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Expression of galanin immunoreactivity in gonadotropin-releasing hormone neurons in mice: a confocal microscopic study
Brain Research 821 Ž1999. 270–276
Research report
Expression of galanin immunoreactivity in gonadotropin-releasing hormone neurons in mice: a confocal microscopic study Gopalan Rajendren, Marie J. Gibson
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Department of Medicine, Mount Sinai School of Medicine, Box 1055, 1 GustaÕe L. LeÕy Place, New York, NY 10029, USA Accepted 15 December 1998
Abstract The expression of galanin immunoreactivity Žgalanin-IR. in gonadotropin-releasing hormone ŽGnRH. neurons was investigated in mice using double label immunohistochemistry combined with confocal laser scanning microscopy. A large proportion of GnRH cells in proestrous mice and very few GnRH cells in male mice exhibited galanin-IR. These results are consistent with earlier reports in rats. Unlike in rats, the proportion of GnRH cells coexpressing galanin in mice was high following ovariectomy ŽOVX. and the treatment of OVX mice with estrogen decreased the number of GnRH cells with galanin-IR. The GnRH system can be considered more active during proestrous and following OVX since the output of luteinizing hormone is elevated during these phases in females. Since the induction of galanin-IR in GnRH cells is more pronounced in OVX and proestrous mice, the expression of galanin-IR in GnRH cells in mice appears to be an activation-dependent phenomenon rather than a direct effect of estrogen. However, in OVX mice treated with steroids to induce an LH surge the number of GnRH cells with galanin-IR was not proportionately increased. The possible reasons for this discrepancy are also discussed. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Galanin; Gonadotropin-releasing hormone; GnRH; Luteinizing hormone-releasing hormone; Estrous cycle; Immunohistochemistry
1. Introduction Successful reproduction and thus survival of the species in mammals is dependent on the few hundred gonadotropin-releasing hormone ŽGnRH. neuronal cell bodies scattered in the ventral forebrain w12,35x. GnRH regulates the gonadotropic functions of the anterior pituitary gland, the control of which is different in the two sexes. The cyclicity inherent in female reproductive function is dictated by the cyclic release of GnRH into the hypothalamo–hypophyseal portal system w28x. The present knowledge of the regulation of the GnRH system is primarily based on studies carried out in the rat. In that species, almost all known neurotransmitters and neuromodulators have been implicated in influencing the GnRH system w13x. However, the precise orchestration of these factors in the control of the synthesis and release of GnRH is largely unknown.
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The gut–brain peptide galanin was originally discovered in porcine intestine w37x. Galanin is heavily concentrated in the hypothalamus and the median eminence w22,24x. It is suggested that galanin functions as a hypothalamic–hypophysiotropic hormone that modulates the release of growth hormone, prolactin and luteinizing hormone w16x. In the central nervous system, galanin is colocalized with vasopressin, dynorphin, tyrosine hydroxylase, g-aminobutyric acid, norepinephrine, serotonin and choline acetyl transferase w4,20–22,36x. A significant number of GnRH neuronal cell bodies are also immunoreactive for galanin w6,23x. The presence of galanin immunoreactivity Žgalanin-IR. w25x and the expression of galanin mRNA w7,19,29x in GnRH cells appear to be sexually dimorphic in rats. A greater proportion of GnRH cells in female rats expresses galanin as compared with males and is dependent on circulating levels of estrogen. Ovariectomy ŽOVX. decreases and estrogen treatment of OVX rats increases the expression of galanin in GnRH cells w7,18,19x. To date there has been no study reported investigating the expression of galanin in GnRH neurons in any species other than the rat. Often, the studies in rats are generalized to include other species as well. There are some indica-
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tions that even closely related species such as mice may vary significantly from rats. For example, sequential injection of estrogen and progesterone induces a preovulatorylike surge of LH in OVX rats; even estrogen alone, if injected in sufficiently high doses, will induce a surge of LH in OVX rats w15x. Similar treatment of OVX mice with steroids rarely induces an LH surge; the time of steroid treatment in relation to OVX and the dose of steroids administered have to be precisely adjusted in order to obtain a consistent surge of LH in OVX mice w1,2x. A tonic suppression of LH release in male rats by endogenous opiates is suggested by the findings that administration of the opioid receptor antagonist naloxone facilitates LH release w5x. In male mice on the other hand, naloxone has no noticeable effect on the release of LH w27x. Thus, the purpose of the present studies was to evaluate galanin colocalization in GnRH neurons in mice to investigate whether the incidence of galanin-IR in GnRH neurons follows a pattern similar to that reported in rats. Since galanin-IR in GnRH cell bodies was faint and the preoptic region exhibited an intense galanin fiber IR, confocal microscopy was employed to identify GnRH cells with galanin-IR.
