBs is apparently caused by an extremely regular polymerization pattern of the SB subunits. The exact thickness of SBs was measured and amounts to 35 nm. There was no homogeneous distribution of SB profiles in any section series. Sometimes even in large areas (>2000 mm2) not a single SB profile occurred whereas in other areas up to

10 SB profiles were located in a few mm2. This was mainly caused by SB fields that were randomly scattered throughout the entire material. The two evaluated areas from animal #9, lying at a distance of some micrometers had nearly the same size (about 10,600 mm2), however SSPC values were quite different 35 versus 56 SB profiles, whereas DC was approximately the same (9 versus 10 SBs). This animal showed, unusually, many different fields of SBs (34). 3.2. Quantitative results Quantity of SBs using the disector counting procedures (DC) as well as of SB profiles applying SSPCs is shown in Table 2. Most differences between the experimental groups were not statistically significant. The only significant differences were seen between groups 6 and 1 as well as 6 and 3 in both, SSPC and DC (6 to 1: SSPC P = 0.002; DC P = 0.006; 6 to 3: SSPC P = 0.006; DC P = 0.028). Group 1 had just significantly (P = 0.050) less SBs than group 5 in DC whereas in SSPC no significance was reached (P = 0.085).

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Fig. 6. Four ribbon- or plate-like SBs and a starting SB close to the membranes of two cells in 3D reconstruction (a) and in a 3D ‘‘true colour’’, i.e. original grey tone image (b) taken from a stereo animation on http://www.drjastrow.de/SBrat.html which shows the five-banded structure (three dense, two light) of SBs that is only visible when they are exactly to be viewed from the direction of their long axis in an exactly 908 position of the section plane. One SB lies in a finger-like process of a pinealocyte completely invaginated in another pinealocyte showing two membrane-associated SBs.

When pooling all groups under the same LD conditions significantly more SB profiles were present under LD 20:4 (P = 0.023; Anova; P = 0.005 in an all pairwise multiple comparison procedure; Fisher LSD method). However, the Tukey test revealed that differences were only caused by the very strong difference between groups 6 and 2. A statistically significant difference was not reached for entire SBs in DC (P = 0.065, Anova). 3.3. SB profile length

Fig. 7. Five plate-like SBs close to the cell membrane (red lines). Note that one is anchored to an attached small electron-dense plaque (arrow).

The length of SB profiles is given in Table 2. There is no statistically significant difference (P = 0.237; Kruskal-Wallis) of the average length of SBs, only the t-test shows a significant increase (P = 0.036) of length of LD 20:4 when pooling all groups of the two different lighting regimes (1–3 to 4–6). When taking all measured lengths into account a statistically significant increase of length occurs during 20:4 conditions (P < 0.001) and a significant nocturnal decrease under LD 20:4 (P < 0.007; 4 to 5). SB profiles are significantly longer in animals killed under 20:4 compared to 4:20, when killed after 4 h of light (P = 0.047; 2 to 5). They are significantly longer

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Table 2 Counting results expressed as means  standard error of the mean Group and condition

DC

SSPC

Length

L 4 D 20 1: +0 h dark, i.e. +4 h light 2: +4 h dark 3: +13 h dark

9.34  1.85 11.34  7.83 11.28  3.19

32.32  6.23 42.48  23.65 37.20  7.20

187.11  38.29 206.41  30.46 199.56  16.30

10.65  4.79

37.37  14.47

197.69  29.15

11.30  6.22 13.20  3.81 18.88  6.51

51.74  25.35 47.78  18.82 71.08  22.79

237.31  53.67 207.89  39.64 231.34  45.10

14.46  6.25

56.87  23.57

Groups 1–3 pooled L 20 D 4 4: +0 h dark, i.e. +20 h light 5: +4 h dark 6: +13 h dark onset, i.e. 9 h light Groups 4–6 pooled

225.52  45.59 3

L: hours of light; D: hours of darkness; +: killed after . . .; DC: number of SBs counted using the disector per standard volume of 1 mm ; SSPC: single section profile count of SBs per standard area of 20,000 mm2; Length: length in nanometers.

after the longest light exposure compared to longest dark exposure (P < 0.001; 3 to 4) or at the end of the light phases when LD 4:20 and 20:4 are compared (P < 0.001; 1 to 4).

disector value). In brief again very considerable errors will occur with the application of a calculation factor 2 that takes profile lengths into account.

