Brain Research, 586 (1992) 171-175 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
171
BRES 25288
c-fos
expression in noradrenergic A2 neurons of the rat during the estrous cycle and after steroid hormone treatments L o t h a r J e n n e s , M a r y E. J e n n e s , Cheryl Purvis a n d M. N e e s
Department of Anatomy and Neurobiology, Universityof Kentucky, Collegeof Medicine, Leal..h:ton, KY 40536.0084 (US~) (Accepted 28 April 1992)
Key words: Noradrenergic A2 cell group; Estrous cycle; Estradiol; c-fos expression; rat
The expression of the proto-oncogene product fos in noradrenergic neurons of the A2 cell group was studied with immunohistochemistry during the estrous cycle of the rat and after ovariectomy and estrogen treatments. During the estrous cycle the percentage of fos.positive norepinephrine containing neurons was highest at proestrus (39%), followed by estrus (36%) while during diestrus only 4% of the A2 neurons contained immunoreactive los protein in their nuclei. Ovariectomy caused a further decrease in the number of fos-positive A2 neurons (2%) while long-term estradiol administration partially reversed the effects of ovarian steroid removal (19%). However, 3 h after a single suhcutanous injection of estradiol into ovariectomised rats, 79% of the noradrenergic neurons in the A2 area showed fos immunoreactivity in their nuclei. The results indicate that fo~-expression in the noradrenergic neurons in the A2 region varies depending upon the circulating estradiol levels. Since norepinephrine stimulates gonadotropin releasing hormone (GnRH) secretion from the median eminence during proestrus and the GnRlt neurons do not contain estrogen receptors, it is suggested that the A2 region is, at least in part, responsible for conveying the estrogen signal to the GnRH neurons.
One of the major neuronal systems involved in the regulation of GnRH release uses norepinephrine (NE) as a stimulatory transmitter, It has been long known that inhibition of NE synthesis or depletion of NE stores suppresses GnRH release from the median eminence and that this effect can be reversed by administration of an a2-adrenergic agonist. Similarly, intraventricular injections of NE cause a significant rise in GnRH release which is prevented by a2-adrenergic receptor antagonists (for review see ref. 11). The stimulatory effects of NE on the GnRH system appear to be exerted in the median eminence where noradrenergic nerve terminals are in juxtaposition to GnRH containing axon terminals as well as at the GnRH perikaryon which is closely apposed by NE containing neurites '~. This hypothesis is supported by the results of studies which describe an increase in the NE turnover rate in the median eminence during proestrus which is paralleled by an increase in GnRH-LH release ml. In the present study we focused on the NE containing cell groups in the area of the nucleus tractus
solitarii (A2) since this cell group has been shown to project to the median eminence and rostral hypothalamus '~,12'~s. The expression of the proto-oncogene product fos was used to identify the subsets of NE neurons which are activated during various stages of the estrous cycle or after steroid hormone treatment. Fos is a protein which is encoded by the immediate early gene c.fos and it is expressed rapidly and transiently in many neurons in response to a variety of stimuli, such as electrical, environmental, chemical or hormonal treatments ~3. Thus, changes in the presence of the fos protein in the nuclei of certain cells can be used as a sensitive indicator of changes in the transcriptional activity and may therefor allow to identify select neurons which are affected by and respond to a changing hormonal environment. Adult female Sprague-Dawley rats (50-60 days old) were used for all experiments. The animals were perfusion fixed under anesthesia via transcardiac puncture with 50 ml phosphate-buffered saline (PBS; 0.1 M, pH 7.4) followed by 5~O ml of 4% paraformaidehyde in
Correspondence: L. Jennes, University of Kentucky, College of Medicine, Department of Anatomy and Neurobiology, MN 210 Medical Center, Lexington, KY 40536-0084, USA. Fax: (1)(606)258-5946.
C~
•
~
~; ;i;ii~ ¸
C7
~
~t
L o
%
0"
L~
0
.~
%
O
e %
,ll
II
..