2. Materials and methods 2.1. Animals C3HrHeH= 101H mice were housed under a partially reversed lightrdark cycle Ž14 h of light and 10 h of dark; lights off at 13:00 h. and controlled temperature Ž24–268C.. Food and drinking water were available ad libitum. All procedures and animal care were in accord with the standard approved by the NIH Guide for the Care and Use of Laboratory Animals and were approved by the IACUC of the Mount Sinai School of Medicine. 2.2. Experimental protocol Pilot studies indicated that treatment of the mice with colchicine was required in order to visualize galanin-IR in GnRH cell bodies. Between 12:00 and 13:00 h, the mice were anesthetized with chloral hydrate Ž380 mgrkg; i.p.. and blood samples were obtained from the orbital sinus for LH assay. They were then stereotaxically injected with 15 mg of colchicine Žin 1 ml sterile distilled water. into the lateral cerebral ventricle. 24 h later, they were anesthetized with an overdose of chloral hydrate Ž760 mgrkg; i.p.., perfused transcardially with 15 ml of saline followed by 60 ml of ice-cold 4% paraformaldehyde and the brains were processed for double label immunostaining for GnRH and galanin. Adult mice, 10–12 weeks old, were divided into five groups and treated as follows.
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2.2.1. Proestrus Vaginal smears were obtained daily for 2 weeks and those exhibiting two consecutive 4–5 days cycles were included Ž n s 4.. On the day of proestrus they were injected with colchicine intracerebroventricularly Ži.c.v.. and the brains were processed the following day for double label immunocytochemistry. 2.2.2. OVX The females were bilaterally OVX Ž n s 5. and 3 weeks later, they were injected with colchicine i.c.v. and the brains were processed for immunostaining as in the previous group. 2.2.3. OVX q estrogen (OVX-E2 ) The females were bilaterally OVX and 2 weeks later they were implanted with a silastic capsule Ž10 mm; i.d., 1.0 mm; o.d., 2.0 mm; Dow Corning, Midland, MI. containing 10 mg estradiol ŽE 2 . in sesame oil ŽSO. Ž n s 4.. One week after implantation of the E 2-containing capsule, they were injected with colchicine i.c.v. and the brains were processed as above. 2.2.4. OVX and E q P with LH surge (OVX-surge) The females were bilaterally OVX and were implanted with a silastic capsule Ždimensions as above. containing 1 mg E 2 in SO and were sequentially primed with estradiol benzoate ŽEB. and progesterone ŽP. to induce a luteinizing hormone surge Ž n s 5. as described earlier w11x. Briefly, 1.0 mg of EB in 0.05 ml SO and 500 mg of P in 0.05 ml SO were administered s.c. at 09:00 h on days 7 and 8 post-OVX, respectively. 4 h after P injection they were injected with colchicine i.c.v. and the brains were processed as above. 2.2.5. Males Male mice Ž n s 5. were injected with colchicine i.c.v. and the brains were processed for galanin and GnRH staining as above. 2.3. Immunohistochemistry The brains were removed, post-fixed in 4% paraformaldehyde overnight and coronal sections, 50 mm thick, were obtained using a Vibratome. The sections were washed and were incubated for 72 h with a dilute anti-galanin antibody Žraised in rabbit; Peninsula Labs, Belmont, CA. in 0.1 M PBS Ž1:3000. containing 1.5% normal donkey serum ŽNDS. and 0.3% Triton X. The sections were washed twice and were incubated for 3 h with anti-rabbit IgG–FITC conjugate Ž5 mgrml in 0.1 M PBS containing 1.5% NDS and 0.3% Triton X. raised in donkey ŽJackson Immunoresearch, West Grove, PA.. The sections were washed twice in PBS and were incubated for 96 h with a dilute Ž1:5000 in 0.1 M PBS containing 1.5% NDS and 0.3% Triton X.
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standard. The coefficient of variances were 3.52% and 5.21% Žintra-assay., and 4.42% Žinter-assay.. The limit of detection was 0.22 ngrml. 2.6. Data analysis The data were analyzed using one-way analysis of variance followed by Fisher’s LSD Žprotected t-test. using the GB Stat program.
3. Results Fig. 1. Plasma LH in male and female mice. Plasma LH was significantly higher in OVX-surge females than in all other groups Ž p- 0.01..
