Neuroendocrinology (DOI:10.1159/000433440)
(Accepted, unedited article not yet assigned to an issue)
© 2015 S. Karger AG, Basel www.karger.com/nen
Advanced Release: May 29, 2015
Received: February 3, 2015 Accepted after revision: May 19, 2015
Impact of melatonin on time of day‐dependent changes in cell proliferation and apoptosis in the adult murine hypothalamic‐ hypophyseal system Michaela Fredricha*, Elmar Christ b, Amin Derouicheb, Horst‐Werner Korf a, b a Dr. Senckenbergisches Chronomedizinisches Institut, Goethe‐Universität, Theodor‐Stern‐Kai 7, D‐60590 Frankfurt/M, Germany b Dr. Senckenbergische Anatomie, Institut für Anatomie II, Goethe‐Universität, Theodor‐Stern‐Kai 7, D‐60590 Frankfurt/M, Germany * Corresponding author: Dr. Senckenbergisches Chronomedizinisches Institut, Goethe‐Universität, Theodor‐Stern‐Kai 7, D‐60590 Frankfurt/M, Germany Fax: +49 6963016017. E‐mail address: [email protected]‐frankfurt.de Short title Regulation of cell proliferation and apoptosis by melatonin Keywords: apoptosis, median eminence, melatonin, pars distalis, pars tuberalis, proliferation
Abstract
Downloaded by: University of Western Ontario 129.100.58.76 - 6/8/2015 11:02:01 AM
Background/Aims: Cell proliferation and apoptosis are known to adjust neuroendocrine circuits to the photoperiod. The latter is communicated by melatonin, the hormone secreted by the pineal organ. The present study investigated time of day‐dependent changes in cell proliferation and apoptosis in the adult murine neuroendocrine system and their regulation by melatonin. Methods: Adult melatonin‐proficient (C3H/HeN) and melatonin‐deficient (C57Bl/6J) mice, as well as melatonin‐ proficient (C3H/HeN) mice with targeted deletion of both melatonin receptor types (MT1 and MT2) were adapted to a 12h light, 12h dark photoperiod and were sacrificed at Zeitgeber times ZT00, ZT06, ZT12 and ZT18. Immunohistochemistry for Ki67 and activated caspase‐3 served to identify and quantify proliferating and apoptotic cells in the median eminence (ME), hypophyseal pars tuberalis (PT) and pars distalis (PD). Results: Time of day‐dependent changes in cell proliferation and apoptosis were found exclusively in melatonin‐proficient mice with functional MTs. Cell proliferation in the ME and the PD showed time of day‐dependent changes indicated by an increase at ZT12 (ME) and a decrease at ZT06 (PD). Apoptosis showed time of day‐dependent changes in all regions analyzed, indicated by an increase at ZT06. Proliferating and apoptotic cells were found in nearly all cell types residing in the regions analyzed. Conclusions: Our results indicate that time of day‐ dependent changes in cell proliferation are counterbalanced by time of day‐dependent changes in apoptosis exclusively in melatonin‐proficient mice with functional MTs. Melatonin signaling appears to be crucial in both generation and timing of proliferation and apoptosis that serve the high rate of physiological cell turnover in the adult neuroendocrine system.
