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Uptake of serotonin and norepinephrine in hypothalamic and limbic brain regions during the estrous cycle and the effect of neurotoxin lesions on estrous cyclicity
Brain Research Bulletin, Vol. 10,
pp.639-645, 1983. 0 Ankho International Inc. Printed in the U.S.A.
Uptake of Serotonin and Norepinephrine in Hypothalamic and Limbic Brain Regions During the Estrous Cycle and the Effect of Neurotoxin Lesions on Estrous Cyclicity DONALD Department
C. MEYER,
JYOTI SINGH AND AGNES E. JIMENEZ
of Physiology and Biophysics, University of Louisville School of Medicine Health Sciences Center, Louisville, KY 40292 Received
15 December
1982
MEYER, D. C., J. SINGH AND A. E. JIMENEZ. Uptake of serotonin and norepinephrine in hypothalamic and limbic brain regions during the estrous cycle and the effect of neurotoxin lesions on estrous cyclicity. BRAIN RES BULL lO(5) 639-645, 1983.-The temporal pattern of hypothalamic and limbic aminetgic activity during the estrous cycle has been measured by changes in the serotonergic and noradrenergic neuroual uptake during the regular estrous cycle. Sign&ant changes @=0.006,0.01) in the uptake of *H 5-HT and SH NE were recorded in the suprachiasmatic nuclei (SCN) at 1200 hours on proestrus. Significant changes (p=O.Ol, 0.018) in the uptake of 8H 5-HT in the preoptic area (FGA) were found at 1200 hours on both diestrus and proestrus while median eminence (ME) 5-HT showed a significant increase only during proestrus (p-0.001). In the amygdala (AMYG) SH 5-HT uptake was significantly different at 1200 hours on die&us and proestrus while SH NE uptake was significant @=O.OOl) at 1200 hours only during proestrus. The plasma proestrous luteinixing hormone surge occurred at 1700 hours. The serotonin neurotoxin, 5,7dihydroxytryptamine, was stereotaxically it&cued through a 30 gauge needle in concentrations of 5-10 &dkninute for 1 minute in various nuckar regions. In the SCN 4 or 5 day estrous cyclicity was interrupted by diestrus for averageperiodsof up to 15days; in the POAfor periodsof 10days; in the ME for periods of 13 days; and in the AMYG for periods of up to 6 days. Sham injections of 5% ascorbic acid’had no effect on cyclicity and following these periods of acyclicity rats resumed normal cycles. These lesion effects and patterns of uptake suggest a common timing mechanism utilixing serotonergic raphe systems for neuroendocriue control. Lesions with 5,7-DHT in all four brain regions disrupt cyclicity, but the greatest delays occur in the SCN and ME regions which are particularly critical to intrinsic neuroendocrine rhythms. Patterns of increase in reuptake capacity in all 4 regions occur 3 hours prior to the critical period for the plasma LH surge and may be an important mechanism for many types of neuroendocrine events including ovulation. Serotonin
Norepinephrine
Hypothalamic nuclei
Uptake
CONSIDERABLE literature evidence shows that serotonin’s effect on gonadotrophin release is anatomically and temporally dependent. Depletion of brain serotonin with parachlorophenylalanine (PCPA) administered 1122 hours prior to the proestrous critical period blocks the luteinixing hormone (LH) surge and ovulation [ 111. Depletion of brain serotonin using PCPA also prevents the daily surge of LH in estradiol treated ovariectomized rats [lo]. Treatment of these animals with 5-hydroxytryptophan (S-HTP) restores the LH surge only when given g-10 hours prior to the expected LH surge. Similarly 5-HTP treatments will counteract the inhibitory effects of PCPA on PMS induced ovulation if given approximately 6 hours prior to the critical period [ 11,261.On the basis of lesion experiments these effects have been shown to be dependent on the suprachiasmatic nucleus [4]. The inhibitory effects of serotonin on induced ovulation have been localized to the median eminence region on the basis of local injections of monoamine oxidase inhibitors [ 141 and require administration within several hours of the critical
639
Neurotoxin lesions
Estrous cyclicity
In adult cycling rats subcutaneous serotonin given during the late critical period was found to block ovulation I271. The present experiments are designed to establish a temporal and anatomic basis for the various pharmacological treatments affecting ovulation. In vitro uptake [23], a specific and sensitive indicator of serotonergic and noradrenergic activity will be used to determine neurotransmitter uptake capacity at key times and locations. These results should reveal the relative changes in neurotransmitter activity among several nuclei and the serotonin neurotoxin lesions will demonstrate the relative importance of these regions in maintaining regular estrous cycles and ovulation. period.
METHOD Animals
Sprague Dawley female rats (160-170 g) were maintained on a 14 L: 10 D photoperiod (0500-1900) at 22% 1°C in metal
.MfJ’i’ER, SINGH
cages and allowed food and water ad lib. Only those females demonstrating at least 2 consecutive estrous cycles, using vaginal histology, were used in the experiments. Dissrction of Fresh Brain Regims
The rats were sacrificed in groups of three and coronal sections of fresh brain were obtained using razor blades in a tissue holding device. A four millimeter section from the junction of the optic tracts posterior to the median eminence includes most of the hypothalamus. The 4 mm coronal section is fixed rostra1 surface up to aluminum blocks (1.6~ 1.4~ 1.3 cm) with a quick setting tissue adhesive (Locktite Corp., Newington CT). The aluminum block containing the tissue was then placed in the chuck of the “Vibratome” and m~nta~ned at 3-4°C in a phosphate buffer at pH 7.4 f13]. Coronal brain sections of a thickness approximating the rostral-caudal dimensions of the relevant nuclei were obtained at speeds of l-5 and amplitude settings of 8-9. Blade angle was 27”. The preoptic area (POA) (Fig. 6B) (570 microns) corresponded to A 7.02-6.57 mm coordinates of the rat brain atlas [14]. The suprachiasmatic nuclear region (SNR) (Fig. 6A) (580 microns) corresponded to A 6.36-5.78. The amygdala, (AMY@ (Fig. 6 C, D) (2003 microns) composing the basolateral, basomedial central, cortical, lateral and medial components corresponded to A 5.78-3.75 mm. The median eminence (ME) (Fig. 6C) corresponds to A 4.89-3.75 mm. The suprachiasmatic nuclei on either side of the infundibular tract was obtained by coring out the tissue with a 20 gauge needle on a cold glass slide under a dissecting microscope. The preoptic area required an I8 gauge needle and the amygdala obtained in punches from two coronal sections required a 13 gauge needle. The median eminence was obtained by dissection with microknives. Protein measurements were made according to the methods of Lowry rv ul. [18].