anti-GnRH antibody Žmouse monoclonal; QED Advanced Research, San Diego, CA.. The sections were then washed twice in PBS and were incubated for 3 h with anti-mouse IgG–Texas red conjugate Ž5 mgrml. raised in donkey ŽJackson Immunoresearch.. The sections were washed, mounted onto gelatin-coated slides, cover-slipped using Vectashield mounting medium ŽVector Labs, Burlingame, CA. and analyzed under fluorescent and confocal microscopes. 2.4. Microscopy GnRH cells were counted from sections obtained from the block of brain encompassing the anterior septum, organum vasculosum of lamina terminalis, preoptic area, hypothalamus and the mammillary bodies. This block of brain contained the majority of the GnRH cell bodies in the brain. The cells were counted using an Olympus fluorescence microscope under a magnification of 400 = . Due to the intense galanin fiber staining in the ventral forebrain, the region where the majority of GnRH cell bodies are located, it was not possible to reliably identify the GnRH cells with galanin-IR under regular fluorescent microscope. Therefore, confocal microscopy was employed for this purpose. Since it was not practical to analyze all the sections under the confocal microscope, two sections were selected from each brain under the fluorescent microscope using anatomical landmarks w9x, one passing through the anterior septum Žq1.10 mm bregma. and the other through organum vasculosum of lamina terminalisranterior preoptic area Žq0.32 mm bregma.. These two sections were examined under the confocal microscope. All the GnRH cells in these sections were individually scanned under a 100 = objective lens and the digital images of thin optical sections were analyzed for dual labeling. 2.5. Radioimmunoassay Plasma samples were assayed in duplicate for LH with a kit provided by the NIDDK using rat LH RP-3 as the
Plasma LH values are shown in Fig. 1. One-way analysis of variance revealed that LH values were significantly different among the groups Ž F s 16.82; df s 4; p - 0.001.. Post-hoc analyses indicated that the value of LH in OVXsurge females was significantly higher than all the other groups Ž p - 0.01.. The total number of GnRH cells in each group is shown in Table 1. The mean number of GnRH cells was significantly different among the groups Ž F s 2.94; df s 4; p 0.05.. Post-hoc comparison revealed that the total number of GnRH cells in proestrous mice was significantly higher Ž p - 0.05. than that in OVX-E 2 and OVX-surge mice. GnRH cell number in male mice also was significantly greater Ž p - 0.05. than that in OVX-E 2 and OVX-surge mice. The differences in the number of GnRH cells among the other groups were not significant. Galanin-IR in cell bodies was variable in intensity, whether or not the cells also contained GnRH. However, the thin optical sectioning capability and extreme sensitivity of confocal microscopy enabled the identification of even faint galanin-IR in GnRH cells ŽFig. 2.. In some cases there was relatively strong staining of galanin-IR in GnRH neurons ŽA and B. while in other instances the galanin-IR in the GnRH cell body would have been difficult to visualize without confocal microscopy, because of the network of intensely stained fibers containing galaninIR in the region ŽC and D..
Table 1 Mean"S.E.M. of the total number of GnRH cells in the experimental groups and the percent analyzed with confocal microscopy Group Ž n.
Total number
Number Žpercent of total. analyzed under confocal microscope
Proestrus Ž4. OVX Ž5. OVX-E 2 Ž4. OVX-surge Ž5. Male Ž5.
431.8"22.1) 367.8"28.9 297.5"24.7 321.6"13.4 414.8"47.6)
42.3"2.2 Ž9.8. 40.2"3.4 Ž10.9. 29.3"1.0 Ž9.8. 26.0"3.7 Ž8.1. 39.8"3.9 Ž9.6.
) p- 0.05 vs. OVX-E 2 and OVX-surge.
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Fig. 2. Confocal images of galanin-IR in GnRH cells. Arrows indicate double staining. The GnRH cell in A has intense galanin-IR as shown in B. In contrast, as seen in D, there is weak galanin-IR in the GnRH cell in C. This would be difficult to detect with a regular fluorescence microscope particularly in an area with intense galanin fiber staining. The second GnRH cell in C is galanin negative. Scale bar s 10 mm.
Since there was no noticeable difference in galanin staining in GnRH cells in the two sections obtained from
Fig. 3. Galanin-IR in GnRH neurons. Confocal microscopy was used to evaluate the percent of GnRH neurons in which galanin-IR was detectable. Significance of difference: Proestrus vs. OVX-E 2 , OVX-surge or male, p- 0.01; OVX vs. OVX-E 2 or male, p- 0.01, and vs. OVXsurge, p- 0.05; OVX-surge vs. OVX-E 2 , p- 0.05, and vs. male, p0.01.
each brain, the data were combined and presented in the results. Approximately 10% of the total number of GnRH cells in each brain were analyzed under the confocal microscope ŽTable 1.. The percent of GnRH cells exhibiting galanin-IR is presented in Fig. 3. The number of GnRH cells exhibiting galanin-IR was significantly different among the groups Ž F s 18.51; df s 4; p - 0.001.. GnRH cells coexpressing galanin-IR in proestrous mice were significantly higher Ž p - 0.01. than that in OVXE 2-primed, OVX-surge, and male mice. The number of GnRH cells exhibiting galanin-IR in OVX mice was significantly higher than that in OVX-E 2-primed Ž p - 0.01., OVX-surge Ž p - 0.05., and male Ž p - 0.01. mice. The expression of galanin-IR in GnRH cells in OVX-surge mice was significantly higher Ž p - 0.05. than that in OVX-E 2-primed and male mice.