0 < X
0 < X
0
0"
°
171
BRES 25288
c-fos
expression in noradrenergic A2 neurons of the rat during the estrous cycle and after steroid hormone treatments L o t h a r J e n n e s , M a r y E. J e n n e s , Cheryl Purvis a n d M. N e e s
Department of Anatomy and Neurobiology, Universityof Kentucky, Collegeof Medicine, Leal..h:ton, KY 40536.0084 (US~) (Accepted 28 April 1992)
Key words: Noradrenergic A2 cell group; Estrous cycle; Estradiol; c-fos expression; rat
The expression of the proto-oncogene product fos in noradrenergic neurons of the A2 cell group was studied with immunohistochemistry during the estrous cycle of the rat and after ovariectomy and estrogen treatments. During the estrous cycle the percentage of fos.positive norepinephrine containing neurons was highest at proestrus (39%), followed by estrus (36%) while during diestrus only 4% of the A2 neurons contained immunoreactive los protein in their nuclei. Ovariectomy caused a further decrease in the number of fos-positive A2 neurons (2%) while long-term estradiol administration partially reversed the effects of ovarian steroid removal (19%). However, 3 h after a single suhcutanous injection of estradiol into ovariectomised rats, 79% of the noradrenergic neurons in the A2 area showed fos immunoreactivity in their nuclei. The results indicate that fo~-expression in the noradrenergic neurons in the A2 region varies depending upon the circulating estradiol levels. Since norepinephrine stimulates gonadotropin releasing hormone (GnRH) secretion from the median eminence during proestrus and the GnRlt neurons do not contain estrogen receptors, it is suggested that the A2 region is, at least in part, responsible for conveying the estrogen signal to the GnRH neurons.
One of the major neuronal systems involved in the regulation of GnRH release uses norepinephrine (NE) as a stimulatory transmitter, It has been long known that inhibition of NE synthesis or depletion of NE stores suppresses GnRH release from the median eminence and that this effect can be reversed by administration of an a2-adrenergic agonist. Similarly, intraventricular injections of NE cause a significant rise in GnRH release which is prevented by a2-adrenergic receptor antagonists (for review see ref. 11). The stimulatory effects of NE on the GnRH system appear to be exerted in the median eminence where noradrenergic nerve terminals are in juxtaposition to GnRH containing axon terminals as well as at the GnRH perikaryon which is closely apposed by NE containing neurites '~. This hypothesis is supported by the results of studies which describe an increase in the NE turnover rate in the median eminence during proestrus which is paralleled by an increase in GnRH-LH release ml. In the present study we focused on the NE containing cell groups in the area of the nucleus tractus
solitarii (A2) since this cell group has been shown to project to the median eminence and rostral hypothalamus '~,12'~s. The expression of the proto-oncogene product fos was used to identify the subsets of NE neurons which are activated during various stages of the estrous cycle or after steroid hormone treatment. Fos is a protein which is encoded by the immediate early gene c.fos and it is expressed rapidly and transiently in many neurons in response to a variety of stimuli, such as electrical, environmental, chemical or hormonal treatments ~3. Thus, changes in the presence of the fos protein in the nuclei of certain cells can be used as a sensitive indicator of changes in the transcriptional activity and may therefor allow to identify select neurons which are affected by and respond to a changing hormonal environment. Adult female Sprague-Dawley rats (50-60 days old) were used for all experiments. The animals were perfusion fixed under anesthesia via transcardiac puncture with 50 ml phosphate-buffered saline (PBS; 0.1 M, pH 7.4) followed by 5~O ml of 4% paraformaidehyde in
Correspondence: L. Jennes, University of Kentucky, College of Medicine, Department of Anatomy and Neurobiology, MN 210 Medical Center, Lexington, KY 40536-0084, USA. Fax: (1)(606)258-5946.
C~
•
~
~; ;i;ii~ ¸
C7
~
~t
L o
%
0"
L~
0
.~
%
O
e %
,ll
II
..
0 < X
0 < X
0
0"
°
