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Gender-specific 5-HT1A receptor changes in BrdU nuclear labeling patterns in neonatal dentate gyrus
Developmental Brain Research 157 (2005) 65 – 73 www.elsevier.com/locate/devbrainres
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Gender-specific 5-HT1A receptor changes in BrdU nuclear labeling patterns in neonatal dentate gyrus John M. Greaves, Sharon S. Russo, Efrain C. Azmitia* Department of Biology, New York University, 10-09 Silver Building, 100 Washington Square East, New York, NY 10003, USA Accepted 10 March 2005 Available online 28 April 2005
Abstract The actions of 5-HT1A receptors on cell proliferation in the rat neonatal dentate gyrus are unknown. We injected a 5-HT1A receptor agonist (ipsapirone) or antagonist (Way 100635) 1 h before injections of BrdU in neonates of both genders between days 2 – 4, a peak time of dentate gyrus granule cell proliferation. The BrdU immunoreactive (IR) nuclei in the granule cell layer and subgranular zone were examined after 2 weeks. The BrdU-IR nuclear staining patterns were classified as being either diffuse (homogenous dark BrdU-staining throughout the nucleus) or punctate (multiple distinct small stained spots within the nucleus). Most BrdU-labeled nuclei with a diffuse pattern were seen in the subgranular zone while the punctate pattern nuclei were seen within the granular cell layer of the dentate gyrus. 5-HT1A antagonist showed no overall change in absolute number or pattern of labeled nuclei compared to control animals. After a 5-HT1A agonist, there was also no differences in the total number of BrdU-IR nuclei (punctate and diffuse pattern). However, in both genders, the proportion of the BrdU-labeled nuclei showing a punctate compared to diffuse pattern increased: 33% in females and 18% in males. In females, the 5-HT1A receptor agonist increased the number of nuclei showing a punctate pattern by 41%, while in males the 5-HT1A receptor agonist decreased the number of nuclei showing a diffuse pattern by 29%. These results indicate gender-specific 5-HT1A receptor action on the state of nuclear DNA in the cells of the dentate gyrus, without increasing the total number of BrdU-labeled nuclei. D 2005 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Genesis of neurons and glia Keywords: Granule cell; Subgranular; Punctate; Diffuse; Ipsapirone; Hippocampus; Differentiation; Neurogenesis
1. Introduction A delay of the serotonin innervations in the developing cortex produces a corresponding delay in neuronal differentiation; including decreased synaptic density and learning deficits [31], final cell proliferation [28], dendritic and spine maturation of cortical neurons [51], barrel field formation [12], and cortical thickness [2]. Serotonin receptor drugs have been shown to have actions on cell proliferation and survival in cultured fibroblasts [48] and peripheral cells [1,11]. The 5-HT1A receptor promotes differentiation, but not survival, of cultured fetal neuronal cells [19,20,34,49]. This receptor has a high expression during early develop* Corresponding author. Fax: +1 212 995 4015. E-mail address: [email protected] (E.C. Azmitia). 0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2005.03.003
ment and shows a marked decrease after birth in female rats and a delayed postnatal rise in males [25]. Injection of a 5HT1A agonist showed accelerated incisor eruption and eyeopening [13] and increased Purkinje cell dendrites [26]. 5HT1A agonists also stimulate neuronal maturation during development after the inhibitory actions of cocaine [2], alcohol [44], or 5-HT depleting drugs [33]. Therefore, during early development, 5-HT acts through a 5-HT1A receptor to promote differentiation and maturation of immature cells. In adult rats, 5-HT1A receptor agonists increase cell proliferation in the hippocampus [9,23,38,41,43]. The term ‘‘cell proliferation’’ is used instead of neurogenesis since the latter has come to include mitosis, migration, and maturation. These studies examined the ability of cells to synthesize DNA in the subgranular zone of the dentate gyrus and used pulse labeling with 5-bromodeoxyuridine
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(BrdU) after the injection of a 5-HT1A agonist. However, no study has previously examined the action of the 5-HT1A receptor on BrdU uptake during peak dentate gyrus cell proliferation in the neonatal rat. The formation of the dentate gyrus in the rat begins at embryonic day 21 and the cell proliferation of granule neurons proceeds laterally from the suprapyramidal limb (adjacent to the hippocampal fissure) to the infrapyramidal limb (next to the deep roots of the alveus) [4,6,7]. This early stage of granule cell proliferation is essentially complete by postnatal day 5 and is replaced by a prominent larger second proliferative zone in the polymorph layer (CA4 or hilus area of the dentate gyrus) beginning at postnatal day 3. This broad proliferation zone of the hilus becomes confined to a narrow subgranular zone at the base of the granular layer by P15. This zone is the source of granule cell proliferation until adult age [4,42]. We selected days 2– 4 of the neonatal rat to focus on the early generation of cells from this second dentate proliferation zone. Previous studies have indicated a clear gender difference in the development and function of the serotonergic system. Females have higher levels of 5-HT and 5-HT1A receptors [17,24,32]. Female rats are more sensitive during development to a variety of 5-HT drugs [22,30,47] and glucocorticoids [29]. 5-HT transporter knockout mutant female mice show a greater decrease in 5-HT1A receptors than males [14,40]. Finally, prenatal cocaine injections produced a transitory decreased in 5-HT1A receptor binding in female at 30 days and a pronounced decrease in 5-HT1A receptor binding from 60 –120 days in male rats [25]. Therefore, both male and female rats were studied and compared in this experiment. Our study describes the effect of 5-HT1A receptor drugs on the maturation of dentate gyrus granule neurons in both the suprapyramidal and infrapyramidal blades of the granule cell layer of the dentate gyrus at 2 weeks survival time. We
Fig. 2. A coronal section of the hippocampus from a two-week old female rat is shown. BrdU-immunoreactive nuclear staining can be seen in all regions of cornu Ammonus (CA) and dentate gyrus following injections made 2 – 4 days of age. The labeling is highest in lacunosum moleculare (LM), the CA fields and the granular cell layer (G). For purpose of analysis, the suprapyramidal (dorsal) and infrapyramidal (ventral) blades of the dentate gyrus were examined independently. Photograph was taken at 63 and the bar represents 200 A. Abbreviations: M: molecular layer of the dentate gyrus; SR: stratum radiatum of the CA fields.
found that ipsapirone treated rats showed no significant difference in the absolute number of BrdU-IR nuclei in either male or female brains. We found a significant increase of punctate nuclear BrdU-staining patterns in the female pups and a significantly decreased number of diffuse nuclear BrdU-staining patterns in the male pups. In both males and females, the ratio of punctate to diffuse nuclear BrdUlabeling patterns was increased after 5-HT1A agonist treatment. Treatment of animals with a 5-HT1A antagonist showed no significant changes compared to control in the absolute number or staining pattern of BrdU-labeled nuclei.