4. Discussion The present findings that galanin-IR is expressed in GnRH neurons in mice is in agreement with the earlier
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reports in rat w6,23x. A greater proportion of GnRH cells with galanin-IR in intact female mice as compared with males in our studies is also similar to that reported in rats w25,26x. Nevertheless, there are differences between the two species in the conditions when peptide coexpression is observed. Removal of the ovaries in rats reduces the number of GnRH cells exhibiting galanin-IR and treatment of OVX rats with estrogen restores it in both young adult w26x and aged w3x animals. This is in sharp contrast to the results in mice in which a large number of GnRH cells in OVX animals coexpressed galanin-IR and estrogen treatment significantly reduced colocalization. As in most of the immunocytochemical studies in rat w3,25,26x colchicine treatment was required to visualize galanin within the cell bodies of the mouse brains. In pilot studies, it appeared that when injections of colchicine were given too early or too late on proestrus, galanin colocalization in GnRH neurons was not evident Ždata not shown.. Since the primary purpose of the present study was to determine whether there was any colocalization in the mouse, this was not pursued. It is uncertain how rapidly colchicine arrests axonal flow, but our pilot studies suggest that galanin peptide is expressed and secreted rapidly at the time of the surge. This may be a factor contributing to the lower expression of galanin in OVX-surge than in proestrous mice. Plasma LH measures obtained approximately 10–15 min prior to colchicine treatment may be imperfectly correlated with GnRH activation. The high LH value in OVX-surge mice suggests that the surge of LH was close to its peak at the time of sampling and colchicine treatment. Had the injection of colchicine been carried out earlier, there may have been a greater proportion of GnRH cells with galanin-IR in these mice. The expression of galanin mRNA in GnRH neurons in the rat follows a similar pattern as peptide expression, with higher expression in females vs. males w7x. It should be borne in mind that strict comparisons cannot be made between detection of galanin peptide in numbers of GnRH cells by immunocytochemistry and of galanin message by averaging intensity of silver grains in all GnRH cells in a given field. Nevertheless, galanin gene expression in GnRH neurons in the rat is decreased with OVX and increases with estrogen treatment w18,31x, as does the peptide. Immunocytochemical analysis of galanin and GnRH coexpression during a steroid-induced LH surge in the OVX rat has not been reported, but galanin mRNA expression in GnRH neurons is increased in this model w19,31x. That this elevated coexpression reflects GnRH activation is supported by the observation that it is blocked by prior administration of adrenergic blockers w19x or of NMDA receptor blockers w31x. Furthermore, triple labeling shows that a high proportion of GnRH neurons that are activated during a steroid-induced surge, as indicated by Fos expression, also contain galanin mRNA w10x. A time course study shows that c-fos mRNA increases significantly in GnRH neurons at the time of the initial increase in steroid-in-
duced LH, while increased galanin mRNA is not seen until the peak LH is attained w8x. Galanin mRNA continues to rise thereafter and remains elevated for at least 24 h, leading the authors to suggest that Fos expression in GnRH neurons induces galanin gene expression, and that the sustained levels may be related to the need to replenish stores of galanin peptide w8x. The strong evidence that galanin expression increases in GnRH neurons at the time of increased activation of those neurons is consistent with our findings in the mouse. Appreciable numbers of GnRH neurons contained detectable galanin-IR at times of increased LH release, as in the proestrous, OVX and OVX-surged mice. In contrast, when plasma LH levels are low, as in the estrogen-treated OVX female and in the male, little colocalization was evident. In the rat, however, the effects of estrogen appear to be overriding, leading to increased galanin coexpression in estrogen-treated OVX animals Žwhen plasma LH is suppressed w18x. as well as at the time of the surge. A significantly lower number of GnRH cells present in female mice in which the LH surge was induced by steroids as compared with proestrous females suggests that in females with the steroid-induced surge some of the GnRH cells might have released their contents such that they were undetectable by immunocytochemistry. This would also contribute to the lower number of GnRH cells exhibiting galanin-IR in these females. In rats of both sexes, the number and staining intensity of immunoreactive GnRH cells fluctuate depending on the activity of the system w14,34x. These reports are in line with the present results that the number of immunocytochemically detectable GnRH cells may vary depending on the circulating levels of gonadal steroids. The role of galanin in GnRH cells is not understood. As discussed above, the expression of galanin in GnRH neurons is associated with periods of enhanced activity of the system. Galanin is suggested to be a hypothalamo–hypophyseal hormone which influences gonadotropic function of the pituitary w16x. Galanin stimulates the release of LH from the pituitary w6x and enhances the GnRH-induced release of LH from dispersed pituitary cells w16x. Infusion of galanin into the cerebral ventricle enhances the release of LH w32x. LH surge in female rats can be blunted by the administration of galanin antibody w17x or a potent galanin antagonist w33x. A possible central effect of galanin on GnRH secretion is supported by the report that galanin induces the release of GnRH from median eminence in vitro w23x. Thus the increase in the expression of galanin in GnRH cell bodies during periods of enhanced GnRH activity supports the hypothesis that galanin released from GnRH cells may act upon the GnRH terminals to further enhance the release of GnRH w30x. The ability of galanin to facilitate the release of LH from the pituitary may further amplify the magnitude of LH surge. In conclusion, the present results indicate that the induction of galanin-IR in GnRH neuronal cell bodies in the
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mouse coincides with enhanced activity of the GnRH system. The contrast in steroid regulation of galanin peptide coexpression in GnRH neurons in mice and rats suggests that there may be differences among even closely related species in such regulation. Further studies, including those evaluating galanin mRNA in mice, may clarify the significance of such differences.
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Acknowledgements w17x
Supported by NIH grants NS 20335, HD19077 and T32DK07645. Confocal laser scanning microscopy was performed at the MSSM-CLSM core facility, supported with funding from NIH shared instrumentation grant Ž1 S10 RR0 9145-01. and NSF major research instrumentation grant ŽDBI-9724504..
