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Neurobiological Mechanisms of the Onset of Puberty in Primates¹

Department of Pediatrics (E.T.), Wisconsin Regional Primate Research Center (E.T., D.L.F.), and Center for Neuroscience (E.T.), University of Wisconsin-Madison, Madison, Wisconsin 53715-1299

	Abstract

Top Abstract I. Introduction II. Description of Puberty III. When Does the... IV. What Determines the... V. Neurobiology of Puberty VI. Master Gene(s) Controlling... VII. Summary and Conclusions References

 
An increase in pulsatile release of LHRH is essential for the onset of puberty. However, the mechanism controlling the pubertal increase in LHRH release is still unclear. In primates the LHRH neurosecretory system is already active during the neonatal period but subsequently enters a dormant state in the juvenile/prepubertal period. Neither gonadal steroid hormones nor the absence of facilitatory neuronal inputs to LHRH neurons is responsible for the low levels of LHRH release before the onset of puberty in primates. Recent studies suggest that during the prepubertal period an inhibitory neuronal system suppresses LHRH release and that during the subsequent maturation of the hypothalamus this prepubertal inhibition is removed, allowing the adult pattern of pulsatile LHRH release. In fact, -aminobutyric acid (GABA) appears to be an inhibitory neurotransmitter responsible for restricting LHRH release before the onset of puberty in female rhesus monkeys. In addition, it appears that the reduction in tonic GABA inhibition allows an increase in the release of glutamate as well as other neurotransmitters, which contributes to the increase in pubertal LHRH release. In this review, developmental changes in several neurotransmitter systems controlling pulsatile LHRH release are extensively reviewed.

I. Introduction

II. Description of Puberty

III. When Does the LHRH Neuronal System Mature?

A. Embryonic and fetal periods

B. Postnatal maturation

IV. What Determines the Timing of Puberty?

A. Hormonal changes during the neonatal and juvenile periods

B. Hormonal changes at the onset of and during puberty

C. Positive feedback effects of estrogen on the LH surge

D. Two hypotheses on the mechanism of the onset of puberty

E. Species differences in the mechanism of the onset of puberty

V. Neurobiology of Puberty

A. Inhibitory neurotransmitters

B. Stimulatory neurotransmitters

C. Role of glia and growth factors

D. Nutritional and metabolic factors

VI. Master Gene(s) Controlling the Onset of Puberty

VII. Summary and Conclusion

	I. Introduction

Top Abstract I. Introduction II. Description of Puberty III. When Does the... IV. What Determines the... V. Neurobiology of Puberty VI. Master Gene(s) Controlling... VII. Summary and Conclusions References

 
PUBERTY is defined as the transient period between childhood and adulthood during which reproductive function is attained. During this period the secondary sexual characteristics appear, the adolescent growth spurt occurs, the gonads start to produce mature gametes (sperm or oocytes) capable of fertilization, and major psychological changes occur (1). In the last two decades, it has become apparent that puberty begins when the pulsatile release of LH releasing hormone (LHRH, also called GnRH) increases. The hypothalamic decapeptide, LHRH, stimulates pituitary release of LH and FSH, which are essential for the production of mature gametes and gonadal steroid secretion. Thus, the question of "what triggers the onset of puberty?" becomes rather the question of "what triggers the pubertal increase in LHRH release?" Since control of pulsatile LHRH release occurs within the hypothalamus, the answer to the latter question requires a neurobiological approach in animal models. Although many excellent reviews on the mechanism of the onset of puberty are available (2, 3, 4, 5, 6), in this article we will examine the mechanism of puberty from a neurobiological perspective with an emphasis on its developmental aspect. Our discussion will focus on events in the nonhuman primate as a model for humans, but we will review data from other species, particularly from the rat, a common laboratory rodent.

Puberty is also associated with several neurological and reproductive disorders. For example, it has been documented that the onset of schizophrenia occurs during late puberty to early young adulthood (7), precocious puberty is often associated with epilepsy in children (8, 9), and the new onset of epileptic seizures tends to occur early in life as well as during the adolescent period (10). In addition, taking into consideration the recent view that polycystic ovarian syndrome originates during the pubertal stage (11, 12), it is possible that an irregularity of the normal control of pulsatile LHRH release during puberty may be a cause of the polycystic ovarian syndrome, although this is highly speculative at this time. Therefore, it is our hope that this article will be helpful for understanding disorders in humans, including precocious puberty and delayed puberty, and the neuropathogenesis of the clinical disorders associated with puberty.

	II. Description of Puberty

Top Abstract I. Introduction II. Description of Puberty III. When Does the... IV. What Determines the... V. Neurobiology of Puberty VI. Master Gene(s) Controlling... VII. Summary and Conclusions References

 
Physical and hormonal changes associated with puberty have been used as signs of sexual maturation. First appearance of pubic hair and enlargement of the breasts occurs at 8–10 yr of age (2, 13, 14). Pubic and axillary hair growth is primarily due to a pubertal increase in adrenal androgen (adrenarche), which occurs independently from pituitary-gonadal maturation [gonadarche (15)], whereas breast development is due to an increase in ovarian estrogens (2). Subsequently, menarche occurs at an average age of 11–13 yr due to a further increase in ovarian estrogens (2, 13, 14). Based on development of the breasts and pubic hair, Tanner (16) has developed standards for puberty in girls. Because an increase in estrogen during the early stage of puberty stimulates epiphyseal growth, accelerated growth (the pubertal increase in height velocity) is the first sign of puberty in individual girls (2).

In boys, an increase in testicular size occurs at 9.5–13.5 yr (average 12 yr) of age (17), which is followed by the growth of pubic hair and of the penis (16). Testicular growth is due to a thickening of the seminiferous tubular lining, the formation of a lumen, and the differentiation and growth of Sertoli and Leydig cells, resulting in a volume increase in the Leydig cells. Tanner (16) also has established standards for pubertal development in boys.

In the female rhesus monkey, the first physical signs of puberty are a slight increase in growth velocity and nipple size, and development of perineal sex-skin color, which occurs at 25–28 months of age (18). The increase in nipple size and perineal sex skin development further progresses, and menarche occurs at 28–32 months of age. Several episodes of menstrual cycles occur after menarche (18) without ovulation, and first ovulation occurs at 42–50 months of age (18, 19, 20, 21, 22). After first ovulation, the ovulatory cycles in the rhesus monkey often exhibit a short luteal phase. Previously, we designated the period before any signs of puberty as the prepubertal stage, the period between first signs of puberty and menarche as the early pubertal stage, the period between menarche and first ovulation as the midpubertal stage, and the period between first ovulation and full maturity as the late pubertal stage (18). In female rats, vaginal opening, which occurs around postnatal (P) day 35–37 (P35-P37), and which is usually accompanied by the first ovulation, is a marker of puberty. However, because vaginal opening often occurs without first ovulation under experimental conditions, direct observations of ova in oviducts or corpora lutea formation on the ovary ensure the documentation of true puberty.

In male rhesus monkeys testicular descent into the scrotum occurs slightly after 30 months of age. Subsequently, testicular volume starts to increase. This increase continues until approximately 48 months of age, when full spermatogenesis is observed (3). Male rats become fertile at P42–P45, which is accounted as the time of puberty (4).

