Ontogenic changes in the hypothalamic levels of amino acid neurotransmitters in the female rat

Del,elopmental Brain Research, 77 (1994) 183-188 © 1994 Elsevier Science B.V. All rights reserved 0165-3806/94/$07.00

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B R E S D 51741

Ontogenic changes in the hypothalamic levels of amino acid neurotransmitters in the female rat Detlef Goroll a, Pablo Arias

b,,

Wolfgang Wuttke

a

" Abteilung fiir klinische und experimentelle Endokrinologie, Frauenklinik, Georg-August Unit,ersitdt, Gdttingen, Germany, and h Departamento de Fisiolog{a, Facultad de Medicina, Unicersidad de Buenos Aires, Paraguay 2155, 7° Piso, 112l Buenos Aires, Argentim~ (Accepted 17 August 1993)

Key words: Puberty; Hypothalamus; A m i n o acid neurotransmitter; Aspartate: Glutamate; Homocysteic acid: Taurine; Glycine; G A B A

In order to evaluate the participation of several amino acid neurotransmitters (AANT) in sexual maturation we measured the hypothalamic concentrations of aspartate (Asp), glutamate (Glu), homocysteic acid (HCA), glycine (Gly), taurine (Tau) and ,/-aminobutyric acid (GABA) in female rats at different ages of sexual development. Animals (15-, 20-, 25-, 30-, 35- and 40-day-old female rats, as well as adult diestrous rats, n = 8 - 1 0 / g r o u p ) w e r e decapitated at noon; each brain was rapidly removed and frozen on dry ice. Preoptic area (POA) and mediobasal hypothalamic (MBH) needle punch samples were obtained from 500-600 /xm thick coronal slices, homogenized and centrifuged. A A N T concentrations were measured in the supernatants following derivatization with phenyl isothiocyanate and reverse-phase t t P L C separation by U V detection. Significant ( P < 0.05) changes in hypothalamic A A N T concentrations can be summarized as follows: P O A Glu, H C A and Gly levels rose transiently at day 20 and then increased steadily in 35- and 40-day-old rats. Asp concentrations rose in 35- and 40-day-old rats. Tau concentrations decreased markedly from day 30 onwards. G A B A levels were lowest in 15-day-old rats, as compared to the other age groups. Asp. Glu, Gly and G A B A concentrations were highest in adult diestrous rats. Changes in MBH A A N T levels were not as relevant: only Gly concentrations rose significantly at day 40; Asp, Glu, HCA, Gly and G A B A concentrations increased in adult diestrous rats and Tau concentrations were lower from day 35 onwards. It is concluded that increased levels of excitatory amino acids (Asp, Glu and HCA) could contribute to sexual maturation as a result of their synaptogenic properties a n d / o r acting directly on the L H R H neurons, stimulating L t t R I t release. The striking decrease of hypothalamic Tau concentrations in peripubertal rats suggests a possible role of this amino acid in thc neuroendocrine events leading to sexual maturation.

INTRODUCTION Excitatory amino acids (EAA), such as glutamate and aspartate, are major neurotransmitters in the mammalian central nervous system l°. In the past 15 years, evidence has accumulated concerning the effects of these EAA and of related agonist substances (such as N-methyl-D-aspartate or NMDA) on the regulation of gonadotropin releaseS't3'14'29'32'38'44; nowadays it is accepted that E A A act at the hypothalamus 38, to enhance luteinizing hormone-releasing hormone ( L H R H ) release s'13. EAA could either act in the p r e o p t i c / anterior hypothalamic area, where most of the L H R H cell bodies are located 45 or in the mediobasal hypotha-

* Corresponding author. Fax: (54) (1) 962-5457.