Neuroendocrinology (DOI:10.1159/000433440)
© 2015 S. Karger AG, Basel
2
Introduction In adult vertebrates, cell proliferation provides a constant supply of new cells required for replacement of lost cells, tissue maintenance, and adaptation to physiological stimuli. Furthermore, apoptosis contributes to adaptation, self‐renewal, and maintenance of tissues by allowing an organism to control cell number and tissue size e.g. by clearing superfluous cells. Cell proliferation, apoptosis, and the balance between them are key mechanisms required for proper tissue architecture, homeostasis, and function [1]. By now it is well known that the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus generate new cells in the adult brain [2, 3]. However, constitutive cell proliferation and apoptosis have also been found in the adult neuroendocrine system, namely the median eminence (ME) at the base of the mediobasal hypothalamus and the pars tuberalis (PT) and pars distalis (PD) of the pituitary [4‐12]. These regions hold key functions in neuroendocrine regulation. The PT is the interface between photoperiodically regulated melatonin signaling and neuroendocrine functions and sends signals to the hypothalamus, including the ME, and to the PD via retrograde and anterograde pathways, respectively [13, 14]. The ME belongs to the circumventricular organs and is open to the portal vessel system. Therefore, it allows hypothalamic releasing hormones to reach the PD of the pituitary [15, 16]. Studies on photoperiodic variations of cell proliferation in the ME, PT, and PD of adult mammals suggest that melatonin plays a crucial role in regulation of diurnal and seasonal cell proliferation rates in the adult [4, 17, 18]. Melatonin is secreted from the pineal gland during the night. The melatonin signal is decoded by cells bearing melatonin receptor type1 (MT1) and type2 (MT2) [19]. Among the regions containing melatonin receptors, the PT stands out due to harboring the highest MT1 density [20, 21]. Melatonin signaling transduces length of day, onset of darkness and onset of light and thereby gates rhythmic, light‐entrained expression of clock genes in the PT [22‐24]. Clock genes in turn regulate clock‐controlled genes and in its entity this molecular clockwork communicates daily (entrained)/circadian (endogenous) rhythmicity to physiological functions [14, 25, 26]. Cell proliferation and apoptosis are regulated and timed by clock genes and several clock‐ controlled genes. A time of day‐dependent regulation of the cell cycle and synchronization of its phases by the circadian clockwork was shown in the liver and other tissues [27, 28; 29, reviewed in 30, 31]. For example, entry of mitosis is restricted to a specific time of day [28]. The goal of the present study was to analyze time of day‐dependent changes in cell proliferation and apoptosis in key regions of the adult neuroendocrine system and to investigate if these changes depend on and are regulated by melatonin‐signaling. For these purposes cell proliferation and apoptosis in the ME, PT, and PD of adult melatonin‐proficient mice (C3H) were analyzed and compared with that of melatonin‐deficient mice (C57Bl) and melatonin‐proficient mice lacking functional melatonin receptors (MT1/2 KO). In addition we identified the cell types of the proliferating and apoptotic cells in the corresponding regions.
Animals All animal experimentations reported here were conducted in accordance with accepted standards of humane animal care, as outlined in the ethical guidelines and were consistent with federal guidelines and the European Communities Council Directive (89/609/EEC). Melatonin‐proficient C3H/HeN (C3H) and Melatonin‐deficient C57Bl/6J (C57Bl) mice were purchased from Charles River laboratories (Sulzfeld, Germany). Mice with a targeted deletion of the MT1 gene (MT1‐/‐) [32] and the MT2 gene (MT2‐/‐) [33] were bred on a melatonin‐proficient C3H/HeN background for at least 10 generations. Mice deficient for both melatonin receptors (MT1/2 KO) were obtained by crossing MT1‐/‐ and MT2‐/‐ mice and breeding the MT1/2 KO offspring for at least 10 generations. Genotypes were determined by PCR amplification of genomic DNA extracted from the tail as described previously [32, 33]. Experiments were performed with adult, male C3H (n=16), C57Bl (n=16) and MT1/2 KO (n=20) mice. All animals were adapted to a photoperiod of 12 h lights (250 lux), 12h dark
Downloaded by: University of Western Ontario 129.100.58.76 - 6/8/2015 11:02:01 AM
Materials and methods
Neuroendocrinology (DOI:10.1159/000433440)
© 2015 S. Karger AG, Basel
3
for 3 weeks before experiments, with lights‐on defined as zeitgeber time (ZT) 00 and lights‐off defined as ZT12. Food and water were available ad libitum. Tissue processing At the age of 8‐10 weeks the animals were euthanized by isoflurane inhalation and perfused transcardially with 1.5ml 0.9% sodium chloride solution for 30s followed by 60ml 4% paraformaldehyde (PFA) in 0.1M phosphate‐buffer (PB, pH 7.4) for 15min at ZT00, ZT06, ZT12 and ZT18, with 4 or 5 animals per ZT and mouse strain/genotype. Perfusions at ZT18 were performed under dim red light ( 620 nm). Brains and pituitaries were removed from the skull and post‐fixed in 4% PFA for 1h and 45min, respectively. Tissues were cryo‐protected in increasing grades of sucrose in PB (10%, 20%, 30%) and cryo‐sectioned into 10µm‐thick horizontal (pituitary) or frontal (brain including PT) serial sections.