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Research report
Expression of galanin immunoreactivity in gonadotropin-releasing hormone neurons in mice: a confocal microscopic study Gopalan Rajendren, Marie J. Gibson
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Department of Medicine, Mount Sinai School of Medicine, Box 1055, 1 GustaÕe L. LeÕy Place, New York, NY 10029, USA Accepted 15 December 1998
Abstract The expression of galanin immunoreactivity Žgalanin-IR. in gonadotropin-releasing hormone ŽGnRH. neurons was investigated in mice using double label immunohistochemistry combined with confocal laser scanning microscopy. A large proportion of GnRH cells in proestrous mice and very few GnRH cells in male mice exhibited galanin-IR. These results are consistent with earlier reports in rats. Unlike in rats, the proportion of GnRH cells coexpressing galanin in mice was high following ovariectomy ŽOVX. and the treatment of OVX mice with estrogen decreased the number of GnRH cells with galanin-IR. The GnRH system can be considered more active during proestrous and following OVX since the output of luteinizing hormone is elevated during these phases in females. Since the induction of galanin-IR in GnRH cells is more pronounced in OVX and proestrous mice, the expression of galanin-IR in GnRH cells in mice appears to be an activation-dependent phenomenon rather than a direct effect of estrogen. However, in OVX mice treated with steroids to induce an LH surge the number of GnRH cells with galanin-IR was not proportionately increased. The possible reasons for this discrepancy are also discussed. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Galanin; Gonadotropin-releasing hormone; GnRH; Luteinizing hormone-releasing hormone; Estrous cycle; Immunohistochemistry
1. Introduction Successful reproduction and thus survival of the species in mammals is dependent on the few hundred gonadotropin-releasing hormone ŽGnRH. neuronal cell bodies scattered in the ventral forebrain w12,35x. GnRH regulates the gonadotropic functions of the anterior pituitary gland, the control of which is different in the two sexes. The cyclicity inherent in female reproductive function is dictated by the cyclic release of GnRH into the hypothalamo–hypophyseal portal system w28x. The present knowledge of the regulation of the GnRH system is primarily based on studies carried out in the rat. In that species, almost all known neurotransmitters and neuromodulators have been implicated in influencing the GnRH system w13x. However, the precise orchestration of these factors in the control of the synthesis and release of GnRH is largely unknown.
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The gut–brain peptide galanin was originally discovered in porcine intestine w37x. Galanin is heavily concentrated in the hypothalamus and the median eminence w22,24x. It is suggested that galanin functions as a hypothalamic–hypophysiotropic hormone that modulates the release of growth hormone, prolactin and luteinizing hormone w16x. In the central nervous system, galanin is colocalized with vasopressin, dynorphin, tyrosine hydroxylase, g-aminobutyric acid, norepinephrine, serotonin and choline acetyl transferase w4,20–22,36x. A significant number of GnRH neuronal cell bodies are also immunoreactive for galanin w6,23x. The presence of galanin immunoreactivity Žgalanin-IR. w25x and the expression of galanin mRNA w7,19,29x in GnRH cells appear to be sexually dimorphic in rats. A greater proportion of GnRH cells in female rats expresses galanin as compared with males and is dependent on circulating levels of estrogen. Ovariectomy ŽOVX. decreases and estrogen treatment of OVX rats increases the expression of galanin in GnRH cells w7,18,19x. To date there has been no study reported investigating the expression of galanin in GnRH neurons in any species other than the rat. Often, the studies in rats are generalized to include other species as well. There are some indica-
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tions that even closely related species such as mice may vary significantly from rats. For example, sequential injection of estrogen and progesterone induces a preovulatorylike surge of LH in OVX rats; even estrogen alone, if injected in sufficiently high doses, will induce a surge of LH in OVX rats w15x. Similar treatment of OVX mice with steroids rarely induces an LH surge; the time of steroid treatment in relation to OVX and the dose of steroids administered have to be precisely adjusted in order to obtain a consistent surge of LH in OVX mice w1,2x. A tonic suppression of LH release in male rats by endogenous opiates is suggested by the findings that administration of the opioid receptor antagonist naloxone facilitates LH release w5x. In male mice on the other hand, naloxone has no noticeable effect on the release of LH w27x. Thus, the purpose of the present studies was to evaluate galanin colocalization in GnRH neurons in mice to investigate whether the incidence of galanin-IR in GnRH neurons follows a pattern similar to that reported in rats. Since galanin-IR in GnRH cell bodies was faint and the preoptic region exhibited an intense galanin fiber IR, confocal microscopy was employed to identify GnRH cells with galanin-IR.
