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Estrogen receptor-α immunoreactivity in parasympathetic preganglionic neurons innervating the bladder in the adult ovariectomized cat
Neuroscience Letters 298 (2001) 147±150
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Estrogen receptor-a immunoreactivity in parasympathetic preganglionic neurons innervating the bladder in the adult ovariectomized cat Veronique G.J.M. VanderHorst*, Ellie Meijer, Gert Holstege Department of Anatomy, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands Received 20 July 2000; received in revised form 15 November 2000; accepted 17 November 2000
Abstract Estrogen affects autonomic functions such as micturition. The sacral cord is important in the control of micturition and contains numerous estrogen receptor-a immnoreactive (ER-a IR) neurons. Therefore, the present double labeling study examines whether sacral parasympathetic preganglionic neurons innervating the bladder are immunoreactive for ER-a. In the sacral cord of seven female ovariectomized cats, the distribution of ER-a IR neurons was studied using the H222 and 1D5 antibodies. Choleratoxin subunit b (CTb) was injected into the bladder wall to visualize its preganglionic neurons. ER-a IR was present in the nuclei of cells in laminae I, II, V, VII, and X, and in nuclei and cytoplasm of neurons in the sacral parasympathetic nucleus. The vast majority of CTb labeled neurons contained ER-a IR nuclei, indicating that preganglionic neurons innervating the bladder express ER-a. The results suggest that estrogen modulates micturition in the cat via ER-a in bladder preganglionic neurons. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sacral cord; Micturition; Incontinence; Reproductive behavior; Immunohistochemistry; Estrogen receptor; Autonomic
Micturition is affected by estrogen [2,10,13]. Both local [7,11] and systemic estrogen treatment [6] in postmenopausal women decrease nocturia and frequency. These reports leave the possibility open that part of the effect of estrogen on micturition is mediated by the central nervous system (CNS). In the CNS, estrogen affects neuronal function through several mechanisms [19]. One of them involves genomic activation via the classical estrogen receptor (ER-a). ER-a immunoreactive (IR) neurons are present in neurons in distinct areas of the forebrain, brainstem and spinal cord (e.g. Refs. [1,12,15±18]). The sacral cord is one of the areas that is involved in the micturition re¯ex [4,5] and contains ER-a IR neurons [15,18]. The present double labeling study examines whether sacral parasympathetic preganglionic neurons innervating the bladder are ER-a IR. Seven adult female cats were used (cases 2364, 2376, 2400, 2408, 2409, 2498 and 2504). They had been ovariectomized 2±4 weeks prior to the perfusion to enhance the intensity of ER-IR [17,20]. In two animals (2400 and * Corresponding author. Tel.: 131-50-363-2460; fax: 131-50363-2461. E-mail address: [email protected] (V.G.J.M. VanderHorst).