In vitro uptake was performed according to the techniques of Shaskan and Snyder [23]. Cores of fresh tissue were immediately placed in cold Krebs Henseleit (1932) buffer solutions (2 ml) and were preincubat~ for 5 minutes at 37°C or 0°C in a “Dubnoff’ shaking incubator under 95% 0, 5% CO, atmosphere. Tritiated serotonin (2 PCi with a specific activity of 27 Ci/mmol) or tritiated norepinephrine (0.05 ,uCi with a specific activity of 3.2 Ci mmol) were added to each of the bleakers and incubated for 25 minutes at 37°C. The total concentration of serotonin and norepineph~ne in the beakers were, respectively, 3.12~ IO-* M and 8.86~ lo-!” M, The serotonin concentration was well within the range required to minimize uptake into other amine& systems. Incubations were stopped by immersion of the beakers in ice water. The incubation media containing the tissue pellets was poured over filter paper in a vacuum filtration apparatus, and washed with 20 ml of ice cold 0.9% saline to remove residual radioactivity. The filter paper discs were sonicated for 20 seconds (Branson Instruments) and 10 ~1 aliquots removed for protein determination by the methods of Lowry [ 181. A 30 ,ul aliquot was then removed and added to liquid scinti~ation vials containing 1 ml of ethanol and IO ml toluene phosphor, and counted with liquid scintillation spectrometry. DPM/mg protein obtained at 0°C were subtracted from 37°C values to
ANI:, .itMEfNt-.rl,
obtain the true active transport of scrotonin gross the synaptic membrane. The 5,7-Dihydroxytryptamine was dissolved in 0.05% (50 mg/lOO ml) ascorbic acid and injected in the following concentration. Five wg of free base in 1 ~1 ascorbic acid into the region of suprachiasmatic nucleus and POA. and 10 _ug free base in 2 yl ascorbic acid into the ME, or AMYG. The 30 gauge stainless steel needle was connected via ~lyethylene tubing to a SO ~1 Hamilton syringe, mounted to a Harvard infusion pump (Harvard Apparatus, Boston, MA). Infusion rates were 1 pl/min. Coordinates for the various nuclear regions were jl4]:
A=&06 A=6.86 A=4.62 A=4.62
mm mm mm mm
H=3.2 Hz2.9 H=3.9 H=2.0
mm mm
mm mm
LzO.3 L=O.4 LzO.3 L-3.5
mm for mm for mm for mm for
the SNR
the POA the ME the AMYG
The standard injection protocol [ 191 consisted of desmethylimipramine (DMI); (USV Pharmaceuticals, NY: 25 mg/kg in 0.9% saline IP) pretreatment approximately 40-60 minutes before lesioning with 5,7-DHT (Regis Chemical Co., Chicago, IL). DMI blocks the uptake of 5,7-DHT into noradrenergic neurons thus preventing destruction of this system and enhancing the specificity of 5,7-DI-IT in lesioning serotonergic cells. Rats were lesioned at various times from 40-60 days. Sham injections consisted of the infusion of l-2 ~1 of ascorbic acid into the nuclear region of interest and subsequent monitoring of estrous cyclicity.
Some animals injected with neurotoxin in the various brain regions were perfused with saline and 1% fo~a~n after sacrifice, 30 days post lesion. The diencephaton was isolated from the brain and 10 pm paraffin secretions were obtained containing the suprachiasmatic, preoptic, median eminence regions, or amygdala. Lesions were found to be localized in the SNR, PQA, or ME on the basis of degeneration at the tip of the needle track. Concentrations of neurotoxin were determined from previous data [I93 in which 4-8 I.tg of toxin produced significafit decreases in uptake of S-I-IT. Thus doses of 5 pg/l were used to lesion the SCN and POA, whife 10 pg were used in large regions such as the median eminence or amygdala. These doses when injected in l-2 ~1 amounts, result in a sphere of specific serotonergic destruction 1.5-2.0 mm in diameter, surrounding a region of non-specific damage 0.5 mm i*ndiameter [ 13. Statistical Analysis
The data were analyzed by a two-way analysis of variance with days and hours as the factors. Significant interactions among days and hours were subjected to a one-way analysis of variance to determine which days showed signiEzcant
ESTROUS CYCLE AND NEUROTOXIN LESIONS
641
TWE ,Hu.ms,
FIG. 1. In vitro uptake of SH S-HT and 8H NE during diestrus and proestrus in the supfachiasmatic nucleus.
FIG. 2. In vitro uptake of SH 5-I-R and ‘NE during diestrus and proestrus preoptic area.
FIG. 3. In vitro uptake of SH S-HT during dies&us and proestrus eminence.
in the
in the median
641
Twf,*ouls,
t9KI4us>
FIG. 4. In vitro uptake of :W S-&IT and “H NE during diestrus and proestrus in the amygdala.
differences among the hours. Significant differences alllO% the hours were further analyzed using the Duncan M~lt~Pie Range test.
Rats were examined by vagiuai histology to determine the extent of disruption to normal 4 or 5 day estrons cycles. These rats exhibiting constant diestrus had not ovulated and examination via laparatomy revealed EIO evidence of fresh corpra iutea, but large mature follicles that had not been ovulated.