	III. When Does the LHRH Neuronal System Mature?

Top Abstract I. Introduction II. Description of Puberty III. When Does the... IV. What Determines the... V. Neurobiology of Puberty VI. Master Gene(s) Controlling... VII. Summary and Conclusions References

 
A. Embryonic and fetal periods
Studies in the mouse (23, 24), rhesus monkey (25, 26), and human (27) indicate that LHRH neurons originate from the olfactory epithelium during the gestational period. In the rhesus monkey, LHRH cells are found in the olfactory pit as early as at embryonic (E) day 32 (E32) and commonly at E34–E36 (Refs. 25, 26, 28 and Fig. 1). LHRH cells migrate along the nasal septum and terminal nerve, enter the forebrain at E38 (26), and subsequently migrate into the basal hypothalamus of the rhesus monkey by E47 (25). The basic distribution pattern of LHRH neurons in the brain is already established at E55 (25, 26, 29), although LHRH cell migration into the preoptic area (POA) and hypothalamus continues after E55 until the last trimester. The gonadotropes are found in the pituitary at E50 (25), and sex-specific gonadal steroids are detectable in the umbilical cord at E70 (30), suggesting that LHRH cells are functioning at E50–E70. However, activity of LHRH neurons may not be high until close to term; pro-LHRH mRNA is detectable in LHRH cells at E38–E50, but does not increase significantly until E135 (25). Gonadectomy at E98–E104 results in the elevation of LH and FSH in male monkeys, but not in female monkeys (31), indicating that the negative feedback system is operative in the male hypothalamo-pituitary-gonadal axis during the second trimester.

View larger version (19K): [in this window] [in a new window]   Figure 1. Ontogeny of the LHRH-pituitary-gonadal system before birth until after the onset of puberty in female rhesus monkeys (top) and in female humans (bottom). Days and months are used as the scale for the gestational period and after birth in monkeys, respectively, whereas weeks and years are used as the scale in humans. The age of events indicated in this figure is approximate.

FSH and LH are detectable in the human pituitary by E week 10, and their content increases until E week 25–29. The pituitary starts to release gonadotropins into the general circulation by E week 11–12. Circulating gonadotropins reach peak levels at midgestation, and subsequently both LH and FSH levels decline during late gestation (34, 35, 36, 37). The gonadotropes in human fetuses respond to LHRH by releasing LH and FSH both in vivo and in vitro (38, 39, 40, 41). A sex difference in gonadotropin levels is seen during midgestation: pituitary content and circulating concentrations of LH and FSH in female fetuses are higher than those in male fetuses (34, 42, 43, 44). Since circulating testosterone levels are high in male fetuses as compared with circulating estrogen levels in female fetuses during midgestation, both the sex difference in gonadotropin levels and the decrease in gonadotropin levels toward late gestation in fetuses are attributed to the development of the negative feedback mechanism by the gonadal steroid hormones from the fetal gonads as well as from the placenta (2). A report that castration in male rhesus monkeys at E98–104 increases circulating gonadotropins to levels as high as those in female monkeys at similar ages (30, 31) supports this notion. Negative feedback by ovarian steroids is operative in the human female fetus during late gestation when estrogen secretion is elevated (32, 34). In sheep fetuses during late gestation, LH release is pulsatile, and orchidectomy in males, but not ovariectomy in females, results in an increase in the pulse amplitude of LH (45).

B. Postnatal maturation
Although the distribution pattern of LHRH neurons is already established before birth in most species in which it has been studied, the function, morphology, and biosynthesis, as well as synaptic connectivity of LHRH neurons, may not be mature until the time of puberty. In rats it has been shown that the number of LHRH neurons with a wrinkled contour increases, while the number of LHRH neurons with a smooth contour decreases at the age of puberty (46). Further, in rats and mice, LHRH mRNA levels increase gradually with age, and significant increases occur at P15–P30 depending on sex and experimental conditions (47, 48, 49, 50, 51). These observations imply that LHRH neurons in rodents may receive more intensive innervation from other neurons, and that LHRH gene expression matures after birth, but before puberty. In contrast, in primates, available data indicate that LHRH neurons are mature well before the onset of puberty: there are no differences in the number (52) or the shape of LHRH neurons (53) or in the LHRH mRNA levels (54) between juvenile and adult monkeys. Further, we have shown that electrical stimulation of the medial basal hypothalamus (MBH) in prepubertal monkeys, in which basal LHRH release is very low, induces LHRH release similar to that observed in pubertal monkeys (55). However, it is possible that LHRH neurons in primates undergo subtle developmental changes during the juvenile period, as shown by two recent reports: 1) perikarya of LHRH neurons located in the MBH of early pubertal female monkeys are invested with more glial sheathes than in adult cyclic female monkeys (56); and 2) a decrease in the area occupied by synapses onto LHRH neurons occurs in adult castrated male monkeys as compared with prepubertal castrated male monkeys (57), indicating that subtle changes may occur in the innervation pattern of LHRH neurons. Nonetheless, it is problematic to discuss the significance of these discrepancies, because ontogenic changes in morphology and gene expression of LHRH neurons in primates have not been systematically studied, and in both rodents and primates the issue as to how the presence or absence of gonadal steroids affects ontogeny of morphology and gene expression of LHRH neurons has not been assessed.

	IV. What Determines the Timing of Puberty?

Top Abstract I. Introduction II. Description of Puberty III. When Does the... IV. What Determines the... V. Neurobiology of Puberty VI. Master Gene(s) Controlling... VII. Summary and Conclusions References

 
A. Hormonal changes during the neonatal and juvenile periods
Circulating LH levels in human male neonates abruptly increase within the first few minutes after birth, followed by an increase in serum concentrations of testosterone during the first 3–21 h (58). High levels of LH in the human male infant decline within 6 months and remain low until the beginning of puberty. FSH levels in human males are slightly elevated for the first 3 postnatal months, after which they become low (59, 60). Circulating levels of testosterone are also elevated for 2–4 months postnatally (61). In contrast, in female neonates LH levels are only slightly elevated during the first few months of life, but FSH levels are high for the first 5 months (59, 60). After the first 6 months of life, circulating levels of FSH, LH, and gonadal steroids are all low, and the hypothalamo-pituitary-gonadal system enters a quiescent stage until the time of puberty. Plasma concentrations of LH and FSH in infants with gonadal dysgenesis are strikingly elevated, because of the absence of the negative feedback loop (59, 60, 62, 63). However, the elevated level of gonadotropin in patients with gonadal dysgenesis declines during the juvenile period, as seen in eugonadal children.

In infantile male monkeys, circulating LH and testosterone are elevated during the first 2–3 postnatal months (64, 65), and diurnal variation of testosterone, quantitatively similar to that seen in sexually mature adults, is observed (66). Bilateral orchidectomy at 1 week of age results in an increase in LH and FSH secretion, with a pulse pattern very similar to that in castrated adult males (67). Plant (68) suggests that the hypothalamo-pituitary axis governing testicular function in males is fully mature by the neonatal stage of development. Both LH and FSH levels in male monkeys decrease after 3–4 months of age (69, 70). By contrast, in female monkeys circulating LH is only slightly elevated or not elevated during the first 3 months (65), and the negative feedback mechanism appears to be only partially operative in the late gestational period through the neonatal period. A moderate elevation of estrogen levels is observed during late gestation through the neonatal period in females (65). Ovariectomy in females at 1 week of age induces a truncated and abbreviated elevation of LH release with slower pulse frequency, when compared with those in orchidectomized male infants (71). It is possible that the LHRH neurosecretory system in female monkeys during the neonatal period is less mature than in males (71). Alternatively, in females, central inhibition to the LHRH neurosecretory system (see below) may start earlier than in males and may not be as complete as in males. Nevertheless, at 4–6 months of age, the LHRH neurosecretory system in both sexes enters a quiescent state, which persists until the time of puberty (18, 68, 70, 72).