3.4. Can a factor be used to calculate DC values from SSPC?

4. Discussion 4.1. Morphology of SBs

A factor 1 (F1) may be calculated by dividing SSPC per 20,000 mm2 with DC per 1000 mm3, for one animal of an identically treated group. If this F1 is applied to the SSPC results of the other animals of the group and calculated DC values are compared to the really counted SBs, differences will range from 14.9 SBs that are calculated too less up to 14.8 SBs which are estimated too much. The range of error in percent (of the true disector value) is from 67.4 to +159.9%. The standard deviation (S.D.) is 3.84 SBs, and 25.4%. The mean F1 calculated on basis of all 36 animals is 0.27  0.07 (range: 0.13–0.46). The given values were determined by calculation of all possible combinations for all groups. When regarding groups of identically treated animals for themselves the S.D. of the values ranges from 1.87 to 5.40 SBs and from 9.98 to 48.86% (of the true disector value). In short very considerable errors will occur in case of applying a calculation factor as proposed above. If length of profiles is also taken into account, a factor 2 (F2) can be calculated by the formula F2 = DC  L/SSPC, where DC is the disector count result per mm3, L the mean length of SB profiles in mm and SSPC is the single section profile count per 20,000 mm2. In case such a factor is calculated for one animal of an identically treated group and resulting DC values of the other animals are compared to the true DC values, differences will range from 12.9 SBs that are calculated too less up to 23.6 SBs which are estimated too much. The range of error in percent (of the true disector value) is from 60.4 to +139.6%. The S.D. is 4.50 SBs, and 25.9%. The mean F2 calculated on basis of all 36 animals is 0.057  0.015 (range: 0.031–0.094). These values result from calculation of all possible combinations for all groups. When analyzing groups of identically treated animals only, the S.D. of the values ranges from 2.66 to 6.00 SBs and from 14.59 to 35.47% (of the true

SBs have not been previously reconstructed three dimensionally from Sprague–Dawley rat pineal gland. The few speculations about their true morphology based on the analysis of serial sections and range from rod-like to plate-like organelles (Spiwoks-Becker, 1995). Our five reconstructed SB fields showed mainly bent, more or less twisted ribbons and few plates. The organelles were arranged less often parallel and were not as large as in the guinea-pig pineal gland (Jastrow et al., 2004) or in rhesus monkey (McNulty et al., 1986). Six singly lying profiles were followed-up and most of them turned out to be small plates. Our reconstructions were performed to give an impression of the true 3D morphology of SD rat pineal SBs, but not show morphological details of reconstructed SBs under the different conditions. However they clearly show two forms of SBs: plates and ribbons that always have two parallel major surfaces at a constant distance of 35 nm from each other, and did not show any thicker areas or protrusions at any of our extreme light conditions. From serial and neighbouring sections we assume that most SBs that lie close to the cell membrane are attached to the latter, over a distance of 50–150 nm, by fine electron-dense material which ends at small plaques (Fig. 7). These plaques were considerably smaller than arciform densities which anchor retinal SBs at the cell membrane. It remains to be elucidated if the protein Bassoon controls the attachment of pineal SBs to these plaques, as has been shown for the retina (Dick et al., 2001, 2003). 4.2. No spheres discovered Contrary to reports of others (Karasek and Vollrath, 1982; Vollrath et al., 1985; McNulty et al., 1989; Spiwoks-Becker, 1995) we did not discover any definite spheres in our SD rat

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material. Our findings may be due to the extreme light–dark conditions applied in this study or the schedule of animal sacrifice. The highest numbers of spheres were observed under experimental and untreated organ culture conditions (SpiwoksBecker, 1995). Other in vivo figures are much lower for the SD rat pineal gland. In contrast to this, spherical SBs were identified and reconstructed in other species, e.g., guinea-pigs (Jastrow et al., 2004). Virtually all TEM projection profiles of these spheres were perfect circles, with large diameters. We found no such profiles in our present material.

length will correspond to the mean size or volume of the SBs. Since, as mentioned before, determination of the true 3D structure of SBs is not simple, it is virtually impossible to generate random SBs and to calculate how many random length measurements would be necessary to get a mean profile length that correlates to the genuine mean volume of the structures with acceptable accuracy. We estimate that at least 500 profiles would be necessary for a reasonable calculation.