The results show temporally significant changes in uptake of both .5-HT (p =0.006) and NE (p=O.Ol) in the SNR (Fig. I) during proestrus prior to the LH surge (Fig. 5). In the POA (Fig. 2) only S-HT showed significant changes (p=O.Of , O.OlS), and these occurred during diestrus and proestrus, again prior to the LH surge. Median eminence (Fig. 3) 5-HT uptake showed a significant change only during proestrus &J=O.OOl) while the amygdala (Fig. 4) had significant changes @=O.OI) in 5-L-U uptake during diestrus and proestrus, but only significant changes in NE uptake @==O.Ol) during proestrus. Plasma LH levels (Fig. 5) were typical for female rats during diestrus and demonstrated the characteristic proestrus surge between 1700 and 1900. The neurotoxin lesions (Table I ) caused significant delays in estrous cyclicity, the severity and duration of which depended on the region Iesioned. Suprachiasmatic-lesions resulted in irregular cycles with as much as 15 day intervals between estrus. Int~cistern~ injections’ which affect the SNR and ME, typically showed 15 day intervals between the cycles while amygdala lesions resulted in 6 days between periods of es&us. Preoptic lesions interrupted cycles for periods of ten days while median eminence iesions typically resulted in delays of 13 days. These delay periods were usually characterized by a continuous diestrous state but normal 4 or 5 day estrous cycles resumed after the period of acyclicity.
TIME IWOUR) FWESFRUS
SEWid LH LEVELS
i9xi I800 Ixx, lb00 Is00 1400 r?m I2wJ 1100 3 flax, P3w J, iim 8600 500 400 300 200 loo D tmo 1200
c.w
,700
SW
ElOO
TlMElHOWI~
FIG. 5. Serum LH levels during diestrus (A) and proestrus (El).
DISCUSSION
These results point to a number of interesting
aspects of
ESTROUS
CYCLE AND NEUROTOXIN
LESIONS
FIG. 6. Punches from fresh brain tissue of various nuclear regions. The other side of the brain has been left undissected for comparison. (A) Suprachiasmatic nuclear region; (B) Preoptic area; (C) Median Eminence and Amygdala; (D) Amygdala. Two punches required to obtain the AMYG in order to facilitate more precise dissection of the ME.
neurotransmitter activity rhythms in general and may have relevance to particular aspects of serotonin’s role in ovulation control. Regularly cycling female rats showed peak uptake capacity for serotonin at 1Mo hours on proestrus, 3 hours prior to the proestrus critical period for LH release. The significant proestrous peak in serotonin uptake occurred in all brain regions studied and also in the POA and AMYG during dies&us. Interestingly, norepinephrine showed peak uptake at 1200 hours but only on proestrus in the SNR and AMYG, again indicating an increase in reuptake capacity for norepinephrine about 3 hours prior to the critical period for LH release. The repetition of this reuptake peak for serotonin at a critical time in several brain regions over two consentive days of the estrous cycle suggests a generalized circadian pattern whose function may be related to rhythmic pituitary hormone release including the LH and prolactin surges. In a previous paper Meyer and Quay [20] showed that this uptake of SNR serotonin also occurred, albeit at a later time in the
photoperiod, in males and females of a different strain and under a 12L:12D photoperiod. Experiments using the ovariectomized, estradiol implanted rat point to the existence of a critical period and role for serotonin in the control of phasic LH release [5,10]. Phasic LH release can be blocked with a serotonin synthesis inhibitor, parachlorophenylalamine (PCPA) and temporarily restored if serotonin synthesis is reestablished with the serotonin precursor, 5-Hydroxytryptophan (54ITP). The diestrous timing of this S-HTP response is important, suggesting that threshold levels of synaptic serotonin are required for this phasic release of LH. Thus increased reuptake capacity may be part of a presynaptic feedback mechanism to remove the increased serotonin release during the serotonin critical period. In these reuptake experiments we were careful to use concentrations of serotonin (3 x lo-9 that would insure specific, active uptake of 3H serotonin into serotonergic nerve
MEYER,SlNGH ANDJIMENEi!
644
1
TABLE EFFECT OF 5.7 DIHYDR~XYTRY~AMINE
Brain
Dose
Region
(Icg)
Lesion SHAM
5
Lesion SHAM
10
Lesion SHAM
5
Lesion SHAM
10
Lesion SHAM
200
Pre Lesion Bodywt
BRAIN LESIONS ON ESTROUS CYCLICITY
Number of Rats With Regular
*Duration of Acyclicity
Estrous Cycles
(Days)
O/S lOi11
17.3 i 1.0
016 414
15.5 t 2.4
l/5 416
11.8 t 1.4
3.4 4.1
2110 5/5
7.4 2 0.4
7.3 12.8
4113 10110
17.8 i 1.5
Post Lesion Bodywt
Suprachiasmatic N (SCN) 277.11 t 6.9 (9) 321 2 2.2 (8) 277.27 + 3.7 (IO) 325 2 2.4(11) Median Eminence (ME) 257.67 t 2.7 (12) 274.89 f 2.5 (6) 259.01 t 3.4 (7) 277.63 + 5.2 (4) Preoptic Area (POA) 202.00 ‘- 3.3 (5) 231.2 _t 7.2 (5) Amygdala (AMYG) 234.6 233.8
2 7.7 (5) & 7.98 (5)
156 154
r 2.7 2 2.1
284.1 + 287.4 i Intracisternal 130.4 t 240.4 2
*p
endings 1231. Based on in vivo invertebrate and in vivo mammalian studies 12,121 the reuptake of serotonin is strongly indicative of increased neuronal activity. Since these rhythms persist under the different hormonal conditions of dies&us and proestrus in different CNS nuclear regions it is probable that hormones are not obligatory for these rhythms. Others have also not found any significant evidence for hormonal generation of these rhythms 1261. Other experiments in which levels 1221 or turnover [S] of serotonin were found to demonstrate a similar circadian periodicity suggests that rhythmic capacity for reuptake is developed in response to a circadian pattern of neural activity. In the rat these rhythms of activity may be manifestations of the LH surge dependent cyclic neural activity which can be blocked by pentobarbital administration [6]. The lesion experiments utilizing the specitic serotonin neurotoxin, 5,7-DHT all interrupted estrous cyclicity and by implication ovulatory LH release during periods of diestrus. Lesions in regions such as the SNR, ME, and POA with a major serotonergic innervation, and role in LH release, exerted greater delays in estrous cyclicity. Van de Kar [24]
1. Bjorklund, A., A. Nobin and U. Stenevi. The use of neurotoxic dihydroxytryptamines as tools for morphological studies and localized lesioning of central indoleamine neurons. 2 Zellforsch 145: 479-501, 1973. 2. Bruinvels, J. Role of sodium in neuronal uptake of monoamines and amino acid precursors. Nature 257: 606, 1975.
showed that 5,7-DHT lesions of dorsal raphe reduced serum LH levels and serotonin levels in various regions including the arcuate, medial preoptic and amygdala nuclei. Wuttke ef al. [28] using intracisternal S,%HDT lesions found similar decreases in several pituitary hormones including luteinizing hormone. The finding that plasma hormone levels returned to normal in one month was attributed to reg~eration of .5-HT terminais and is probably the reason that estrous cyclicity eventually returned to our lesioned female rats. Thus it appears that rhythmic serotonergic synaptic transmission is required for the cyclic activation of pituitary hormone release. The raphe nuclei, suprachiasmatic nucleus and possibly other midbrain structures are parts of a central clock which synchronizes neuroendocrine rhythms.