Light microscopy Images were taken with an Axioskop 2 epifluorescence microscope (Zeiss, Oberkochen, Germany) and a digital camera (SPOT insight CCD camera; Diagnostic Instruments, Sterling Heights, Michigan, USA) at 16bit gray tone depth using the SPOT 5.0 software (Diagnostic Instruments). For quantitative analysis, images of ME and PT were taken at 20x magnification. The PD was captured by two images per section taken at 10x magnification and mounted in Photoshop CS (Ver. 10; Adobe, San Jose, CA, USA). For identification of the cell types bearing Ki67 or activated caspase‐3 immunoreactivity, images were taken at 40x magnification for green, red and blue fluorescence separately, assigned an appropriate color (always green for Ki67 and red for activated caspase‐3) and assembled with SPOT
Downloaded by: University of Western Ontario 129.100.58.76 - 6/8/2015 11:02:01 AM
Immunohistochemistry Per animal and timepoint 12 sections of the ME, PT and PD were used for quantitative analyses described below. Half of them were incubated with an antibody against Ki67 and the other half with an antibody against activated caspase‐3 (table 1). Ki67 is a marker for cell proliferation because it is located in the cell nucleus during all active phases of the cell cycle, except for G0 [34‐37]. Activated caspase‐3 is the key effector caspase during the execution phase of apoptosis [38‐40]. Ki67 and activated caspase‐3 were visualized by immunofluorescence in alternating sections to prevent double counting of cells immunoreactive for Ki67 or activated caspase‐3, respectively. For identification of proliferating and apoptotic cells, Ki67 and activated caspase‐3 were co‐visualized with GFAP, S100β, Iba1, CNPase, OLIG2, DARPP‐32, Connexin 43, HuC/D, CD31, ACTH, FSHβ, LHβ, Prolactin, TSHβ, or GH (table 1) by immunofluorescence double staining. Each double staining was performed in 5 sections of C3H mice sacrificed at the ZTs with the highest number of proliferating (ME: ZT12, PT: ZT06, PD: ZT00) or apoptotic cells (ME, PT, PD: ZT06). Nonspecific staining was reduced by pre‐incubating all sections with 10% normal horse serum in PBST for 30min at room temperature. If primary antibodies produced in mice were used the sections were in addition pre‐ incubated with 3% mouse‐on‐mouse blocking reagent (MKB‐2213; Vector Laboratories, Burlingame, CA, USA) in phosphate‐buffered saline with 0.3% Triton X‐100 (PBST) for 1h at room temperature. After these pretreatments, sections were incubated with the primary antibodies (table 1) diluted in 1% normal horse serum in PBST over night at room temperature. Immunoreactivity was visualized with secondary antibodies labeled with fluorochromes, either Alexa 488 or Cy3 (table 2). Sections were incubated with the secondary antibodies for 1h at room temperature. Finally all sections were stained with the DNA‐dye Hoechst (Hoechst 33258, Pentahydrate (bis‐Benzimide); 1:5000 in PBST; Life Technologies, San Diego, CA, USA) for 5min at room temperature. The stained sections were cover‐slipped with fluorescent mounting medium (S3023; Dako, Copenhagen, Denmark). For each type of staining and region analyzed incubation parameters were kept strictly constant. To verify the specificity of secondary antibodies, negative controls were run by omitting the primary antibodies in the corresponding incubation step. Specificity of the antibody raised against activated caspase‐3 was additionally verified by pre‐incubation with the corresponding caspase‐3 blocking peptide (1050, Cell Signaling, Danvers, MA, USA) for 2h at room temperature before exposing sections for immunofluorescence staining as described above.