4.3. SB profile lengths

SBs are formed by side-to-side aggregation of RIBEYE dimers, creating in a very regular 2D pattern (Schmitz et al., 2000). Thus, it may be postulated that the outer electron-dense laminae of SBs correspond to the RIBEYE B domain, the intermediate less electron-dense layers to the RIBEYE A domain and the central electron-dense lamina to the dimer subunit which connects two RIBEYE A molecules. However, further smaller components are also present, not in but along the surface of SBs, at least in the retina: Rim1 (RAB-3 interacting molecule1, Wang et al., 1997) is located on the surface of entire SBs, Caveolin-1 (Kachi et al., 2001) and KIF3a (Muresan et al., 1999) are closely associated with SBs, Piccolo is present in tip regions of SBs and Bassoon in the basal part for connection to membrane anchoring plaques or arciform densities (Dick et al., 2001). Most recently it has been demonstrated that RIM2 and CAST also localize at the base of SBs, whereas ELKS is present around entire SBs (Deguchi-Tawarada et al., 2006). Most of the aforementioned proteins are components of active zones and not solely present in or at SBs (tom Dieck et al., 2005). Their presence has been demonstrated at rod and cone synaptic bodies as well as in/on SBs of rod- and cone bipolar cells, but remains to be investigated in the pineal gland.

There are only few data available on SB profile length (Table 1). In general profiles are significantly longer at night. The increase was 23% in Spiwoks-Becker (1995) and 27.6% in Jastrow et al. (1997a). Using serial sections the latter authors calculated surface areas and volume of SBs, which also increased (both by 19.3%). The previous data are derived from LD 12:12 animals. Under the extreme light conditions of this study, the only comparable significant change was a nocturnal decrease under LD 20:4, when all animals were pooled. This indicates only a minor general influence of light on SB profiles in this study. SB profiles were significantly longer after longest light compared to longest dark exposure. This is contrary to what might be expected from the LD 12:12 data. The very long exposure to light (20 h) results in very significantly longer SB profiles than the quite short one (4 h) under LD 4:20, indicating again that longer light exposure markedly increases SB profile length. This is also in accordance with the finding that when pooling all groups of the two different lighting regimes the LD 20:4 animals showed significantly longer SB profiles. There are no data on SB profile lengths after constant light exposure in SD rats. An increase of SB profile length was present in pineal glands of guinea-pigs kept under permanent light for 4 months. However this was only statistically significant in the intermediate and distal regions but not the major part of the gland (Vollrath, 1986). When considering the length of the SB profiles, one has to be aware that these lengths are simply random distances measured inside plates or long ribbon-like structures. The true length of the 3D objects can only be determined when the latter are reconstructed from serial sections and even then one length will not be enough for a reasonable description of the whole structure, due to the fact that SBs are never ideal rectangular cuboids and may further be bent. Thus the lengths of the four (more accurately 8) main long edges are all different (Figs. 6 and 7). In some cases there are even more than four edges, for example when one large SB is formed by fusion of two plates (Fig. 5). Some of the plates also exhibit holes, as demonstrated in Jastrow et al. (1997a). The only constant part of the organelles is their thickness that can be correctly determined only when it is oriented exactly at right angle to the section plane, i.e. when the five laminae are clearly visible, as shown in Fig. 6b; it is exactly 35 nm. On the assumption that SBs are randomly oriented it can be assumed that if the length of a very large number of SB profiles is measured the resulting mean

4.4. Molecular composition of SBs

4.5. Comparison of quantitative data A summary of quantitative data on SB frequency calculated to the established standard area (20,000 mm2) detailing all available quantitative information on Sprague–Dawley (SD) rats in vivo in the literature is given in Table 1 (for other rat strains, culture or other experimental conditions, and further species see McNulty and Fox, 1992; Bhatnagar, 1994; Karasek and Zielinska, 2000). In general, SB profile numbers did not show differences at different stages of the oestrous cycle or between males and females (Saidapur et al., 1990). The comparison of data shows considerable variations of results in SSPC even under quite similar conditions. Possibly unmentioned factors, like the intensity of light (lux value), the origin and number of the animals per cage, temperature, month of the year, geographical location and further still unknown conditions could have a major influence on the SSPC of SBs. In this context, a limited investigation at our Department may also be of relevance: to check in how far SSPC depend on the investigator, six persons (one professor, two assistants, two students and one technical assistant) who all were used to count SB for at least 6 months, evaluated exactly the same five grids of a randomly chosen SD rat pineal gland. The results had a