2409), during the same laparotomy, injections with 80 ml 0.5% cholera toxin subunit b (CTb) were placed into the bladder wall using a Hamilton syringe. The surgical procedures, pre- and postoperative care, and handling and housing of the animals occurred according to the protocols approved by the Faculty of Medicine of the University of Groningen. After 2±4 weeks, the animals were transcardially perfused with 2 l of heparinized phosphate buffered saline, followed by 2 l of ®xative in 0.1 M phosphate buffer; pH 7.2±7.4, containing 4% paraformaldehyde and 0.5% glutaraldehyde, or 4% paraformaldehyde (cases 2498 and 2504). Brains and spinal cords were removed, and post®xed for 2±10 h. The L7-S3 segments were cut using a vibratome into ®ve series of 60 mm sections. The sections were subsequently pretreated in 0.01 M NaIO4, 1% NaBH4, 0.5±1% Triton X-100, and 1% H2O2 (in Tris-buffered phosphate, TBS) and incubated with the estrogen receptor-a antibody H222 (1 mg/ml; 1:1000; gift from F. van Leeuwen, Netherlands Institute for Brain Research, Amsterdam; cases 2364, 2376, 2400, 2408, 2409) or 1D5 (1:800; DAKO, Carpinteria, CA; case 2498) for 4 nights at 48C. After a standard ABC procedure [1,15,16,17], the sections were stained with 0.04% diaminobenzidine (DAB), 0.2% nickel ammonium sulphate, and 0.012% H2O2, which resulted into a blue-
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 71 3- 4
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V.G.J.M. VanderHorst et al. / Neuroscience Letters 298 (2001) 147±150
black precipitate. In order to control for method speci®city, incubations were performed as described above, but without the ®rst antibody. The control sections did not contain any labeled cells. In cases 2400 and 2409, tissue was further processed to visualize CTb as described previously [15]. This method allows visualization of ER-a with antibodies that are not recognized by any of the antibodies that are required for the elucidation of CTb. After the ER-immunohistochemistry, sections of the L7-S3 segments were treated with methanol-TBS-H2O2 to inhibit peroxidase activity (see Ref. [15]), incubated in a blocking solution (5% normal rabbit serum (NRS; Vector, Burlingame, CA) in TBS containing 0.5% Triton X-100; for 30 min), and transferred to the ®rst antibody, goat anti-CTb (List Biological Labs, CA; 1:4000 in TBS containing 2% NRS, 2% Triton X-100; for 2 days at 48C). Rabbit anti-goat immunoglobulin (DAKO; 1:100 in TBS containing 2% NRS, and 1% Triton X-100; for 1 h at room temperature) was applied, followed by goat peroxidase anti-peroxidase (PAP goat, DAKO, Carpinteria, CA;1:100 in TBS containing 1% Triton X-100). The tissue was then soaked in 0.025% DAB in TBS, and 0.004% H2O2 was added, resulting in a brown precipitate in the cytoplasm. TBS was used to wash sections between all steps of the immunohistochemical procedures. In the sacral cord of all seven cases, a similar distribution
Fig. 1. Schematic overview of ER-a IR neurons in the S1±S3 segments (case 2364). Each dot indicates an ER-a IR nucleus. The open circles indicate the distinct group of ER-a IR neurons in the lateral intermediolateral cell column. A few ER-a IR neurons are located just outside the gray matter. dh, dorsal horn; SPN, sacral parasympathetic nucleus.
pattern of ER-a IR neurons was found (Fig. 1). The majority of ER-a IR neurons were present in neuronal nuclei in the super®cial layers of the dorsal horn and in the lateral and medial intermediate zone. Nuclei in the lateral part of lamina I, in the lateral and medial intermediate zone (laminae V and VII), and in lamina X were densely stained, whereas ER-a-IR nuclei in laminae II, and in the dorsomedial part of the ventral horn (medial lamina VII and lamina VIII) were lightly stained. In the lateral intermediate zone of the sacral segments, another distinct group of ER-a IR neurons was present. The immunoreactive nuclei in this group were larger, and, in addition to nuclear labeling, considerable cytoplasmic labeling was present (Fig. 2A). The ER-a labeled dendrites extended dorsomedially into the intermediomedial cell column and to a lesser extent dorsolaterally into the lateral dorsal horn. In cases 2400 and 2409, respectively, 481 (5.6/ section) and 455 (6.0/section) ER-a IR neurons with such morphological characteristics were counted in the sacral segments (bilaterally). In both cases the majority of these neurons (254 in case 2400; 316 in cases 2409) were present in the S2 segment. Injections