2. Materials and methods The research protocol for animal use was approved by the NYU animal welfare committee (02-1176). Pregnant Table 1 The number of nuclei (average T SEM) for the four groups of rats Diffuse Control male
Ipsapirone male
Control female Fig. 1. A coronal section of the hippocampus from a two-week old male rat is shown. BrdU-immunoreactive nuclear staining can be seen in all regions of cornu Ammonus (CA) and dentate gyrus following injections made 2 – 4 days of age. The BrdU nuclear labeling is highest in lacunosum moleculare (LM), the CA fields and the granular cell layer (G). For purpose of analysis, the suprapyramidal (dorsal) and infrapyramidal (ventral) blades of the dentate gyrus were examined independently. Photograph was taken at 63 and the bar represents 200 A. Abbreviations: M: molecular layer of the dentate gyrus; SR: stratum radiatum of the CA fields.
Ipsapirone female
Average Avg. infra. Avg. supra. Average Avg. infra. Avg. supra. Average Avg. infra. Avg. supra. Average Avg. infra. Avg. supra.
41.3 41.9 40.8 29.7 30.9 28.4 36.3 39.4 33.3 33.9 36.7 31.0
T T T T T T T T T T T T
2.6 2.4 3.0 1.4 4.8 2.4 10.3 14.8 5.8 3.9 5.3 3.7
Punctate 36.0 38.7 33.3 30.6 33.6 27.6 27.2 31.9 22.5 39.4 44.6 34.3
T T T T T T T T T T T T
3.1 3.2 3.9 1.8 0.8 3.2 4.6 6.3 2.9 5.8 5.0 7.6
Total cells 77.3 80.5 74.1 60.3 64.6 56.1 63.5 71.2 55.8 73.3 81.3 65.3
T T T T T T T T T T T T
5.6 5.6 6.9 2.9 5.6 4.2 14.0 19.9 8.2 9.7 10.3 11.2
The values are divided by regions to present the total counts for the dentate gyrus (Average) and the counts for the suprapyramidal (supra.; dorsal) and infrapyramidal (infra.; ventral) arms of the granule cell layer of the dentate gyrus. The cells are also divided by the pattern of BrdU label seen in the nucleus: diffuse or punctate. There were four rats in each group.
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female rats (16 day of gestation) were kept in our animal care facilities on a 12/12 light dark cycle for 5 days before use. The pups were combined at birth and separated into three equal groups that were injected subcutaneously on P2, P3, P4 with either vehicle, ipsapirone (5-HT1A agonist 1 mg/kg Bayer), or WAY 100635 (5-HT1A antagonist 1 mg/ kg, Sigma Chemical, Inc, St. Louis). All these animals were injected 1 h after the drug or saline injections with 5bromodeoxyuridine (50 mg/kg, Roche) subcutaneously. Two weeks after the final injections, the rats were anesthetized with a ketamine/xylazine combination (300 mg/180 mg in 30 ml saline) and transcardially perfused with 25 ml of 0.9% saline followed by 400 ml of 4% paraformaldehyde in pH 7.4 phosphate buffer. The heads were removed and placed in fixative for 24 h. The following day, the brains were removed and coronally sectioned at 70 Am using a vibratome series 1000 (Oxford) and serially collected for BrdU-staining.
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The sections were washed in distilled water, dehydrated and re-hydrated through a series of ethanol solutions. Sections were then rinsed, heated in 65 -C water bath for 10 min and treated with pepsin (0.4 mg/ml) in 0.1 N HCl for 12 min. The sections were washed again in phosphate buffer (pH 7.4) with 0.85% saline (PBS) and then incubated with 2 N HCl at 37 -C for 20 min. Sections were washed in PBS 5 times and put in blocking solution (PBS with 5% Normal Goat Serum (NGS) and 1% Triton X-100) for 90 min. Next, the sections were incubated in mouse anti-BrdU antibody (NCL-BrdU; 1:500: Novacastro) in 2.5% NGS and 1% Triton X-100 (TX) in PBS with agitation for 48 h in a cold room. The sections were than processed for DAB using the Vectastain Kit (Vector) and mounted. The dentate gyrus at either extreme of the hippocampus (septal or temporal poles) was not included in the measurements. Sections were coded and photographed with a Kodak DCS 760 (resolution of 3032 X 2800 pixels) digital camera
Fig. 3. The numbers of BrdU-labeled nuclei from male and female rats are shown. The top panel shows the total number of labeled nuclei in either the suprapyramidal (supra) or infrapyramidal (infra) blade of the dentate gyrus. The next two panels show the number of BrdU labeled nuclei with either a punctate or diffuse labeling pattern. *Indicates P < .05 using a Student’s t test for comparison of the adjacent bars, after ANOVA showed P < .01. n = 4 for all groups. The values can be seen in table one.
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and the image captured in TIFF format. Pictures were taken of both the suprapyramidal and infrapyramidal blades of the dentate gyrus. These pictures were then transferred to a Dell Pentium 4 computer running Microsoft Windows XP. The sections were analyzed using a morphometric program called the University of Texas Health Science Center San Antonio Image Tool (UTHSCSA, http://ddsdx.uthscsa. edu/dig/itdesc.html). Pictures were analyzed utilizing the manual count feature of the program to count the nuclei exhibiting a diffuse or punctate labeling pattern. These data were then entered into Microsoft Excel XP and the code broken to group animal by experimental condition and sex. Descriptive statistics were performed for each blade of the dentate gyrus, and the sum averaged for each animal to give a single value. These values were used for group statistics using ANOVA and Student’s t test. Data were organized by sex (male and female), experimental group (saline, ipsapirone, and WAY 100635), brain region (suprapyramidal and infrapyramidal blades of the dentate gyrus), and nuclear BrdU-staining pattern (diffuse and punctate).