Neuroendocrinology (DOI:10.1159/000433440)
© 2015 S. Karger AG, Basel
4
5.0 to produce multicolor overlays. For each type of staining and region analyzed, microscope settings, exposure times and global rendering of the images were held strictly constant. Image processing and acquisition of quantitative data To distinguish between background and specific staining, background intensity was determined in negative control sections and subtracted from the overall staining intensity using the ‘adjust levels’ function in Photoshop CS. From each animal 12 sections of the ME, PT and PD were used to determine the “total” number of cells (stained with Hoechst) in the three regions. Six of these sections were used to count the number of proliferating cells (immunoreactive for Ki67) or apoptotic cells (immunoreactive for activated caspase‐3), respectively. The cell numbers were determined by an automated process using the particle counter plugin and the cluster indicator (BioVoxxel, Mutterstadt, Germany) of ImageJ/Fiji (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, 1997‐2014) [41]. Proliferation or apoptosis were quantified by forming the ratio of the counted numbers of Ki67 immunoreactive or activated caspase‐3 immunoreactive cells per 1000 cells in the ME, PT or PD. To test for changes in the “total” number of cells, the number of cells stained with Hoechst per mm² of the ME, PT and PD were calculated at different ZTs. Statistical analysis was performed using BIAS (Version 10.11, Epsilon‐Verlag). To assess whether the data come from a Gaussian distribution the Kolmogoroff‐Smirnoff test with the Dallal‐Wilkinson correction were used. Two‐way ANOVAs followed by Bonferroni post‐test were performed to determine significant time of day‐dependent changes in cell proliferation and apoptosis and to assess the effects of the strain/genotype on cell proliferation and apoptosis at different ZTs. Values were considered significantly different with p
(Accepted, unedited article not yet assigned to an issue)
© 2015 S. Karger AG, Basel www.karger.com/nen
Advanced Release: May 29, 2015
Received: February 3, 2015 Accepted after revision: May 19, 2015
Impact of melatonin on time of day‐dependent changes in cell proliferation and apoptosis in the adult murine hypothalamic‐ hypophyseal system Michaela Fredricha*, Elmar Christ b, Amin Derouicheb, Horst‐Werner Korf a, b a Dr. Senckenbergisches Chronomedizinisches Institut, Goethe‐Universität, Theodor‐Stern‐Kai 7, D‐60590 Frankfurt/M, Germany b Dr. Senckenbergische Anatomie, Institut für Anatomie II, Goethe‐Universität, Theodor‐Stern‐Kai 7, D‐60590 Frankfurt/M, Germany * Corresponding author: Dr. Senckenbergisches Chronomedizinisches Institut, Goethe‐Universität, Theodor‐Stern‐Kai 7, D‐60590 Frankfurt/M, Germany Fax: +49 6963016017. E‐mail address: [email protected]‐frankfurt.de Short title Regulation of cell proliferation and apoptosis by melatonin Keywords: apoptosis, median eminence, melatonin, pars distalis, pars tuberalis, proliferation
Abstract
Downloaded by: University of Western Ontario 129.100.58.76 - 6/8/2015 11:02:01 AM
Background/Aims: Cell proliferation and apoptosis are known to adjust neuroendocrine circuits to the photoperiod. The latter is communicated by melatonin, the hormone secreted by the pineal organ. The present study investigated time of day‐dependent changes in cell proliferation and apoptosis in the adult murine neuroendocrine system and their regulation by melatonin. Methods: Adult melatonin‐proficient (C3H/HeN) and melatonin‐deficient (C57Bl/6J) mice, as well as melatonin‐ proficient (C3H/HeN) mice with targeted deletion of both melatonin receptor types (MT1 and MT2) were adapted to a 12h light, 12h dark photoperiod and were sacrificed at Zeitgeber times ZT00, ZT06, ZT12 and ZT18. Immunohistochemistry for Ki67 and activated caspase‐3 served to identify and quantify proliferating and apoptotic cells in the median eminence (ME), hypophyseal pars tuberalis (PT) and pars distalis (PD). Results: Time of day‐dependent changes in cell proliferation and apoptosis were found exclusively in melatonin‐proficient mice with functional