with CTb into the bladder wall resulted in retrogradely labeled neurons in the sacral parasympathetic nucleus. In cases 2400 and 2409, respectively, 74 and 76 CTb-labeled neurons were counted. The majority of them contained ER-a IR (74% in case 2400; 68% in case 2409), and only in a minor portion of the CTb labeled neurons did not contain an ER-a IR nucleus (16% in case 2400; 4% in case 2409). In the remaining CTb labeled pro®les, it could not be determined with certainty whether they contained ER-a IR, for example because a nucleus was not present in the section or because dense retrograde labeling obscured the nucleus. The distribution of ER-a IR neurons in the sacral dorsal horn and lamina X is in line with previous immunohistochemical studies in rat [18] and cat [15]. The presence of ER-a IR in sacral parasympathetic preganglionic neurons is surprising, because in the rat [18] the sacral parasympathetic nucleus does not contain ER-a IR neurons. Moreover, ER-a IR is mostly present in neurons involved in the processing of sensory information (i.e. neurons in the dorsal horn) and in the regulation of endocrine and reproductive functions (i.e. neurons in the hypothalamus and midbrain). In the adult CNS, ER-a is neither expressed in somatic motoneurons, nor in parasympathetic and sympathetic preganglionic neurons in the brainstem and thoracic and upper lumbar cord (in the cat: VanderHorst et al., personal communication). A special characteristic of the ER-a IR in the sacral parasympathetic neurons is that it is not only present in nuclei, but also in the cytoplasm of their perikarya and dendrites. Cytoplasmic labeling has been reported to be occasionally present around densely stained nuclei, using different types of antibodies (Ref. [3] see also Ref. [1]). However, the intensity of cytoplasmic labeling in sacral parasympathetic preganglionic neurons is high, and the
V.G.J.M. VanderHorst et al. / Neuroscience Letters 298 (2001) 147±150
149
Fig. 2. Photomicrographs of transverse sections of the S2 segment. A shows an overview of ER-a IR nuclei in the dorsal horn and the distinct group of ER-a IR neurons in the lateral intermediolateral cell column, characterized by cytoplasmic labeling and larger nuclei (blue-black reaction product). B shows CTb labeled bladder preganglionic neurons (brown reaction product) containing ER-a IR nuclei (blue-black reaction product) marked with arrows. Unidenti®ed ER-a IR neurons are marked by arrowheads. Some of the double labeled cells show a brownish hue over their nuclei, probably due to brown (CTb labeled) cytoplasm overshadowing the blue-black nuclei in the thick (60 mm) sections. Dh, dorsal horn; LF, lateral funiculus; SPN, sacral parasympathetic nucleus. Scale bars A,B, 100 mm.
labeling is present in more than one occasional neuron. Cytoplasmic staining in neurites may indicate that the overall immunoreactivity for ER-a was so high that even neurites became visible. Because all animals in this study were ovariectomized, it cannot be excluded that ER-a IR might have appeared more exclusively in nuclei in the presence of estrogen, i.e. after translocation of the ER-a to the nucleus. The distribution of bladder preganglionic parasympathetic neurons in the ventral portion of the sacral parasympathetic nucleus is in line with previous studies in the cat [5,8,14]. The double labeling experiments showed that the majority of labeled bladder preganglionic motoneurons were ER-a IR. However, only part of the population of ER-a IR parasympathetic preganglionic neurons was labeled with CTb. Possible explanations are that (1) the CTb injection did not label the entire population of bladder preganglionic motoneurons; (2) preganglionic motoneurons innervating other organs than the bladder also contain ER-a IR. This latter possibility is not very likely. Preganglionic neurons innervating the colon/rectum are located more dorsally, forming the `dorsal band' [5,8]. Although the dorsal band region also contains ER-a IR neurons, their morphology is not the same as that of the ER-a IR bladder preganglionic neurons, but strongly resembles the morphology of ER-a IR neurons in the super®cial layers of the sacral dorsal horn. Outside the CNS, ER-a mRNA has been found in a subpopulation of neurons in pelvic autonomic ganglia [9]. It is not known whether it is present in other autonomic ganglia as well. Altogether, estrogen appears to directly modulate autonomic control especially of the urogenital organs.