SSDI t l 1 6 5 - 3 8 ( ) 6 ( 9 3 ) E 0 1 3 9 - C

lamus, where the peptidergic neurons release L H R H into the capillary plexus of the median eminence. Inhibitory amino acid neurotransmitters are also involved in the control of LH secretion. Considerable evidence indicates that y-aminobutyric acid (GABA) inhibits LH release; however, some investigators have demonstrated a stimulatory effect of GABA on LH release (for a review, see ref. 43). It has been suggested that taurine, a putative inhibitory neurotransmitter ubiquitous in the mammalian brain C N S 2s, also inhibits LH release 33. Previous studies have shown that the systemic administration of N M D A advances the onset of puberty in female rats and, vice versa, that such event is delayed by the injection of NMDA-antagonist d r u g s 22'42'4~'. There is indirect evidence to suggest that activation of hypothalamic N M D A receptors is significantly enhanced in peripubertal male rats ~. These experiments

strongly indicate that the EAA system is involved in the neuroendocrine events that lead to the onset of puberty. It is generally accepted that regional changes in the concentration of neurotransmitters reflect different densities of innervation, thus providing indirect information on neurotransmitter activity. Therefore, in order to evaluate the participation of some hypothalamic amino acid neurotransmitters (AANT) in sexual maturation and puberty, we measured the concentrations of aspartate, glutamate, homocysteic acid, glycine, taurine and GABA in the preoptic area and in the mediobasal hypothalamus of female rats at different stages of sexual development.

400

800

MATERIALS

AND METHODS

Animals. A total of 54 immature and 10 adult female Sprague-Dawley rats, housed under controlled conditions of lighting and temperature (lights on 7.00-19.00 h, 22-24°C), were used in these experiments. Three days post partum, litters were reduced to eight p u p s / cage. Weaning took place on day 21. Thereafter, animals (three per cage) had free access to laboratory chow and tap water. Cycling young adult animals (controlled by daily vaginal smears) were studied in the diestrous phase. Preparation of tissue samples. Young animals aged 15, 20, 25, 30, 35 and 40 days, as well as adult diestrous rats (8-10 animals/group), were decapitated at noon. Each brain was rapidly dissected within 30 s and frozen on dry ice. Coronal slices (500 p~rn thick in 15-, 20- and 25-day-old rats, 600 tzm thick in the other groups) were cut on a cryostate and the investigated regions (preoptic area or P O A and posterior mediobasal hypothalamus or MBH, including the n. arcuatus and the median eminence) were punched out using 0.7 m m i.d. needles, according to the stereotaxic atlas of de Grootll; in rats aged

70

Glu

HCA

o

6oo

5o

3

.0 U

0

O~ 200 m 0 30 300

10

0

200

0

600

Tau

600

400

400

200

200

v

0

0 C~ --~ 1 0 0 0

i

Age (days) Fig. 1. Preoptic area concentrations of neurally active amino acids evaluated in 15-40 day-old female rats and in adult diestrous (D) animals. Asp, aspartate; Glu, glutamate; HCA, homocysteic acid; GIy, glycine; Tau, taurine; G A B A , y-aminobutyric acid. a, p < 0.05 vs. 15-day-old rats; b, p < 0.05 vs. 20-day-old rats; c, p < 0.05 vs. 25-day-old rats; d p < 0.05 vs. 30-day-old rats; e, p < 0.05 vs. 35-day-old rats; f, P < 0.05 vs. 40-day-old rats.

185 40 days or less, the anatomical description of the developing rat brain by Sherwood and Timiras 37 was used as reference. Tissue samples were homogenized in 0.1 M acetic acid and centrifuged (12,000 g, 5 min); the supernatant was kept frozen at -30°C until determination of neurotransmitters. Analytics. The concentrations of aspartate (Asp), glutamate (Glu), homocysteic acid (HCA), taurine (Tau) glycine (Gly) and GABA were estimated after derivatization with phenyl isothiocyanate by reverse-phase HPLC separation and UV detection at 254 nm, as previously described 16. The mean inter- and intraassay coefficients of variation for the measured amino acids varied between 4.0% and 8.0%; the detection limit was 20 pmol for Asp and Glu, 30 pmol for HCA and Gly and 50 pmol for Tau and GABA. Protein concentrations of the tissue samples were measured according to Lowry et al. zt ' Statistical eraluation. Unless otherwise stated, results are presented as mean values + S.E.M. Differences between age groups were tested for statistical significance using a one way analysis of variance and Tukey's multiple comparison test 41.