B. Hormonal changes at the onset of and during puberty
Before the onset of puberty, LH and FSH levels are low, but a highly sensitive assay indicates that circulating LH and FSH levels in prepubertal children are pulsatile, with slightly higher values at night than morning (73, 74, 75, 76, 77, 78, 79, 80). In both boys and girls, preceding the physical signs of puberty, LH and FSH levels become elevated, pulsatility of these hormones becomes more pronounced, and the nocturnal increase in gonadotropin release is enhanced (75, 76, 77, 78, 81, 82). Both pulse frequency and amplitude of LH release increase at this stage as well (77, 78).

Similarly, the first hormonal sign of puberty in female rhesus monkeys appears several months earlier than menarche (18, 83). During the early pubertal period, mean FSH levels increase, followed by an increase in mean LH levels. Basal levels of LH and the amplitude and frequency of pulsatile LH release increase, and a circadian fluctuation (nocturnal increase) of LH emerges. The pubertal increase in gonadotropin release stimulates estrogen secretion from the ovary, resulting in nipple growth, sex skin development, and subsequently menarche (18, 83). Due to an increase in circulating estrogen, FSH levels are suppressed. During the midpubertal period, the nocturnal increase in LH becomes more pronounced, and basal LH levels and the amplitude of LH pulses further increase. A periodic increase in estrogen causes menstrual cycles, although data with laparoscopy indicate that periodic vaginal bleeding is not due to cyclic ovulation (83). Circulating estrogen during this developmental period sometimes reaches the preovulatory level, but there is no LH surge until the time of first ovulation (18). This is called pubescent infertility. After the age of first ovulation, both basal LH levels and LH pulse amplitude decline, and the nocturnal increase in LH release disappears (18).

These changes in the LH release pattern during puberty are the result of developmental changes in pulsatile LHRH release, since pulsatile LHRH release also increases (84). The pulse frequency of LHRH release increases at the onset of puberty and remains elevated throughout the pubertal period (Refs. 84, 85 and Fig. 2), while the pulse amplitude of LHRH release starts to increase at the transition period between prepuberty and puberty and continues to increase throughout puberty until first ovulation occurs (84, 85). The nocturnal increase in LHRH release is readily observed during the early pubertal period and becomes increasingly prominent during the midpubertal period, while during the prepubertal period, nocturnal increases in LHRH release are seldom seen (84, 85). These facts indicate that the hormonal profiles appear as gradual changes throughout puberty and that the development of the LHRH neurosecretory system or its regulatory systems is essential for the onset of puberty.

View larger version (24K): [in this window] [in a new window]   Figure 2. Schematic illustration of postnatal changes in LHRH release and neurotransmitter events that occur in the hypothalamus in association with puberty in nonhuman female primates. The LHRH neurosecretory system appears to be active during the infantile period, but is suppressed by an inhibitory mechanism comprising GABA neurons. At the onset of puberty, a reduction in GABA inhibition and a subsequent increase in glutamate excitation of LHRH neurons result in the pubertal increase in the pulse frequency, pulse amplitude, and baseline levels of LHRH, which trigger puberty. Furthermore, a higher nocturnal level of LHRH release, shown by hatched bars, becomes particularly prominent. After the onset of puberty, other stimulatory neurotransmitters such as NPY and NE take part in control of LHRH neurons, resulting in further increases in the pulse amplitude and baseline level of LHRH release, until the time of first ovulation. The nocturnal increase in LHRH release and LHRH pulse amplitude are reduced to the adult level, perhaps due to participation of inhibitory neurotransmitters, such as opioids. Stippled bars indicate morning levels of LHRH. In males the LHRH neurosecretory system is fully mature during the infantile period, but is suppressed completely during the juvenile period. Recently, it has been hypothesized that NPY is an inhibitory neurotransmitter before puberty in males. (See text for details.)

There is a controversy as to the sites of action of estrogen in nonhuman primates. In a series of studies in adult female monkeys, Knobil and colleagues (104, 105, 106, 107, 108, 109, 110) have shown that the estrogen-induced LH surge does not require the hypothalamus other than as a supply source of pulsatile LHRH release: 1) the positive feedback effects of estrogen occur in monkeys with complete deafferentation or isolation of the MBH (105, 106); 2) whereas lesions of the MBH abolish LH surges (107); 3) pulsatile infusion of LHRH into animals with lesions in the MBH is sufficient to result in the estrogen-induced LH surge (108); and 4) estrogen can induce an LH surge in animals with lesions in the MBH up to 48 h after cessation of pulsatile LHRH infusion (109, 110). In support of the pituitary site of estrogen action in primates, Ferin et al. (111) report that isolation of the pituitary gland from the hypothalamus by stalk section with a Silastic barrier in monkeys fails to block the estrogen-induced LH surge. In contrast, Spies and colleagues (112) report that the hypothalamus is essential for estrogen action in monkeys, based on the observation that stalk section with a Teflon barrier blocks the estrogen-induced LH surge (112), and the estrogen-induced LH surge or the preovulatory LH surge is accompanied by an increase in pulsatile LHRH release (113, 114). The estrogeninduced LHRH increase has also been reported by another group in OVX monkeys (115) as well as by us in pubertal monkeys (99). Without going into further detail on this controversy, it is safe to conclude that, in the rhesus monkey, estrogen stimulates an increase in LHRH release from the hypothalamus, but estrogen also stimulates an LH surge directly from the gonadotropes. Nonetheless, stimulation of pulsatile LHRH release is essential for the functional gonadotropes, and an increase in pulsatile LHRH release triggers puberty; the establishment of the neuronal mechanism responsible for the adult type of pulsatile LHRH release is obligatory to the positive feedback effect of estrogen during the pubertal period.

D. Two hypotheses on the mechanism of the onset of puberty
Two hypotheses have been proposed for the mechanism of the onset of puberty. The first one is commonly called the "gonadostat" hypothesis, or "differential sensitivity to ovarian steroids" hypothesis. More than a half-century ago, this hypothesis was proposed by Dohrn and Hohlweg (117), based on the observation that, compared with adults, in immature rats a smaller dose of estrogen can suppress the castration-induced changes in the gonadotropes. According to this hypothesis, puberty occurs when the regulating system for gonadotropin secretion becomes desensitized to steroid feedback during sexual maturation, and the shift in sensitivity to steroids permits gonadotropin secretion. It has been shown that in rats and sheep, "escape of suppressed LH secretion by gonadal steroids" occurs at the onset of puberty (118, 119), although Ojeda and colleagues (120) argue that the shift in the sensitivity to gonadal steroids does not occur until after the first proestrous surge in rats. Escape from the negative feedback effect of estrogen occurs in monkeys during the midpubertal stage, but not at the onset of puberty (121, 122). Thus, the hypothesis is not applicable to primates (see below), but it appears to be applicable to sheep (see Ref. 5).

The second hypothesis is that sexual quiescence before the onset of puberty is due to central inhibition of LHRH release, independent of the negative feedback of gonadal steroids, and that an increase in pulsatile LHRH release triggers puberty (18, 123, 124). This hypothesis is based on the fact that gonadotropin secretion in gonadectomized monkeys and in human subjects with gonadal dysgenesis is elevated during the neonatal period, but subsequently suppressed until the time of puberty. This hypothesis has been supported by the direct measurement of LHRH in the stalk-median eminence (S-ME) of rhesus monkeys using a push-pull perfusion method. For example, ovariectomy of prepubertal female monkeys fails to stimulate pulsatile LHRH release, whereas ovariectomy of early and midpubertal female monkeys induces the postcastration-induced LHRH release (85). The age of the pubertal increase in LHRH release in OVX monkeys is essentially similar to that in ovarian intact counterparts (85), indicating that the pubertal increase in LHRH release is independent of the ovarian steroids. Therefore, maturational changes in the hypothalamic mechanism that controls pulsatile LHRH release, independent of the negative steroid feedback mechanism, are responsible for the onset of puberty. In addition, estrogen injection in gonadectomized females of pubertal age readily suppresses pulsatile LHRH release, whereas the same treatment does not cause any significant change in LHRH release in gonadectomized females of prepubertal age (125), suggesting that the LHRH neurosecretory system is insensitive to negative feedback effects of estrogen before the onset of puberty. In fact, the LHRH neurosecretory system increases its sensitivity to ovarian steroid feedback after the onset of puberty.