Supported by NIH 12886 01 Al. We wish to thank Dr. A. F. Parlow and the NIAMD for their generous gift of the components used in the rat LH assay. The skillful technical assistance of Mary Jo Clark is also acknowledged.
3. Coen, C. W., M. Franklin, R. W. Laynes and P. C. B. MacKinnon. Effects of manipulating serotottin on the incidence of ovulation in the rat. J Endocrinol87: 195-201, 19%0. 4. Coen, C. W. and P. C. B. MacKinnon. Lesions of the suprachiasmatic nuclei and the serotonin dependent release of luteinizing hormone in the rat: Effects on drinking rhytbmicity and on the consequences of preoptic area stimulation. J Endocrinol 84: 231-236, 1980.
ESTROUS CYCLE AND NEUROTOXIN LESIONS
5. Coen, C. W. and P. C. B. MacKinnon. Serotonin involvement in the control of phasic luteinizing hormone release in the rat: Evidence for a critical period.J En~~~noi 82: 105-l 13, 1979. 6. Everett, J. W. Neuroendocrine aspects of mammalian reproduction. Annu Rev Physiol31: 38-16, 1969. 7. Gerschenfeld, H. hi., M. Hamon and D. Paupardin-Tritsch. Release of endogenous serotonin from two identified serotonincontaining neurones and the physiological role of serotonin reuptake.-J Physiol (Lo& 274; 265-278, 1978. 8. Hert. F.. E. Rower and J. Glowinski. Effect of 6-hydroxydopamine on daily variations of 5-HT synthesis in the hydrous of the rat. Brain Res 58: 135-146, 1973. 9. Her+, F., E. Rower and J. Glowinski. Daily variations of serotonin metabolism in the rat brain. Bruin Res 43s 445-465, 1972. 10. Hery, hf., E. LaPlante and C. Kordon. Participation of serotonin in the phasic release of LH. 1. Evidence from pharmacological experiments. Endocrinology 99: 496-503, 1976. 11. Her$, M., E. LaPlante, E. Patton and C. Kordon. Interaction de la serotonine cerebrate over la liberation cyclique de LH chez la ratte. Ann Endocrinol (Paris) 36: 123-130. 1975. 12. Iversen, L. L. Uptake mechanisms for ne~o~s~tter amines. Bi~~~~ ~~ar~uco~ 23: 1927-1935, 1974. 13. Jacobowitz, D. M. Removal of discrete fresh regions of the rat brain. Brain Res 88: 1I i-1 15. 1974. 14. Kiinig, F. R. and R. A. Klippel. A Stereofaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Baltimore: Williams and Wilkins Co., 1%3. IS. Kordon, C. Effect of selective experimental chauges in regional hypothalamic monoamine levels on superovulation in the immature rat. Neuroendocrinology 4: 129-138. 1969. 16. Kordon. C., M. Her$, A. Szafarczyk, G. Ixart and I. Assenmacher. Serotonin and the regulation of pituitary hormone secretion and of neuroendocrine rhythms. J Physiot (Paris) 77s 489-4%, 1981. 17. Krebs,
H. D. and K. Henselet. Untersuckliug uber de Harmstoffidung in Tierkorpen. Hoppe Seylers 2 Physiol Chem 210: 33-66, 1932.
645
18. Lowry, 0. H., N. J. Rosehrough, A. L. Farr and R. J. Randall. Protein measurement with the folin phenol reagent. J Biof Chem 1% 26X75, 1951. 19. Meyer, D. C. Hypothalamic and mphe serotonerglc systems in ovulation control. Endocrinology 143: 1067-1874,1978. 20. Meyer, D. C. and W. B. Quay. Hypothalamic and suprachiasmatic uptake of serotonin in vitro: Twenty-four hour changes in male and proestrous female rats. Endocrinology 98: 1160-l 165, 1976. 21. O’Steen, W. K. Suppression of ovarian activity in immature rats by serotonin. Endocrinology 77: 937-939, 1965. 22. Quay, W. B. Di&mnces in circadian rhythm of S-hydroxytryptamine accordin to bmin region. Am J PhysioI215: 1448 1453, 1968. 23. Shaskan, E. and S. Snyder. Kinetics of serotonin accumulation into slices from rat brain: relationship to catecholamine uptake. J Pharmacol Exp Ther 175: 404-418, 1970. 24. Van de Kar, L. D., S. A. Lorens, A. Vodraska, G. Allers, M.
Green, D. E. Van Grden and L. S. Van Orden, III. Effect of selective midbrain and diencephalic 5,7Dihydroxytryptamine lesions on serotonin content in individual preopticohy~th~~ nuclei and on serum luteinizing hormone level. ~euroendocrino~ogy 31: 389-315, 1988. 25. Wilson, C. A., M. Andrews, J, D. Hadley and M. Lenon. The role of hypothalamic serotouin (S-HI’) before ovulation in immature rats treated with pregnant mare serum (PMS). Psychoneuroendocrinology 2: 267-274, 1977. 26. Wilson, C. A., M. Andrews, J. L. Hadley, M. Lenon and T.
Yeo. The role of hypothalamic serotonin (S-HT) before ovulation in immature rats treated with pregnant mare serum (PMS). Psychoneuroendocrinoiogy 2: 267-274, 1977. 27. Wilson, C. A. and P. G. MacDonald. Inhibitory effect of serotonin on ovulation in adult rats. .I Endocrine 60: 253-260, 1974. 28. Wuttke, W., A. Bjorklund, H. G. Baumgarten, L. Lachen-
mayer, M. Fenske and H. P. Klemm. De and Regeneration of brain serotonin neurons following 5,7-DHT treatment: Effects of serum LH, FSH and prolactin levels in male rats. Bmin Res 134: 317-331, 1977.
pp.639-645, 1983. 0 Ankho International Inc. Printed in the U.S.A.