3. Results 3.1. Normal rats We examined a total of 34 dentate gyri from 17 animals for this analysis. The BrdU-IR nuclei were distributed throughout dentate gyrus in both males and female brains (Figs. 1 and 2), but the highest density of BrdU-labeled nuclei occurred in the granule cell layer. The cells with a diffuse nuclear BrdU-staining pattern were located chiefly in the subgranular zone and those with a punctate pattern within the granule cell layer in both the suprapyramidal and infrapyramidal blades (Figs. 1 and 2 and Table 1). The total number of BrdU-IR nuclei in both blades of the dentate gyrus averaged 153 T 12 for males and 129 T 24 in females. There was no significant gender difference either in the total number of BrdU-IR nuclei or in their distribution within the suprapyramidal and infrapyramidal blades of the dentate gyrus (Fig. 3). There were more BrdU-IR nuclei with a diffuse BrdU-IR staining pattern than punctate BrdU-IR staining pattern (Figs. 4 and 6). The labeling pattern could be easily
Fig. 4. Coronal sections from male rats of the granular cell layer in suprapyramidal and infrapyramidal blades of the dentate gyrus are shown. The animals were injected with BrdU and perfused at 2 weeks of age as described in Fig. 1 and either saline (control), ipsapirone (5-HT1A agonist) or WAY100635 (5-HT2A antagonist) was given 1 hour prior to injection of the BrdU at all three time points. Note that there is no visible difference in the number of labeled nuclei in the three groups. Photograph was taken at 250 and the bar represents 50 A.
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Fig. 5. Coronal sections from female rats of the granular cell layer in suprapyramidal and infrapyramidal blades of the dentate gyrus are shown. The animals were injected with BrdU and perfused at 2 weeks of age as described in Fig. 1 and either saline (control), ipsapirone (5-HT1A agonist) or WAY100635 (5HT2A antagonist) was given 1 hour prior to injection of the BrdU at all three time points. Note that there is no visible difference in the number of labeled nuclei in the three groups. Photograph was taken at 250 and the bar represents 50 A.
characterized and the diffuse BrdU-IR staining nuclei were most abundant in the subgranular zone, while the punctate nuclei were mainly seen within the granular cell layer (Figs. 5 and 7). This separation was more pronounced in the suprapyramidal than in the infrapyramidal blade (Fig. 3). In males, the diffuse pattern was 25% higher in the suprapyramidal and 16% higher in the infrapyramidal than the punctate pattern. In females, the diffuse pattern was 43% higher than the punctate pattern in the suprapyramidal and 27% higher in the infrapyramidal. Thus, in both sexes, the majority of BrdU-labeled nuclei showed a diffuse BrdUstaining pattern within the subgranular zone. 3.2. 5-HT1A receptor agonist treated neonates No significant difference in the number of BrdU-labeled nuclei compared to saline injections was found after multiple injection of ipsapirone, the 5-HT1A receptor agonist (Fig. 3). The average number of BrdU-IR nuclei in both blades of the dentate gyrus was 119 T 6 (78% of control) for males and 145 T 17 (112% of control) for females. There were more BrdU-IR nuclei in the infrapyramidal blade than the supra-
pyramidal blades for female rats but not males. In males, the number was 63 T 7 in the infrapyramidal layer and 56 T 4 suprapyramidal layer (Table 1). 3.2.1. Male rats A clear gender difference was seen in the relative distribution of the punctate and diffuse nuclear BrdU-IR staining patterns after injection with a 5-HT1A receptor agonist (Figs. 3 –7). In ipsapirone treated males, a decrease in the number of nuclei having a diffuse BrdU-labeling pattern was found in the subgranular zone (Figs. 4 and 6). There was no consistent change in the number of punctate BrdU-labeled nuclei in males after 5-HT1A receptor agonist treatment. Compared to controls, the total number of BrdU-IR nuclei (punctate + diffuse) located in the granular layers of the dentate gyrus in ipsapirone treated males was reduced by 24%, this was entirely due to a decrease in the number of diffuse patterned BrdU-IR nuclei in the subgranular zone as there was no significant change in punctate nuclei compared to controls (Fig. 3). In ipsapirone treated males, the number of diffuse patterned BrdU-IR nuclei decreased by 26% in the infrapyramidal blade and 32% in the suprapyramidal
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Fig. 6. Coronal sections from male rats of the granular cell layer in suprapyramidal and infrapyramidal blades of the dentate gyrus are shown. The animals were injected and treated as described in Fig. 1. The nuclear labeling obtained after injection of BrdU and immunocytochemical staining of the sections reveals two distinct patterns: punctate (arrow head) and diffuse (arrow). In males there is no visible change in the nuclear pattern ratios (punctate/diffuse) after the various drug treatments. Photograph was taken at 630 and the bar represents 20 A.
blade, while punctate BrdU-IR nuclei showed little or no change from saline injected animals. We calculated a 24% increase in the ratio of punctate to diffuse patterned BrdUIR nuclei in ipsapirone treated males in comparison to controls (0.86 to 1.06, respectively). 3.2.2. Female rats In females, the number of BrdU-IR nuclei was 80 T 5 in the infrapyramidal and 65 T 6 in the suprapyramidal blade of the dentate gyrus ( P < .05). We found more punctate BrdUlabeled nuclei after 5-HT1A receptor agonist in both the infrapyramidal (+10%) and the suprapyramidal layer (+15%) compared to saline injected animals (Fig. 3; Table 1). The punctate pattern was seen almost exclusively in the granular cells layer. Compared to controls, the BrdU-IR cell number in the granular layer of the dentate gyrus of ipsapirone treated female animals was not significantly changed (Fig. 3). However, the number of punctate BrdU-IR nuclei was significantly increased in the infrapyramidal blade (40%) compared to controls. This increase was exclusively due to an increase in the number of punctate BrdU-IR nuclei since we did not detect significant change in the number of diffusely BrdU-labeled nuclei in females.