The authors would like to thank Peter van der Sijde for digitizing the photographs. [1] Axelson, J.F. and van Leeuwen, F.W., Differential localization of estrogen receptors in various vasopressin synthesizing nuclei of the rat brain, J. Neuroendocrinol., 2 (1990) 209±216. [2] Beach, F.A., Effects of gonadal hormones on urinary behavior in dogs, Physiol. Behav., 12 (1974) 1005±10013. [3] Blaustein, J.D., Cytoplasmic estrogen receptors in rat brain: immunocytochemical evidence using three antibodies with distinct epitopes, Endocrinology, 131 (1992) 1336±1342. [4] Blok, B.F.M. and Holstege, G., The neuronal control of micturition and its relation to the emotional motor system, Prog. Brain. Res., 107 (1996) 113±126. [5] DeGroat, W.C., Vizzard, M.A., Araki, I. and Roppolo, J., Spinal interneurons and preganglionic neurons in sacral autonomic re¯ex pathways, Prog. Brain. Res., 107 (1996) 97±112. [6] Kok, A.L., Burger, C.W., van de Weijer, P.H., Voetberg, G.A., Peters-Muller, E.R. and Kenemans, P., Micturition complaints in postmenopausal women treated with continuously combined hormone replacement therapy: a prospective study, Maturitas, 31 (1999) 143±149. [7] Kurz, C., Nagele, F., Sevelda, P. and Enzelsberger, H., Intravesical administration of estriol in sensory urge incontinence±a prospective study, Geburtshilfe Frauenheilkd, 53 (1993) 535±538. [8] Nadelhaft, I., De Groat, W.C. and Morgan, C., Location and morphology of parasympathetic preganglionic neurons in the sacral spinal cord of the cat revealed by retrograde axonal transport of horseradish peroxidase, J. Comp. Neurol., 193 (1980) 265±281. [9] Papka, R.E., Srinivasan, B., Miller, K.E. and Hayashi, S., Localization of estrogen receptor protein and estrogen receptor messenger RNA in peripheral autonomic and sensory neurons, Neuroscience, 79 (1997) 1153±1163. [10] Samsioe, G., Urogenital aging±a hidden problem, Am. J. Obstet. Gynecol., 178 (1998) S245±S239.
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[11] Schar, G., Kochli, O.R., Fritz, M. and Haller, U., Effect of vaginal estrogen therapy on urinary incontinence in postmenopause, Zentralbl. Gynakol., 117 (1995) 77±80. [12] Scott, C.J., Rawson, J.A., Pereira, A.M. and Clarke, I.J., The distribution of estrogen receptors in the brainstem of female sheep, Neurosci. Lett., 241 (1998) 29±32. [13] Tansy, M.F. and Kaufman, R., In¯uence of sex hormones on frequency of micturition, Nature, 211 (1966) 1184±1185. [14] VanderHorst, V.G. and Holstege, G., Organization of lumbosacral motoneuronal cell groups innervating hindlimb, pelvic ¯oor, and axial muscles in the cat, J. Comp. Neurol., 382 (1997) 46±76. [15] VanderHorst, V.G., Meijer, E., Schasfoort, F.C., Van Leeuwen, F.W. and Holstege, G., Estrogen receptor-immunoreactive neurons in the lumbosacral cord projecting to the periaqueductal gray in the ovariectomized female cat, Neurosci. Lett., 236 (1997) 25±28.