Uptake of Serotonin and Norepinephrine in Hypothalamic and Limbic Brain Regions During the Estrous Cycle and the Effect of Neurotoxin Lesions on Estrous Cyclicity DONALD Department
C. MEYER,
JYOTI SINGH AND AGNES E. JIMENEZ
of Physiology and Biophysics, University of Louisville School of Medicine Health Sciences Center, Louisville, KY 40292 Received
15 December
1982
MEYER, D. C., J. SINGH AND A. E. JIMENEZ. Uptake of serotonin and norepinephrine in hypothalamic and limbic brain regions during the estrous cycle and the effect of neurotoxin lesions on estrous cyclicity. BRAIN RES BULL lO(5) 639-645, 1983.-The temporal pattern of hypothalamic and limbic aminetgic activity during the estrous cycle has been measured by changes in the serotonergic and noradrenergic neuroual uptake during the regular estrous cycle. Sign&ant changes @=0.006,0.01) in the uptake of *H 5-HT and SH NE were recorded in the suprachiasmatic nuclei (SCN) at 1200 hours on proestrus. Significant changes (p=O.Ol, 0.018) in the uptake of 8H 5-HT in the preoptic area (FGA) were found at 1200 hours on both diestrus and proestrus while median eminence (ME) 5-HT showed a significant increase only during proestrus (p-0.001). In the amygdala (AMYG) SH 5-HT uptake was significantly different at 1200 hours on die&us and proestrus while SH NE uptake was significant @=O.OOl) at 1200 hours only during proestrus. The plasma proestrous luteinixing hormone surge occurred at 1700 hours. The serotonin neurotoxin, 5,7dihydroxytryptamine, was stereotaxically it&cued through a 30 gauge needle in concentrations of 5-10 &dkninute for 1 minute in various nuckar regions. In the SCN 4 or 5 day estrous cyclicity was interrupted by diestrus for averageperiodsof up to 15days; in the POAfor periodsof 10days; in the ME for periods of 13 days; and in the AMYG for periods of up to 6 days. Sham injections of 5% ascorbic acid’had no effect on cyclicity and following these periods of acyclicity rats resumed normal cycles. These lesion effects and patterns of uptake suggest a common timing mechanism utilixing serotonergic raphe systems for neuroendocriue control. Lesions with 5,7-DHT in all four brain regions disrupt cyclicity, but the greatest delays occur in the SCN and ME regions which are particularly critical to intrinsic neuroendocrine rhythms. Patterns of increase in reuptake capacity in all 4 regions occur 3 hours prior to the critical period for the plasma LH surge and may be an important mechanism for many types of neuroendocrine events including ovulation. Serotonin
Norepinephrine
Hypothalamic nuclei
Uptake
CONSIDERABLE literature evidence shows that serotonin’s effect on gonadotrophin release is anatomically and temporally dependent. Depletion of brain serotonin with parachlorophenylalanine (PCPA) administered 1122 hours prior to the proestrous critical period blocks the luteinixing hormone (LH) surge and ovulation [ 111. Depletion of brain serotonin using PCPA also prevents the daily surge of LH in estradiol treated ovariectomized rats [lo]. Treatment of these animals with 5-hydroxytryptophan (S-HTP) restores the LH surge only when given g-10 hours prior to the expected LH surge. Similarly 5-HTP treatments will counteract the inhibitory effects of PCPA on PMS induced ovulation if given approximately 6 hours prior to the critical period [ 11,261.On the basis of lesion experiments these effects have been shown to be dependent on the suprachiasmatic nucleus [4]. The inhibitory effects of serotonin on induced ovulation have been localized to the median eminence region on the basis of local injections of monoamine oxidase inhibitors [ 141 and require administration within several hours of the critical
639
Neurotoxin lesions
Estrous cyclicity
In adult cycling rats subcutaneous serotonin given during the late critical period was found to block ovulation I271. The present experiments are designed to establish a temporal and anatomic basis for the various pharmacological treatments affecting ovulation. In vitro uptake [23], a specific and sensitive indicator of serotonergic and noradrenergic activity will be used to determine neurotransmitter uptake capacity at key times and locations. These results should reveal the relative changes in neurotransmitter activity among several nuclei and the serotonin neurotoxin lesions will demonstrate the relative importance of these regions in maintaining regular estrous cycles and ovulation. period.
METHOD Animals
Sprague Dawley female rats (160-170 g) were maintained on a 14 L: 10 D photoperiod (0500-1900) at 22% 1°C in metal
.MfJ’i’ER, SINGH
cages and allowed food and water ad lib. Only those females demonstrating at least 2 consecutive estrous cycles, using vaginal histology, were used in the experiments. Dissrction of Fresh Brain Regims
The rats were sacrificed in groups of three and coronal sections of fresh brain were obtained using razor blades in a tissue holding device. A four millimeter section from the junction of the optic tracts posterior to the median eminence includes most of the hypothalamus. The 4 mm coronal section is fixed rostra1 surface up to aluminum blocks (1.6~ 1.4~ 1.3 cm) with a quick setting tissue adhesive (Locktite Corp., Newington CT). The aluminum block containing the tissue was then placed in the chuck of the “Vibratome” and m~nta~ned at 3-4°C in a phosphate buffer at pH 7.4 f13]. Coronal brain sections of a thickness approximating the rostral-caudal dimensions of the relevant nuclei were obtained at speeds of l-5 and amplitude settings of 8-9. Blade angle was 27”. The preoptic area (POA) (Fig. 6B) (570 microns) corresponded to A 7.02-6.57 mm coordinates of the rat brain atlas [14]. The suprachiasmatic nuclear region (SNR) (Fig. 6A) (580 microns) corresponded to A 6.36-5.78. The amygdala, (AMY@ (Fig. 6 C, D) (2003 microns) composing the basolateral, basomedial central, cortical, lateral and medial components corresponded to A 5.78-3.75 mm. The median eminence (ME) (Fig. 6C) corresponds to A 4.89-3.75 mm. The suprachiasmatic nuclei on either side of the infundibular tract was obtained by coring out the tissue with a 20 gauge needle on a cold glass slide under a dissecting microscope. The preoptic area required an I8 gauge needle and the amygdala obtained in punches from two coronal sections required a 13 gauge needle. The median eminence was obtained by dissection with microknives. Protein measurements were made according to the methods of Lowry rv ul. [18].