This preferential increase in the number of punctate BrdUIR nuclei produced a decrease in the ratio of diffuse to punctate BrdU-IR nuclei ( 33%), which was seen in both the infrapyramidal blade ( 31%) and in the suprapyramidal ( 35%). Overall, the ratio of punctate to diffuse BrdU-IR nuclei increased by 44%, from 0.80 in saline treated compared to 1.16 in ipsapirone treated female animals. 3.3. 5-HT1A receptor antagonist The BrdU-IR cell number in the granular layer of the dentate gyrus for rats treated with Way 100635 was 133 T 6 for males and 130 T 12 for females (data not shown). A higher number of BrdU-IR nuclei appeared in the infrapyramidal blade than the suprapyramidal with males showing 74 T 8 in the infrapyramidal and 59 T 13 in the suprapyramidal layers and females showing 71 T 4 in the infrapyramidal and 60 T 10 in the suprapyramidal layer of the dentate gyrus. These values were similar to controls. In both sexes, the suprapyramidal blade showed a difference in the ratio of diffuse to punctate BrdU-IR nuclei; however no difference was seen in the infrapyramidal blade. Males showed 49% more diffuse than punctate BrdU-IR
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Fig. 7. Coronal sections from female rats of the granular cell layer in suprapyramidal and infrapyramidal blades of the dentate gyrus are shown. The animals were injected and treated as described in Fig. 2. The nuclear labeling obtained after injection of BrdU and immunocytochemical staining of the sections reveals two distinct patterns: punctate (arrow head) and diffuse (arrow). In females there is a visible increase in the punctate pattern ratios after the various drug treatments. Photograph was taken at 630 and the bar represents 20 A.
nuclei in the suprapyramidal blade (compared to 25% in control animals), the absolute numbers were not significantly different from control counts. No significant changes in the number or labeling pattern were found for the female rats compared to controls.
4. Discussion New proliferative neurons in the granular layer are generated at a high rate from embryonic day 10 to postnatal day 20 in the mouse and rat [5,6,46]. The gradient of time of granule neuronal origin proceeds from ‘‘outside – inside’’ order, with the newer neurons added to the inner edge of the granular cell layer from a proliferate zone of germinal cells in the subgranular zone [7]. In our study, diffuse BrdUlabeled nuclei remain for at least 2 weeks within the subgranular zone. The location of these cells in the subgranular zone suggests they are undifferentiated primitive cells [4,5] and may continue to be active within the proliferative layer. Nuclei with a punctate BrdU-labeling pattern were larger and situated in cells within the granular
cell layer. The preponderance of the punctate BrdU-labeling pattern in cells within the granular zone hints at the more differentiated state of these cells. The appearance of a punctate BrdU-labeling pattern in mature cells has been previously shown [35] and probably reflects chromatin remodeling accompanied by gene sequestration which is an important step in cell maturation [39]. The BrdU nuclear aggregates seen after 2 weeks should not be confused with those described occurring during the active process of DNA synthesis [3]. The distribution of newly generated cells in the dentate gyrus was similar in male and female control rats, but different after treatment with a 5-HT1A agonist. Males responded to the 5-HT1A agonist by showing a 28% decrease in the number of diffusely BrdU-labeled nuclei in the subgranular zone while females showed a 45% increase in punctate BrdU-labeled nuclei in the granular layer. Thus, although both genders showed a similar shift in the overall ratio of diffuse to punctate pattern of labeling (diffuse/ punctate), it occurred by a different sequence. In females, the number of mature nuclei increased in the granular cell layer without affecting the number of BrdU-labeled nuclei
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in the subgranular zones. In males, the number of nuclei with a diffuse pattern decreased in the subgranular zone, without any significant change in the number of punctate patterned nuclei in the granular cell layer. These sex differences suggest an action by sex hormones of the 5-HT1A receptor’s action on neurogenesis. Gender differences in brain serotonin have been seen in all animals, including humans [15]. During development, gender differences are found not only in the absolute levels of brain 5HT1A receptor, but also in their response to cocaine [25]. Sex hormones are present during the first week of life when the animals were injected with the 5-HT1A receptor drugs. Testosterone and estradiol have differential effects of on 5HT1A receptor [14]. Estrogen exerts a neuroprotective effect that prevents apoptosis and selectively increases the action of the 5-HT1A receptor [16,17]. On the other hand, males have a lower number of 5-HT1A receptors in the hippocampus which has been attributed to testosterone [52]. Nevertheless, the results of 5-HT1A receptors on BrdU labeling in the dentate gyrus cannot simply be explained by differences in receptor number between the genders. Additional work is necessary to understand the differential gender-specific actions of this receptor on cell maturation. There was no statistically significant effect on BrdU labeling in the dentate gyrus after the administration of Way 100635. WAY 100635 is an antagonist and might be expected to have produced opposite changes to 5-HT1A receptor agonist. It is possible that injections of WAY 100635 might have produced a compensatory increase in 5HT1A receptor expression as seen in culture [36]. Alternatively, the lack of an effect with the antagonist might indicate that the 5-HT1A receptor changes with the agonist are indirect and leave open the possibility that a variety of other factors may be responsible for the altered BrdU nuclear labeling patterns. Further work is necessary to understand what compensatory or indirect changes might be acting over the duration of the agonist treatment. The relevance of the neonatal injections of 5-HT1A agonist to adult cell proliferation is not clear. 5-HT, and in particular, the 5-HT1A receptor, appears to increase adult cell proliferation in the mature dentate gyrus [10,23,41]. These studies demonstrated that, in juvenile rats, injecting a 5-HT1A receptor agonist increased the number of BrdUlabeled nuclei 1 h later. BrdU is a thymidine analog that is incorporated into the DNA during the S phase of the cell cycle prior to the division of the cell [18]. Thus, a drug could stimulate mitosis of a neuron in the S phase of a single cell cycle (progression rate) without actually increasing the proliferating rate which would depend on several cycles of division [8,31]. Furthermore, studies looking at long-term incorporation of BrdU in the juvenile brain indicate poor survival rates of the cells [21, 27,37,45,50]. Further work using specific markers of cell cycle and differentiation state is needed to explain the neonatal actions of the 5-HT1A receptors on granule cell development.
Acknowledgments The research was supported by a grant from the NIMH (R01 MH 88250) and a career scientist award (K5 MH 1838) to ECA. Thanks are given to Dr. Maria Ines Nogueira and Xiao Ping Hou for technical assistance.