MTs. Cell proliferation in the ME and the PD showed time of day‐dependent changes indicated by an increase at ZT12 (ME) and a decrease at ZT06 (PD). Apoptosis showed time of day‐dependent changes in all regions analyzed, indicated by an increase at ZT06. Proliferating and apoptotic cells were found in nearly all cell types residing in the regions analyzed. Conclusions: Our results indicate that time of day‐ dependent changes in cell proliferation are counterbalanced by time of day‐dependent changes in apoptosis exclusively in melatonin‐proficient mice with functional MTs. Melatonin signaling appears to be crucial in both generation and timing of proliferation and apoptosis that serve the high rate of physiological cell turnover in the adult neuroendocrine system.
Neuroendocrinology (DOI:10.1159/000433440)
© 2015 S. Karger AG, Basel
2
Introduction In adult vertebrates, cell proliferation provides a constant supply of new cells required for replacement of lost cells, tissue maintenance, and adaptation to physiological stimuli. Furthermore, apoptosis contributes to adaptation, self‐renewal, and maintenance of tissues by allowing an organism to control cell number and tissue size e.g. by clearing superfluous cells. Cell proliferation, apoptosis, and the balance between them are key mechanisms required for proper tissue architecture, homeostasis, and function [1]. By now it is well known that the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus generate new cells in the adult brain [2, 3]. However, constitutive cell proliferation and apoptosis have also been found in the adult neuroendocrine system, namely the median eminence (ME) at the base of the mediobasal hypothalamus and the pars tuberalis (PT) and pars distalis (PD) of the pituitary [4‐12]. These regions hold key functions in neuroendocrine regulation. The PT is the interface between photoperiodically regulated melatonin signaling and neuroendocrine functions and sends signals to the hypothalamus, including the ME, and to the PD via retrograde and anterograde pathways, respectively [13, 14]. The ME belongs to the circumventricular organs and is open to the portal vessel system. Therefore, it allows hypothalamic releasing hormones to reach the PD of the pituitary [15, 16]. Studies on photoperiodic variations of cell proliferation in the ME, PT, and PD of adult mammals suggest that melatonin plays a crucial role in regulation of diurnal and seasonal cell proliferation rates in the adult [4, 17, 18]. Melatonin is secreted from the pineal gland during the night. The melatonin signal is decoded by cells bearing melatonin receptor type1 (MT1) and type2 (MT2) [19]. Among the regions containing melatonin receptors, the PT stands out due to harboring the highest MT1 density [20, 21]. Melatonin signaling transduces length of day, onset of darkness and onset of light and thereby gates rhythmic, light‐entrained expression of clock genes in the PT [22‐24]. Clock genes in turn regulate clock‐controlled genes and in its entity this molecular clockwork communicates daily (entrained)/circadian (endogenous) rhythmicity to physiological functions [14, 25, 26]. Cell proliferation and apoptosis are regulated and timed by clock genes and several clock‐ controlled genes. A time of day‐dependent regulation of the cell cycle and synchronization of its phases by the circadian clockwork was shown in the liver and other tissues [27, 28; 29, reviewed in 30, 31]. For example, entry of mitosis is restricted to a specific time of day [28]. The goal of the present study was to analyze time of day‐dependent changes in cell proliferation and apoptosis in key regions of the adult neuroendocrine system and to investigate if these changes depend on and are regulated by melatonin‐signaling. For these purposes cell proliferation and apoptosis in the ME, PT, and PD of adult melatonin‐proficient mice (C3H) were analyzed and compared with that of melatonin‐deficient mice (C57Bl) and melatonin‐proficient mice lacking functional melatonin receptors (MT1/2 KO). In addition we identified the cell types of the proliferating and apoptotic cells in the corresponding regions.
