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Effects of estradiol, progesterone, and norethisterone on regional concentrations of galanin in the rat brain1
Peptides 20 (1999) 743–748
Effects of estradiol, progesterone, and norethisterone on regional concentrations of galanin in the rat brain1 Olof Rugarna,*, Annette Theodorssonb, Mats Hammara, Elvar Theodorssonc a
Division of Obstetrics and Gynecology, Faculty of Health Sciences, Linko¨ping University, S-581 85 Linko¨ping, Sweden b Division of Neurosurgery, Faculty of Health Sciences, Linko¨ping University, S-581 85 Linko¨ping, Sweden c Division of Clinical Chemistry, Faculty of Health Sciences, Linko¨ping University, S-581 85 Linko¨ping, Sweden Received 15 October 1998; accepted 26 January 1999
Abstract Concentrations of immunoreactive galanin were compared in eight gross brain regions of ovariectomized female rats treated with either estradiol, estradiol ⫹ progesterone, estradiol ⫹ norethisterone, or placebo. Higher concentrations with estradiol treatment compared with placebo were found in the pituitary (357%), frontal cortex (162%), occipital cortex (174%), hippocampus (170%), and median eminence (202%). A more profound difference with addition of progesterone or norethisterone was seen in the pituitary (529% and 467%, respectively). Sex steroids, particularly estradiol, modulate galanin concentrations not only in reproductive, but also in nonreproductive, brain regions. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Galanin; Estradiol; Progesterone; Norethisterone; Hippocampus; Pituitary; Frontal cortex
1. Introduction The neuropeptide galanin modulates several different neural and neuroendocrine events such as neurotransmitter release [5], food intake [21], learning and memory processes [10,27], and pituitary hormone secretion [15]. Galanin is present in many systems containing other neuroactive compounds, including dopamine, noradrenaline, serotonin, ␥-aminobutyric acid, and other neuropeptides [24]. An example of the latter are hypothalamic-pituitary gonadotropinreleasing hormone neurons, where galanin mRNA is present and can be induced by estrogen [2,31]. The process is facilitated by progesterone [31]. In this case, galanin seems to function as a mediator of sex steroid effects. Consideration of the widespread distribution of both galanin-like immunoreactivity and galanin-receptors in the central nervous system [12,35,36] suggests that galanin may mediate sex steroid action in other parts of the brain. This hypothesis is further warranted by the finding of a functional estrogen * Corresponding author. Tel.: ⫹46-13-22-31-22; fax: ⫹46-13-14-8156. E-mail address: [email protected] (O. Rugarn) This study was supported by the Swedish Medical Research Council (12X-07464). 1
response element sequence within the human galanin gene, which could potentially function as a regulatory element for estrogen action in galanin-expressing neurons [16]. Thus, nonreproductive functions could also be influenced by sex steroids like estrogen through galanin mediation. Brain areas associated with cognition and emotion are of special interest, because the influence of sex steroids on these functions is now well supported [4,13,19,23,34]. A positive regulation of galanin gene expression by estrogen has in fact been demonstrated outside of the pituitary and hypothalamus, for instance, in the locus ceruleus [38]. A regulation of gene expression across puberty was found in the bed nucleus of stria terminalis and medial amygdala [28]. In a previous experiment, we found higher galanin concentrations in adult than prepubertal female rats in the frontal cortex and hippocampus [32]. Progesterone receptors have been demonstrated in hypothalamic galanin-immunoreactive neurons [40], but little is known about progesterone and galanin interactions in other brain areas. The aim of this study was to assess if sex steroids have any effect on galanin immunoreactivity in the brain regionwise. We reasoned that if sex steroids indeed have a potential to regulate galanin in diverse parts of the brain, it could have implications for understanding how the brain is affected by puberty, menopause, and by exogenous steroids
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Table 1 Plasma levels of estradiol (pM) and progesterone (nM) Group
Estradiol median
IQR*
Progesterone median
IQR*
Sham Ovx Estr Estr ⫹ Prog Estr ⫹ NET
62 56 177 188 196
59–64 49–68 173–199 142–235 142–217
19 2.9 15 26 16
12–32 2.6–4.2 9.0–32 15–36 15–16
*Interquartile range. The groups are sham-operated rats (Sham), ovariectomized and placebotreated (Ovx), ovariectomized and estradiol-treated (Estr), ovariectomized and estradiol ⫹ progesterone-treated (Estr ⫹ Prog), and ovariectomized and estradiol ⫹ norethisterone treated (Estr ⫹ NET).
used in oral contraceptives and postmenopausal hormone replacement therapy. Many women report changes in general well-being, emotional state, and eating behavior while using contraceptive pills or menopausal hormone replacement [4,22,37]. The synthetic progestin norethisterone is a commonly used component in both contraceptive pills and menopausal replacement therapy. We have therefore included a study of the effects of this steroid as well, and compared it with those of progesterone. We treated ovariectomized rats with slow-release pellets of estradiol, estradiol and progesterone, or estradiol and norethisterone (Table 1). Extended treatment periods of 5 to 8 weeks were chosen because peptide concentrations depend on synthesis, transport, and storage, which take a much longer time than the corresponding events for monoamines. Control groups were ovariectomized placebo-treated rats and sham-operated placebo-treated rats. After treatment, the concentrations of immunoreactive galanin were measured by using radioimmunoassay in frontal cortex, occipital cortex, hippocampus, striatum, cerebellum, hypothalamus, median eminence, and the pituitary.