The gonadostat hypothesis is, in part, a historical product of the time during which the brain-pituitary unit had been considered as a "black box." The pubertal increase in release of LH and LHRH can occur independent of the presence or absence of gonadal steroids, as seen in rhesus monkeys. The pubertal increase in LH release can also be influenced by the presence of gonadal steroids, as shown in sheep, although the degree to which the pubertal increase in LHRH release is influenced by the gonadal steroids has not been quantitatively assessed. Perhaps, the term "gonadal steroid-dependent LHRH increase," first introduced by Reiter and Grumbach (123), reflects the gonadostat hypothesis most accurately.

In humans, Grumbach and colleagues (2, 123) suggest that a gonadal steroid-dependent LHRH increase also occurs at the onset of puberty, since smaller amounts of gonadal steroids are effective in suppressing FSH and LH levels in prepubertal children than in adults (126, 127). It is possible during the juvenile period in humans that a small amount of LHRH release from the hypothalamus maintains the minimum levels of gonadotropin secretion, which is susceptible to the negative feedback effect of steroid hormones. In contrast, we observed no changes in either LH or LHRH suppression after estrogen treatments in OVX prepubertal monkeys (99, 128). Therefore, the difference in humans and rhesus monkeys can be attributed to a subtle species difference in the mechanism of the onset of puberty.

E. Species differences in the mechanism of the onset of puberty
An increase in pulsatile LHRH release is essential for the pubertal increase in gonadotropin secretion and subsequent gonadal stimulation. A pulsatile infusion of LHRH into sexually immature monkeys (129) and guinea pigs (130) results in precocious puberty. Further, an increase in pulsatile LHRH release has been shown to occur at the onset of puberty not only in monkeys (84), in which the removal of central inhibition triggers puberty (see below), but also in sheep (5) and rats (131), in which a shift of the sensitivity to ovarian steroids appears to be responsible for the onset of puberty.

Nevertheless, the mechanism initiating puberty appears to differ between primates and other species: 1) in monkeys, castration induces an elevation of gonadotropin release during the neonatal period and after the onset of puberty, but not during the juvenile period before the onset of puberty (3), while in rats and sheep the castration-induced elevation of gonadotropin release occurs from the neonatal period throughout life (4, 5); and 2) the N-methyl-D-aspartic acid (NMDA)-induced precocious puberty in monkeys does not lead to normal reproductive cycles as adults after cessation of the drug infusion (132), while in rats the NMDA-induced precocious puberty continues on to cyclic ovulation (133, 134). However, this may occur because by the time the treatment ends, these animals are near the normal age of puberty and not because of an actual species difference. In primates, LHRH neurons are under tonic central inhibition during the juvenile period, and removal of this inhibition is necessary before the activation of central excitations can allow the initiation of puberty (128, 135). In contrast, in rats, tonic central inhibition, equivalent to that in primates, may not exist, and the establishment of glutamatergic and other facilitatory neuronal systems is required for the onset of puberty (136).

Species differences between the rhesus monkey and rat may also be seen in synaptogenesis. Synaptogenesis in the hypothalamus of the rat increases from the neonatal period to puberty (137, 138, 139, 140) as it does in the cerebral and cerebellar cortices (141, 142, 143), although a part of the pubertal increase in synaptogenesis in rat hypothalamus is probably due to the pubertal increase in estrogen (140), as observed in estrogen-primed OVX rats (144). In contrast, according to the studies by Rakic and colleagues (145, 146, 147, 148), synaptogenesis in the cerebral cortex of rhesus monkeys dramatically increases after birth, reaches a plateau at 2–4 months of age, and declines slightly through the juvenile period followed by a substantial decrease at the age of puberty, to adult levels (145). Interestingly, the developmental changes in synaptogenesis are uniform among the different cortical regions (146) and the dentate gyrus (147) as well as different layers of the neocortex (148). Moreover, the density of neurotransmitter receptors such as dopaminergic, adrenergic, serotoninergic, cholinergic, and -aminobutyric acid (GABA)ergic receptors in the cortex also reaches a maximum level between 2 and 4 months of age and declines thereafter, similar to changes in synaptogenesis (145). These data are interpreted by the authors to indicate that overproduction of synapses and neurotransmitter receptors in the neocortex are eliminated by a gene-regulated mechanism and that this elimination is critical to the establishment of specific cortical functions (145, 149). Although the studies by Rakic and colleagues are primarily focused on the perinatal period, and although synaptogenesis in the hypothalamus with emphasis on the onset of puberty has yet to be investigated, the developmental time course of synaptogenesis in the neocortex is strikingly similar to that of gonadotropin release in the neonatally gonadectomized rhesus monkey. It is possible that a similar decrease in synaptogenesis in the hypothalamus occurs during sexual maturation in primates. If the elimination of predominantly stimulatory synapses to LHRH neurons occurs during the first few months of life, this could lead to the suppression of LHRH release during the first few months of life, and if the elimination of predominantly inhibitory synapses to LHRH neurons occurs before the onset of puberty, this could lead to the pubertal increase in LHRH release. Moreover, a decrease in inhibitory synapses at the onset of puberty may allow the synaptogenesis of facilitatory neurons [e.g., glutamate and norepinephrine (NE)] to establish a specialized function. In support of our view, Perera and Plant (57) reported that a decrease in the area occupied by synapses onto LHRH neurons occurred in adult castrated male monkeys as compared with prepubertal male castrated monkeys, although these authors did not qualify the type of synapses. Nonetheless, the hypothesis that the onset of the pubertal increase in LHRH release is due to the elimination of inhibitory neurons and the subsequent establishment of selective neuronal pathways and/or neuronal networks that are responsible for the adult pattern of LHRH release in primates is yet to be tested. Certainly, it is possible that developmental changes in synaptogenesis in female primates is also influenced by the pubertal increase in estrogen, as recently described in adult OVX monkeys by Horvath and Naftolin and colleagues (150). The degree to which changes in synaptogenesis are attributed to developmental or to the pubertal increase in estrogen should be differentiated.

	V. Neurobiology of Puberty

Top Abstract I. Introduction II. Description of Puberty III. When Does the... IV. What Determines the... V. Neurobiology of Puberty VI. Master Gene(s) Controlling... VII. Summary and Conclusions References

 
Most neurotransmitters and neuromodulators possess both excitatory and inhibitory properties depending on several factors, such as composition of the neurocircuit, developmental stage, and hormonal environment. Therefore, the classification of inhibitory and stimulatory neurotransmitters in this article is based on the general feature of control of pulsatile LHRH release in the adult, and no neuroactive substance should be confined to a single category.

A. Inhibitory neurotransmitters
In primates, central inhibition of the LHRH neurons is present before the onset of puberty, and the removal or diminution of central inhibition triggers the onset of puberty. This concept is based upon the following: 1) gonadotropin secretion in agonadal humans (62, 63, 151) and neonatally gonadectomized monkeys remains suppressed until the onset of puberty despite the absence of the gonadal steroids (18, 70); and 2) precocious puberty occurs in humans and primates with hypothalamic lesions (see Ref. 83). Therefore, the search for neuronal substrates responsible for inhibition of LHRH release in the prepubertal period has begun. The first neuromodulators that were extensively studied were endogenous opioids, but evidence indicates that these were not likely candidates for the LHRH suppresser before the onset of puberty. Subsequently, the role of GABA was studied.