Animals All animal experimentations reported here were conducted in accordance with accepted standards of humane animal care, as outlined in the ethical guidelines and were consistent with federal guidelines and the European Communities Council Directive (89/609/EEC). Melatonin‐proficient C3H/HeN (C3H) and Melatonin‐deficient C57Bl/6J (C57Bl) mice were purchased from Charles River laboratories (Sulzfeld, Germany). Mice with a targeted deletion of the MT1 gene (MT1‐/‐) [32] and the MT2 gene (MT2‐/‐) [33] were bred on a melatonin‐proficient C3H/HeN background for at least 10 generations. Mice deficient for both melatonin receptors (MT1/2 KO) were obtained by crossing MT1‐/‐ and MT2‐/‐ mice and breeding the MT1/2 KO offspring for at least 10 generations. Genotypes were determined by PCR amplification of genomic DNA extracted from the tail as described previously [32, 33]. Experiments were performed with adult, male C3H (n=16), C57Bl (n=16) and MT1/2 KO (n=20) mice. All animals were adapted to a photoperiod of 12 h lights (250 lux), 12h dark
Downloaded by: University of Western Ontario 129.100.58.76 - 6/8/2015 11:02:01 AM
Materials and methods
Neuroendocrinology (DOI:10.1159/000433440)
© 2015 S. Karger AG, Basel
3
for 3 weeks before experiments, with lights‐on defined as zeitgeber time (ZT) 00 and lights‐off defined as ZT12. Food and water were available ad libitum. Tissue processing At the age of 8‐10 weeks the animals were euthanized by isoflurane inhalation and perfused transcardially with 1.5ml 0.9% sodium chloride solution for 30s followed by 60ml 4% paraformaldehyde (PFA) in 0.1M phosphate‐buffer (PB, pH 7.4) for 15min at ZT00, ZT06, ZT12 and ZT18, with 4 or 5 animals per ZT and mouse strain/genotype. Perfusions at ZT18 were performed under dim red light ( 620 nm). Brains and pituitaries were removed from the skull and post‐fixed in 4% PFA for 1h and 45min, respectively. Tissues were cryo‐protected in increasing grades of sucrose in PB (10%, 20%, 30%) and cryo‐sectioned into 10µm‐thick horizontal (pituitary) or frontal (brain including PT) serial sections.
Light microscopy Images were taken with an Axioskop 2 epifluorescence microscope (Zeiss, Oberkochen, Germany) and a digital camera (SPOT insight CCD camera; Diagnostic Instruments, Sterling Heights, Michigan, USA) at 16bit gray tone depth using the SPOT 5.0 software (Diagnostic Instruments). For quantitative analysis, images of ME and PT were taken at 20x magnification. The PD was captured by two images per section taken at 10x magnification and mounted in Photoshop CS (Ver. 10; Adobe, San Jose, CA, USA). For identification of the cell types bearing Ki67 or activated caspase‐3 immunoreactivity, images were taken at 40x magnification for green, red and blue fluorescence separately, assigned an appropriate color (always green for Ki67 and red for activated caspase‐3) and assembled with SPOT
Downloaded by: University of Western Ontario 129.100.58.76 - 6/8/2015 11:02:01 AM
Immunohistochemistry Per animal and timepoint 12 sections of the ME, PT and PD were used for quantitative analyses described below. Half of them were incubated with an antibody against Ki67 and the other half with an antibody against activated caspase‐3 (table 1). Ki67 is a marker for cell proliferation because it is located in the cell nucleus during all active phases of the cell cycle, except for G0 [34‐37]. Activated caspase‐3 is the key effector caspase during the execution phase of apoptosis [38‐40]. Ki67 and activated caspase‐3 were visualized by immunofluorescence in alternating sections to prevent double counting of cells immunoreactive for Ki67 or activated caspase‐3, respectively. For identification of proliferating and apoptotic cells, Ki67 and activated caspase‐3 were co‐visualized with GFAP, S100β, Iba1, CNPase, OLIG2, DARPP‐32, Connexin 