tive hormone and placebo was administered with slowrelease pellets (Innovation Research of America, Sarasota, FL, USA), which were placed subcutaneously in the neck fold under anesthesia. The different groups were treated as follows: Group 1 (n ⫽ 8): the sham-operated group. The ovaries were simply identified and the animals were treated with placebo pellets for 6 weeks. Group 2 (n ⫽ 7): the placebo group. The animals were ovariectomized and treated with placebo pellets for 6 weeks. Group 3 (n ⫽ 9): the estradiol group. The animals were ovariectomized and treated with slow-release pellets containing 0.1 mg of 17-estradiol, yielding a constant rate of 1.2 g/day (data supplied by manufacturer) for 8 weeks. During weeks 3 to 5, a pellet containing 50 mg of progesterone, yielding 600 g/day (data supplied by manufacturer), was added. Then, during weeks 6 to 8, 17-estradiol 1.2 g/day was again the sole hormone administration. Group 4 (n ⫽ 8): the estradiol ⫹ progesterone group. The animals were ovariectomized and treated with slowrelease pellets containing 0.1 mg of 17-estradiol, yielding a constant rate of 1.2 g/day for 5 weeks. During the last 2 weeks, a pellet containing 50 mg of progesterone, yielding 600 g/day, was added. Group 5 (n ⫽ 8): the estradiol ⫹ norethisterone group. The animals were ovariectomized and treated with slowrelease pellets containing 0.1 mg of 17-estradiol, yielding a constant rate of 1.2 g/day for 5 weeks. During the last 2 weeks, a pellet containing 50 mg of norethisterone, yielding 600 g/day, was added. The rats were killed by guillotine and the brains dissected [11]. The individual brain regions were weighed immediately and frozen on dry ice. The study and its experimental protocol was approved by the local animal research ethics committee and found to be in accordance with the guidelines issued by the Central Committee For Animal Research.
2. Materials and methods
2.2. Extraction of tissue samples
2.1. Animals and test procedure
The frozen tissue samples were cut into small pieces, and 10 ml of 1 M acetic acid was added for each gram of tissue. After boiling for 10 min, the samples were homogenized by using a steel rod and a Vortex mixer, then centrifuged, whereafter the supernatants were removed. A second extraction with an equal amount of distilled water was then performed to maximize the solubility of galanin and possible neutral homologs. The combined supernatants were lyophilized and stored at ⫺20°C before analysis. The samples were reconstituted in 1 ml of phosphate buffer (0.05 M, pH 7.4) and then divided before further analysis.
Experiments were performed on virgin female rats (ALAB, Sollentuna, Sweden) weighing 250 to 270 g at the start of the experiment. Forty rats divided into five study groups were used. The rats were housed five to a cage at constant room temperature (21 ⫾ 1°C) for 3 days, with free access to water and standard rat food, and with 12-h light/ dark cycles before the experiments. All surgery was performed with the animals under general anesthesia. The animals were premedicated with phentanyl 10 mg/ml ⫹ fluanison 0.2 mg/ml (Hypnorm) 0.2 ml/kg intramuscularly and atropine 0.05 mg/kg. Anesthesia was achieved with xylazine 12 mg/kg intraperitoneally and ketamine 80 mg/kg intraperitoneally. After surgery, buprenorphine 0.05 mg/kg was given subcutaneously for postoperative analgesia. Ac-
2.3. Radioimmunoassays Galanin was analyzed by using antiserum RatGala4 raised against conjugated synthetic rat galanin. The anti-
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serum does not cross-react with neurokinin A, neuropeptide K, substance P, neurokinin B, neuropeptide Y, gastrin, pancreatic polypeptide, glucagon, or neurotensin. High-performance liquid chromatography-purified 125I-rat galanin was used as radioligand and rat galanin as standard. The volume of the sample used for the assay was 100 l. The detection limit of the assay was 8 pM of galanin in the sample. Intraand interassay coefficients of variation were 6% and 10%, respectively, at 50 pM. Reverse-phase high-performance liquid chromatography (20 –50% acetonitrile with 0.1% trifluoroacetic acid) of the immunoreactive material from rat brains revealed a single immunoreactive component for the assay that coeluted with the calibrator. 2.4. Hormone analysis Plasma levels of progesterone and estradiol were measured by time-resolved fluoroimmunoassay (AutoDELFIA, Wallac, Gaithersburg, MD, USA). 2.5. Statistical analysis The median and interquartile ranges were used as measures of central tendency and variation, respectively. Multivariate analysis of variance with Tukey multiple comparisons and correlation test was performed with SYSTAT (Version 5, SYSTAT, Inc, Evanston, IL, USA). If more than half of the measurements for a particular analyte in a particular region were below the limit of detection, the data for that region are neither shown nor used in drawing conclusions. 