Neurons that produce gonadotropin-releasing hormone (GnRH) are the final common pathway by which the brain regulates reproduction. GnRH neurons are regulated by an afferent network of kisspeptin-producing neurons. Kisspeptin binds to its cognate receptor on GnRH neurons and stimulates their activity, which in turn provides an obligatory signal for GnRH secretion, thus gating down-stream events supporting reproduction. We have developed kisspeptin antagonists to facilitate the direct determination of the role of kisspeptin neurons in the neuroendocrine regulation of reproduction. In vitro and in vivo studies of analogues of kisspeptin-10 with amino substitutions have identified several potent and specific antagonists. A selected antagonist was shown to inhibit the firing of GnRH neurons in the brain of the mouse and to reduce pulsatile GnRH secretion in female pubertal monkeys; the later supporting a key role of kisspeptin in puberty onset. This analog also inhibited the kisspeptin-induced release of luteinizing hormone (LH) in rats and mice and blocked the postcastration rise in LH in sheep, rats, and mice, suggesting that kisspeptin neurons mediate the negative feedback effect of sex steroids on gonadotropin secretion in mammals. The development of kisspeptin antagonists provides a valuable tool for investigating the physiological and pathophysiological roles of kisspeptin in the regulation of reproduction and could offer a unique therapeutic agent for treating hormone-dependent disorders of reproduction, including precocious puberty, endometriosis, and metastatic prostate cancer.

Photoreceptor degeneration is one of the most prevalent causes of blindness. Despite photoreceptor loss, the inner retina and central visual pathways remain intact over an extended time period, which has led to creative optogenetic approaches to restore light sensitivity in the surviving inner retina. The major drawbacks of all optogenetic tools recently developed and tested in mouse models are their low light sensitivity and lack of physiological compatibility. Here we introduce a next-generation optogenetic tool, Opto-mGluR6, designed for retinal ON-bipolar cells, which overcomes these limitations. We show that Opto-mGluR6, a chimeric protein consisting of the intracellular domains of the ON-bipolar cell–specific metabotropic glutamate receptor mGluR6 and the light-sensing domains of melanopsin, reliably recovers vision at the retinal, cortical, and behavioral levels under moderate daylight illumination.

GnRH neurons play a pivotal role in the central regulation of fertility. Kisspeptin greatly increases GnRH/LH release and GnRH neuron firing activity and may be involved in estradiol feedback, but the neurobiological mechanisms for these actions are unknown. G protein-coupled receptor 54, the receptor for kisspeptin, is expressed by GnRH neurons as well as other hypothalamic neurons, suggesting both direct and indirect effects are possible. To investigate this and determine whether kisspeptin activation of GnRH neurons is estradiol sensitive, we recorded the firing rate of GnRH neurons in brain slices from adult female mice that were ovariectomized (OVX) and either treated with estradiol (E) capsules (OVX+E) or left without further treatment. Kisspeptin increased GnRH neuronal activity in a dose-dependent manner in cells from both OVX and OVX+E mice, and estradiol significantly potentiated the response. To begin to distinguish direct from indirect actions of kisspeptin, fast synaptic transmission mediated by ionotropic gamma-aminobutyric acid and glutamate receptors was pharmacologically blocked (blockade). Blockade reduced GnRH response to kisspeptin in OVX+E but not in OVX mice. Actions of kisspeptin were also assessed using whole-cell voltage- and current-clamp recording in slices from OVX animals. Kisspeptin application depolarized GnRH neurons in current-clamp and generated inward current in voltage-clamp recordings, even after blocking action potential-dependent neural communication, consistent with a direct effect. Blockers of potassium channels abolished the inward current. Together our data indicate that kisspeptin activates GnRH neurons via both direct and transsynaptic mechanisms and that transsynaptic mechanisms are either enabled and/or potentiated by estradiol.

