The extracellular signal-regulated kinases (ERK) signalling cascade is a key pathway that mediates the NMDA receptor (NMDAR)-dependent neuronal plasticity and survival. However, it is not clear yet how NMDARs regulate ERK activity. Stimulation of the NMDARs induces a complex modification of ERK that includes both ERK activation and inactivation and depends on particular experimental conditions. Here we show that there exists a differential restriction in the regulation of ERK activity that depends on the pool of NMDAR that was activated. The synaptic pool of NMDARs activates ERK whereas the extrasynaptic pool does not; on the contrary, it triggers a signalling pathway that results in the inactivation of ERK. As a result, simultaneous activation of both extrasynaptic and synaptic NMDAR using bath application of NMDA or glutamate (a typical protocol explored in the majority of studies) produced ERK activation that depended on the concentration of agonists and was always significantly weaker than those mediated by synaptic NMDARs. Since the activation of the extrasynaptic NMDA is attributed mainly to global release of glutamate occurring at pathological conditions including hypoxic/ischaemic insults, traumas and epileptic brain damage, the reported differential regulation of ERK cascade by NMDARs provides a unique mechanism for an early identification of the physiological and/or pathophysiological consequences of NMDAR activation. The negative regulation of the ERK activity might be one of the first signalling events determining brain injury and constitutes a putative target of new pharmacological applications. The NMDA subtype of glutamate receptors (NMDAR) is a key receptor involved in the regulation of multiple processes related to synaptic plasticity including learning, memory, neuron development, spine formation, long-term potentiation (LTP) and long-term depression (LTD). The mechanisms underlying such diversity of neuronal responses to the activation of a single receptor are not known. At least some of these processes are associated with the NMDA-dependent activation of the extracellular signal-regulated kinases ERK1 and ERK2 (ERK) signalling cascade whose inhibition modifies spine formation, long-term memory, LTP and cell survival (Adams & Sweatt, 2002;Hardingham & Bading, 2003;Goldin & Segal, 2003;Thomas & Huganir, 2004). Stimulation of NMDA receptors by specific agonists (Bading & Greenberg, 1991;Kurino et al. 1995) or via an increase of synaptic A. Ivanov and C. Pellegrino contributed equally to this work. activity (Hardingham et al. 2001) results in strong ERK phosphorylation. On the other hand, applications of the high concentrations of NMDA (70-100 μm) to neuronal cultures provokes a complex effect; it induces both activation and inactivation of ERK (Chandler et al. 2001;Kim et al. 2005). Although different suggestions were put forward to explain the complex effects of NMDAR agonists on ERK activity (Chandler et al. 2001;Kim et al. 2005), none of them provided compelling evidence supporting their hypo...

Activation of the G-protein-coupled receptor GPR54 by kisspeptins during normal puberty promotes the central release of gonadotropinreleasing hormone (GnRH) that, in turn, leads to reproductive maturation. In humans and mice, a loss of function mutations of GPR54 prevents the onset of puberty and leads to hypogonadotropic hypogonadism and infertility. Using electrophysiological, morphological, molecular, and retrograde-labeling techniques in brain slices prepared from vGluT2-GFP and GnRH-GFP mice, we demonstrate the existence of two physiologically distinct subpopulations of GnRH neurons. The first subpopulation is comprised of septal GnRH neurons that colocalize vesicular glutamate transporter 2 and green fluorescent protein and is insensitive to metabotropic glutamate receptor agonists, but is exquisitely sensitive to kisspeptin which closes potassium channels to dramatically initiate a long-lasting activation in neurons from prepubertal and postpubertal mice of both sexes. A second subpopulation is insensitive to kisspeptin but is uniquely activated by group I metabotropic glutamate receptor agonists. These two physiologically distinct classes of GnRH cells may subserve different functions in the central control of reproduction and fertility.