3. Results 3.1. Galanin immunoreactivity Multivariate analyses of variance (factors ⫽ group, region) revealed overall differences with regard to group [F(4) ⫽ 10.54, P ⬍ 0.001], region [F(7) ⫽ 289.7, P ⬍ 0.001], and group–region [F(28) ⫽ 7.65, P ⬍ 0.001]. In particular (Fig. 1), ovariectomized/estradiol-treated rats had higher galanin concentrations compared with the ovariectomized/placebo-treated in frontal cortex (162% of placebo treated; P ⬍ 0.01), occipital cortex (174%; P ⬍ 0.001), hippocampus (170%; P ⬍ 0.01), pituitary (357%; P ⬍ 0.05), and the median eminence (202%; P ⬍ 0.001). The groups that were treated with estradiol ⫹ progesterone or estradiol ⫹ norethisterone showed results similar to those of the estradiol-only-treated rats in most regions. Accordingly, in the estradiol ⫹ progesterone group, galanin concentrations were higher than the placebo-treated in frontal cortex (138%; P ⬍ 0.05), occipital cortex (157%; P ⬍ 0.001), hippocampus (167%; P ⬍ 0.05), the pituitary (529%; P ⬍ 0.001), and the median eminence (186%; P ⬍ 0.001). Estradiol ⫹ norethisterone-treated rats had higher galanin concentrations compared with placebo-treated in occipital cortex (163%; P ⬍ 0.001), hippocampus (156%; P ⬍
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0.01), the median eminence (185%; P ⬍ 0.001), and in the pituitary (467%; P ⬍ 0.001), whereas no significant difference was found in the frontal cortex. On the other hand, galanin concentrations in the frontal cortex were significantly lower in rats treated with estradiol ⫹ norethisterone compared with rats treated with only estradiol (75% of estrogen treated; P ⬍ 0.05). The sham-operated, gonadally intact, animals had higher concentrations compared with ovariectomized/placebo-treated in the pituitary (P ⬍ 0.05) but did not differ significantly in any other region. In comparison with the estradiol-only–treated group, significant differences were found in frontal cortex (P ⬍ 0.001), occipital cortex (P ⬍ 0.001), hippocampus (P ⬍ 0.05), and the median eminence (P ⬍ 0.05). Compared with the estradiol ⫹ progesterone group, there were significant differences only in frontal cortex (P ⬍ 0.01) and occipital cortex (P ⬍ 0.01). The estradiol ⫹ norethisterone group differed significantly in occipital cortex (P ⬍ 0.01), hippocampus (P ⬍ 0.05), and the median eminence (P ⬍ 0.01). No significant differences between the groups were found in hypothalamus or cerebellum. 3.2. Plasma hormone concentrations The groups that received estrogen pellets had similar plasma levels as near-term pregnant rats [1] and were in accordance with those expected according to the manufacturer of the slow-release pellets. The progesterone levels are lower than expected, which according to the manufacturer should be ⬇60 nM. The animals that were treated with estrogen only for the last 3 weeks (Group 3) and the animals that received estrogen ⫹ norethisterone have noticeably higher progesterone levels compared with the placebo treated. We have no satisfactory explanation for these discrepancies. It is possible that our method, for some reason, is not suitable for rat plasma or that the results are caused by analytical interference (e.g. hemolysis, which occurred in the samples). 4. Discussion Sex steroid treatment and/or ovariectomy affected tissue concentrations of galanin in several brain regions. The effect of estradiol to increase the concentrations of galanin in the pituitary is previously well established [7,18] and was confirmed in our study. One likely source for the galanin found in the pituitary are the hypothalamic gonadotropin-releasing hormone neurons that project to the anterior pituitary. These colocalize galanin and are regulated by estrogen [2]. Gene expression of galanin mRNA within the anterior pituitary, but not the neurointermediate lobe, is also highly stimulated by estradiol treatment [18]. The elevated concentrations of pituitary galanin mRNA in estrogen-treated animals were associated with high tissue concentrations of galanin. We also showed that estradiol in combination with pro-
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Fig. 1. Tissue concentrations of galanin in eight different brain regions showing box plots of concentrations in each treatment group. The groups are sham-operated rats (Sham), ovariectomized and placebo treated (Ovx), ovariectomized and estradiol treated (Estr), ovariectomized and estradiol ⫹ progesterone treated (Estr⫹Prog), and ovariectomized and estradiol ⫹ norethisterone treated (Estr⫹NET). The box includes the 25th to the 75th percentiles with the median value indicated as a line within the box. The ‘whiskers’ above and below the box indicate the 10th and 90th percentiles. The rings show the highest and lowest values. Significant differences between the study groups are stated in the main text.