1. Endogenous opioids. The role of opioids in reproductive function has been well reviewed by others (152, 153, 154, 155, 156, 157). Because opioid substances suppress pulsatile LH release (158, 159, 160), and because the low frequency of LH pulses during the luteal phase, pregnancy, or the negative feedback phase of estrogen can be reversed in adults by the opioid antagonist, naloxone (161, 162, 163, 164, 165, 166, 167), a role for endogenous opioids in puberty has been proposed. In fact, it has been shown that the administration of naloxone stimulates LH release in prepubertal sheep as well as fetal sheep (168, 169, 170). However, a careful study by Foster and colleagues (171) concludes that in sheep the pubertal decrease in sensitivity to inhibitory feedback effects of gonadal steroids before the pubertal increase in LH release is not causally related to opioid inhibition. In addition, both LHRH and ß-endorphin (ß-END) levels in the median eminence of lambs with delayed puberty induced by food restriction are lower than in normal lambs (172), indicating that ß-END is not an inhibitory neurotransmitter responsible for low LHRH release before the onset of puberty.

Similarly, in primates, among possible opioid neurotransmitters, ß-END is an unlikely candidate for the prepubertal inhibition of LHRH release: 1) naloxone, an opioid antagonist, does not increase LH release until after the onset of puberty in chimpanzees (173); 2) effectiveness of naloxone in stimulating LH release increases greatly with the progress of puberty (174); 3) expression of POMC mRNA in the arcuate nucleus increases after puberty (54); and 4) the release of ß-END from the S-ME increases during puberty concomitant with LHRH release (175). It is conceivable that the opioid system may not be active before the pubertal increase in estrogen, since the components of this system, such as the µ-opioid receptor, are activated by estrogen (176, 177). Hence, opioids seem to be a neuronal component controlling LHRH release in mature, but not in immature, animals and humans (Table 1 and Fig. 2).

a. Biochemistry and molecular biology of GABA.

Synthesis of GABA: In presynaptic neurons GABA is synthesized from glutamate by decarboxylation in the presence of glutamic acid decarboxylase (GAD), stored in vesicles, and released by exocytosis upon depolarization in the presence of extracellular Ca²⁺ (179). Although GABA is also synthesized through other pathways, such as putrescine and - hydroxybutyrate, they are considered to play a minor role (see Refs. 180, 181). GABA synthesis occurs in two compartments: 1) GABAergic neuroterminals and 2) perikarya and dendrites. Glia do not synthesize GABA, but play a role in GABA metabolism by providing precursors and by taking up or degrading GABA overflows from the synapse (182). GABA synthesis is controlled by the concentration of glutamate in the GAD-containing cellular compartments, although it is unclear how the glutamate concentration is linked to neuronal activity to control GABA synthesis and maintain the neurotransmitter pool, and how the availability of glutamate regulates physiological GABA synthesis in vivo (180, 181).

Two different forms of proteins have been reported as the GABA-synthetic enzyme GAD. Based on molecular weight they are named GAD67 and GAD65, which are derived from two respective genes (see Refs. 180, 181, 183). The two cDNAs contain no contiguous identical sequences longer than 17 nucleotides (183), and the amino acid sequences of GAD65 and GAD67 from the rat brain are less similar than are sequences of each form in different mammalian species (184). Although both GAD forms are present in most brain regions, the distribution of the two GADs in the brain is not identical (185, 186). Immunocytochemical and in situ hybridization studies suggest that GAD65 is more consistently localized in neuroterminals, whereas GAD67 is found throughout neuronal cells (184, 187). However, recent reports indicating that 1) GAD67 and GAD65 can form a heteromultimer (188); 2) GAD67 may be transported to neuroterminals together with GAD65 (189); and 3) immunoreactive GAD67, but not GAD65, is found in the neuroterminals of hippocampal granular cells (190). The presence of GADs is not limited to the brain. GAD67 mRNA was found in the rat testis, while GAD65 mRNA was found in the rat oviduct (191). Further, both GAD67 and GAD65 are present in pancreatic ß-cells in humans, and a titer of GAD65 antibodies is elevated in patients with insulin-dependent diabetes mellitus (192, 193, 194).

GAD67 and GAD65 exist in the enzymatically active holo form and inactive apo form, and conversion of holo-GAD67 or holo-GAD65 to and from apo-GAD67 or apo-GAD65 is determined by the presence of the cofactor, pyridoxal-5'-phosphate, which is influenced by physiological states as well as by experimental conditions (180, 183).

Recently, it has been hypothesized that vesicular release of GABA may be associated with GAD65, whereas nonvesicular release of GABA, which is mediated by the reversal of GABA transporters (see below), may be associated with GAD67 (199A ).

Reuptake of GABA: Elevated local concentrations of amino acid neurotransmitters in the synaptic cleft are actively removed by specific transporters, located on presynaptic terminals and surrounding glial cells, where the neurotransmitters are recycled. GABA transporters (GAT) are classified in the Na⁺ and Cl^--coupled transporter family. Four GABA transporters (GAT-1, GAT-2, GAT-3, and BGT-1) have been described (195, 196, 197). GAT-1 is the predominant GABA transporter in the mammalian brain (197), and specific GAT-1 antagonists increase extracellular GABA concentration in the rat thalamus (198). Interestingly, the GAT-1 transcript contains an estrogen-responsive element (199). It is possible, therefore, that the reduction of GABA concentration in the S-ME at the onset of puberty might be due to a developmentally regulated increase in GABA transporter activity. At this time there is no information on the role of GAT in puberty.

b. Molecular biology and pharmacology of GABA receptors.
Three GABA receptors, GABA_A, GABA_B, and GABA_C have been identified in the brain. The GABA_A receptor is a pentameric membrane-spanning ligand-gated ion channel that permits Cl^- entry into the cell upon binding with the neurotransmitter GABA (200, 201, 202), whereas the GABA_B receptor is coupled to calcium and potassium channels, as well as an intracellular signaling pathway, via GTP binding proteins (203, 204). The GABA_C receptor is a Cl^- pore and, according to current reports, is found predominantly in the vertebrate retina. GABA_A receptors are modulated by allosteric agonists, benzodiazepines, barbiturates, neurosteroids (primarily metabolites of pregnane steroids), and ethanol, as well as polyvalent cations (205, 206). GABA_A receptors are composed of at least 18 genetically distinct subunits (1–6, ß1–ß4, 1–4, , , 1–2). An -subunit, ß-subunit, and -subunit are all required to form a fully functional GABA_A receptor (207, 208), and the 1ß22 arrangement is the most common in the brain.