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considerable range and there was a statistical significant difference between the maximal and minimal count. Discussion and joint re-evaluation demonstrated that virtually all larger or clearly visible SB profiles were noted by all participants, in some cases very small profiles were not included, in fewer cases similar structures were mistaken as SB profiles. Since disector counts always involve at least two consecutive sections, in cases of uncertainties further ones, the probability of overlooking small SB profiles is reduced, but not abolished. Thus, even the results of the most skilful investigator could produce minimal figures. In all quantitative studies on SB profiles it was assumed, though never mentioned, that the person(s) who did the evaluation will always make the same mistakes, so results remain comparable. A further fact also needs to be mentioned: the considerable and significant differences in SSPC from the two different section pairs of animal #9. Though the evaluated areas were both only about half of the desired minimum (20,000 mm2) the results show that there is no constant distribution of SBs in the tissue inside one pineal gland. This is of relevance for validity of application of disector counts (see below). There are few investigations of SBs in pineal glands of SD rats exposed to LD conditions other than 12:12. Karasek (1976) and Karasek et al. (1983a,b) exposed SD rats to LD 14:10; SSPC are within the same range as in 12:12 studies (Table 1). Vollrath and Maitra (1986) noted a very significant, over twofold increase, of SB profiles in animals kept under constant light for 63 days. SR numbers and NAT activity significantly decreased when animals were exposed to light at night (Maitra et al., 1986). The pineal glands of Wistar rats kept under a constant photoperiod (12:12) showed statistical significant differences in SB profile number when animals were killed at monthly intervals at 01:00 and 13:00 h (Karasek et al., 1988a). SB profile number was highest in October and lowest in April. This may also be true for SD rats. Thus the animal killed at noon in October by Jastrow et al. (1997b) showed very high SB profile numbers (90/20,000 mm2, 33 SBs in disector count/ 1000 mm3) when compared to other data (Table 1). Adult male cotton rats were kept under LD 8:16 (n = 5) or LD 16:8 (n = 5) by Karasek et al. (1986). Animals kept in the short photoperiod showed larger relative volumes of mitochondria, RER, ribosomes, Golgi apparatuses, inclusion bodies (characteristic only for this rat strain) and dense core vesicles which can be interpreted as characteristic for higher active cells, compared to the LD 16:8 animals. However, SBs were not mentioned in this study. A study on the brush mouse, that has a pineal gland relatively similar to that of SD rats, comparing LD 8:16 versus 16:8 (four animals each; Karasek et al., 1983b) showed changes similar to those mentioned previously but did not give any quantitative data on SBs. There was no statistically significant change in SB profile number on different days in summer from the end of July to mid-August under LD 12:12, but day night differences were observed by McNulty et al. (1985). Studies on pineal glands of Wistar rats kept under natural light conditions demonstrated, apart from a significant increase of SB profiles from photo- to scotophase, a significant increase of SB profiles from September to February (Cimas