gesterone, as well as with norethisterone, yielded higher pituitary concentration of galanin compared with estradiol only. The additive effect of progesterone was previously shown for galanin mRNA [31] but has not, to our knowledge, been shown for the peptide. The higher concentrations of galanin in the median eminence in hormone-treated animals are probably caused by a
stimulation of galanin synthesis in dopaminergic tuberoinfundibular neurons originating in the arcuate nucleus. Galanin is found in large quantities in the cell bodies of these neurons [24] and also in nerve endings in the median eminence, where it inhibits the release of dopamine [26]. Estrogen has been shown to decrease tyrosine hydroxylase activity and dopamine concentrations in this system [17],
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and stimulation of galanin synthesis may be an additional mechanism for estrogenergic modulation of dopamine activity. The present study corroborates our hypothesis that sex hormones influence the concentrations of galanin, not only in brain regions associated with reproductive functions, but also in regions that do not directly participate in the hypothalamic–pituitary– gonadal system. The higher galanin concentration in hippocampus in animals treated with sex hormones may be the result of a stimulation of synthesis in one or several systems known to localize galanin. A recent study of the distribution of immunohistochemically detectable galanin within the hippocampal formation has shown that the majority of the galanin fibers were noradrenergic, originating in the locus ceruleus [41]. The locus ceruleus has a robust galanin synthesis, and its gene expression is positively regulated by estrogen [38]. It is suggested that the high concentrations of hippocampal galanin in the sex hormone-treated rats is at least partially the result of a stimulation of galanin synthesis in noradrenergic neurons in the locus ceruleus with projections to hippocampus. The significance of this finding is unclear. It is possible, however, that galanin is an inhibitory modulator of noradrenergic transmission and that estrogen, through galanin mediation, reduces noradrenergic tone in hippocampus. This hypothesis is supported by the findings that galanin reduces stimulation-evoked noradrenaline release from slices of the dorsal spinal cord [29] and from slices of medulla oblongata [39], although the effects in hippocampus have not been studied. Estrogen treatment does not have any effect on tyrosine hydroxylase gene expression in the locus ceruleus [38], and neither did estrogen, in nonhuman primates, regulate noradrenaline transporter mRNA in the locus ceruleus [33]. These negative findings on a direct effect of estrogen, together with the up-regulation of galanin expression with estrogen treatment, seem to accentuate the role for galanin as a target for estrogenic influence on noradrenergic hippocampal neurons. In the ventral hippocampus, there are also nonnoradrenergic galanin-positive fibers, scarcer, however, than the noradrenergic [41]. Nevertheless, it is possible that an upregulation by sex hormones in these neurons also contributes to the higher concentration of galanin that was seen in the hormone-treated groups. There are several neuron groups that are known to have the ability to synthesize galanin and that could give rise to galanin fibers in the hippocampus. These are, in addition to the noradrenergic fibers from the locus ceruleus, the cholinergic forebrain neurons [25], the dorsal raphe serotonin neurons [24], and the magnocellular neurons, which express ␥-aminobutyric acid and/or histamine [20]. The cholinergic forebrain neurons are relevant to memory and are also affected in dementias. They are sensitive to estrogen in several ways and estrogen generally enhances the functional status of these neurons as well as reduces cognitive deficits associated with cholinergic impairment [8]. In particular, estrogen increases
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potassium-stimulated acetylcholine release in the hippocampus [9]. In contrast, centrally administered galanin inhibits acetylcholine release in the rat ventral hippocampus, and produces deficits in learning and memory tasks [3,30]. Whether galanin expression in cholinergic septohippocampal pathways is affected by hormone status is unknown, but because the effects of estrogen and galanin are divergent, it seems less likely that galanin is mediating the estrogen effects. In the frontal cortex, we had previously found higher galanin concentrations in adult female rats compared with prepubertal [32]. In the present study, the estradiol-treated rats had galanin concentrations that were higher than both placebo-treated and sham-operated controls. With the addition of progesterone, a significant difference remained, although it was smaller. Thus, it is probable that estrogen causes the higher galanin concentration found in adult female frontal cortex in comparison with prepubertal. An interesting observation is that animals treated with norethisterone, which has some androgenic properties, had significantly lower galanin concentrations compared with those treated with estradiol only. Thus, norethisterone apparently counteracted the estradiol effect in this brain region. This finding can be related to the unfavorable effects on mood, mental status, and well-being that was found in women who also received progesterone during estrogen replacement therapy [14]. In this study, the women received lynestrenol, which is rapidly metabolized to norethisterone. Studies with subcortical lesions suggest that a substantial portion of cortical galanin may also derive from noradrenergic neurons [6]. The same hypothesis that was made for hippocampus, that galanin functions as an inhibitory modulator of noradrenergic transmission and that estrogen, through galanin mediation, reduces noradrenergic tone, is made for cortex. If galanin affects noradrenaline transmission in the hippocampus and frontal cortex, it suggests that estrogen has the ability to influence noradrenergic regulation of mood states through galanin mediation. Some mood changes in women are considered to be associated with changes in plasma concentrations of ovarian hormones, e.g. premenstrual tension and postpartum depression. These mood changes often appear several days after the changes in hormone plasma concentrations. This observation implies that a peptide is involved, because a change in peptide concentrations in nerve terminals depends on upor down-regulation of synthesis in the soma, storage, and axonal transport. These events require more time than events leading to a change in monoamine concentrations and would therefore accord with a delayed response to hormone plasma variations. In conclusion, sex hormones modulate galanin concentrations, not only in reproductive, but also in nonreproductive, regions. This observation may have implications for the understanding of how sex hormones effect cognition and emotion.