Ocular dominance (OD) plasticity in mouse primary visual cortex (V1) declines during postnatal development and is absent beyond postnatal day 110 if mice are raised in standard cages (SCs). An enriched environment (EE) promotes OD plasticity in adult rats. Here, we explored cellular mechanisms of EE in mouse V1 and the therapeutic potential of EE to prevent impairments of plasticity after a cortical stroke. Using in vivo optical imaging, we observed that monocular deprivation in adult EE mice (i) caused a very strong OD plasticity previously only observed in 4-wk-old animals, (ii) restored already lost OD plasticity in adult SC-raised mice, and (iii) preserved OD plasticity after a stroke in the primary somatosensory cortex. Using patch-clamp electrophysiology in vitro, we also show that (iv) local inhibition was significantly reduced in V1 slices of adult EE mice and (v) the GABA/AMPA ratio was like that in 4-wk-old SC-raised animals. These observations were corroborated by in vivo analyses showing that diazepam treatment significantly reduced the OD shift of EE mice after monocular deprivation. Taken together, EE extended the sensitive phase for OD plasticity into late adulthood, rejuvenated V1 after 4 mo of SCrearing, and protected adult mice from stroke-induced impairments of cortical plasticity. The EE effect was mediated most likely by preserving low juvenile levels of inhibition into adulthood, which potentially promoted adaptive changes in cortical circuits. O cular dominance (OD) plasticity induced by monocular deprivation (MD) is one of the best studied models of experience-dependent plasticity in the mammalian cortex (1, 2). OD plasticity in primary visual cortex (V1) of C57BL/6J mice is maximal at 4 wk of age, declines after 2-3 mo, and is absent beyond postnatal day 110 (PD110) if animals are raised in standard cages (SCs) (3-6). In 4-wk-old mice, 4 d of MD are sufficient to induce an OD shift to the open eye; therefore, neurons in the binocular V1, which are usually dominated by the contralateral eye in rodents (3, 7), become activated more equally by both eyes (5,8). This juvenile OD shift is predominantly mediated by a decrease in the visual cortical responses to the deprived eye (1, 9-11), whereas significant OD shifts in older animals up to PD110 need 7 d of MD and are mediated primarily by increased openeye responses in V1. Raising animals in an enriched environment (EE) gives them the opportunity of enhanced physical, social, and cognitive stimulation and influences brain physiology and behavior in many ways (12, 13). It has been shown previously that EE enhances visual system development in rats (14) and mice (15-17), increases levels of the brain-derived neurotrophic factor and serotonin (18), reduces both extracellular GABA levels (18, 19) and the density of ECM perineuronal nets (PNNs) (19), and promotes OD plasticity in adult and aging rats (18-21). Here, we explored cellular mechanisms of EE in V1 of mice and the therapeutic potential of EE to prevent impairments of plasticity after a c...

GnRH neurons are the final central pathway controlling fertility. Kisspeptin potently activates GnRH release via G protein-coupled receptor 54 (GPR54). GnRH neurons express GPR54, and kisspeptin can act directly; however, GPR54 is broadly expressed, suggesting indirect actions are possible. Transsynaptic mechanisms are involved in estradiol-induced potentiation of GnRH neuron response to kisspeptin. To investigate these mechanisms, separate whole-cell voltage-clamp recordings were performed of γ-aminobutyric acid (GABA)-ergic and glutamatergic transmission to GnRH neurons in brain slices before and during kisspeptin treatment. To determine whether estradiol alters the effect of kisspeptin on synaptic transmission, mice were ovariectomized and either left with no further treatment (OVX) or treated with estradiol implants (OVX+E). Cells were first studied in the morning when estradiol exerts negative feedback. Kisspeptin increased frequency and amplitude of GABAergic postsynaptic currents (PSCs) in GnRH neurons from OVX+E mice. Blocking action potentials eliminated the effect on frequency, indicating presynaptic actions. Amplitude changes were due to postsynaptic actions. Kisspeptin also increased frequency of glutamatergic excitatory PSCs in cells from OVX+E animals. Kisspeptin did not affect either GABAergic or glutamatergic transmission to GnRH neurons in cells from OVX mice, indicating effects on transmission are estradiol dependent. In contrast to stimulatory effects on GABAergic PSC frequency during negative feedback, kisspeptin had no effect during positive feedback. These data suggest estradiol enables kisspeptin-mediated increases in GABA and glutamate transmission to GnRH neurons. Furthermore, the occlusion of the response during positive feedback implies one consequence of estradiol positive feedback is an increase in transmission to GnRH neurons mediated by endogenous kisspeptin.