[16] VanderHorst, V.G., Schasfoort, F.C., Meijer, E., van Leeuwen, F.W. and Holstege, G., Estrogen receptor-a-immunoreactive neurons in the periaqueductal gray of the adult ovariectomized female cat, Neurosci. Lett., 240 (1998) 13±16. [17] Van Leeuwen, F.W., Chouham, S., Axelson, J.F., Swaab, D.F. and Van Eerdenburg, F.J., Sex differences in the distribution of estrogen receptors in the septal area and hypothalamus of the domestic pig (Sus scrofa), Neuroscience, 64 (1995) 261±275. [18] Williams, S.J. and Papka, R.E., Estrogen receptor-immunoreactive neurons are present in the female rat lumbosacral spinal cord, J. Neurosci. Res., 46 (1996) 492±501. [19] Woolley, C.S., Effects of estrogen in the CNS, Curr. Opin. Neurobiol., 9 (1999) 349±354. [20] Yuri, K. and Kawata, M., The effect of estrogen on the estrogen receptor-immunoreactive cells in the rat medial preoptic nucleus, Brain Res., 548 (1991) 50±54.
www.elsevier.com/locate/neulet
Estrogen receptor-a immunoreactivity in parasympathetic preganglionic neurons innervating the bladder in the adult ovariectomized cat Veronique G.J.M. VanderHorst*, Ellie Meijer, Gert Holstege Department of Anatomy, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands Received 20 July 2000; received in revised form 15 November 2000; accepted 17 November 2000
Abstract Estrogen affects autonomic functions such as micturition. The sacral cord is important in the control of micturition and contains numerous estrogen receptor-a immnoreactive (ER-a IR) neurons. Therefore, the present double labeling study examines whether sacral parasympathetic preganglionic neurons innervating the bladder are immunoreactive for ER-a. In the sacral cord of seven female ovariectomized cats, the distribution of ER-a IR neurons was studied using the H222 and 1D5 antibodies. Choleratoxin subunit b (CTb) was injected into the bladder wall to visualize its preganglionic neurons. ER-a IR was present in the nuclei of cells in laminae I, II, V, VII, and X, and in nuclei and cytoplasm of neurons in the sacral parasympathetic nucleus. The vast majority of CTb labeled neurons contained ER-a IR nuclei, indicating that preganglionic neurons innervating the bladder express ER-a. The results suggest that estrogen modulates micturition in the cat via ER-a in bladder preganglionic neurons. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sacral cord; Micturition; Incontinence; Reproductive behavior; Immunohistochemistry; Estrogen receptor; Autonomic
Micturition is affected by estrogen [2,10,13]. Both local [7,11] and systemic estrogen treatment [6] in postmenopausal women decrease nocturia and frequency. These reports leave the possibility open that part of the effect of estrogen on micturition is mediated by the central nervous system (CNS). In the CNS, estrogen affects neuronal function through several mechanisms [19]. One of them involves genomic activation via the classical estrogen receptor (ER-a). ER-a immunoreactive (IR) neurons are present in neurons in distinct areas of the forebrain, brainstem and spinal cord (e.g. Refs. [1,12,15±18]). The sacral cord is one of the areas that is involved in the micturition re¯ex [4,5] and contains ER-a IR neurons [15,18]. The present double labeling study examines whether sacral parasympathetic preganglionic neurons innervating the bladder are ER-a IR. Seven adult female cats were used (cases 2364, 2376, 2400, 2408, 2409, 2498 and 2504). They had been ovariectomized 2±4 weeks prior to the perfusion to enhance the intensity of ER-IR [17,20]. In two animals (2400 and * Corresponding author. Tel.: 131-50-363-2460; fax: 131-50363-2461. E-mail address: [email protected] (V.G.J.M. VanderHorst).