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Effects of estradiol, progesterone, and norethisterone on regional concentrations of galanin in the rat brain1 Olof Rugarna,*, Annette Theodorssonb, Mats Hammara, Elvar Theodorssonc a
Division of Obstetrics and Gynecology, Faculty of Health Sciences, Linko¨ping University, S-581 85 Linko¨ping, Sweden b Division of Neurosurgery, Faculty of Health Sciences, Linko¨ping University, S-581 85 Linko¨ping, Sweden c Division of Clinical Chemistry, Faculty of Health Sciences, Linko¨ping University, S-581 85 Linko¨ping, Sweden Received 15 October 1998; accepted 26 January 1999
Abstract Concentrations of immunoreactive galanin were compared in eight gross brain regions of ovariectomized female rats treated with either estradiol, estradiol ⫹ progesterone, estradiol ⫹ norethisterone, or placebo. Higher concentrations with estradiol treatment compared with placebo were found in the pituitary (357%), frontal cortex (162%), occipital cortex (174%), hippocampus (170%), and median eminence (202%). A more profound difference with addition of progesterone or norethisterone was seen in the pituitary (529% and 467%, respectively). Sex steroids, particularly estradiol, modulate galanin concentrations not only in reproductive, but also in nonreproductive, brain regions. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Galanin; Estradiol; Progesterone; Norethisterone; Hippocampus; Pituitary; Frontal cortex
1. Introduction The neuropeptide galanin modulates several different neural and neuroendocrine events such as neurotransmitter release [5], food intake [21], learning and memory processes [10,27], and pituitary hormone secretion [15]. Galanin is present in many systems containing other neuroactive compounds, including dopamine, noradrenaline, serotonin, ␥-aminobutyric acid, and other neuropeptides [24]. An example of the latter are hypothalamic-pituitary gonadotropinreleasing hormone neurons, where galanin mRNA is present and can be induced by estrogen [2,31]. The process is facilitated by progesterone [31]. In this case, galanin seems to function as a mediator of sex steroid effects. Consideration of the widespread distribution of both galanin-like immunoreactivity and galanin-receptors in the central nervous system [12,35,36] suggests that galanin may mediate sex steroid action in other parts of the brain. This hypothesis is further warranted by the finding of a functional estrogen * Corresponding author. Tel.: ⫹46-13-22-31-22; fax: ⫹46-13-14-8156. E-mail address: [email protected] (O. Rugarn) This study was supported by the Swedish Medical Research Council (12X-07464). 1
response element sequence within the human galanin gene, which could potentially function as a regulatory element for estrogen action in galanin-expressing neurons [16]. Thus, nonreproductive functions could also be influenced by sex steroids like estrogen through galanin mediation. Brain areas associated with cognition and emotion are of special interest, because the influence of sex steroids on these functions is now well supported [4,13,19,23,34]. A positive regulation of galanin gene expression by estrogen has in fact been demonstrated outside of the pituitary and hypothalamus, for instance, in the locus ceruleus [38]. A regulation of gene expression across puberty was found in the bed nucleus of stria terminalis and medial amygdala [28]. In a previous experiment, we found higher galanin concentrations in adult than prepubertal female rats in the frontal cortex and hippocampus [32]. Progesterone receptors have been demonstrated in hypothalamic galanin-immunoreactive neurons [40], but little is known about progesterone and galanin interactions in other brain areas. The aim of this study was to assess if sex steroids have any effect on galanin immunoreactivity in the brain regionwise. We reasoned that if sex steroids indeed have a potential to regulate galanin in diverse parts of the brain, it could have implications for understanding how the brain is affected by puberty, menopause, and by exogenous steroids
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Table 1 Plasma levels of estradiol (pM) and progesterone (nM) Group
Estradiol median
IQR*
Progesterone median
IQR*
Sham Ovx Estr Estr ⫹ Prog Estr ⫹ NET
62 56 177 188 196
59–64 49–68 173–199 142–235 142–217
19 2.9 15 26 16
12–32 2.6–4.2 9.0–32 15–36 15–16
*Interquartile range. The groups are sham-operated rats (Sham), ovariectomized and placebotreated (Ovx), ovariectomized and estradiol-treated (Estr), ovariectomized and estradiol ⫹ progesterone-treated (Estr ⫹ Prog), and ovariectomized and estradiol ⫹ norethisterone treated (Estr ⫹ NET).
used in oral contraceptives and postmenopausal hormone replacement therapy. Many women report changes in general well-being, emotional state, and eating behavior while using contraceptive pills or menopausal hormone replacement [4,22,37]. The synthetic progestin norethisterone is a commonly used component in both contraceptive pills and menopausal replacement therapy. We have therefore included a study of the effects of this steroid as well, and compared it with those of progesterone. We treated ovariectomized rats with slow-release pellets of estradiol, estradiol and progesterone, or estradiol and norethisterone (Table 1). Extended treatment periods of 5 to 8 weeks were chosen because peptide concentrations depend on synthesis, transport, and storage, which take a much longer time than the corresponding events for monoamines. Control groups were ovariectomized placebo-treated rats and sham-operated placebo-treated rats. After treatment, the concentrations of immunoreactive galanin were measured by using radioimmunoassay in frontal cortex, occipital cortex, hippocampus, striatum, cerebellum, hypothalamus, median eminence, and the pituitary.