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Research report
Gender-specific 5-HT1A receptor changes in BrdU nuclear labeling patterns in neonatal dentate gyrus John M. Greaves, Sharon S. Russo, Efrain C. Azmitia* Department of Biology, New York University, 10-09 Silver Building, 100 Washington Square East, New York, NY 10003, USA Accepted 10 March 2005 Available online 28 April 2005
Abstract The actions of 5-HT1A receptors on cell proliferation in the rat neonatal dentate gyrus are unknown. We injected a 5-HT1A receptor agonist (ipsapirone) or antagonist (Way 100635) 1 h before injections of BrdU in neonates of both genders between days 2 – 4, a peak time of dentate gyrus granule cell proliferation. The BrdU immunoreactive (IR) nuclei in the granule cell layer and subgranular zone were examined after 2 weeks. The BrdU-IR nuclear staining patterns were classified as being either diffuse (homogenous dark BrdU-staining throughout the nucleus) or punctate (multiple distinct small stained spots within the nucleus). Most BrdU-labeled nuclei with a diffuse pattern were seen in the subgranular zone while the punctate pattern nuclei were seen within the granular cell layer of the dentate gyrus. 5-HT1A antagonist showed no overall change in absolute number or pattern of labeled nuclei compared to control animals. After a 5-HT1A agonist, there was also no differences in the total number of BrdU-IR nuclei (punctate and diffuse pattern). However, in both genders, the proportion of the BrdU-labeled nuclei showing a punctate compared to diffuse pattern increased: 33% in females and 18% in males. In females, the 5-HT1A receptor agonist increased the number of nuclei showing a punctate pattern by 41%, while in males the 5-HT1A receptor agonist decreased the number of nuclei showing a diffuse pattern by 29%. These results indicate gender-specific 5-HT1A receptor action on the state of nuclear DNA in the cells of the dentate gyrus, without increasing the total number of BrdU-labeled nuclei. D 2005 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Genesis of neurons and glia Keywords: Granule cell; Subgranular; Punctate; Diffuse; Ipsapirone; Hippocampus; Differentiation; Neurogenesis
1. Introduction A delay of the serotonin innervations in the developing cortex produces a corresponding delay in neuronal differentiation; including decreased synaptic density and learning deficits [31], final cell proliferation [28], dendritic and spine maturation of cortical neurons [51], barrel field formation [12], and cortical thickness [2]. Serotonin receptor drugs have been shown to have actions on cell proliferation and survival in cultured fibroblasts [48] and peripheral cells [1,11]. The 5-HT1A receptor promotes differentiation, but not survival, of cultured fetal neuronal cells [19,20,34,49]. This receptor has a high expression during early develop* Corresponding author. Fax: +1 212 995 4015. E-mail address: [email protected] (E.C. Azmitia). 0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.devbrainres.2005.03.003
ment and shows a marked decrease after birth in female rats and a delayed postnatal rise in males [25]. Injection of a 5HT1A agonist showed accelerated incisor eruption and eyeopening [13] and increased Purkinje cell dendrites [26]. 5HT1A agonists also stimulate neuronal maturation during development after the inhibitory actions of cocaine [2], alcohol [44], or 5-HT depleting drugs [33]. Therefore, during early development, 5-HT acts through a 5-HT1A receptor to promote differentiation and maturation of immature cells. In adult rats, 5-HT1A receptor agonists increase cell proliferation in the hippocampus [9,23,38,41,43]. The term ‘‘cell proliferation’’ is used instead of neurogenesis since the latter has come to include mitosis, migration, and maturation. These studies examined the ability of cells to synthesize DNA in the subgranular zone of the dentate gyrus and used pulse labeling with 5-bromodeoxyuridine
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(BrdU) after the injection of a 5-HT1A agonist. However, no study has previously examined the action of the 5-HT1A receptor on BrdU uptake during peak dentate gyrus cell proliferation in the neonatal rat. The formation of the dentate gyrus in the rat begins at embryonic day 21 and the cell proliferation of granule neurons proceeds laterally from the suprapyramidal limb (adjacent to the hippocampal fissure) to the infrapyramidal limb (next to the deep roots of the alveus) [4,6,7]. This early stage of granule cell proliferation is essentially complete by postnatal day 5 and is replaced by a prominent larger second proliferative zone in the polymorph layer (CA4 or hilus area of the dentate gyrus) beginning at postnatal day 3. This broad proliferation zone of the hilus becomes confined to a narrow subgranular zone at the base of the granular layer by P15. This zone is the source of granule cell proliferation until adult age [4,42]. We selected days 2– 4 of the neonatal rat to focus on the early generation of cells from this second dentate proliferation zone. Previous studies have indicated a clear gender difference in the development and function of the serotonergic system. Females have higher levels of 5-HT and 5-HT1A receptors [17,24,32]. Female rats are more sensitive during development to a variety of 5-HT drugs [22,30,47] and glucocorticoids [29]. 5-HT transporter knockout mutant female mice show a greater decrease in 5-HT1A receptors than males [14,40]. Finally, prenatal cocaine injections produced a transitory decreased in 5-HT1A receptor binding in female at 30 days and a pronounced decrease in 5-HT1A receptor binding from 60 –120 days in male rats [25]. Therefore, both male and female rats were studied and compared in this experiment. Our study describes the effect of 5-HT1A receptor drugs on the maturation of dentate gyrus granule neurons in both the suprapyramidal and infrapyramidal blades of the granule cell layer of the dentate gyrus at 2 weeks survival time. We
Fig. 2. A coronal section of the hippocampus from a two-week old female rat is shown. BrdU-immunoreactive nuclear staining can be seen in all regions of cornu Ammonus (CA) and dentate gyrus following injections made 2 – 4 days of age. The labeling is highest in lacunosum moleculare (LM), the CA fields and the granular cell layer (G). For purpose of analysis, the suprapyramidal (dorsal) and infrapyramidal (ventral) blades of the dentate gyrus were examined independently. Photograph was taken at 63 and the bar represents 200 A. Abbreviations: M: molecular layer of the dentate gyrus; SR: stratum radiatum of the CA fields.
found that ipsapirone treated rats showed no significant difference in the absolute number of BrdU-IR nuclei in either male or female brains. We found a significant increase of punctate nuclear BrdU-staining patterns in the female pups and a significantly decreased number of diffuse nuclear BrdU-staining patterns in the male pups. In both males and females, the ratio of punctate to diffuse nuclear BrdUlabeling patterns was increased after 5-HT1A agonist treatment. Treatment of animals with a 5-HT1A antagonist showed no significant changes compared to control in the absolute number or staining pattern of BrdU-labeled nuclei.