The functional and pharmacological characteristics of a GABA_A receptor are determined by the subunit composition (209, 210, 211). The spatial distribution of subunits in the brain differs, and the subunit composition varies from neuron to neuron (209, 210). Further, the pattern of the subunit composition varies during prenatal and postnatal development (212, 213, 214, 215, 216). Since GABA_A receptors mediate GABA inhibition of LHRH release (217), it is particularly important to determine whether the subunit composition in the hypothalamus, especially on LHRH neurons, changes during maturation, related to the onset of puberty. It has been shown that in adult rats some LHRH neurons express the ß₃-, but not ₁- or ß₂-, GABA_A subunits (218), and in peripubertal female rats some LHRH neurons express ₁-, ₂-, ß₃-, and ₂-, but not ₁-subunits (219).

c. Developmental changes in GABA and GABA receptors.
GABA and GAD: The concentration of GABA and the number of GABAergic neurons in the rat spinal cord increase from E13 to the second postnatal week and then decline by the third postnatal week (220). Similarly, GAD67 and GAD65 mRNAs in the rat spinal cord increase exponentially during embryonic development starting on E11 until the first postnatal week, but then decline during the second postnatal week to adult levels (221). In the rat striatum GAD65 mRNA increases until 2 weeks postnatally, and then declines toward adult levels, whereas GAD67 mRNA keeps increasing from 1 week postnatally until adulthood; thus, the ratio of GAD65 to GAD67 mRNAs is high at 1–2 weeks postnatally, and then levels off to the adult ratio by 3 weeks (222). In the mouse cortex, the number of GABA-positive cells decreases postnatally, although the timing of the declining pattern differs depending on the layers of the cortex examined (223). The increase in GAD and GABA concentrations during embryonic development is due to neurogenesis, whereas the postnatal decline is not likely due to cell death (224).

GAD65 and GAD67 mRNA levels and GABA concentrations in the rat POA increase with age between P1 and P15 (225, 226, 227) and increase further between P15 and P35–P40 (228, 229). However, developmental changes in GAD mRNA expression and GABA concentrations within the MBH require additional studies investigating the precise location of GABA neurons and a wider age window. Some reports show that GAD mRNA in the rat arcuate nucleus, but not dorsomedial nucleus, increases with age between P1 and P20 (227), and GABA concentrations in most areas of the brain, including the MBH, caudate-putamen, hippocampus, and amygdala, increase with age (225, 226), whereas other reports indicate GABA concentrations in the rat MBH (229) or in the rat POA/MBH (230) do not change with age between P15–P40.

Developmental changes in GABA and GAD in primates are not well studied, and there are conflicting data from the postnatal period. In the Japanese macaque, GAD concentration in the fetal cerebellum is very low during the second trimester, quickly increases 6.3-fold at full term, and further increases 2.2-fold by adulthood (231). GABA concentrations in the occipital cortex of rhesus monkeys increase 2-fold from the fetal period (E70–E150) to the neonatal period, but do not change further in adults (232). The number of GABA neurons in the visual cortex increases gradually between E110 and P160 in rhesus monkeys, but there are no descriptions at older ages (233, 234). GAD activity in the human neocortex sharply increases at birth and continues to increase until 1 yr of age, after which it declines gradually until pubertal age and then slightly increases at adulthood (235, 236). Recently, Urbanski et al. (237) reported that the distribution pattern and concentration of GAD67 and GAD65 mRNAs in hypothalamic nuclei assessed by in situ hybridization in juvenile (0.6 yr of age) gonadally intact male rhesus monkeys were not different from those in adult (10 yr of age) male monkeys, and a report by Plant and colleagues (238) indicates that GAD mRNA levels in the basal hypothalamus of juvenile castrated male rhesus monkeys did not differ from those in adult castrated male monkeys. Although these data do not appear to support the hypothesis that GAD plays an important role in puberty (see below), more precise developmental studies with the exact regional distribution pattern of GABA neurons in nonhuman primates, including in females, are needed before conclusions can be drawn.

GABA release: GABA release in the POA measured by push-pull perfusion decreases between P26 and P35 in female rats (239). Similarly, in female rhesus monkeys GABA release in the S-ME decreases concomitant with the pubertal increase in LHRH release (217, 240). These findings suggest that elevated GABA in the POA in female rats and in the S-ME in female rhesus monkeys may contribute to the suppression of LHRH release before the onset of puberty.

GABA_A receptors and their subunit composition: Rakic and colleagues (145) report that the density of GABA_A receptors in all five layers of the cerebral cortex of rhesus monkeys increases after birth, reaching a maximum level between 2 and 4 months of age, and then declines gradually to adult levels at 36 months of age. Because similar patterns of developmental changes in the receptor density of ₂- and ß-adrenergic, D₂-dopaminergic, 5HT₂-serotoninergic, and M₁-muscarinic receptors are observed in the cerebral cortex, these authors postulate that synaptic circuitry formation may be orchestrated by a common diffusible factor or a synchronous activation by a common gene.

The developmental pattern of each GABA_A receptor subunit is complex and differs from region to region or neuron to neuron, which ensures the functional heterogeneity of GABA input. Nonetheless, it has been reported consistently that, in general, ₂-subunit expression is very high before birth to shortly after birth, and decreases gradually toward adult levels, whereas ₁ expression is minimal before birth and then gradually increases after birth until adulthood (213, 214, 215, 216, 241). In fact, it appears that GABA_A receptors containing ₁-subunits gradually replace GABA_A receptors containing the ₂-subunit during postnatal maturation in the rat, monkey, and human brain, and that the increase in the ₁-subunit is an indication of brain maturation, i.e., the onset of synaptic GABA inhibition, whereas ß-subunits do not generally undergo developmental changes (213, 214, 241). Recently, changes in subunit composition with functional alteration were demonstrated by using a combination of patch-clamp recording and single cell RT-PCR methods. Brooks-Kayal et al. (242) have shown that changes in GABA_A receptor subunit composition in hippocampal neurons preceded the onset of epilepsy by weeks in epileptic rats. They further hypothesize that retention of an immature pattern of GABA_A receptor subunit composition may be responsible for prolonged childhood epilepsy, which normal children spontaneously outgrow.

It is probable, therefore, that developmental changes in the GABA_A subunit composition in neuronal cells may occur before the onset of puberty. Analysis of the literature provides support for the hypothesis that GABA disinhibition of LHRH neurons through GABA_A receptors at the onset of puberty in female monkeys (217) is due to changes in GABA_A receptor subunit composition. For example, the discrepancy that some LHRH neurons in adult rats express the ß₃-, but not ₁- or ß₂-, GABA_A subunits (218), whereas in peripubertal female rats a subset of LHRH neurons express ₁-, ₂-, ß₃-, and ₂-, but not ₁-subunits (219), may be due to technical differences in the two studies, but it may also be due to developmental changes in subunit composition. In fact, a recent report by Herbison and colleagues (243) using single cell RT-PCR suggest that GABA_A subunit composition in LHRH neurons located in the POA and medial septum of sexually immature mice at neonatal and juvenile ages is more heterogeneous than that in adults, and it becomes homogeneous when the mice mature, supporting this notion. Interestingly, the same authors have reported that sensitivity to GABA in LHRH neurons of juvenile mice is lower than that in adult mice and that the pattern of the dose-response curve to GABA in prepubertal LHRH neurons is more heterogeneous than that in adult LHRH neurons (243). At this time, we have little knowledge of developmental changes in the GABA_A subunit composition of LHRH neurons in nonhuman primates.

d. Direct and indirect control of LHRH neurons by GABA.
GABA input can change activity of LHRH neurons directly. Perikarya of LHRH neurons are innervated by GAD- containing axons (presumably GABA neurons) in rats (244, 245, 246), but not in monkeys (247). LHRH neurons in the POA of adult rats and GT-1 neurons express the GABA_A ß₂/ß₃-subunit (218, 248), and the ₁- subunit colocalizes with LHRH in GT-1 neurons (248, 249). Although abundant distribution of ₂-subunit mRNA in the rat POA has been reported (250), only a small number of LHRH neurons express the ₂-subunit (219). El-Etr et al. (248) report that the GABA_{A 2}-subunit mRNA is not expressed in GT-1 cells. It is, however, possible that the negative data are in part due to technical problems. In fact, a recent study with single cell RT-PCR indicates that GABA_A receptors on the LHRH membrane in adult mice are assembled from ₁-, ₃-, ₅-, ß₁-, and ₂-subunits (243) and 100% of adult mouse LHRH neurons respond to GABA (243).