tive hormone and placebo was administered with slowrelease pellets (Innovation Research of America, Sarasota, FL, USA), which were placed subcutaneously in the neck fold under anesthesia. The different groups were treated as follows: Group 1 (n ⫽ 8): the sham-operated group. The ovaries were simply identified and the animals were treated with placebo pellets for 6 weeks. Group 2 (n ⫽ 7): the placebo group. The animals were ovariectomized and treated with placebo pellets for 6 weeks. Group 3 (n ⫽ 9): the estradiol group. The animals were ovariectomized and treated with slow-release pellets containing 0.1 mg of 17-estradiol, yielding a constant rate of 1.2 g/day (data supplied by manufacturer) for 8 weeks. During weeks 3 to 5, a pellet containing 50 mg of progesterone, yielding 600 g/day (data supplied by manufacturer), was added. Then, during weeks 6 to 8, 17-estradiol 1.2 g/day was again the sole hormone administration. Group 4 (n ⫽ 8): the estradiol ⫹ progesterone group. The animals were ovariectomized and treated with slowrelease pellets containing 0.1 mg of 17-estradiol, yielding a constant rate of 1.2 g/day for 5 weeks. During the last 2 weeks, a pellet containing 50 mg of progesterone, yielding 600 g/day, was added. Group 5 (n ⫽ 8): the estradiol ⫹ norethisterone group. The animals were ovariectomized and treated with slowrelease pellets containing 0.1 mg of 17-estradiol, yielding a constant rate of 1.2 g/day for 5 weeks. During the last 2 weeks, a pellet containing 50 mg of norethisterone, yielding 600 g/day, was added. The rats were killed by guillotine and the brains dissected [11]. The individual brain regions were weighed immediately and frozen on dry ice. The study and its experimental protocol was approved by the local animal research ethics committee and found to be in accordance with the guidelines issued by the Central Committee For Animal Research.
2. Materials and methods
2.2. Extraction of tissue samples
2.1. Animals and test procedure
The frozen tissue samples were cut into small pieces, and 10 ml of 1 M acetic acid was added for each gram of tissue. After boiling for 10 min, the samples were homogenized by using a steel rod and a Vortex mixer, then centrifuged, whereafter the supernatants were removed. A second extraction with an equal amount of distilled water was then performed to maximize the solubility of galanin and possible neutral homologs. The combined supernatants were lyophilized and stored at ⫺20°C before analysis. The samples were reconstituted in 1 ml of phosphate buffer (0.05 M, pH 7.4) and then divided before further analysis.
Experiments were performed on virgin female rats (ALAB, Sollentuna, Sweden) weighing 250 to 270 g at the start of the experiment. Forty rats divided into five study groups were used. The rats were housed five to a cage at constant room temperature (21 ⫾ 1°C) for 3 days, with free access to water and standard rat food, and with 12-h light/ dark cycles before the experiments. All surgery was performed with the animals under general anesthesia. The animals were premedicated with phentanyl 10 mg/ml ⫹ fluanison 0.2 mg/ml (Hypnorm) 0.2 ml/kg intramuscularly and atropine 0.05 mg/kg. Anesthesia was achieved with xylazine 12 mg/kg intraperitoneally and ketamine 80 mg/kg intraperitoneally. After surgery, buprenorphine 0.05 mg/kg was given subcutaneously for postoperative analgesia. Ac-
2.3. Radioimmunoassays Galanin was analyzed by using antiserum RatGala4 raised against conjugated synthetic rat galanin. The anti-
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serum does not cross-react with neurokinin A, neuropeptide K, substance P, neurokinin B, neuropeptide Y, gastrin, pancreatic polypeptide, glucagon, or neurotensin. High-performance liquid chromatography-purified 125I-rat galanin was used as radioligand and rat galanin as standard. The volume of the sample used for the assay was 100 l. The detection limit of the assay was 8 pM of galanin in the sample. Intraand interassay coefficients of variation were 6% and 10%, respectively, at 50 pM. Reverse-phase high-performance liquid chromatography (20 –50% acetonitrile with 0.1% trifluoroacetic acid) of the immunoreactive material from rat brains revealed a single immunoreactive component for the assay that coeluted with the calibrator. 2.4. Hormone analysis Plasma levels of progesterone and estradiol were measured by time-resolved fluoroimmunoassay (AutoDELFIA, Wallac, Gaithersburg, MD, USA). 2.5. Statistical analysis The median and interquartile ranges were used as measures of central tendency and variation, respectively. Multivariate analysis of variance with Tukey multiple comparisons and correlation test was performed with SYSTAT (Version 5, SYSTAT, Inc, Evanston, IL, USA). If more than half of the measurements for a particular analyte in a particular region were below the limit of detection, the data for that region are neither shown nor used in drawing conclusions. 