2. Materials and methods The research protocol for animal use was approved by the NYU animal welfare committee (02-1176). Pregnant Table 1 The number of nuclei (average T SEM) for the four groups of rats Diffuse Control male
Ipsapirone male
Control female Fig. 1. A coronal section of the hippocampus from a two-week old male rat is shown. BrdU-immunoreactive nuclear staining can be seen in all regions of cornu Ammonus (CA) and dentate gyrus following injections made 2 – 4 days of age. The BrdU nuclear labeling is highest in lacunosum moleculare (LM), the CA fields and the granular cell layer (G). For purpose of analysis, the suprapyramidal (dorsal) and infrapyramidal (ventral) blades of the dentate gyrus were examined independently. Photograph was taken at 63 and the bar represents 200 A. Abbreviations: M: molecular layer of the dentate gyrus; SR: stratum radiatum of the CA fields.
Ipsapirone female
Average Avg. infra. Avg. supra. Average Avg. infra. Avg. supra. Average Avg. infra. Avg. supra. Average Avg. infra. Avg. supra.
41.3 41.9 40.8 29.7 30.9 28.4 36.3 39.4 33.3 33.9 36.7 31.0
T T T T T T T T T T T T
2.6 2.4 3.0 1.4 4.8 2.4 10.3 14.8 5.8 3.9 5.3 3.7
Punctate 36.0 38.7 33.3 30.6 33.6 27.6 27.2 31.9 22.5 39.4 44.6 34.3
T T T T T T T T T T T T
3.1 3.2 3.9 1.8 0.8 3.2 4.6 6.3 2.9 5.8 5.0 7.6
Total cells 77.3 80.5 74.1 60.3 64.6 56.1 63.5 71.2 55.8 73.3 81.3 65.3
T T T T T T T T T T T T
5.6 5.6 6.9 2.9 5.6 4.2 14.0 19.9 8.2 9.7 10.3 11.2
The values are divided by regions to present the total counts for the dentate gyrus (Average) and the counts for the suprapyramidal (supra.; dorsal) and infrapyramidal (infra.; ventral) arms of the granule cell layer of the dentate gyrus. The cells are also divided by the pattern of BrdU label seen in the nucleus: diffuse or punctate. There were four rats in each group.
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female rats (16 day of gestation) were kept in our animal care facilities on a 12/12 light dark cycle for 5 days before use. The pups were combined at birth and separated into three equal groups that were injected subcutaneously on P2, P3, P4 with either vehicle, ipsapirone (5-HT1A agonist 1 mg/kg Bayer), or WAY 100635 (5-HT1A antagonist 1 mg/ kg, Sigma Chemical, Inc, St. Louis). All these animals were injected 1 h after the drug or saline injections with 5bromodeoxyuridine (50 mg/kg, Roche) subcutaneously. Two weeks after the final injections, the rats were anesthetized with a ketamine/xylazine combination (300 mg/180 mg in 30 ml saline) and transcardially perfused with 25 ml of 0.9% saline followed by 400 ml of 4% paraformaldehyde in pH 7.4 phosphate buffer. The heads were removed and placed in fixative for 24 h. The following day, the brains were removed and coronally sectioned at 70 Am using a vibratome series 1000 (Oxford) and serially collected for BrdU-staining.
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The sections were washed in distilled water, dehydrated and re-hydrated through a series of ethanol solutions. Sections were then rinsed, heated in 65 -C water bath for 10 min and treated with pepsin (0.4 mg/ml) in 0.1 N HCl for 12 min. The sections were washed again in phosphate buffer (pH 7.4) with 0.85% saline (PBS) and then incubated with 2 N HCl at 37 -C for 20 min. Sections were washed in PBS 5 times and put in blocking solution (PBS with 5% Normal Goat Serum (NGS) and 1% Triton X-100) for 90 min. Next, the sections were incubated in mouse anti-BrdU antibody (NCL-BrdU; 1:500: Novacastro) in 2.5% NGS and 1% Triton X-100 (TX) in PBS with agitation for 48 h in a cold room. The sections were than processed for DAB using the Vectastain Kit (Vector) and mounted. The dentate gyrus at either extreme of the hippocampus (septal or temporal poles) was not included in the measurements. Sections were coded and photographed with a Kodak DCS 760 (resolution of 3032 X 2800 pixels) digital camera
Fig. 3. The numbers of BrdU-labeled nuclei from male and female rats are shown. The top panel shows the total number of labeled nuclei in either the suprapyramidal (supra) or infrapyramidal (infra) blade of the dentate gyrus. The next two panels show the number of BrdU labeled nuclei with either a punctate or diffuse labeling pattern. *Indicates P < .05 using a Student’s t test for comparison of the adjacent bars, after ANOVA showed P < .01. n = 4 for all groups. The values can be seen in table one.
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and the image captured in TIFF format. Pictures were taken of both the suprapyramidal and infrapyramidal blades of the dentate gyrus. These pictures were then transferred to a Dell Pentium 4 computer running Microsoft Windows XP. The sections were analyzed using a morphometric program called the University of Texas Health Science Center San Antonio Image Tool (UTHSCSA, http://ddsdx.uthscsa. edu/dig/itdesc.html). Pictures were analyzed utilizing the manual count feature of the program to count the nuclei exhibiting a diffuse or punctate labeling pattern. These data were then entered into Microsoft Excel XP and the code broken to group animal by experimental condition and sex. Descriptive statistics were performed for each blade of the dentate gyrus, and the sum averaged for each animal to give a single value. These values were used for group statistics using ANOVA and Student’s t test. Data were organized by sex (male and female), experimental group (saline, ipsapirone, and WAY 100635), brain region (suprapyramidal and infrapyramidal blades of the dentate gyrus), and nuclear BrdU-staining pattern (diffuse and punctate).