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RESULTS

Preoptic area The

acid

preoptic

concentrations

measured

area samples obtained

in t h e

from imma-

t u r e a n d a d u l t d i e s t r o u s r a t s a r e d e p i c t e d in Fig. 1; t h e most relevant changes can be summarized

as f o l l o w s :

Asp content showed a transitory, insignificant elevation at day

20, as c o m p a r e d

concentrations

to day

15; G l u

and

HCA

also rose transiently, but significantly at

d a y 20 ( P < 0.05 vs. d a y 15); a t d a y 30, H C A , A s p a n d G l u l e v e l s in t h e P O A measured

at

were not different from those

15 d a y s o f age.

content of these EAA

80

600

Asp

amino

punched

At

d a y 35, t h e

POA

increased significantly; elevated

-

"-~

HCA

~" ¢.) m

u 60 m

o

"~ I

400

200

40

0

E 200 20

0

200

600

Gly

600

Tau

GABA

o • --

o

u d ~~

400

400 d

100

200

0

200

0 ~omo~o ~ i~4 N ¢9 ¢0 ,~ a

0 ~ o ~ o ~ o P c~l N ~

Q

~omo~o

a

Age (days) Fig. 2. Mediobasal hypothalamic concentrations of neurally active amino acids evaluated in 15-40 day-old female rats and in adult diestrous (D) animals. Asp, aspartate; Glu, glutamate; HCA, homocysteic acid; Gly, glycine; Tau, taurine; GABA, y-aminobutyric acid. ", P < 0.05 vs. 15-day-old rats; b p < 0.05 vs. 20-day-old rats; c, p < 0.05 vs. 25-day-old rats; d, p < 0.05 vs. 30-day-old rats; ~', P < 0.05 vs. 35-day-old rats; f, P < 0.05 vs. 40-day-old rats.

18(1

levels persisted at 40 days of age. Adult diestrous rats showed similar Glu and Asp concentrations, but significantly (P < 0.05) lower HCA concentrations, as compared to 35- and 40-day-old animals. Fig. 1 also shows the age-related changes of Tau, Gly and GABA content observed in the POA. Tau levels decreased ( P < 0 . 0 5 ) from day 25 onwards, reaching lowest concentrations at day 30. No further changes were detected thereafter. Conversely, POA GABA concentrations rose significantly ( P < 0.05) as early as day 20; 40-day-old rats showed even higher GABA levels and these increased further in adult diestrous rats. POA Gly levels rose transiently (P < 0.05) in 20-day-old-rats; 40-day-old and adult rats showed significantly higher (P < 0.05) Gly levels as compared to immature animals.

Mediobasal hypothalamus Fig. 2 reflects the amino acid concentrations measured in the MBH. A slight (insignificant) increase in Asp, Glu and HCA concentrations was detected at the end of sexual maturation (days 35-40). In adult animals, Asp, Glu and HCA concentrations were significantly higher ( P < 0.05), as compared to immature rats. In these region again, Tau levels were significantly (P < 0.05) higher in the younger animals (15-25-days old), as compared to the peripubertal rats. Adult diestrous rats showed higher ( P < 0.05 vs. 15-35-day-old animals) MBH GABA levels. MBH Gly levels rose slightly (P <0.05 vs. 15- and 25-day-old animals) in 40-day-old-rats; adult diestrous rats showed significantly higher (P < 0.05) Gly levels as compared to the immature animals. DISCUSSION It is not possible, by the methodology employed in these experiments, to distinguish between metabolic and transmitter amino acid pools. This fact should be considered when interpreting our results, showing significant ontogenic differences in the amino acid concentrations in two hypothalamic areas of the female rat, known to play a role in the regulation of LH release: (1) The preoptic amino acid content displays significant age-related changes in immature female rats. EAA (Glu, Asp, HCA) levels rise transiently at day 20-25 and then increase consistently from day 35 onwards, whereas the concentrations of Tau decrease markedly by day 30. Gly levels also rise at day 20 and 40. GABA levels are very low in 15-day-old rats, as compared to the other age groups.