3. Results 3.1. Galanin immunoreactivity Multivariate analyses of variance (factors ⫽ group, region) revealed overall differences with regard to group [F(4) ⫽ 10.54, P ⬍ 0.001], region [F(7) ⫽ 289.7, P ⬍ 0.001], and group–region [F(28) ⫽ 7.65, P ⬍ 0.001]. In particular (Fig. 1), ovariectomized/estradiol-treated rats had higher galanin concentrations compared with the ovariectomized/placebo-treated in frontal cortex (162% of placebo treated; P ⬍ 0.01), occipital cortex (174%; P ⬍ 0.001), hippocampus (170%; P ⬍ 0.01), pituitary (357%; P ⬍ 0.05), and the median eminence (202%; P ⬍ 0.001). The groups that were treated with estradiol ⫹ progesterone or estradiol ⫹ norethisterone showed results similar to those of the estradiol-only-treated rats in most regions. Accordingly, in the estradiol ⫹ progesterone group, galanin concentrations were higher than the placebo-treated in frontal cortex (138%; P ⬍ 0.05), occipital cortex (157%; P ⬍ 0.001), hippocampus (167%; P ⬍ 0.05), the pituitary (529%; P ⬍ 0.001), and the median eminence (186%; P ⬍ 0.001). Estradiol ⫹ norethisterone-treated rats had higher galanin concentrations compared with placebo-treated in occipital cortex (163%; P ⬍ 0.001), hippocampus (156%; P ⬍
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0.01), the median eminence (185%; P ⬍ 0.001), and in the pituitary (467%; P ⬍ 0.001), whereas no significant difference was found in the frontal cortex. On the other hand, galanin concentrations in the frontal cortex were significantly lower in rats treated with estradiol ⫹ norethisterone compared with rats treated with only estradiol (75% of estrogen treated; P ⬍ 0.05). The sham-operated, gonadally intact, animals had higher concentrations compared with ovariectomized/placebo-treated in the pituitary (P ⬍ 0.05) but did not differ significantly in any other region. In comparison with the estradiol-only–treated group, significant differences were found in frontal cortex (P ⬍ 0.001), occipital cortex (P ⬍ 0.001), hippocampus (P ⬍ 0.05), and the median eminence (P ⬍ 0.05). Compared with the estradiol ⫹ progesterone group, there were significant differences only in frontal cortex (P ⬍ 0.01) and occipital cortex (P ⬍ 0.01). The estradiol ⫹ norethisterone group differed significantly in occipital cortex (P ⬍ 0.01), hippocampus (P ⬍ 0.05), and the median eminence (P ⬍ 0.01). No significant differences between the groups were found in hypothalamus or cerebellum. 3.2. Plasma hormone concentrations The groups that received estrogen pellets had similar plasma levels as near-term pregnant rats [1] and were in accordance with those expected according to the manufacturer of the slow-release pellets. The progesterone levels are lower than expected, which according to the manufacturer should be ⬇60 nM. The animals that were treated with estrogen only for the last 3 weeks (Group 3) and the animals that received estrogen ⫹ norethisterone have noticeably higher progesterone levels compared with the placebo treated. We have no satisfactory explanation for these discrepancies. It is possible that our method, for some reason, is not suitable for rat plasma or that the results are caused by analytical interference (e.g. hemolysis, which occurred in the samples). 4. Discussion Sex steroid treatment and/or ovariectomy affected tissue concentrations of galanin in several brain regions. The effect of estradiol to increase the concentrations of galanin in the pituitary is previously well established [7,18] and was confirmed in our study. One likely source for the galanin found in the pituitary are the hypothalamic gonadotropin-releasing hormone neurons that project to the anterior pituitary. These colocalize galanin and are regulated by estrogen [2]. Gene expression of galanin mRNA within the anterior pituitary, but not the neurointermediate lobe, is also highly stimulated by estradiol treatment [18]. The elevated concentrations of pituitary galanin mRNA in estrogen-treated animals were associated with high tissue concentrations of galanin. We also showed that estradiol in combination with pro-
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Fig. 1. Tissue concentrations of galanin in eight different brain regions showing box plots of concentrations in each treatment group. The groups are sham-operated rats (Sham), ovariectomized and placebo treated (Ovx), ovariectomized and estradiol treated (Estr), ovariectomized and estradiol ⫹ progesterone treated (Estr⫹Prog), and ovariectomized and estradiol ⫹ norethisterone treated (Estr⫹NET). The box includes the 25th to the 75th percentiles with the median value indicated as a line within the box. The ‘whiskers’ above and below the box indicate the 10th and 90th percentiles. The rings show the highest and lowest values. Significant differences between the study groups are stated in the main text.