In vitro uptake was performed according to the techniques of Shaskan and Snyder [23]. Cores of fresh tissue were immediately placed in cold Krebs Henseleit (1932) buffer solutions (2 ml) and were preincubat~ for 5 minutes at 37°C or 0°C in a “Dubnoff’ shaking incubator under 95% 0, 5% CO, atmosphere. Tritiated serotonin (2 PCi with a specific activity of 27 Ci/mmol) or tritiated norepinephrine (0.05 ,uCi with a specific activity of 3.2 Ci mmol) were added to each of the bleakers and incubated for 25 minutes at 37°C. The total concentration of serotonin and norepineph~ne in the beakers were, respectively, 3.12~ IO-* M and 8.86~ lo-!” M, The serotonin concentration was well within the range required to minimize uptake into other amine& systems. Incubations were stopped by immersion of the beakers in ice water. The incubation media containing the tissue pellets was poured over filter paper in a vacuum filtration apparatus, and washed with 20 ml of ice cold 0.9% saline to remove residual radioactivity. The filter paper discs were sonicated for 20 seconds (Branson Instruments) and 10 ~1 aliquots removed for protein determination by the methods of Lowry [ 181. A 30 ,ul aliquot was then removed and added to liquid scinti~ation vials containing 1 ml of ethanol and IO ml toluene phosphor, and counted with liquid scintillation spectrometry. DPM/mg protein obtained at 0°C were subtracted from 37°C values to
ANI:, .itMEfNt-.rl,
obtain the true active transport of scrotonin gross the synaptic membrane. The 5,7-Dihydroxytryptamine was dissolved in 0.05% (50 mg/lOO ml) ascorbic acid and injected in the following concentration. Five wg of free base in 1 ~1 ascorbic acid into the region of suprachiasmatic nucleus and POA. and 10 _ug free base in 2 yl ascorbic acid into the ME, or AMYG. The 30 gauge stainless steel needle was connected via ~lyethylene tubing to a SO ~1 Hamilton syringe, mounted to a Harvard infusion pump (Harvard Apparatus, Boston, MA). Infusion rates were 1 pl/min. Coordinates for the various nuclear regions were jl4]:
A=&06 A=6.86 A=4.62 A=4.62
mm mm mm mm
H=3.2 Hz2.9 H=3.9 H=2.0
mm mm
mm mm
LzO.3 L=O.4 LzO.3 L-3.5
mm for mm for mm for mm for
the SNR
the POA the ME the AMYG
The standard injection protocol [ 191 consisted of desmethylimipramine (DMI); (USV Pharmaceuticals, NY: 25 mg/kg in 0.9% saline IP) pretreatment approximately 40-60 minutes before lesioning with 5,7-DHT (Regis Chemical Co., Chicago, IL). DMI blocks the uptake of 5,7-DHT into noradrenergic neurons thus preventing destruction of this system and enhancing the specificity of 5,7-DI-IT in lesioning serotonergic cells. Rats were lesioned at various times from 40-60 days. Sham injections consisted of the infusion of l-2 ~1 of ascorbic acid into the nuclear region of interest and subsequent monitoring of estrous cyclicity.
Some animals injected with neurotoxin in the various brain regions were perfused with saline and 1% fo~a~n after sacrifice, 30 days post lesion. The diencephaton was isolated from the brain and 10 pm paraffin secretions were obtained containing the suprachiasmatic, preoptic, median eminence regions, or amygdala. Lesions were found to be localized in the SNR, PQA, or ME on the basis of degeneration at the tip of the needle track. Concentrations of neurotoxin were determined from previous data [I93 in which 4-8 I.tg of toxin produced significafit decreases in uptake of S-I-IT. Thus doses of 5 pg/l were used to lesion the SCN and POA, whife 10 pg were used in large regions such as the median eminence or amygdala. These doses when injected in l-2 ~1 amounts, result in a sphere of specific serotonergic destruction 1.5-2.0 mm in diameter, surrounding a region of non-specific damage 0.5 mm i*ndiameter [ 13. Statistical Analysis
The data were analyzed by a two-way analysis of variance with days and hours as the factors. Significant interactions among days and hours were subjected to a one-way analysis of variance to determine which days showed signiEzcant
ESTROUS CYCLE AND NEUROTOXIN LESIONS
641
TWE ,Hu.ms,
FIG. 1. In vitro uptake of SH S-HT and 8H NE during diestrus and proestrus in the supfachiasmatic nucleus.
FIG. 2. In vitro uptake of SH 5-I-R and ‘NE during diestrus and proestrus preoptic area.
FIG. 3. In vitro uptake of SH S-HT during dies&us and proestrus eminence.
in the
in the median
641
Twf,*ouls,
t9KI4us>
FIG. 4. In vitro uptake of :W S-&IT and “H NE during diestrus and proestrus in the amygdala.
differences among the hours. Significant differences alllO% the hours were further analyzed using the Duncan M~lt~Pie Range test.
Rats were examined by vagiuai histology to determine the extent of disruption to normal 4 or 5 day estrons cycles. These rats exhibiting constant diestrus had not ovulated and examination via laparatomy revealed EIO evidence of fresh corpra iutea, but large mature follicles that had not been ovulated.
The results show temporally significant changes in uptake of both .5-HT (p =0.006) and NE (p=O.Ol) in the SNR (Fig. I) during proestrus prior to the LH surge (Fig. 5). In the POA (Fig. 2) only S-HT showed significant changes (p=O.Of , O.OlS), and these occurred during diestrus and proestrus, again prior to the LH surge. Median eminence (Fig. 3) 5-HT uptake showed a significant change only during proestrus &J=O.OOl) while the amygdala (Fig. 4) had significant changes @=O.OI) in 5-L-U uptake during diestrus and proestrus, but only significant changes in NE uptake @==O.Ol) during proestrus. Plasma LH levels (Fig. 5) were typical for female rats during diestrus and demonstrated the characteristic proestrus surge between 1700 and 1900. The neurotoxin lesions (Table I ) caused significant delays in estrous cyclicity, the severity and duration of which depended on the region Iesioned. Suprachiasmatic-lesions resulted in irregular cycles with as much as 15 day intervals between estrus. Int~cistern~ injections’ which affect the SNR and ME, typically showed 15 day intervals between the cycles while amygdala lesions resulted in 6 days between periods of es&us. Preoptic lesions interrupted cycles for periods of ten days while median eminence iesions typically resulted in delays of 13 days. These delay periods were usually characterized by a continuous diestrous state but normal 4 or 5 day estrous cycles resumed after the period of acyclicity.