2409), during the same laparotomy, injections with 80 ml 0.5% cholera toxin subunit b (CTb) were placed into the bladder wall using a Hamilton syringe. The surgical procedures, pre- and postoperative care, and handling and housing of the animals occurred according to the protocols approved by the Faculty of Medicine of the University of Groningen. After 2±4 weeks, the animals were transcardially perfused with 2 l of heparinized phosphate buffered saline, followed by 2 l of ®xative in 0.1 M phosphate buffer; pH 7.2±7.4, containing 4% paraformaldehyde and 0.5% glutaraldehyde, or 4% paraformaldehyde (cases 2498 and 2504). Brains and spinal cords were removed, and post®xed for 2±10 h. The L7-S3 segments were cut using a vibratome into ®ve series of 60 mm sections. The sections were subsequently pretreated in 0.01 M NaIO4, 1% NaBH4, 0.5±1% Triton X-100, and 1% H2O2 (in Tris-buffered phosphate, TBS) and incubated with the estrogen receptor-a antibody H222 (1 mg/ml; 1:1000; gift from F. van Leeuwen, Netherlands Institute for Brain Research, Amsterdam; cases 2364, 2376, 2400, 2408, 2409) or 1D5 (1:800; DAKO, Carpinteria, CA; case 2498) for 4 nights at 48C. After a standard ABC procedure [1,15,16,17], the sections were stained with 0.04% diaminobenzidine (DAB), 0.2% nickel ammonium sulphate, and 0.012% H2O2, which resulted into a blue-
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 71 3- 4
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V.G.J.M. VanderHorst et al. / Neuroscience Letters 298 (2001) 147±150
black precipitate. In order to control for method speci®city, incubations were performed as described above, but without the ®rst antibody. The control sections did not contain any labeled cells. In cases 2400 and 2409, tissue was further processed to visualize CTb as described previously [15]. This method allows visualization of ER-a with antibodies that are not recognized by any of the antibodies that are required for the elucidation of CTb. After the ER-immunohistochemistry, sections of the L7-S3 segments were treated with methanol-TBS-H2O2 to inhibit peroxidase activity (see Ref. [15]), incubated in a blocking solution (5% normal rabbit serum (NRS; Vector, Burlingame, CA) in TBS containing 0.5% Triton X-100; for 30 min), and transferred to the ®rst antibody, goat anti-CTb (List Biological Labs, CA; 1:4000 in TBS containing 2% NRS, 2% Triton X-100; for 2 days at 48C). Rabbit anti-goat immunoglobulin (DAKO; 1:100 in TBS containing 2% NRS, and 1% Triton X-100; for 1 h at room temperature) was applied, followed by goat peroxidase anti-peroxidase (PAP goat, DAKO, Carpinteria, CA;1:100 in TBS containing 1% Triton X-100). The tissue was then soaked in 0.025% DAB in TBS, and 0.004% H2O2 was added, resulting in a brown precipitate in the cytoplasm. TBS was used to wash sections between all steps of the immunohistochemical procedures. In the sacral cord of all seven cases, a similar distribution
Fig. 1. Schematic overview of ER-a IR neurons in the S1±S3 segments (case 2364). Each dot indicates an ER-a IR nucleus. The open circles indicate the distinct group of ER-a IR neurons in the lateral intermediolateral cell column. A few ER-a IR neurons are located just outside the gray matter. dh, dorsal horn; SPN, sacral parasympathetic nucleus.