(2) In the mediobasal hypothalamus, EAA, Gly and GABA concentrations rise significantly only in mature animals and the decrease in Tau concentrations occurs in peripubertal rats aged 35 days. As far as we are aware, information concerning ontogenic changes in the hypothalamic concentration of AANT is scarce. Previous publications '~'2~' reported significant ( ~ 30%) increases in hypothalamic Asp and Glu content and marked (---50%) decreases in hypothalamic GABA content in 30-day-old female rats, as compared to 15-day-old pups. However, these investigators measured AANT concentrations in homogenates of hypothalamic fragments containing large amounts of tissue other than arcuate nucleus, eminentia mediana and POA. In the present study we demonstrate clearly that regional differences exist in the ontogenetic pattern of AANT content; such differences might account for the observed discrepancies. There is now conclusive evidence that EAA are involved in the positive feedback effect exerted by estradiol on LH release 7'16'2°. The hypothalamus of the immature rat becomes fully responsive to the positive feedback effect of estradiol between postnatal days 21-22 s'35. The increased Glu and HCA concentrations measured in the POA at day 20-25 are temporally, and perhaps causally related to this relevant neuroendocrine event. Several studies have indicated that EAA are involved in neuronal growth and synaptogenesis, including processes of development and stabilization of neuronal connections (for review, see ref. 24). Significant ontogenic alterations in the activity of EAA have been detected by binding and autoradiographic studies in cortical and subcortical structuresa'34'3e"4°; important increases in local EAA activity seem to be associated with periods of heightened synaptic plasticity 4'24'4°. In the rat, the period around days 20-25 of postnatal life represents a crucial phase in sexual development, characterized by important neuroendocrine changes, e.g. the maturation of the positive feedback effect of ovarian steroids on LH secretion s'35 and qualitative as well as quantitative modifications in the effect of different neurotransmitters on LH release 225 27. It might therefore be hypothesized that the early increase in Glu and HCA levels (20-25 days) detected in the POA could be related to a neuroendocrine reorganization process of the hypothalamus that results in the acquisition of the 'adult' type of control of gonadotropin secretion by gonadal steroids and hypothalamic neurotransmitters. As demonstrated before, the administration of NMDA-receptor antagonists delay the time of vaginal opening in the ratZ2'42'46; present experiments show that the hypothalamic content of Glu and Asp, endoge-

187 nous l i g a n d s to t h e N M D A r e c e p t o r , i n c r e a s e p r i o r to a n d at the t i m e of p u b e r t y . T h e s e n e u r o c h e m i c a l m o d i fications might be decisive for initiating the first p r e ovulatory surge. T h e s a m e a r g u m e n t a p p l i e s to the o b s e r v e d i n c r e a s e in H C A levels. It was s u g g e s t e d that this a m i n o acid r e p r e s e n t s an e n d o g e n o u s ligand to N M D A r e c e p t o r s ~2 a n d t h a t it s t i m u l a t e s L H r e l e a s e 32, I n t e r e s t i n g l y , P O A H C A c o n c e n t r a t i o n s a r e h i g h e r in 35- a n d 40-day-old rats, as c o m p a r e d to those m e a s u r e d in a d u l t rats; this w o u l d suggest that an excitatory o v e r d r i v e is n e c e s s a r y d u r i n g the last days o f sexual d e v e l o p m e n t , p e r h a p s in o r d e r to o v e r c o m e a previously existing inhibitory ( o p i a t e r g i c ? , d o p a m i n e r g i c ? ) t o n e on L H s e c r e t i o n ~'s. It was shown e a r l i e r that T a u , a p u t a t i v e i n h i b i t o r y a m i n o acid n e u r o t r a n s m i t t e r 28, d e c r e a s e s L H r e l e a s e in a d u l t rats 33. T h e o b s e r v e d fall in T a u c o n c e n t r a t i o n s m a y thus r e p r e s e n t a r e d u c t i o n of a h y p o t h a l a m i c inhibition acting on L H R H n e u r o n s , l e a d i n g to the first p r e o v u l a t o r y L H surge. A s d e m o n s t r a t e d r e c e n t l y 26, the G A B A e r g i c c o n t r o l of g o n a d o t r o p i n s e c r e t i o n c h a n g e s d u r i n g sexual develo p m e n t in the f e m a l e rat. A c c o r d i n g to t h e s e results, G A B A w o u l d exert a s t i m u l a t o r y t o n e on L H r e l e a s e in p r e p u b e r t a l a n d an inhibitory o n e in p e r i p u b e r t a l rats. T h e h i g h e r p r e o p t i c G A B A c o n t e n t o b s e r v e d in 2 0 - 3 0 d a y - o l d a n i m a l s could c o n t r i b u t e to t h e relative inhibition o f L H R H r e l e a s e o b s e r v e d in p e r i p u b e r t a l a n i m a l s ~5. In a d u l t rats p r e o p t i c G A B A e r g i c activity is high d u r i n g d i e s t r u s 23, a n d G A B A r e l e a s e r a t e s a r e m a r k e d l y r e d u c e d p r i o r to a L H episode~6'~s; consequently, it has b e e n p o s t u l a t e d that the synchronic d e s i n h i b i t i o n of L H R H n e u r o n s by a d e c r e a s e in G A B A r e l e a s e r a t e s results in p u l s a t i l e L H R H 17 release. C h a n g e s in h y p o t h a l a m i c E A A levels a r e p a r a l l e l e d by f l u c t u a t i o n s in Gly c o n t e n t . T h e role of this a m i n o acid as a n e u r o t r a n s m i t t e r has b e e n q u e s t i o n e d by the fact that b a s a l Gly r e l e a s e is not e n h a n c e d by the a d d i t i o n of KC139; besides, t h e i n t r a v e n t r i c u l a r a d m i n istration o f this a m i n o acid has no effect on L H secretion 3°. H o w e v e r , this a m i n o acid strongly p o t e n t i a t e s the d e p o l a r i z a t i o n i n d u c e d by N M D A in c u l t u r e d m o u s e b r a i n n e u r o n s , acting p r o b a b l y at an allosteric r e g u l a t o r y site ~9. H e n c e , Gly c o u l d m a g n i f y t h e signals e v o k e d by t h e f l u c t u a t i o n s in E A A activity, as far as these a r e m e d i a t e d by N M D A r e c e p t o r s . A c c o r d i n g to o u r e x p e r i m e n t s , m o s t r e l e v a n t a g e - r e l a t e d m o d i f i c a t i o n s in A A N T levels t a k e p l a c e in the P O A a n d few in the M B H . P u b l i c a t i o n s c o n c e r n i n g t h e p r e c i s e h y p o t h a l a m i c site of action of t h e A A N T a r e n e i t h e r a b u n d a n t nor conclusive. T h e injection o f N M D A into the P O A , w h e r e the vast m a j o r i t y of