43, HuC/D, CD31, ACTH, FSHβ, LHβ, Prolactin, TSHβ, or GH (table 1) by immunofluorescence double staining. Each double staining was performed in 5 sections of C3H mice sacrificed at the ZTs with the highest number of proliferating (ME: ZT12, PT: ZT06, PD: ZT00) or apoptotic cells (ME, PT, PD: ZT06). Nonspecific staining was reduced by pre‐incubating all sections with 10% normal horse serum in PBST for 30min at room temperature. If primary antibodies produced in mice were used the sections were in addition pre‐ incubated with 3% mouse‐on‐mouse blocking reagent (MKB‐2213; Vector Laboratories, Burlingame, CA, USA) in phosphate‐buffered saline with 0.3% Triton X‐100 (PBST) for 1h at room temperature. After these pretreatments, sections were incubated with the primary antibodies (table 1) diluted in 1% normal horse serum in PBST over night at room temperature. Immunoreactivity was visualized with secondary antibodies labeled with fluorochromes, either Alexa 488 or Cy3 (table 2). Sections were incubated with the secondary antibodies for 1h at room temperature. Finally all sections were stained with the DNA‐dye Hoechst (Hoechst 33258, Pentahydrate (bis‐Benzimide); 1:5000 in PBST; Life Technologies, San Diego, CA, USA) for 5min at room temperature. The stained sections were cover‐slipped with fluorescent mounting medium (S3023; Dako, Copenhagen, Denmark). For each type of staining and region analyzed incubation parameters were kept strictly constant. To verify the specificity of secondary antibodies, negative controls were run by omitting the primary antibodies in the corresponding incubation step. Specificity of the antibody raised against activated caspase‐3 was additionally verified by pre‐incubation with the corresponding caspase‐3 blocking peptide (1050, Cell Signaling, Danvers, MA, USA) for 2h at room temperature before exposing sections for immunofluorescence staining as described above.
Neuroendocrinology (DOI:10.1159/000433440)
© 2015 S. Karger AG, Basel
4
5.0 to produce multicolor overlays. For each type of staining and region analyzed, microscope settings, exposure times and global rendering of the images were held strictly constant. Image processing and acquisition of quantitative data To distinguish between background and specific staining, background intensity was determined in negative control sections and subtracted from the overall staining intensity using the ‘adjust levels’ function in Photoshop CS. From each animal 12 sections of the ME, PT and PD were used to determine the “total” number of cells (stained with Hoechst) in the three regions. Six of these sections were used to count the number of proliferating cells (immunoreactive for Ki67) or apoptotic cells (immunoreactive for activated caspase‐3), respectively. The cell numbers were determined by an automated process using the particle counter plugin and the cluster indicator (BioVoxxel, Mutterstadt, Germany) of ImageJ/Fiji (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, 1997‐2014) [41]. Proliferation or apoptosis were quantified by forming the ratio of the counted numbers of Ki67 immunoreactive or activated caspase‐3 immunoreactive cells per 1000 cells in the ME, PT or PD. To test for changes in the “total” number of cells, the number of cells stained with Hoechst per mm² of the ME, PT and PD were calculated at different ZTs. Statistical analysis was performed using BIAS (Version 10.11, Epsilon‐Verlag). To assess whether the data come from a Gaussian distribution the Kolmogoroff‐Smirnoff test with the Dallal‐Wilkinson correction were used. Two‐way ANOVAs followed by Bonferroni post‐test were performed to determine significant time of day‐dependent changes in cell proliferation and apoptosis and to assess the effects of the strain/genotype on cell proliferation and apoptosis at different ZTs. Values were considered significantly different with p