In primates, direct innervation of LHRH neurons by GABA neurons has not been found (247), although inhibitory synapses on LHRH perikarya, their phenotype yet to be determined, have been reported recently (150). However, GABA inhibition of LHRH neurons could be through glutamatergic neurons, since there is a reciprocal innervation between GABAergic and glutamatergic neurons in monkeys (247). In addition, GABA may alter LHRH release through LHRH neuroterminals or dendritic input, since direct control of oxytocin-vasopressin neuroterminals by GABA has been described (251). Currently, GABA innervation at dendrites and neuroterminals of LHRH neurons in primates has not been investigated. The interaction between GABA neurons and other neurons involved in the control of LHRH release in rats was described in detail by Leranth et al. (252) and Silverman et al. (253).

e. Role of GABA in LHRH release.
The role of GABA in the control of LHRH release appears to be both inhibitory and stimulatory. For example, some early studies indicate that systemic administration or intracerebroventricular infusion of GABA, and application of GABA onto hypothalamic explants, stimulates LHRH and LH release (254, 255, 256), while others show inhibitory or biphasic/triphasic effects (257, 258, 259, 260, 261).

Recent studies using GT-1 cells (249, 262, 263) and LHRH neurons from the mouse olfactory pit (264) suggest that GABA induces excitatory effects on LHRH release, intracellular Ca²⁺ oscillations, and membrane potentials, although the study by Martinez de la Escalera et al. (262) demonstrates that an initial stimulatory effect on LHRH release is followed by prolonged inhibition.

It is possible that GABA is an inhibitory neurotransmitter through GABA_A receptors, whereas it is a stimulatory neurotransmitter through GABA_B receptors for pulsatile LHRH/LH release in adult female animals. GABA release in the medial preoptic area (MPOA) is pulsatile, and a reduction of GABA release precedes an increase in LH release in OVX rats (265, 266). Direct infusion of GABA into the POA suppresses LH release in rats and sheep (267, 268, 269, 270), and GABA and the GABA_A agonist, muscimol, inhibit LH pulses when injected into the third ventricle or the MPOA in rats (257, 271). Similarly, muscimol suppresses, and the GABA_A antagonist bicuculline stimulates, the pulse frequency, but not the pulse amplitude, of LHRH pulse-generator activity, as determined by multiunit activity recording in the hypothalamus of OVX female rats (272). In contrast, infusion of baclofen, a GABA_B agonist, into the arcuate-ventromedial nucleus stimulates LH pulses in castrated rams during the nonbreeding season (268, 273, 274). There are, however, reports indicating that GABA_B receptors are inhibitory for basal LHRH release (258, 260, 261). In addition, the GABA_B agonist baclofen iontophoretically applied to adult LHRH neurons in guinea pigs causes immediate hyperpolarization via opening of potassium channels (274A ), indicating that the direct, GABA_B-mediated action of GABA in these cells is inhibitory.

GABA is also an important inhibitory neurotransmitter before the preovulatory LH surge, and in the positive feedback action of ovarian steroids in females. GABA release as well as GAD67 and GAD65 mRNA levels in the MPOA decrease before the initiation of an estrogen-induced LH surge in OVX female rats and ewes (265, 266, 269, 275, 276, 277, 278) and before the LH surge in female rats (269, 270). The GABA turnover rate also decreases before the estrogen-induced LH surge (276). GABA or muscimol infusion into the third ventricle or MPOA blocks the preovulatory LH surge in female rats and reduces the size of the estrogen-induced LH surge in OVX rats (276, 279, 280, 281), while both intraventricular and intravenous infusions of bicuculline advance the timing of the LH surge in estrogen-primed OVX rats and proestrous rats (271, 282). There is a report, however, that GABA_B, not GABA_A, receptors mediate inhibitory action of GABA during the LHRH surge (274B ).

In males, GABA neurons are involved in the negative feedback effects of testosterone. The GABA turnover rate as well as GAD67 and GAD65 mRNA levels in the MPOA decrease after castration concomitant with an increase in LH release, whereas testosterone administration prevents these changes in adult male rats (283, 284). Moreover, the GABA turnover rate in the MPOA decreases with an increase in LH release after implantation of the androgen receptor antagonist, hydroxyflutamide, into the rostral portion of the MPOA (285).

The conflicting stimulatory and inhibitory roles of GABA in LHRH release may be, in part, explained by the following hypotheses. First, GABA action may differ depending on the receptor subtype. In some studies the receptor subtype is not examined. Second, since GABA effects on LHRH neurons may occur not only directly, but also indirectly through interneurons, results may depend on alterations in the population and/or sensitivity of interneurons, differences in the location of GABA infusion in in vivo studies, and the loss of input from other brain areas in in vitro hypothalamic explant studies. For instance, in in vitro studies Nikolarakis et al. (259) suggest that GABA is inhibitory at LHRH neuroterminals, but stimulatory at LHRH perikarya through inhibitory opioid neurons. GABA input to LHRH neurons in the monkey hypothalamus appears to occur indirectly through glutamate neurons (247, 286).

Third, GABA_A receptor properties may change during maturation, such that GABA is excitatory when LHRHsecreting cells are immature, whereas GABA is inhibitory when LHRH neurons become mature. GABA is stimulatory in immature neurons, such as GT-1 cells and cultured LHRH cells from embryonic mice (249, 263, 264). Similar developmental switches of GABA action have been shown not only in the hypothalamus (287, 288), but also in the spinal cord, hippocampus, cerebellum, cortex, and olfactory bulb (see Refs. 289, 290). So far, two possible mechanisms of developmental changes in GABA_A receptor properties have been shown. 1) Changes in the subunit composition of GABA_A receptors occur during maturation of the brain (215, 241, 291) or under different physiological conditions (292), whereby the response to GABA varies, as discussed in a previous section. A recent report, however, indicates that although the changes in the subunit composition of GABA_A receptors in mouse LHRH neurons occurs between the juvenile and adult stages, GABA input to LHRH neurons is excitatory regardless of the age of mice when tested using pipettes filled with a high chloride solution (243). 2) Changes in the response to GABA could be explained by chloride homeostasis during maturation (290) or some physiological conditions, such as diurnal rhythmicity (293). There is a differential chloride gradient across the neuronal membrane, which is reversed between the immature state and the mature state (294, 295, 296). A recent report further suggests that the neuronal Cl^--extruding K⁺/Cl^- cotransporter, KCC2, responsible for hyperpolarizing postsynaptic inhibition (297), undergoes developmental changes when hippocampal pyramidal neurons developmentally switch GABA_A-mediated responses from the depolarizing state to the hyperpolarizing state (298).

Fourth, interaction with glutamatergic input and/or receptors due to physiological changes, including neuronal maturation, may alter receptor sensitivity to GABA or the relative importance of the inhibitory GABAergic input to the stimulatory glutamatergic input. This was clearly shown in the hippocampus (290) as well as in the POA-hypothalamus (306, 307). Fifth, GABA receptor subunit composition can be altered by the presence of steroid hormones. For example, estrogen injection in OVX rats altered GABA_A receptor subunit composition (250, 299).