pattern of ER-a IR neurons was found (Fig. 1). The majority of ER-a IR neurons were present in neuronal nuclei in the super®cial layers of the dorsal horn and in the lateral and medial intermediate zone. Nuclei in the lateral part of lamina I, in the lateral and medial intermediate zone (laminae V and VII), and in lamina X were densely stained, whereas ER-a-IR nuclei in laminae II, and in the dorsomedial part of the ventral horn (medial lamina VII and lamina VIII) were lightly stained. In the lateral intermediate zone of the sacral segments, another distinct group of ER-a IR neurons was present. The immunoreactive nuclei in this group were larger, and, in addition to nuclear labeling, considerable cytoplasmic labeling was present (Fig. 2A). The ER-a labeled dendrites extended dorsomedially into the intermediomedial cell column and to a lesser extent dorsolaterally into the lateral dorsal horn. In cases 2400 and 2409, respectively, 481 (5.6/ section) and 455 (6.0/section) ER-a IR neurons with such morphological characteristics were counted in the sacral segments (bilaterally). In both cases the majority of these neurons (254 in case 2400; 316 in cases 2409) were present in the S2 segment. Injections with CTb into the bladder wall resulted in retrogradely labeled neurons in the sacral parasympathetic nucleus. In cases 2400 and 2409, respectively, 74 and 76 CTb-labeled neurons were counted. The majority of them contained ER-a IR (74% in case 2400; 68% in case 2409), and only in a minor portion of the CTb labeled neurons did not contain an ER-a IR nucleus (16% in case 2400; 4% in case 2409). In the remaining CTb labeled pro®les, it could not be determined with certainty whether they contained ER-a IR, for example because a nucleus was not present in the section or because dense retrograde labeling obscured the nucleus. The distribution of ER-a IR neurons in the sacral dorsal horn and lamina X is in line with previous immunohistochemical studies in rat [18] and cat [15]. The presence of ER-a IR in sacral parasympathetic preganglionic neurons is surprising, because in the rat [18] the sacral parasympathetic nucleus does not contain ER-a IR neurons. Moreover, ER-a IR is mostly present in neurons involved in the processing of sensory information (i.e. neurons in the dorsal horn) and in the regulation of endocrine and reproductive functions (i.e. neurons in the hypothalamus and midbrain). In the adult CNS, ER-a is neither expressed in somatic motoneurons, nor in parasympathetic and sympathetic preganglionic neurons in the brainstem and thoracic and upper lumbar cord (in the cat: VanderHorst et al., personal communication). A special characteristic of the ER-a IR in the sacral parasympathetic neurons is that it is not only present in nuclei, but also in the cytoplasm of their perikarya and dendrites. Cytoplasmic labeling has been reported to be occasionally present around densely stained nuclei, using different types of antibodies (Ref. [3] see also Ref. [1]). However, the intensity of cytoplasmic labeling in sacral parasympathetic preganglionic neurons is high, and the
V.G.J.M. VanderHorst et al. / Neuroscience Letters 298 (2001) 147±150
149
Fig. 2. Photomicrographs of transverse sections of the S2 segment. A shows an overview of ER-a IR nuclei in the dorsal horn and the distinct group of ER-a IR neurons in the lateral intermediolateral cell column, characterized by cytoplasmic labeling and larger nuclei (blue-black reaction product). B shows CTb labeled bladder preganglionic neurons (brown reaction product) containing ER-a IR nuclei (blue-black reaction product) marked with arrows. Unidenti®ed ER-a IR neurons are marked by arrowheads. Some of the double labeled cells show a brownish hue over their nuclei, probably due to brown (CTb labeled) cytoplasm overshadowing the blue-black nuclei in the thick (60 mm) sections. Dh, dorsal horn; LF, lateral funiculus; SPN, sacral parasympathetic nucleus. Scale bars A,B, 100 mm.
labeling is present in more than one occasional neuron. Cytoplasmic staining in neurites may indicate that the overall immunoreactivity for ER-a was so high that even neurites became visible. Because all animals in this study were ovariectomized, it cannot be excluded that ER-a IR might have appeared more exclusively in nuclei in the presence of estrogen, i.e. after translocation of the ER-a to the nucleus. The distribution of bladder preganglionic parasympathetic neurons in the ventral portion of the sacral parasympathetic nucleus is in line with previous studies in the cat [5,8,14]. The double labeling experiments showed that the majority of labeled bladder preganglionic motoneurons were ER-a IR. However, only part of the population of ER-a IR parasympathetic preganglionic neurons was labeled with CTb. Possible explanations are that (1) the CTb injection did not label the entire population of bladder preganglionic motoneurons; (2) preganglionic motoneurons innervating other organs than the bladder also contain ER-a IR. This latter possibility is not very likely. Preganglionic neurons innervating the colon/rectum are located more dorsally, forming the `dorsal band' [5,8]. Although the dorsal band region also contains ER-a IR neurons, their morphology is not the same as that of the ER-a IR bladder preganglionic neurons, but strongly resembles the morphology of ER-a IR neurons in the super®cial layers of the sacral dorsal horn. Outside the CNS, ER-a mRNA has been found in a subpopulation of neurons in pelvic autonomic ganglia [9]. It is not known whether it is present in other autonomic ganglia as well. Altogether, estrogen appears to directly modulate autonomic control especially of the urogenital organs.