gesterone, as well as with norethisterone, yielded higher pituitary concentration of galanin compared with estradiol only. The additive effect of progesterone was previously shown for galanin mRNA [31] but has not, to our knowledge, been shown for the peptide. The higher concentrations of galanin in the median eminence in hormone-treated animals are probably caused by a
stimulation of galanin synthesis in dopaminergic tuberoinfundibular neurons originating in the arcuate nucleus. Galanin is found in large quantities in the cell bodies of these neurons [24] and also in nerve endings in the median eminence, where it inhibits the release of dopamine [26]. Estrogen has been shown to decrease tyrosine hydroxylase activity and dopamine concentrations in this system [17],
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and stimulation of galanin synthesis may be an additional mechanism for estrogenergic modulation of dopamine activity. The present study corroborates our hypothesis that sex hormones influence the concentrations of galanin, not only in brain regions associated with reproductive functions, but also in regions that do not directly participate in the hypothalamic–pituitary– gonadal system. The higher galanin concentration in hippocampus in animals treated with sex hormones may be the result of a stimulation of synthesis in one or several systems known to localize galanin. A recent study of the distribution of immunohistochemically detectable galanin within the hippocampal formation has shown that the majority of the galanin fibers were noradrenergic, originating in the locus ceruleus [41]. The locus ceruleus has a robust galanin synthesis, and its gene expression is positively regulated by estrogen [38]. It is suggested that the high concentrations of hippocampal galanin in the sex hormone-treated rats is at least partially the result of a stimulation of galanin synthesis in noradrenergic neurons in the locus ceruleus with projections to hippocampus. The significance of this finding is unclear. It is possible, however, that galanin is an inhibitory modulator of noradrenergic transmission and that estrogen, through galanin mediation, reduces noradrenergic tone in hippocampus. This hypothesis is supported by the findings that galanin reduces stimulation-evoked noradrenaline release from slices of the dorsal spinal cord [29] and from slices of medulla oblongata [39], although the effects in hippocampus have not been studied. Estrogen treatment does not have any effect on tyrosine hydroxylase gene expression in the locus ceruleus [38], and neither did estrogen, in nonhuman primates, regulate noradrenaline transporter mRNA in the locus ceruleus [33]. These negative findings on a direct effect of estrogen, together with the up-regulation of galanin expression with estrogen treatment, seem to accentuate the role for galanin as a target for estrogenic influence on noradrenergic hippocampal neurons. In the ventral hippocampus, there are also nonnoradrenergic galanin-positive fibers, scarcer, however, than the noradrenergic [41]. Nevertheless, it is possible that an upregulation by sex hormones in these neurons also contributes to the higher concentration of galanin that was seen in the hormone-treated groups. There are several neuron groups that are known to have the ability to synthesize galanin and that could give rise to galanin fibers in the hippocampus. These are, in addition to the noradrenergic fibers from the locus ceruleus, the cholinergic forebrain neurons [25], the dorsal raphe serotonin neurons [24], and the magnocellular neurons, which express ␥-aminobutyric acid and/or histamine [20]. The cholinergic forebrain neurons are relevant to memory and are also affected in dementias. They are sensitive to estrogen in several ways and estrogen generally enhances the functional status of these neurons as well as reduces cognitive deficits associated with cholinergic impairment [8]. In particular, estrogen increases
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potassium-stimulated acetylcholine release in the hippocampus [9]. In contrast, centrally administered galanin inhibits acetylcholine release in the rat ventral hippocampus, and produces deficits in learning and memory tasks [3,30]. Whether galanin expression in cholinergic septohippocampal pathways is affected by hormone status is unknown, but because the effects of estrogen and galanin are divergent, it seems less likely that galanin is mediating the estrogen effects. In the frontal cortex, we had previously found higher galanin concentrations in adult female rats compared with prepubertal [32]. In the present study, the estradiol-treated rats had galanin concentrations that were higher than both placebo-treated and sham-operated controls. With the addition of progesterone, a significant difference remained, although it was smaller. Thus, it is probable that estrogen causes the higher galanin concentration found in adult female frontal cortex in comparison with prepubertal. An interesting observation is that animals treated with norethisterone, which has some androgenic properties, had significantly lower galanin concentrations compared with those treated with estradiol only. Thus, norethisterone apparently counteracted the estradiol effect in this brain region. This finding can be related to the unfavorable effects on mood, mental status, and well-being that was found in women who also received progesterone during estrogen replacement therapy [14]. In this study, the women received lynestrenol, which is rapidly metabolized to norethisterone. Studies with subcortical lesions suggest that a substantial portion of cortical galanin may also derive from noradrenergic neurons [6]. The same hypothesis that was made for hippocampus, that galanin functions as an inhibitory modulator of noradrenergic transmission and that estrogen, through galanin mediation, reduces noradrenergic tone, is made for cortex. If galanin affects noradrenaline transmission in the hippocampus and frontal cortex, it suggests that estrogen has the ability to influence noradrenergic regulation of mood states through galanin mediation. Some mood changes in women are considered to be associated with changes in plasma concentrations of ovarian hormones, e.g. premenstrual tension and postpartum depression. These mood changes often appear several days after the changes in hormone plasma concentrations. This observation implies that a peptide is involved, because a change in peptide concentrations in nerve terminals depends on upor down-regulation of synthesis in the soma, storage, and axonal transport. These events require more time than events leading to a change in monoamine concentrations and would therefore accord with a delayed response to hormone plasma variations. In conclusion, sex hormones modulate galanin concentrations, not only in reproductive, but also in nonreproductive, regions. This observation may have implications for the understanding of how sex hormones effect cognition and emotion.
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