Gamma-aminobutyric acid (GABA), acting through GABA(A) receptors (GABA(A)R), is hypothesized to suppress reproduction by inhibiting GnRH secretion, but GABA actions directly on GnRH neurons are not well established. In green fluorescent protein-identified adult mouse GnRH neurons in brain slices, gramicidin-perforated-patch-clamp experiments revealed the reversal potential (E(GABA)) for current through GABA(A)Rs was depolarized relative to the resting potential. Furthermore, rapid GABA application elicited action potentials in GnRH neurons but not controls. The consequence of GABA(A)R activation depends on intracellular chloride levels, which are maintained by homeostatic mechanisms. Membrane proteins that typically extrude chloride (KCC-2 cotransporter, CLC-2 channel) were absent from the GT1-7 immortalized GnRH cell line and GnRH neurons in situ or were not localized to the proper cell compartment for function. In contrast, GT1-7 cells and some GnRH neurons expressed the chloride-accumulating cotransporter, NKCC-1. Patch-clamp experiments showed that blockade of NKCC hyperpolarized E(GABA) by lowering intracellular chloride. Regardless of reproductive state, rapid GABA application excited GnRH neurons. In contrast, bath application of the GABA(A)R agonist muscimol transiently increased then suppressed firing; suppression persisted 4-15 min. Rapid activation of GABA(A)R thus excites GnRH neurons whereas prolonged activation reduces excitability, suggesting the physiological consequence of synaptic activation of GABA(A)R in GnRH neurons is excitation.

The initiation of mammalian puberty requires an increase in pulsatile release of GnRH from the hypothalamus. This increase is brought about by coordinated changes in transsynaptic and glial-neuronal communication. As the neuronal and glial excitatory inputs to the GnRH neuronal network increase, the transsynaptic inhibitory tone decreases, leading to the pubertal activation of GnRH secretion. The excitatory neuronal systems most prevalently involved in this process use glutamate and the peptide kisspeptin for neurotransmission/neuromodulation, whereas the most important inhibitory inputs are provided by gamma-aminobutyric acid (GABA)ergic and opiatergic neurons. Glial cells, on the other hand, facilitate GnRH secretion via growth factor-dependent cell-cell signaling. Coordination of this regulatory neuronal-glial network may require a hierarchical arrangement. One level of coordination appears to be provided by a host of unrelated genes encoding proteins required for cell-cell communication. A second, but overlapping, level might be provided by a second tier of genes engaged in specific cell functions required for productive cell-cell interaction. A third and higher level of control involves the transcriptional regulation of these subordinate genes by a handful of upper echelon genes that, operating within the different neuronal and glial subsets required for the initiation of the pubertal process, sustain the functional integration of the network. The existence of functionally connected genes controlling the pubertal process is consistent with the concept that puberty is under genetic control and that the genetic underpinnings of both normal and deranged puberty are polygenic rather than specified by a single gene. The availability of improved high-throughput techniques and computational methods for global analysis of mRNAs and proteins will allow us to not only initiate the systematic identification of the different components of this neuroendocrine network but also to define their functional interactions.