A surge of gonadotropin-releasing hormone (GnRH) release from the brain triggers the luteinizing hormone (LH) surge that causes ovulation. The GnRH surge is initiated by a switch in estradiol action from negative to positive feedback. Estradiol signals critical for the surge are likely transmitted to GnRH neurons at least in part via estradiol-sensitive afferents. Using an ovariectomized estradiol-treated (OVX+E) mouse model that exhibits daily LH surges, we examined changes in glutamate transmission to GnRH neurons during negative feedback and positive feedback. Spontaneous glutamatergic excitatory postsynaptic currents (EPSCs) mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainate receptors (AMPA/ KA Rs) or N-methyl-D-aspartate receptors (NMDARs) were recorded in GnRH neurons from OVX+E and OVX mice. There were no diurnal changes in the percentage of GnRH neurons from OVX mice exhibiting EPSCs. In cells from OVX+E mice, the profile of AMPA/KA R-mediated and NMDAR-mediated EPSCs showed changes dependent on time of day. Comparison of AMPA/KA R-mediated EPSC frequency in OVX+E and OVX cells showed that estradiol suppressed transmission during negative feedback but had no effect during positive feedback. Tetrodotoxin treatment to block action potential firing did not affect AMPA/KA R-mediated EPSC frequency in OVX cells during negative feedback or in OVX+E cells during positive feedback, suggesting that estradiol-induced suppression of glutamate transmission may be primarily due to activity-independent changes. The diurnal removal of estradiol-induced suppression of AMPA/KA R-mediated glutamate transmission to GnRH neurons during positive feedback suggests that the primary role for estradiol-induced changes in glutamate transmission may be in mediating negative feedback.

Estradiol has both negative and positive feedback actions upon gonadotropin-releasing hormone (GnRH) release; the latter actions trigger the preovulatory GnRH surge. Although neurobiological mechanisms of the transitions between feedback modes are becoming better understood, the roles of voltage-gated potassium currents, major contributors to neuronal excitability, are unknown. Estradiol alters two components of potassium currents in these cells: a transient current, I(A), and a sustained current, I(K). Kisspeptin is a potential mediator between estradiol and GnRH neurons and can act directly on GnRH neurons. We examined how estradiol, time of day, and kisspeptin interact to regulate these conductances in a mouse model exhibiting daily switches between estradiol negative (morning) and positive feedback (evening). Whole-cell voltage clamp recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice and from OVX mice treated with estradiol (OVX+E). There were no diurnal changes in either I(A) or I(K) in GnRH neurons from OVX mice. In contrast, in GnRH neurons from OVX+E mice, I(A) and I(K) were greater during the morning when GnRH neuron activity is low and smaller in the evening when GnRH neuron activity is high. Estradiol increased I(A) in the morning and decreased it in the evening, relative to that in cells from OVX mice. Exogenously applied kisspeptin reduced I(A) regardless of time of day or estradiol status. Estradiol, interacting with time of day, and kisspeptin both depolarized I(A) activation. These findings extend our understanding of both the neurobiological mechanisms of estradiol negative vs. positive regulation of GnRH neurons and of kisspeptin action on these cells.

The ability of the adult brain to undergo plastic changes is of particular interest in medicine, especially regarding recovery from injuries or improving learning and cognition. Matrix metalloproteinases (MMPs) have been associated with juvenile experience-dependent primary visual cortex (V1) plasticity, yet little is known about their role in this process in the adult V1. Activation of MMPs is a crucial step facilitating structural changes in a healthy brain; however, upon brain injury, upregulated MMPs promote the spread of a lesion and impair recovery. To clarify these seemingly opposing outcomes of MMP-activation, we examined the effects of MMP-inhibition on experience-induced plasticity in healthy and stoke-affected adult mice. In healthy animals, 7-day application of MMP-inhibitor prevented visual plasticity. Additionally, treatment with MMP-inhibitor once but not twice following stroke rescued plasticity, normally lost under these conditions. Our data imply that an optimal level of MMP-activity is crucial for adult visual plasticity to occur.DOI: http://dx.doi.org/10.7554/eLife.11290.001