2. Materials and methods 2.1. Animals C3HrHeH= 101H mice were housed under a partially reversed lightrdark cycle Ž14 h of light and 10 h of dark; lights off at 13:00 h. and controlled temperature Ž24–268C.. Food and drinking water were available ad libitum. All procedures and animal care were in accord with the standard approved by the NIH Guide for the Care and Use of Laboratory Animals and were approved by the IACUC of the Mount Sinai School of Medicine. 2.2. Experimental protocol Pilot studies indicated that treatment of the mice with colchicine was required in order to visualize galanin-IR in GnRH cell bodies. Between 12:00 and 13:00 h, the mice were anesthetized with chloral hydrate Ž380 mgrkg; i.p.. and blood samples were obtained from the orbital sinus for LH assay. They were then stereotaxically injected with 15 mg of colchicine Žin 1 ml sterile distilled water. into the lateral cerebral ventricle. 24 h later, they were anesthetized with an overdose of chloral hydrate Ž760 mgrkg; i.p.., perfused transcardially with 15 ml of saline followed by 60 ml of ice-cold 4% paraformaldehyde and the brains were processed for double label immunostaining for GnRH and galanin. Adult mice, 10–12 weeks old, were divided into five groups and treated as follows.
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2.2.1. Proestrus Vaginal smears were obtained daily for 2 weeks and those exhibiting two consecutive 4–5 days cycles were included Ž n s 4.. On the day of proestrus they were injected with colchicine intracerebroventricularly Ži.c.v.. and the brains were processed the following day for double label immunocytochemistry. 2.2.2. OVX The females were bilaterally OVX Ž n s 5. and 3 weeks later, they were injected with colchicine i.c.v. and the brains were processed for immunostaining as in the previous group. 2.2.3. OVX q estrogen (OVX-E2 ) The females were bilaterally OVX and 2 weeks later they were implanted with a silastic capsule Ž10 mm; i.d., 1.0 mm; o.d., 2.0 mm; Dow Corning, Midland, MI. containing 10 mg estradiol ŽE 2 . in sesame oil ŽSO. Ž n s 4.. One week after implantation of the E 2-containing capsule, they were injected with colchicine i.c.v. and the brains were processed as above. 2.2.4. OVX and E q P with LH surge (OVX-surge) The females were bilaterally OVX and were implanted with a silastic capsule Ždimensions as above. containing 1 mg E 2 in SO and were sequentially primed with estradiol benzoate ŽEB. and progesterone ŽP. to induce a luteinizing hormone surge Ž n s 5. as described earlier w11x. Briefly, 1.0 mg of EB in 0.05 ml SO and 500 mg of P in 0.05 ml SO were administered s.c. at 09:00 h on days 7 and 8 post-OVX, respectively. 4 h after P injection they were injected with colchicine i.c.v. and the brains were processed as above. 2.2.5. Males Male mice Ž n s 5. were injected with colchicine i.c.v. and the brains were processed for galanin and GnRH staining as above. 2.3. Immunohistochemistry The brains were removed, post-fixed in 4% paraformaldehyde overnight and coronal sections, 50 mm thick, were obtained using a Vibratome. The sections were washed and were incubated for 72 h with a dilute anti-galanin antibody Žraised in rabbit; Peninsula Labs, Belmont, CA. in 0.1 M PBS Ž1:3000. containing 1.5% normal donkey serum ŽNDS. and 0.3% Triton X. The sections were washed twice and were incubated for 3 h with anti-rabbit IgG–FITC conjugate Ž5 mgrml in 0.1 M PBS containing 1.5% NDS and 0.3% Triton X. raised in donkey ŽJackson Immunoresearch, West Grove, PA.. The sections were washed twice in PBS and were incubated for 96 h with a dilute Ž1:5000 in 0.1 M PBS containing 1.5% NDS and 0.3% Triton X.
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standard. The coefficient of variances were 3.52% and 5.21% Žintra-assay., and 4.42% Žinter-assay.. The limit of detection was 0.22 ngrml. 2.6. Data analysis The data were analyzed using one-way analysis of variance followed by Fisher’s LSD Žprotected t-test. using the GB Stat program.
3. Results Fig. 1. Plasma LH in male and female mice. Plasma LH was significantly higher in OVX-surge females than in all other groups Ž p- 0.01..