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Garcı´a et al., 1987; Martı´nez-Soriano et al., 1992), a significant decrease from winter to spring (Martı´nez-Soriano et al., 1996, 2002) and significant differences between full- and new-moon phase (Martı´nez-Soriano et al., 2002). In general, these studies confirmed that SB profile numbers are inversely proportional to environmental luminosity as it was first noted by Vollrath (1973) for the guinea-pig pineal gland. Pinealocytes of Djungarian hamsters kept under LD 16:8 or 8:16 conditions did not reveal striking differences except an increase of dense core vesicles (P < 0.3) and an insignificant (P < 1.6) decrease of SB profiles in short-day animals compared to long-day animals (n = 4 per group; Fechner, 1986). They showed a significant decrease in animals killed after 67 days of continuous light whereas in SD rats a significant increase was noted, as mentioned before (Vollrath and Maitra, 1986). 4.6. Disector It is generally agreed that SBs are somewhat randomly but nevertheless homogenously distributed in pineal tissue when large enough areas are investigated. All studies on SB quantity carried out so far are based on this tacit understanding. In this context it was demonstrated that the number of SB profiles and fields of such profiles is not statistically different for the proximal, intermediate and distal part of the gland in SD rats (Kurumado and Mori, 1977), Wistar rats (Kosaras et al., 1983) or guinea-pigs (Vollrath, 1973). However our disector results demonstrate that either the investigated areas were too small or a general requirement for the application of the disector (Gundersen et al., 1988), i.e. the constant distribution of structures under scrutinity in the reference volume was not fulfilled. This could be an explanation for the fact that calculation of factors to estimate disector count values from SSPCs, as was theoretically proposed in Jastrow et al. (1997b), does not make sense in practice. As documented in Section 3, the errors, at least in the material of this investigation, are unacceptable (>25%), even when taking length of profiles into account. Thus, for reliable statements about the quantity of SBs in a volume of pineal tissue true disector counts have to be performed for each animal. Due to the random appearance larger fields of SBs a truly valid quantification, like the fractionator (Gundersen et al., 1988; West, 1993), would require disector counts of SBs on thousands of TEM screens, involving dozens of section pairs taken in systematic random from just one gland. The reason for this is the high variation of fractionator estimates in comparable random point clouds with irregular distribution of sampled objects. This variation can only be reduced by very large disector counts (Schmitz, 1998), which can hardly be realized in practice using TEM technology. In this context, the most reasonable procedure at present would be to label SBs with antibodies against their known components, e.g. RIBEYE A or B (Schmitz et al., 2000), and then use immunofluorescence and laser scanning microscopy to perform optical disector counts in systematic random samples according to the requirements of the fractionator. Preliminary investigations are in progress and

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confirm the above mentioned extremely irregular distribution of SBs and the presence of more fields than expected by regular electron microscopy of the SD rat pineal gland (SpiwoksBecker, personal communication). 4.7. Chronobiology of SBs A day/night rhythm of SB profile number is well established in the literature (Vollrath, 1973; Kurumado and Mori, 1977, 1980; Spiwoks-Becker, 1995; Martı´nez-Soriano et al., 2002). This circadian rhythm develops shortly after initial sympathetic innervation of the pineal parenchyma, between postnatal days 10 and 20 (King and Dougherty, 1980). However, our data are not in accordance with any clear rhythm, neither in single section profile- nor in disector counts. The fact that the only statistically significant quantitative differences were seen between group 6 (LD 20:4, 9 h of light) and group 1 (LD 4:20; 4 h of light) and group 3 (LD 4:20; 13 h of dark) in SSPC and DC are difficult to interpret, especially since after 9 h of light SB number was at its maximum. There is a clear contrast to other studies: under LD 12:12 pinealocytes showed significant minima of SB profile numbers 6–10 h after lighton followed by a slight increase beginning a few hours before light-off (Kurumado and Mori, 1977). These authors’ results from SD rats correspond very well to data from male guineapigs under same LD regimen (Vollrath, 1973). Our animals, sacrificed at the end of the light period (group 4), had less instead of more SBs and SB profiles, however, statistical significance was not achieved (P = 0.067). In short, our data are quite unusual in so far as on the one hand under light SBs first increased and then decreased and on the other hand only very few significant changes were observed concerning SB- and SB profile numbers: groups 1:6 and 3:6, and the borderline significance in DC (groups 1–5; cf. Section 3). In addition, there was only a statistical significant increase in SB profile number from LD 4:20 to 20:4, when all animals under these conditions were pooled. Adly et al. (1999) and Spiwoks-Becker et al. (2004) demonstrated under light conditions retinal SBs of BALB/c mice, loose material showing protrusions and regain it in the dark phase. In our investigation of the rat pineal we could not detect any material that seemed to bud off from or fuse with SBs. Therefore we assume that at least in the case of rat pineal SBs, the changes in size are based on molecular diffusion of small components that are beneath TEM resolution. Such mechanisms were also assumed for the guinea-pig pineal SBs by Vollrath (1973). In contrast to the retina the vast majority of SBs is much smaller in the pineal gland and thus changes may not need to be as quick as in rods and cones. Continuous illumination caused bizarre alterations of SBs in guinea-pig pineal glands (Vollrath and Huss, 1973; Vollrath, 1986; Jastrow et al., 2004), but not in the SD rat pineal gland (Vollrath and Maitra, 1986). Spiwoks-Becker (1995) demonstrated that after 6 h of light under LD 12:12 at 12.00 37.5% of all SBs in SD rat pinealocytes lie in fields, this is significantly higher than at 24.00 and agrees with results from guinea-pig (Vollrath, 1973). We did not try to give data on SB fields since we had the