3. Results 3.1. Normal rats We examined a total of 34 dentate gyri from 17 animals for this analysis. The BrdU-IR nuclei were distributed throughout dentate gyrus in both males and female brains (Figs. 1 and 2), but the highest density of BrdU-labeled nuclei occurred in the granule cell layer. The cells with a diffuse nuclear BrdU-staining pattern were located chiefly in the subgranular zone and those with a punctate pattern within the granule cell layer in both the suprapyramidal and infrapyramidal blades (Figs. 1 and 2 and Table 1). The total number of BrdU-IR nuclei in both blades of the dentate gyrus averaged 153 T 12 for males and 129 T 24 in females. There was no significant gender difference either in the total number of BrdU-IR nuclei or in their distribution within the suprapyramidal and infrapyramidal blades of the dentate gyrus (Fig. 3). There were more BrdU-IR nuclei with a diffuse BrdU-IR staining pattern than punctate BrdU-IR staining pattern (Figs. 4 and 6). The labeling pattern could be easily
Fig. 4. Coronal sections from male rats of the granular cell layer in suprapyramidal and infrapyramidal blades of the dentate gyrus are shown. The animals were injected with BrdU and perfused at 2 weeks of age as described in Fig. 1 and either saline (control), ipsapirone (5-HT1A agonist) or WAY100635 (5-HT2A antagonist) was given 1 hour prior to injection of the BrdU at all three time points. Note that there is no visible difference in the number of labeled nuclei in the three groups. Photograph was taken at 250 and the bar represents 50 A.
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Fig. 5. Coronal sections from female rats of the granular cell layer in suprapyramidal and infrapyramidal blades of the dentate gyrus are shown. The animals were injected with BrdU and perfused at 2 weeks of age as described in Fig. 1 and either saline (control), ipsapirone (5-HT1A agonist) or WAY100635 (5HT2A antagonist) was given 1 hour prior to injection of the BrdU at all three time points. Note that there is no visible difference in the number of labeled nuclei in the three groups. Photograph was taken at 250 and the bar represents 50 A.
characterized and the diffuse BrdU-IR staining nuclei were most abundant in the subgranular zone, while the punctate nuclei were mainly seen within the granular cell layer (Figs. 5 and 7). This separation was more pronounced in the suprapyramidal than in the infrapyramidal blade (Fig. 3). In males, the diffuse pattern was 25% higher in the suprapyramidal and 16% higher in the infrapyramidal than the punctate pattern. In females, the diffuse pattern was 43% higher than the punctate pattern in the suprapyramidal and 27% higher in the infrapyramidal. Thus, in both sexes, the majority of BrdU-labeled nuclei showed a diffuse BrdUstaining pattern within the subgranular zone. 3.2. 5-HT1A receptor agonist treated neonates No significant difference in the number of BrdU-labeled nuclei compared to saline injections was found after multiple injection of ipsapirone, the 5-HT1A receptor agonist (Fig. 3). The average number of BrdU-IR nuclei in both blades of the dentate gyrus was 119 T 6 (78% of control) for males and 145 T 17 (112% of control) for females. There were more BrdU-IR nuclei in the infrapyramidal blade than the supra-
pyramidal blades for female rats but not males. In males, the number was 63 T 7 in the infrapyramidal layer and 56 T 4 suprapyramidal layer (Table 1). 3.2.1. Male rats A clear gender difference was seen in the relative distribution of the punctate and diffuse nuclear BrdU-IR staining patterns after injection with a 5-HT1A receptor agonist (Figs. 3 –7). In ipsapirone treated males, a decrease in the number of nuclei having a diffuse BrdU-labeling pattern was found in the subgranular zone (Figs. 4 and 6). There was no consistent change in the number of punctate BrdU-labeled nuclei in males after 5-HT1A receptor agonist treatment. Compared to controls, the total number of BrdU-IR nuclei (punctate + diffuse) located in the granular layers of the dentate gyrus in ipsapirone treated males was reduced by 24%, this was entirely due to a decrease in the number of diffuse patterned BrdU-IR nuclei in the subgranular zone as there was no significant change in punctate nuclei compared to controls (Fig. 3). In ipsapirone treated males, the number of diffuse patterned BrdU-IR nuclei decreased by 26% in the infrapyramidal blade and 32% in the suprapyramidal
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Fig. 6. Coronal sections from male rats of the granular cell layer in suprapyramidal and infrapyramidal blades of the dentate gyrus are shown. The animals were injected and treated as described in Fig. 1. The nuclear labeling obtained after injection of BrdU and immunocytochemical staining of the sections reveals two distinct patterns: punctate (arrow head) and diffuse (arrow). In males there is no visible change in the nuclear pattern ratios (punctate/diffuse) after the various drug treatments. Photograph was taken at 630 and the bar represents 20 A.
blade, while punctate BrdU-IR nuclei showed little or no change from saline injected animals. We calculated a 24% increase in the ratio of punctate to diffuse patterned BrdUIR nuclei in ipsapirone treated males in comparison to controls (0.86 to 1.06, respectively). 3.2.2. Female rats In females, the number of BrdU-IR nuclei was 80 T 5 in the infrapyramidal and 65 T 6 in the suprapyramidal blade of the dentate gyrus ( P < .05). We found more punctate BrdUlabeled nuclei after 5-HT1A receptor agonist in both the infrapyramidal (+10%) and the suprapyramidal layer (+15%) compared to saline injected animals (Fig. 3; Table 1). The punctate pattern was seen almost exclusively in the granular cells layer. Compared to controls, the BrdU-IR cell number in the granular layer of the dentate gyrus of ipsapirone treated female animals was not significantly changed (Fig. 3). However, the number of punctate BrdU-IR nuclei was significantly increased in the infrapyramidal blade (40%) compared to controls. This increase was exclusively due to an increase in the number of punctate BrdU-IR nuclei since we did not detect significant change in the number of diffusely BrdU-labeled nuclei in females.