The authors would like to thank Peter van der Sijde for digitizing the photographs. [1] Axelson, J.F. and van Leeuwen, F.W., Differential localization of estrogen receptors in various vasopressin synthesizing nuclei of the rat brain, J. Neuroendocrinol., 2 (1990) 209±216. [2] Beach, F.A., Effects of gonadal hormones on urinary behavior in dogs, Physiol. Behav., 12 (1974) 1005±10013. [3] Blaustein, J.D., Cytoplasmic estrogen receptors in rat brain: immunocytochemical evidence using three antibodies with distinct epitopes, Endocrinology, 131 (1992) 1336±1342. [4] Blok, B.F.M. and Holstege, G., The neuronal control of micturition and its relation to the emotional motor system, Prog. Brain. Res., 107 (1996) 113±126. [5] DeGroat, W.C., Vizzard, M.A., Araki, I. and Roppolo, J., Spinal interneurons and preganglionic neurons in sacral autonomic re¯ex pathways, Prog. Brain. Res., 107 (1996) 97±112. [6] Kok, A.L., Burger, C.W., van de Weijer, P.H., Voetberg, G.A., Peters-Muller, E.R. and Kenemans, P., Micturition complaints in postmenopausal women treated with continuously combined hormone replacement therapy: a prospective study, Maturitas, 31 (1999) 143±149. [7] Kurz, C., Nagele, F., Sevelda, P. and Enzelsberger, H., Intravesical administration of estriol in sensory urge incontinence±a prospective study, Geburtshilfe Frauenheilkd, 53 (1993) 535±538. [8] Nadelhaft, I., De Groat, W.C. and Morgan, C., Location and morphology of parasympathetic preganglionic neurons in the sacral spinal cord of the cat revealed by retrograde axonal transport of horseradish peroxidase, J. Comp. Neurol., 193 (1980) 265±281. [9] Papka, R.E., Srinivasan, B., Miller, K.E. and Hayashi, S., Localization of estrogen receptor protein and estrogen receptor messenger RNA in peripheral autonomic and sensory neurons, Neuroscience, 79 (1997) 1153±1163. [10] Samsioe, G., Urogenital aging±a hidden problem, Am. J. Obstet. Gynecol., 178 (1998) S245±S239.
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V.G.J.M. VanderHorst et al. / Neuroscience Letters 298 (2001) 147±150
[11] Schar, G., Kochli, O.R., Fritz, M. and Haller, U., Effect of vaginal estrogen therapy on urinary incontinence in postmenopause, Zentralbl. Gynakol., 117 (1995) 77±80. [12] Scott, C.J., Rawson, J.A., Pereira, A.M. and Clarke, I.J., The distribution of estrogen receptors in the brainstem of female sheep, Neurosci. Lett., 241 (1998) 29±32. [13] Tansy, M.F. and Kaufman, R., In¯uence of sex hormones on frequency of micturition, Nature, 211 (1966) 1184±1185. [14] VanderHorst, V.G. and Holstege, G., Organization of lumbosacral motoneuronal cell groups innervating hindlimb, pelvic ¯oor, and axial muscles in the cat, J. Comp. Neurol., 382 (1997) 46±76. [15] VanderHorst, V.G., Meijer, E., Schasfoort, F.C., Van Leeuwen, F.W. and Holstege, G., Estrogen receptor-immunoreactive neurons in the lumbosacral cord projecting to the periaqueductal gray in the ovariectomized female cat, Neurosci. Lett., 236 (1997) 25±28.
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