GABA neurons or GABA_A receptors seem to mediate progesterone action as follows: 1) Progesterone receptorcontaining neurons in the monkey and rat hypothalamus are GABA neurons (246, 300); and 2) GABA_A receptors contain steroid recognition sites (301), i.e., neurosteroids, which include some progesterone metabolites and derivatives, exert their action through GABA_A receptors located on the cell membrane (301). Recently, Herbison et al. (302) demonstrated that allopregnanolone, a neurosteroid derived from circulating progesterone, directly exerts a facilitatory action on GABA_A receptor-chloride channels in LHRH neurons. GABA neurons also appear to mediate estrogen action. In rats, estrogen-receptive cells in the arcuate nucleus are also GABA neurons (245), and estrogen is necessary for an increase in the number of progesterone receptors in immunostained GABA neurons of OVX monkeys (246, 300). Moreover, the fact that a decline in GABA levels in the MPOA occurs before the estrogen-induced LH surge in rats and sheep (275, 303), while an elevation of GABA levels in the MPOA occurs during the negative feedback action of estrogen in sheep (275), further supports the possible role of GABA neurons in estrogen action. Conversely, estrogen priming in OVX rats increases GABA_A receptor ₂-, and ₁-, but not ß₃-, subunit mRNAs in the POA and bed nucleus of the stria terminalis (250).

f. Role of GABA in puberty.
There are reports that GABA is stimulatory on LH release in male and female rats during the juvenile stage, whereas GABA is inhibitory around the age of puberty (304, 305, 306, 307). Mitsushima and Kimura (308) report that neither bicuculline nor muscimol causes any significant effect on LH release in sexually immature male rats, whereas bicuculline stimulates and muscimol inhibits LH release in pubertal male rats. In contrast, our studies in nonhuman primates using push-pull perfusion suggest that the LHRH neuronal system is tonically inhibited by GABA neurons before the onset of puberty (Fig. 2). First, GABA levels in the S-ME in prepubertal rhesus monkeys are much higher than in midpubertal monkeys (217, 240). Second, bicuculline, a GABA_A antagonist, stimulates LHRH release in prepubertal monkeys by removing endogenous GABA inhibition, whereas exogenous GABA is not effective in suppressing LHRH release until after the onset of puberty, when endogenous GABAergic tone is reduced (217). Third, infusion of antisense oligodeoxynucleotides for GAD67 and GAD65 mRNAs into the S-ME of prepubertal monkeys results in a dramatic increase in LHRH release (309, 310), presumably due to the reduction in GABA synthesis and subsequent release (240, 309). Finally, pulsatile infusion of bicuculline into the third ventricle of prepubertal monkeys results in precocious menarche, which occurs 6–8 weeks after the initiation of bicuculline infusion, and in precocious first ovulation, which occurs by 30 months, the age of menarche in control females (311). However, since the interval between menarche and first ovulation is not shortened by bicuculline infusion, additional mechanisms, such as the establishment of the stimulatory neuronal system for pulsatile LHRH release, are necessary for the pubertal transition in female primates. Nonetheless, disinhibition of LHRH neurons from GABA appears to be critical for the onset of puberty in primates (Table 1).

g. The role of GABA in sexual differentiation in the brain.
Sexual differentiation of the rodent brain is dependent upon exposure to estrogen or testosterone during the perinatal critical period, beginning in late gestation and ending early in neonatal life (312, 313). Sexual dimorphism of the brain, including the male dominant sexually dimorphic nuclei (314, 315), and the female dominant anterior ventral periventricular nucleus [or medial preoptic nucleus (316, 317, 318)], has been extensively described. According to a series of studies by McCarthy, Selmanoff, and colleagues (226, 227, 319), GABA neurons appear to mediate steroid action on the sexual differentiation of the brain during the perinatal period. The GABA turnover rate in the medial preoptic nucleus in the MPOA and ventromedial nucleus of the hypothalamus (VMH) in male rats is 2-fold higher than in female rats (319), and GAD67 and GAD65 mRNA levels in the MPOA and VMH in males are higher than in females on P1, but not on P15 (226, 227). Neonatal castration of male rats results in the female type of GAD mRNA responses, whereas neonatal treatment of female rats with testosterone results in the male type of GAD mRNA responses (227). Further, a higher level of GABA in the VMH and arcuate nucleus in males than in females is observed only during the first few days of life (226), and this dimorphism is lost as GABA levels increase developmentally in both males and females (320). A similar sex difference in GABA concentrations in the MPOA of P10 rats has also been reported by others (225). Since GABA has a potent growth factor-like property for neuronal survival and migration, growth cone guidance, and neurite branching (224, 321, 322, 323, 324, 325, 326, 327, 328), and since GABA is excitatory in the neonatal period, McCarthy and colleagues (328) propose the hypothesis that a rise in GABA release induced by testosterone during the critical period of sexual differentiation permanently alters the structure and function of the POA and hypothalamus.

h. GABA and human diseases associated with puberty.
There is evidence indicating that GABA neurons may be responsible for diseases associated with puberty. First, it has been documented that the onset of schizophrenia often occurs between late puberty and early young adulthood (7). A significant decrease in GAT-1 immunoreactivity on axonal terminals of a subset of GABA neurons that innervate pyramidal cells in the frontal cortex is observed in patients with schizophrenia when compared with normal human subjects (329). Second, the new onset of epileptic seizures tends to occur early in life and during the adolescent period (10). Precocious puberty is often associated with epilepsy in children (8, 9). Furthermore, treatment with sodium valproic acid, a GABA agonist—the mechanism of action has yet to be determined—delays the timing of puberty in children with seizure disorders (330, 331) and in genetically epilepsy-prone mice (332). It is possible that the pubertal increase in gonadal steroids may sensitize neurocircuits involved in epileptic seizures, but it is also possible that there is a common mechanism of developmental deficiency, i.e., weakened GABA inhibition in the LHRH neuronal system resulting in precocious puberty and weakened GABAergic inhibition in the brain at the pubertal age resulting in epilepsy (333). Recently, Bourguignon and colleagues (334) treated an 11-month-old child, who exhibited severe epileptic seizures and precocious puberty, with loreclezole and vigabatrin, GABA agonists. At an earlier stage, traditional treatment for epilepsy with phenobarbital was not effective in this patient. However, treatment with loreclezole followed by vigabatrin not only caused all signs of precocious puberty to regress, but also settled seizure attacks. Finally, a study indicates that treatment with the GABA agonist valproate results in a high rate of polycystic ovaries with elevated serum testosterone concentrations among women with epilepsy, especially if the patients start to receive the valproate treatment before the age of 20 yr (335). Taking into consideration the recent view that polycystic ovarian syndrome originates during the pubertal stage (11, 12), it is possible that an irregularity of the normal control of pulsatile LHRH release by the GABA neurotransmitter system during puberty and/or failure of opioid inhibition during the postpubertal period may be a cause of the polycystic ovarian syndrome, although this is highly speculative.

3. Other inhibitory neuropeptides and neurotransmitters. The neuropeptides, such as vasoactive intestinal peptide (VIP), CRH, and melatonin, are inhibitory to neuronal activity (336, 337) and could be potential inhibitory neurotransmitters before the onset of puberty. However, to date, the role of hypothalamic VIP in puberty has not been systematically studied, and available evidence indicates that changes in CRH are not likely to play a major role in determining the timing of puberty. ß-END, ACTH, and cortisol levels in response to CRH in children and adult humans are independent from age and sexual maturity (338, 339), and there is no evidence for direct inhibitory effects of CRH on LHRH release. Therefore, in this section we will only discuss the role of melatonin in puberty.

a. Melatonin.
Melatonin has been suggested as an inhibitory neurotransmitter for puberty. This is based on the observations that 1) some human patients with pineal tumors exhibit precocious puberty (340); 2) circulating melatonin concentration is elevated in early childhood, declines during late childhood, and remains stable from the early pubertal period to adulthood in humans and monkeys (