impression that (a) they were too few for a reasonable statistic evaluation, (b) they had different numbers of SBs, (c) fields which just show their first outer SB profile will not be detected and (d) there was no clear but a random relation to any experimental conditions. It has to be assumed that many of the singly lying SB profiles in the evaluated sections of all studies belonged in fact to fields that would have appeared when further sections would have been followed up. The same goes for paired profiles. A reasonable evaluation of SB fields would at any rate require serial section analysis of all profiles. This was not done totally in this investigation, since for certain disector counts check of adjacent sections was performed, until it was decided whether close profiles derived from one or more structures. 4.8. Functional considerations The number of SBs and SB profile length that apparently correlate with SB size, surface area or volume were determined in many studies. However, despite 40 years of research work, SB function and the functional relevance of such data still remains to be elucidated in the pineal gland. SBs may serve in intercellular communication (Vollrath, 1973), but many of them lie distant to the cell membrane and instead of postsynaptic other pinealocytes it was not rare to find connective tissue structures next to membrane-close SBs. Maybe glutamate is released in such instances to diffuse to vessels, in a paracrine way. Pinealocytes communicate via a paracrine signal system involving GABA, glycine, aspartat and glutamate. These substances are stored in synaptic-like microvesicles (Redecker et al., 2001), probably in contact with SBs. On the contrary, it has become evident that in the retina and sensory organs SBs are present wherever synaptic exocytosis is evoked by graded depolarization (Sterling and Matthews, 2005). However, the pineal gland does not show any graded depolarization, but action potentials with a mean discharge rate of 1.5  0.3 impulses/s (Schenda and Vollrath, 1997). It is a neuroendocrine organ with a characteristically fluctuating melatonin-secretion activity. It is clear that SBs are involved in excitatory glutamatergic synapses, whereby transmitter release alters the physio-chemical properties of postsynaptic cells. Vollrath (1973) postulated that this could induce changes in activity of enzymes involved in the melatonin synthesis, of which the aaNAT is the most important. However, there is no correlation of pineal SB profile numbers with melatonin synthesis (Vollrath and Welker, 1984). Spessert et al. (1992) showed that a non-adrenergic cGMP metabolic pathway is involved in the regulation of SR numbers in the rat pineal gland and that stimulation of cytosolic guanylate cyclase increased SB profile numbers in a dose- and time-dependent way. Karasek et al. (1983a) demonstrated an inverse correlation of the density of adrenergic nerve endings with SB profile numbers in several mammalian species. Accordingly, McNulty et al. (1996) emphasized that neural mechanisms play an important role in regulating SB numbers as their co-culture of pineal gland and superior cervical ganglia demonstrated a significant decline of SBs compared to isolated pineal organ

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culture. It is most likely that further still unknown factors influence SB formation and decay. There are three major models of SB function proposed for SBs of sensory organs: 1. SBs serve as conveyor belts and transport vesicles to the cell membrane, to resupply the active zones of retinal ribbon synapses (Bunt, 1971; Lenzi and von Gersdorff, 2001; Parsons and Sterling, 2003). In this context ELKS- and Piccolo-mediated protein complexes may be responsible for the binding or transport of vesicles, whereas CAST- and Bassoon-mediated complexes could serve in fusion and release vesicles at the nearby active zones (DeguchiTawarada et al., 2006). In our opinion, a vesicle conveyor belt function of SBs is quite improbable for the pineal gland, since synaptic architecture is different to the retina, i.e. a considerable number of SBs are oriented nearly parallel to the cell membrane and in fields only the very few directly membrane-attached SBs could plausibly transport synaptic vesicles (SVs) to their release sites. It appears unlikely that there is a movement of SB subunits (RIBEYE A, B) in one specific direction, as would be required for a conveyor belt, because then SB material must be rapidly depolymerized at the base of the SBs. Where can the fine electron-dense material that anchors most SBs to the underlying cell membrane-associated electron-dense plaque (or arciform density in the retina) find robust binding sites to prevent detachment of the SB? If this is the case, the basal region would be exclusively responsible for both anchoring and SB dispersion. If nevertheless a conveyor belt does exist the vesicle movement most probably would be managed by proteins associated with the outer RibeyeB domain. Perhaps measurement of glutamate concentration in single SVs at the SBs can help to solve this problem: the vesicular glutamate transporter type I (Scherry et al., 2003) located in the membrane of SVs pumps glutamate into the vesicles; provided it is present in approximately equal concentration in all SV membranes, the longer the time it works, the higher would be the glutamate concentration inside the SVs. In case of a conveyor belt SV, glutamate concentration should then increase from tip to base of the SB. Some further arguments against a conveyor belt function of SBs are noted in Parsons and Sterling (2003). A conveyor belt makes sense when ultrafast vesicle release is required, as in the retina. However, pinealocyte electrical and neurotransmission activity is significantly lower than that of rods, cones or bipolar cells (Schenda and Vollrath, 1998). Another suggested function for SBs, i.e. that of 2. Storage sites of vesicles. This sounds more reasonable especially because glutamate-containing vesicles are not quite as abundant in pinealocytes as in the terminals of rods or cones. If SBs capture SVs and prevent their exocytosis (Vollrath and Spiwoks-Becker, 1996), SBs could serve as the sites at which recycling vesicles are located (Parsons and Sterling, 2003; Singer and Diamond, 2006) that serve to refill the readily-releasable pool of SVs.