L H R H n e u r o n s are l o c a t e d 4~, but not into the M B H , results in e n h a n c e d L H s e c r e t i o n -~. T h e e s t r a d i o l - i n d u c e d L H p e a k is p r e c e d e d by an i n c r e a s e in G l u and A s p r e l e a s e in the P O A , as shown r e c e n t l y by p u s h - p u l l e x p e r i m e n t s ~. H o w e v e r , d i f f e r e n t investigators, using p e r i f u s e d or i n c u b a t e d h y p o t h a l a m i c e x p l a n t s 5'j3, have d e m o n s t r a t e d that N M D A a n d o t h e r g l u t a m a t e r e c e p tor agonists e n h a n c e L H R H r e l e a s e from r e t r o c h i a s m a t i c h y p o t h a l a m i c f r a g m e n t s , suggesting that E A A can act on L H R H t e r m i n a l s . In conclusion, d e v e l o p i n g f e m a l e rats display relevant c h a n g e s in the h y p o t h a l a m i c c o n c e n t r a t i o n s of a m i n o acid n e u r o t r a n s m i t t e r s . T h e p r e o p t i c a r e a shows the most significant o n t o g e n i c changes: a b i m o d a l inc r e a s e in the c o n c e n t r a t i o n s of excitatory a m i n o acids (Glu, A s p a n d HCA)~ an a g e - r e l a t e d i n c r e m e n t in G A B A c o n t e n t and a striking d e c r e a s e in T a u levels. T h e i n c r e a s e d levels of A s p , G l u a n d H C A could then c o n t r i b u t e to sexual m a t u r a t i o n as a result of their s y n a p t o g e n i c p r o p e r t i e s a n d / o r acting directly on the L H R H n e u r o n s , s t i m u l a t i n g L H R H release. T h e possible role of T a u in the n e u r o e n d o c r i n e events l e a d i n g to sexual m a t u r a t i o n d e s e r v e s f u r t h e r investigation. Supported by the Volkswagen Foundation, Germany. P.A. is a fellow of the Alexander wm 11umboldt Foundation.

Acknowledgements.

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