TIME IWOUR) FWESFRUS
SEWid LH LEVELS
i9xi I800 Ixx, lb00 Is00 1400 r?m I2wJ 1100 3 flax, P3w J, iim 8600 500 400 300 200 loo D tmo 1200
c.w
,700
SW
ElOO
TlMElHOWI~
FIG. 5. Serum LH levels during diestrus (A) and proestrus (El).
DISCUSSION
These results point to a number of interesting
aspects of
ESTROUS
CYCLE AND NEUROTOXIN
LESIONS
FIG. 6. Punches from fresh brain tissue of various nuclear regions. The other side of the brain has been left undissected for comparison. (A) Suprachiasmatic nuclear region; (B) Preoptic area; (C) Median Eminence and Amygdala; (D) Amygdala. Two punches required to obtain the AMYG in order to facilitate more precise dissection of the ME.
neurotransmitter activity rhythms in general and may have relevance to particular aspects of serotonin’s role in ovulation control. Regularly cycling female rats showed peak uptake capacity for serotonin at 1Mo hours on proestrus, 3 hours prior to the proestrus critical period for LH release. The significant proestrous peak in serotonin uptake occurred in all brain regions studied and also in the POA and AMYG during dies&us. Interestingly, norepinephrine showed peak uptake at 1200 hours but only on proestrus in the SNR and AMYG, again indicating an increase in reuptake capacity for norepinephrine about 3 hours prior to the critical period for LH release. The repetition of this reuptake peak for serotonin at a critical time in several brain regions over two consentive days of the estrous cycle suggests a generalized circadian pattern whose function may be related to rhythmic pituitary hormone release including the LH and prolactin surges. In a previous paper Meyer and Quay [20] showed that this uptake of SNR serotonin also occurred, albeit at a later time in the
photoperiod, in males and females of a different strain and under a 12L:12D photoperiod. Experiments using the ovariectomized, estradiol implanted rat point to the existence of a critical period and role for serotonin in the control of phasic LH release [5,10]. Phasic LH release can be blocked with a serotonin synthesis inhibitor, parachlorophenylalamine (PCPA) and temporarily restored if serotonin synthesis is reestablished with the serotonin precursor, 5-Hydroxytryptophan (54ITP). The diestrous timing of this S-HTP response is important, suggesting that threshold levels of synaptic serotonin are required for this phasic release of LH. Thus increased reuptake capacity may be part of a presynaptic feedback mechanism to remove the increased serotonin release during the serotonin critical period. In these reuptake experiments we were careful to use concentrations of serotonin (3 x lo-9 that would insure specific, active uptake of 3H serotonin into serotonergic nerve
MEYER,SlNGH ANDJIMENEi!
644
1
TABLE EFFECT OF 5.7 DIHYDR~XYTRY~AMINE
Brain
Dose
Region
(Icg)
Lesion SHAM
5
Lesion SHAM
10
Lesion SHAM
5
Lesion SHAM
10
Lesion SHAM
200
Pre Lesion Bodywt
BRAIN LESIONS ON ESTROUS CYCLICITY
Number of Rats With Regular
*Duration of Acyclicity
Estrous Cycles
(Days)
O/S lOi11
17.3 i 1.0
016 414
15.5 t 2.4
l/5 416
11.8 t 1.4
3.4 4.1
2110 5/5
7.4 2 0.4
7.3 12.8
4113 10110
17.8 i 1.5
Post Lesion Bodywt
Suprachiasmatic N (SCN) 277.11 t 6.9 (9) 321 2 2.2 (8) 277.27 + 3.7 (IO) 325 2 2.4(11) Median Eminence (ME) 257.67 t 2.7 (12) 274.89 f 2.5 (6) 259.01 t 3.4 (7) 277.63 + 5.2 (4) Preoptic Area (POA) 202.00 ‘- 3.3 (5) 231.2 _t 7.2 (5) Amygdala (AMYG) 234.6 233.8
2 7.7 (5) & 7.98 (5)
156 154
r 2.7 2 2.1
284.1 + 287.4 i Intracisternal 130.4 t 240.4 2
*p
endings 1231. Based on in vivo invertebrate and in vivo mammalian studies 12,121 the reuptake of serotonin is strongly indicative of increased neuronal activity. Since these rhythms persist under the different hormonal conditions of dies&us and proestrus in different CNS nuclear regions it is probable that hormones are not obligatory for these rhythms. Others have also not found any significant evidence for hormonal generation of these rhythms 1261. Other experiments in which levels 1221 or turnover [S] of serotonin were found to demonstrate a similar circadian periodicity suggests that rhythmic capacity for reuptake is developed in response to a circadian pattern of neural activity. In the rat these rhythms of activity may be manifestations of the LH surge dependent cyclic neural activity which can be blocked by pentobarbital administration [6]. The lesion experiments utilizing the specitic serotonin neurotoxin, 5,7-DHT all interrupted estrous cyclicity and by implication ovulatory LH release during periods of diestrus. Lesions in regions such as the SNR, ME, and POA with a major serotonergic innervation, and role in LH release, exerted greater delays in estrous cyclicity. Van de Kar [24]
1. Bjorklund, A., A. Nobin and U. Stenevi. The use of neurotoxic dihydroxytryptamines as tools for morphological studies and localized lesioning of central indoleamine neurons. 2 Zellforsch 145: 479-501, 1973. 2. Bruinvels, J. Role of sodium in neuronal uptake of monoamines and amino acid precursors. Nature 257: 606, 1975.
showed that 5,7-DHT lesions of dorsal raphe reduced serum LH levels and serotonin levels in various regions including the arcuate, medial preoptic and amygdala nuclei. Wuttke ef al. [28] using intracisternal S,%HDT lesions found similar decreases in several pituitary hormones including luteinizing hormone. The finding that plasma hormone levels returned to normal in one month was attributed to reg~eration of .5-HT terminais and is probably the reason that estrous cyclicity eventually returned to our lesioned female rats. Thus it appears that rhythmic serotonergic synaptic transmission is required for the cyclic activation of pituitary hormone release. The raphe nuclei, suprachiasmatic nucleus and possibly other midbrain structures are parts of a central clock which synchronizes neuroendocrine rhythms.