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3. SBs were suggested as coordinators of multivesicular release, vesicle traps and essential place for vesicle priming (Prescott and Zenisek, 2005). Such functions make sense for SBs in sensory organs where ultrafast release is essential. At such locations Parsons and Sterling (2003) proposed a compound exocytosis which results from the fusion of vesicles tethered to the ribbon with docked vesicles, either preceding or following their fusion with the cell membrane. But, compared to synaptic terminals of primary sensory cells, retinal rods and cones, activity of the few small SBs with their relatively sparse vesicles in the pineal gland is very low. Signs of compound fusion, that is fusion of vesicles tethered to SBs, of multivesicular release or of highly organized ribbon synapses as typical for most SBs in sensory organs, were never seen in any pineal gland (Jastrow, unpublished observations). 4. In hair cells SBs were suggested to position vesicles for ultrafast release near calcium channels (Khimich et al., 2005). These authors concluded from their experiments with Bassoon-deficient mice that SBs are required for a precisely timed release of several synaptic vesicles. Perhaps the surface of SBs is the morphological parameter of greatest importance concerning SB function, because it binds SVs. Due to the uniform thickness of SBs, at least in the material of this study, the surface is proportional to volume and has a relationship to the mean profile length. Larger SBs bind more SVs, resulting in more SVs ready for release, primed for rapid calcium-dependent fusion, regardless of their proximity to the plasma membrane (Heidelberger et al., 2005). Further, the number of SBs is of interest for it correlates with the number of synapses and in conjunction with SB surface area it is related to the number of SB-bound vesicles available to act in neural transmission. Acknowledgements The authors thank Mrs. I.v. Graevenitz for her excellent technical assistance. Data in this study are parts of the thesis to be presented for the degree of Dr. Med. (J.R.). References Adly, M.A., Spiwoks-Becker, I., Vollrath, L., 1999. Ultrastructural changes of photoreceptor synaptic ribbons in relation to time of day and illumination. IOVIS 40, 2165–2172. Austin, A., Fagan, D.G., Mayhew, T.M., 1995. A stereological method for estimating the total number of myocyte nuclei in fetal and postnatal hearts. J. Anat. 197, 641–647. Bhatnagar, K.P., 1994. Synaptic ribbons of the mammalian pineal gland: enigmatic organelles of poorly understood function. Adv. Struct. Biol. 3, 47–94. Bunt, A.H., 1971. Enzymatic digestion of synaptic ribbons in amphibian retinal photoreceptors. Brain Res. 25, 571–577. Calvo, J., Boya, J., 1984. Ultrastructure of the pineal gland in the adult rat. J. Anat. 138 (3), 405–409. Cimas Garcı´a, C., Martı´nez-Soriano, F., Ruiz Torner, A., 1987. Circadian and photoperiodic correlation between number of pineal gland synaptic ribbons and serum melatonin levels in the rat. Acta Anat. 130, 228–231. Cos, S., Bardasano, J.L., Mediavilla, M.D., Sa´nchez Barcelo´, E.J., 1989. Synaptische Ba¨nder in den Pinealocyten bulbektomierter Ratten unter experimentellen Bedingungen. J. Hirnforsch. 30, 91–98.

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