This preferential increase in the number of punctate BrdUIR nuclei produced a decrease in the ratio of diffuse to punctate BrdU-IR nuclei ( 33%), which was seen in both the infrapyramidal blade ( 31%) and in the suprapyramidal ( 35%). Overall, the ratio of punctate to diffuse BrdU-IR nuclei increased by 44%, from 0.80 in saline treated compared to 1.16 in ipsapirone treated female animals. 3.3. 5-HT1A receptor antagonist The BrdU-IR cell number in the granular layer of the dentate gyrus for rats treated with Way 100635 was 133 T 6 for males and 130 T 12 for females (data not shown). A higher number of BrdU-IR nuclei appeared in the infrapyramidal blade than the suprapyramidal with males showing 74 T 8 in the infrapyramidal and 59 T 13 in the suprapyramidal layers and females showing 71 T 4 in the infrapyramidal and 60 T 10 in the suprapyramidal layer of the dentate gyrus. These values were similar to controls. In both sexes, the suprapyramidal blade showed a difference in the ratio of diffuse to punctate BrdU-IR nuclei; however no difference was seen in the infrapyramidal blade. Males showed 49% more diffuse than punctate BrdU-IR
J.M. Greaves et al. / Developmental Brain Research 157 (2005) 65 – 73
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Fig. 7. Coronal sections from female rats of the granular cell layer in suprapyramidal and infrapyramidal blades of the dentate gyrus are shown. The animals were injected and treated as described in Fig. 2. The nuclear labeling obtained after injection of BrdU and immunocytochemical staining of the sections reveals two distinct patterns: punctate (arrow head) and diffuse (arrow). In females there is a visible increase in the punctate pattern ratios after the various drug treatments. Photograph was taken at 630 and the bar represents 20 A.
nuclei in the suprapyramidal blade (compared to 25% in control animals), the absolute numbers were not significantly different from control counts. No significant changes in the number or labeling pattern were found for the female rats compared to controls.
4. Discussion New proliferative neurons in the granular layer are generated at a high rate from embryonic day 10 to postnatal day 20 in the mouse and rat [5,6,46]. The gradient of time of granule neuronal origin proceeds from ‘‘outside – inside’’ order, with the newer neurons added to the inner edge of the granular cell layer from a proliferate zone of germinal cells in the subgranular zone [7]. In our study, diffuse BrdUlabeled nuclei remain for at least 2 weeks within the subgranular zone. The location of these cells in the subgranular zone suggests they are undifferentiated primitive cells [4,5] and may continue to be active within the proliferative layer. Nuclei with a punctate BrdU-labeling pattern were larger and situated in cells within the granular
cell layer. The preponderance of the punctate BrdU-labeling pattern in cells within the granular zone hints at the more differentiated state of these cells. The appearance of a punctate BrdU-labeling pattern in mature cells has been previously shown [35] and probably reflects chromatin remodeling accompanied by gene sequestration which is an important step in cell maturation [39]. The BrdU nuclear aggregates seen after 2 weeks should not be confused with those described occurring during the active process of DNA synthesis [3]. The distribution of newly generated cells in the dentate gyrus was similar in male and female control rats, but different after treatment with a 5-HT1A agonist. Males responded to the 5-HT1A agonist by showing a 28% decrease in the number of diffusely BrdU-labeled nuclei in the subgranular zone while females showed a 45% increase in punctate BrdU-labeled nuclei in the granular layer. Thus, although both genders showed a similar shift in the overall ratio of diffuse to punctate pattern of labeling (diffuse/ punctate), it occurred by a different sequence. In females, the number of mature nuclei increased in the granular cell layer without affecting the number of BrdU-labeled nuclei
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in the subgranular zones. In males, the number of nuclei with a diffuse pattern decreased in the subgranular zone, without any significant change in the number of punctate patterned nuclei in the granular cell layer. These sex differences suggest an action by sex hormones of the 5-HT1A receptor’s action on neurogenesis. Gender differences in brain serotonin have been seen in all animals, including humans [15]. During development, gender differences are found not only in the absolute levels of brain 5HT1A receptor, but also in their response to cocaine [25]. Sex hormones are present during the first week of life when the animals were injected with the 5-HT1A receptor drugs. Testosterone and estradiol have differential effects of on 5HT1A receptor [14]. Estrogen exerts a neuroprotective effect that prevents apoptosis and selectively increases the action of the 5-HT1A receptor [16,17]. On the other hand, males have a lower number of 5-HT1A receptors in the hippocampus which has been attributed to testosterone [52]. Nevertheless, the results of 5-HT1A receptors on BrdU labeling in the dentate gyrus cannot simply be explained by differences in receptor number between the genders. Additional work is necessary to understand the differential gender-specific actions of this receptor on cell maturation. There was no statistically significant effect on BrdU labeling in the dentate gyrus after the administration of Way 100635. WAY 100635 is an antagonist and might be expected to have produced opposite changes to 5-HT1A receptor agonist. It is possible that injections of WAY 100635 might have produced a compensatory increase in 5HT1A receptor expression as seen in culture [36]. Alternatively, the lack of an effect with the antagonist might indicate that the 5-HT1A receptor changes with the agonist are indirect and leave open the possibility that a variety of other factors may be responsible for the altered BrdU nuclear labeling patterns. Further work is necessary to understand what compensatory or indirect changes might be acting over the duration of the agonist treatment. The relevance of the neonatal injections of 5-HT1A agonist to adult cell proliferation is not clear. 5-HT, and in particular, the 5-HT1A receptor, appears to increase adult cell proliferation in the mature dentate gyrus [10,23,41]. These studies demonstrated that, in juvenile rats, injecting a 5-HT1A receptor agonist increased the number of BrdUlabeled nuclei 1 h later. BrdU is a thymidine analog that is incorporated into the DNA during the S phase of the cell cycle prior to the division of the cell [18]. Thus, a drug could stimulate mitosis of a neuron in the S phase of a single cell cycle (progression rate) without actually increasing the proliferating rate which would depend on several cycles of division [8,31]. Furthermore, studies looking at long-term incorporation of BrdU in the juvenile brain indicate poor survival rates of the cells [21, 27,37,45,50]. Further work using specific markers of cell cycle and differentiation state is needed to explain the neonatal actions of the 5-HT1A receptors on granule cell development.
Acknowledgments The research was supported by a grant from the NIMH (R01 MH 88250) and a career scientist award (K5 MH 1838) to ECA. Thanks are given to Dr. Maria Ines Nogueira and Xiao Ping Hou for technical assistance.
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