anti-GnRH antibody Žmouse monoclonal; QED Advanced Research, San Diego, CA.. The sections were then washed twice in PBS and were incubated for 3 h with anti-mouse IgG–Texas red conjugate Ž5 mgrml. raised in donkey ŽJackson Immunoresearch.. The sections were washed, mounted onto gelatin-coated slides, cover-slipped using Vectashield mounting medium ŽVector Labs, Burlingame, CA. and analyzed under fluorescent and confocal microscopes. 2.4. Microscopy GnRH cells were counted from sections obtained from the block of brain encompassing the anterior septum, organum vasculosum of lamina terminalis, preoptic area, hypothalamus and the mammillary bodies. This block of brain contained the majority of the GnRH cell bodies in the brain. The cells were counted using an Olympus fluorescence microscope under a magnification of 400 = . Due to the intense galanin fiber staining in the ventral forebrain, the region where the majority of GnRH cell bodies are located, it was not possible to reliably identify the GnRH cells with galanin-IR under regular fluorescent microscope. Therefore, confocal microscopy was employed for this purpose. Since it was not practical to analyze all the sections under the confocal microscope, two sections were selected from each brain under the fluorescent microscope using anatomical landmarks w9x, one passing through the anterior septum Žq1.10 mm bregma. and the other through organum vasculosum of lamina terminalisranterior preoptic area Žq0.32 mm bregma.. These two sections were examined under the confocal microscope. All the GnRH cells in these sections were individually scanned under a 100 = objective lens and the digital images of thin optical sections were analyzed for dual labeling. 2.5. Radioimmunoassay Plasma samples were assayed in duplicate for LH with a kit provided by the NIDDK using rat LH RP-3 as the
Plasma LH values are shown in Fig. 1. One-way analysis of variance revealed that LH values were significantly different among the groups Ž F s 16.82; df s 4; p - 0.001.. Post-hoc analyses indicated that the value of LH in OVXsurge females was significantly higher than all the other groups Ž p - 0.01.. The total number of GnRH cells in each group is shown in Table 1. The mean number of GnRH cells was significantly different among the groups Ž F s 2.94; df s 4; p 0.05.. Post-hoc comparison revealed that the total number of GnRH cells in proestrous mice was significantly higher Ž p - 0.05. than that in OVX-E 2 and OVX-surge mice. GnRH cell number in male mice also was significantly greater Ž p - 0.05. than that in OVX-E 2 and OVX-surge mice. The differences in the number of GnRH cells among the other groups were not significant. Galanin-IR in cell bodies was variable in intensity, whether or not the cells also contained GnRH. However, the thin optical sectioning capability and extreme sensitivity of confocal microscopy enabled the identification of even faint galanin-IR in GnRH cells ŽFig. 2.. In some cases there was relatively strong staining of galanin-IR in GnRH neurons ŽA and B. while in other instances the galanin-IR in the GnRH cell body would have been difficult to visualize without confocal microscopy, because of the network of intensely stained fibers containing galaninIR in the region ŽC and D..
Table 1 Mean"S.E.M. of the total number of GnRH cells in the experimental groups and the percent analyzed with confocal microscopy Group Ž n.
Total number
Number Žpercent of total. analyzed under confocal microscope
Proestrus Ž4. OVX Ž5. OVX-E 2 Ž4. OVX-surge Ž5. Male Ž5.
431.8"22.1) 367.8"28.9 297.5"24.7 321.6"13.4 414.8"47.6)
42.3"2.2 Ž9.8. 40.2"3.4 Ž10.9. 29.3"1.0 Ž9.8. 26.0"3.7 Ž8.1. 39.8"3.9 Ž9.6.
) p- 0.05 vs. OVX-E 2 and OVX-surge.
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Fig. 2. Confocal images of galanin-IR in GnRH cells. Arrows indicate double staining. The GnRH cell in A has intense galanin-IR as shown in B. In contrast, as seen in D, there is weak galanin-IR in the GnRH cell in C. This would be difficult to detect with a regular fluorescence microscope particularly in an area with intense galanin fiber staining. The second GnRH cell in C is galanin negative. Scale bar s 10 mm.
Since there was no noticeable difference in galanin staining in GnRH cells in the two sections obtained from
Fig. 3. Galanin-IR in GnRH neurons. Confocal microscopy was used to evaluate the percent of GnRH neurons in which galanin-IR was detectable. Significance of difference: Proestrus vs. OVX-E 2 , OVX-surge or male, p- 0.01; OVX vs. OVX-E 2 or male, p- 0.01, and vs. OVXsurge, p- 0.05; OVX-surge vs. OVX-E 2 , p- 0.05, and vs. male, p0.01.
each brain, the data were combined and presented in the results. Approximately 10% of the total number of GnRH cells in each brain were analyzed under the confocal microscope ŽTable 1.. The percent of GnRH cells exhibiting galanin-IR is presented in Fig. 3. The number of GnRH cells exhibiting galanin-IR was significantly different among the groups Ž F s 18.51; df s 4; p - 0.001.. GnRH cells coexpressing galanin-IR in proestrous mice were significantly higher Ž p - 0.01. than that in OVXE 2-primed, OVX-surge, and male mice. The number of GnRH cells exhibiting galanin-IR in OVX mice was significantly higher than that in OVX-E 2-primed Ž p - 0.01., OVX-surge Ž p - 0.05., and male Ž p - 0.01. mice. The expression of galanin-IR in GnRH cells in OVX-surge mice was significantly higher Ž p - 0.05. than that in OVX-E 2-primed and male mice.