A considerable number of patients with central precocious puberty (CPP) treated with depot GnRH agonists have reached final height (FH). The aim of this prospective, multicentric study was the evaluation of the benefits, side-effects, and long term outcome of depot GnRH agonist therapy. We investigated 50 young women (mean +/- SD age, 16.7+/-2.6 yr; range, 12.9-23.4 yr) at FH. They received depot triptorelin over a period of 4.4+/-2.1 yr (range, 1.0-9.7 yr). Target height (TH) and predicted adult height (PAH) at the start of treatment were 163.6+/-6.2 and 154.9+/-9.6 cm, respectively (P < 0.05). FH was 160.6+/-8.0 cm (FH vs. TH, P = NS; FH vs. PAH, P < 0.05). Young patients showed the highest height gain (FH minus initial PAH). Seventy-eight percent of all patients reached a FH within their TH range. Even in young patients and those with an unfavorable initial PAH below the TH range, 60% reached a FH within their individual TH range. Standardized bone mineral density and standardized bone mineral density SD score investigated by dual energy x-ray absorptiometry of the lumbar spine (L1-L4) were 1040.9+/-124.2 mg/cm2 and 0.0+/-1.0; those of the femoral neck were 902.2+/-115.4 mg/cm2 and 0.2+/-1.0, respectively. The SD score of the ratio of sitting height over lower leg length was normal (0.3+/-1.2). Body mass index SD scores at pretreatment, at the end of treatment, and at FH were not significantly different (2.0+/-2.0, 2.0+/-2.0, and 1.7+/-2.2, respectively). Menarche or remenarche started at age 12.3+/-1.4 yr (range, 9.3-15.8 yr) in all patients. In conclusion, long term depot GnRH agonist treatment of CPP girls preserved genetic height potential and improved FH significantly combined with normal body proportions. No negative effect on bone mineral density and reproductive function was seen. Treatment neither caused nor aggravated obesity.

The initiation of mammalian puberty requires the activation of hypothalamic neurons secreting the neuropeptide luteinizing hormone-releasing hormone (LHRH). It is thought that this activation is caused by changes in trans-synaptic input to LHRH neurons. More recently, it has been postulated that the pubertal increase in LHRH secretion in female animals also requires neuron-glia signaling mediated by growth factors of the epidermal growth factor (EGF) family and their astrocytic erbB receptors. Although it appears clear that functional astrocytic erbB1 receptors are necessary for the timely advent of puberty, the physiological contribution that erbB4 receptors may make to this process has not been established. To address this issue, we generated transgenic mice expressing a dominant-negative erbB4 receptor (DN-erbB4) under the control of the GFAP promoter, which targets transgene expression to astrocytes. DN-erbB4 expression is most abundant in hypothalamic astrocytes, where it blocks the ligand-dependent activation of glial erbB4 and erbB2 receptors, without affecting erbB1 (EGF) receptor signaling. Mice carrying the transgene exhibit delayed sexual maturation and a diminished reproductive capacity in early adulthood. These abnormalities are related to a deficiency in pituitary gonadotropin hormone secretion, caused by impaired release of LHRH, the hypothalamic neuropeptide that controls sexual development. In turn, the reduction in LHRH release is caused by the inability of hypothalamic astrocytes to respond to neuregulin (NRG) with production of prostaglandin E(2), which in wild-type animals mediates the stimulatory effect of astroglial erbB receptor activation on neuronal LHRH release. Thus, neuron-astroglia communication via NRG-erbB4/2 receptor signaling appears to be essential for the timely unfolding of the developmental program by which the brain controls mammalian sexual maturation.

The initiation of mammalian puberty and the maintenance of female reproductive cycles are events controlled by hypothalamic neurons that secrete the decapeptide gonadotropin-releasing hormone (GnRH). GnRH secretion is, in turn, controlled by changes in neuronal and glial inputs to GnRH-producing neurons. The hierarchical control of the process is unknown, but it requires coordinated regulation of these cell-cell interactions. Here we report the functional characterization of a gene (termed enhanced at puberty 1 [EAP1]) that appears to act as an upstream transcriptional regulator of neuronal networks controlling female reproductive function. EAP1 expression increased selectively at puberty in both the nonhuman primate and rodent hypothalamus. EAP1 encoded a nuclear protein expressed in neurons involved in the inhibitory and facilitatory control of reproduction. EAP1 transactivated genes required for reproductive function, such as GNRH1, and repressed inhibitory genes, such as preproenkephalin. It contained a RING finger domain of the C3HC4 subclass required for this dual transcriptional activity. Inhibition of EAP1 expression, targeted to the rodent hypothalamus via lentivirusmediated delivery of EAP1 siRNAs, delayed puberty, disrupted estrous cyclicity, and resulted in ovarian abnormalities. These results suggest that EAP1 is a transcriptional regulator that, acting within the neuroendocrine brain, contributes to controlling female reproductive function.