Supported by NIH 12886 01 Al. We wish to thank Dr. A. F. Parlow and the NIAMD for their generous gift of the components used in the rat LH assay. The skillful technical assistance of Mary Jo Clark is also acknowledged.
3. Coen, C. W., M. Franklin, R. W. Laynes and P. C. B. MacKinnon. Effects of manipulating serotottin on the incidence of ovulation in the rat. J Endocrinol87: 195-201, 19%0. 4. Coen, C. W. and P. C. B. MacKinnon. Lesions of the suprachiasmatic nuclei and the serotonin dependent release of luteinizing hormone in the rat: Effects on drinking rhytbmicity and on the consequences of preoptic area stimulation. J Endocrinol 84: 231-236, 1980.
ESTROUS CYCLE AND NEUROTOXIN LESIONS
5. Coen, C. W. and P. C. B. MacKinnon. Serotonin involvement in the control of phasic luteinizing hormone release in the rat: Evidence for a critical period.J En~~~noi 82: 105-l 13, 1979. 6. Everett, J. W. Neuroendocrine aspects of mammalian reproduction. Annu Rev Physiol31: 38-16, 1969. 7. Gerschenfeld, H. hi., M. Hamon and D. Paupardin-Tritsch. Release of endogenous serotonin from two identified serotonincontaining neurones and the physiological role of serotonin reuptake.-J Physiol (Lo& 274; 265-278, 1978. 8. Hert. F.. E. Rower and J. Glowinski. Effect of 6-hydroxydopamine on daily variations of 5-HT synthesis in the hydrous of the rat. Brain Res 58: 135-146, 1973. 9. Her+, F., E. Rower and J. Glowinski. Daily variations of serotonin metabolism in the rat brain. Bruin Res 43s 445-465, 1972. 10. Hery, hf., E. LaPlante and C. Kordon. Participation of serotonin in the phasic release of LH. 1. Evidence from pharmacological experiments. Endocrinology 99: 496-503, 1976. 11. Her$, M., E. LaPlante, E. Patton and C. Kordon. Interaction de la serotonine cerebrate over la liberation cyclique de LH chez la ratte. Ann Endocrinol (Paris) 36: 123-130. 1975. 12. Iversen, L. L. Uptake mechanisms for ne~o~s~tter amines. Bi~~~~ ~~ar~uco~ 23: 1927-1935, 1974. 13. Jacobowitz, D. M. Removal of discrete fresh regions of the rat brain. Brain Res 88: 1I i-1 15. 1974. 14. Kiinig, F. R. and R. A. Klippel. A Stereofaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Baltimore: Williams and Wilkins Co., 1%3. IS. Kordon, C. Effect of selective experimental chauges in regional hypothalamic monoamine levels on superovulation in the immature rat. Neuroendocrinology 4: 129-138. 1969. 16. Kordon. C., M. Her$, A. Szafarczyk, G. Ixart and I. Assenmacher. Serotonin and the regulation of pituitary hormone secretion and of neuroendocrine rhythms. J Physiot (Paris) 77s 489-4%, 1981. 17. Krebs,
H. D. and K. Henselet. Untersuckliug uber de Harmstoffidung in Tierkorpen. Hoppe Seylers 2 Physiol Chem 210: 33-66, 1932.
645
18. Lowry, 0. H., N. J. Rosehrough, A. L. Farr and R. J. Randall. Protein measurement with the folin phenol reagent. J Biof Chem 1% 26X75, 1951. 19. Meyer, D. C. Hypothalamic and mphe serotonerglc systems in ovulation control. Endocrinology 143: 1067-1874,1978. 20. Meyer, D. C. and W. B. Quay. Hypothalamic and suprachiasmatic uptake of serotonin in vitro: Twenty-four hour changes in male and proestrous female rats. Endocrinology 98: 1160-l 165, 1976. 21. O’Steen, W. K. Suppression of ovarian activity in immature rats by serotonin. Endocrinology 77: 937-939, 1965. 22. Quay, W. B. Di&mnces in circadian rhythm of S-hydroxytryptamine accordin to bmin region. Am J PhysioI215: 1448 1453, 1968. 23. Shaskan, E. and S. Snyder. Kinetics of serotonin accumulation into slices from rat brain: relationship to catecholamine uptake. J Pharmacol Exp Ther 175: 404-418, 1970. 24. Van de Kar, L. D., S. A. Lorens, A. Vodraska, G. Allers, M.
Green, D. E. Van Grden and L. S. Van Orden, III. Effect of selective midbrain and diencephalic 5,7Dihydroxytryptamine lesions on serotonin content in individual preopticohy~th~~ nuclei and on serum luteinizing hormone level. ~euroendocrino~ogy 31: 389-315, 1988. 25. Wilson, C. A., M. Andrews, J, D. Hadley and M. Lenon. The role of hypothalamic serotouin (S-HI’) before ovulation in immature rats treated with pregnant mare serum (PMS). Psychoneuroendocrinology 2: 267-274, 1977. 26. Wilson, C. A., M. Andrews, J. L. Hadley, M. Lenon and T.
Yeo. The role of hypothalamic serotonin (S-HT) before ovulation in immature rats treated with pregnant mare serum (PMS). Psychoneuroendocrinoiogy 2: 267-274, 1977. 27. Wilson, C. A. and P. G. MacDonald. Inhibitory effect of serotonin on ovulation in adult rats. .I Endocrine 60: 253-260, 1974. 28. Wuttke, W., A. Bjorklund, H. G. Baumgarten, L. Lachen-
mayer, M. Fenske and H. P. Klemm. De and Regeneration of brain serotonin neurons following 5,7-DHT treatment: Effects of serum LH, FSH and prolactin levels in male rats. Bmin Res 134: 317-331, 1977.