4. Discussion The present findings that galanin-IR is expressed in GnRH neurons in mice is in agreement with the earlier
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reports in rat w6,23x. A greater proportion of GnRH cells with galanin-IR in intact female mice as compared with males in our studies is also similar to that reported in rats w25,26x. Nevertheless, there are differences between the two species in the conditions when peptide coexpression is observed. Removal of the ovaries in rats reduces the number of GnRH cells exhibiting galanin-IR and treatment of OVX rats with estrogen restores it in both young adult w26x and aged w3x animals. This is in sharp contrast to the results in mice in which a large number of GnRH cells in OVX animals coexpressed galanin-IR and estrogen treatment significantly reduced colocalization. As in most of the immunocytochemical studies in rat w3,25,26x colchicine treatment was required to visualize galanin within the cell bodies of the mouse brains. In pilot studies, it appeared that when injections of colchicine were given too early or too late on proestrus, galanin colocalization in GnRH neurons was not evident Ždata not shown.. Since the primary purpose of the present study was to determine whether there was any colocalization in the mouse, this was not pursued. It is uncertain how rapidly colchicine arrests axonal flow, but our pilot studies suggest that galanin peptide is expressed and secreted rapidly at the time of the surge. This may be a factor contributing to the lower expression of galanin in OVX-surge than in proestrous mice. Plasma LH measures obtained approximately 10–15 min prior to colchicine treatment may be imperfectly correlated with GnRH activation. The high LH value in OVX-surge mice suggests that the surge of LH was close to its peak at the time of sampling and colchicine treatment. Had the injection of colchicine been carried out earlier, there may have been a greater proportion of GnRH cells with galanin-IR in these mice. The expression of galanin mRNA in GnRH neurons in the rat follows a similar pattern as peptide expression, with higher expression in females vs. males w7x. It should be borne in mind that strict comparisons cannot be made between detection of galanin peptide in numbers of GnRH cells by immunocytochemistry and of galanin message by averaging intensity of silver grains in all GnRH cells in a given field. Nevertheless, galanin gene expression in GnRH neurons in the rat is decreased with OVX and increases with estrogen treatment w18,31x, as does the peptide. Immunocytochemical analysis of galanin and GnRH coexpression during a steroid-induced LH surge in the OVX rat has not been reported, but galanin mRNA expression in GnRH neurons is increased in this model w19,31x. That this elevated coexpression reflects GnRH activation is supported by the observation that it is blocked by prior administration of adrenergic blockers w19x or of NMDA receptor blockers w31x. Furthermore, triple labeling shows that a high proportion of GnRH neurons that are activated during a steroid-induced surge, as indicated by Fos expression, also contain galanin mRNA w10x. A time course study shows that c-fos mRNA increases significantly in GnRH neurons at the time of the initial increase in steroid-in-
duced LH, while increased galanin mRNA is not seen until the peak LH is attained w8x. Galanin mRNA continues to rise thereafter and remains elevated for at least 24 h, leading the authors to suggest that Fos expression in GnRH neurons induces galanin gene expression, and that the sustained levels may be related to the need to replenish stores of galanin peptide w8x. The strong evidence that galanin expression increases in GnRH neurons at the time of increased activation of those neurons is consistent with our findings in the mouse. Appreciable numbers of GnRH neurons contained detectable galanin-IR at times of increased LH release, as in the proestrous, OVX and OVX-surged mice. In contrast, when plasma LH levels are low, as in the estrogen-treated OVX female and in the male, little colocalization was evident. In the rat, however, the effects of estrogen appear to be overriding, leading to increased galanin coexpression in estrogen-treated OVX animals Žwhen plasma LH is suppressed w18x. as well as at the time of the surge. A significantly lower number of GnRH cells present in female mice in which the LH surge was induced by steroids as compared with proestrous females suggests that in females with the steroid-induced surge some of the GnRH cells might have released their contents such that they were undetectable by immunocytochemistry. This would also contribute to the lower number of GnRH cells exhibiting galanin-IR in these females. In rats of both sexes, the number and staining intensity of immunoreactive GnRH cells fluctuate depending on the activity of the system w14,34x. These reports are in line with the present results that the number of immunocytochemically detectable GnRH cells may vary depending on the circulating levels of gonadal steroids. The role of galanin in GnRH cells is not understood. As discussed above, the expression of galanin in GnRH neurons is associated with periods of enhanced activity of the system. Galanin is suggested to be a hypothalamo–hypophyseal hormone which influences gonadotropic function of the pituitary w16x. Galanin stimulates the release of LH from the pituitary w6x and enhances the GnRH-induced release of LH from dispersed pituitary cells w16x. Infusion of galanin into the cerebral ventricle enhances the release of LH w32x. LH surge in female rats can be blunted by the administration of galanin antibody w17x or a potent galanin antagonist w33x. A possible central effect of galanin on GnRH secretion is supported by the report that galanin induces the release of GnRH from median eminence in vitro w23x. Thus the increase in the expression of galanin in GnRH cell bodies during periods of enhanced GnRH activity supports the hypothesis that galanin released from GnRH cells may act upon the GnRH terminals to further enhance the release of GnRH w30x. The ability of galanin to facilitate the release of LH from the pituitary may further amplify the magnitude of LH surge. In conclusion, the present results indicate that the induction of galanin-IR in GnRH neuronal cell bodies in the
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mouse coincides with enhanced activity of the GnRH system. The contrast in steroid regulation of galanin peptide coexpression in GnRH neurons in mice and rats suggests that there may be differences among even closely related species in such regulation. Further studies, including those evaluating galanin mRNA in mice, may clarify the significance of such differences.
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Acknowledgements w17x
Supported by NIH grants NS 20335, HD19077 and T32DK07645. Confocal laser scanning microscopy was performed at the MSSM-CLSM core facility, supported with funding from NIH shared instrumentation grant Ž1 S10 RR0 9145-01. and NSF major research instrumentation grant ŽDBI-9724504..
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