The novel hypothalamic peptides avian gonadotropin inhibitory hormone (GnIH) and its mammalian analogue RFRP-3, are emerging as key negative regulators of reproductive functions across species. GnIH/RFRP-3 reduces gonadotropin release and may play an inhibitory role in ovulation and seasonal reproduction, actions opposite to that of the puberty-promoting kisspeptins. GnIH directly inhibits gonadotropin release from the anterior pituitary in birds. GnIH/RFRP-3-immunoreactive fibres also abut the preoptic-septal gonadotropin-releasing hormone (GnRH) neurons, suggesting an additional site of action that has not been studied at the cellular level. Using anatomical labelling and electrophysiological recordings in septal brain slices from GnRH-GFP, vGluT2-GFP and GAD67-GFP mice, we report inhibitory actions of GnIH/RFRP-3 on kisspeptin-activated vGluT2 (vesicular glutamate transporter 2)-GnRH neurons as well as on kisspeptin-insensitive GnRH neurons, but not on cholinergic or GABAergic neurons (n = 531). GnIH and RFRP-3 produced a strikingly similar non-desensitizing hyperpolarization following brief 15 s applications (GnIH: 9.3 ± 1.9 mV; RFRP-3: 9.0 ± 0.9 mV) with IC 50 values of 34 and 37 nm, respectively. The inhibitory effect was mediated via a direct postsynaptic Ba 2+ -sensitive K + current mechanism and could prevent or interrupt kisspeptin-induced activation of vGluT2-GnRH neurons. GnIH-immunoreactive fibres were in apparent contact with vGluT2-GFP neurons. Thus, GnIH/RFRP-3 could reduce GnRH and glutamate release in target brain regions and in the median eminence via a direct inhibition of vGluT2-GnRH neurons. This in turn could suppress gonadotropin release, influence reproductive development and alter sex behaviour.

A link between energy balance and reproduction is critical for the survival of all species. Energy-consuming reproductive processes need to be aborted in the face of a negative energy balance, yet knowledge of the pathways mediating this link remains limited. Fasting and food restriction that inhibit fertility also upregulate the hypothalamic melanin-concentrating hormone (MCH) system that promotes feeding and decreases energy expenditure; MCH knockout mice are lean and have a higher metabolism but remain fertile. MCH also modulates sleep, drug abuse behavior, and mood, and MCH receptor antagonists are currently being developed as antiobesity and antidepressant drugs. Despite the clinical implications of MCH, the direct postsynaptic effects of MCH have never been reported in CNS neurons. Using patch-clamp recordings in brain slices from multiple lines of transgenic GFP mice, we demonstrate a strong inhibitory effect of MCH on an exclusive population of septal vGluT2-GnRH neurons that is activated by the puberty-triggering and preovulatory luteinizing hormone surge-mediating peptide, kisspeptin. MCH has no effect on kisspeptin-insensitive GnRH, vGluT2, cholinergic, or GABAergic neurons located within the same nucleus. The inhibitory effects of MCH are reproducible and nondesensitizing and are mediated via a direct postsynaptic Ba 2؉ -sensitive K ؉ channel mechanism involving the MCHR1 receptor. MCH immunoreactive fibers are in close proximity to vGluT2-GFP and GnRH-GFP neurons. Importantly, MCH blocks the excitatory effect of kisspeptin on vGluT2-GnRH neurons. Considering the role of MCH in regulating energy balance and of GnRH and kisspeptin in triggering puberty and maintaining fertility, MCH may provide a critical link between energy balance and reproduction directly at the level of the kisspeptin-activated vGluT2-GnRH neuron.fertility ͉ gonadotropins ͉ HPG axis ͉ obesity ͉ starvation N utritional status and availability of energy stores exert a profound impact on reproductive function (1-3). Reproduction is an expensive energy-consuming process, and thus it is important that puberty, pregnancy, and lactation occur only when metabolic fuel is available (4). Availability of metabolic fuel is conveyed to the brain by peripherally generated signals, such as leptin, insulin, and ghrelin, as well as by centrally released peptides such as neuropeptide Y, melanocortins, and melanin-concentrating hormone (MCH). One or more of these signals can directly or indirectly link energy balance with reproduction at one or more levels of the hypothalamic-pituitary-gonadal (HPG) axis. Thus, insulin and leptin may indirectly influence GnRH neurons (2, 5-7); leptin could regulate the HPG axis via kisspeptin-containing hypothalamic neurons (8, 9). Kisspeptin and its receptor are critical for reproduction (10-12); both kisspeptin and kisspeptin receptor knockout mice fail to enter puberty, and humans with loss of function mutations in the kisspeptin receptor exhibit hypogonadotropic hypogonadism and are infertile (13-15). Mechanisti...