Live-Cell Imaging of Single Neurotrophin Receptor Molecules on Human Neurons in Alzheimer's Disease.

Neurotrophin receptors such as the tropomyosin receptor kinase A receptor (TrkA) and the low-affinity binding p75 neurotrophin receptor p75NTR play a critical role in neuronal survival and their functions are altered in Alzheimer's disease (AD). Changes in the dynamics of receptors on the plasma membrane are essential to receptor function. However, whether receptor dynamics are affected in different pathophysiological conditions is unexplored. Using live-cell single-molecule imaging, we examined the surface trafficking of TrkA and p75NTR molecules on live neurons that were derived from human-induced pluripotent stem cells (hiPSCs) of presenilin 1 (PSEN1) mutant familial AD (fAD) patients and non-demented control subjects. Our results show that the surface movement of TrkA and p75NTR and the activation of TrkA- and p75NTR-related phosphoinositide-3-kinase (PI3K)/serine/threonine-protein kinase (AKT) signaling pathways are altered in neurons that are derived from patients suffering from fAD compared to controls. These results provide evidence for altered surface movement of receptors in AD and highlight the importance of investigating receptor dynamics in disease conditions. Uncovering these mechanisms might enable novel therapies for AD.

High-Fidelity NIR-II Multiplexed Lifetime Bioimaging with Bright Double Interfaced Lanthanide Nanoparticles.

Fluorescence lifetime imaging provides more possibility of in vivo multiplexing in second near infrared (NIR-II) window. However, it still faces the obstacle that fluorescent probes with differentiable lifetime often exhibit quite different fluorescence intensity, especially the short lifetime usually accompanies with a weak fluorescence intensity, resulting in the difficulty for simultaneously decoding multiplexed lifetime information due to the interference of background noise. To facilitate high-fidelity lifetime multiplexed imaging, we developed a series of Er3+ doped double interface fluorescent nanoprobes (Er-DINPs): α-NaYF4 @NaErF4 : Ce@NaYbF4 @NaErF4 : Ce@NaYF4 with strong fluorescence intensity and easily distinguishable fluorescence lifetime. Both in vitro and in vivo experimental results confirmed the advantage of these probes with comparable fluorescence intensity for high-fidelity multiplexed lifetime bioimaging.

The Role of Estradiol in Traumatic Brain Injury: Mechanism and Treatment Potential.

Patients surviving traumatic brain injury (TBI) face numerous neurological and neuropsychological problems significantly affecting their quality of life. Extensive studies over the past decades have investigated pharmacological treatment options in different animal models, targeting various pathological consequences of TBI. Sex and gender are known to influence the outcome of TBI in animal models and in patients, respectively. Apart from its well-known effects on reproduction, 17ß-estradiol (E2) has a neuroprotective role in brain injury. Hence, in this review, we focus on the effect of E2 in TBI in humans and animals. First, we discuss the clinical classification and pathomechanism of TBI, the research in animal models, and the neuroprotective role of E2. Based on the results of animal studies and clinical trials, we discuss possible E2 targets from early to late events in the pathomechanism of TBI, including neuroinflammation and possible disturbances of the endocrine system. Finally, the potential relevance of selective estrogenic compounds in the treatment of TBI will be discussed.

Estradiol-Induced Epigenetically Mediated Mechanisms and Regulation of Gene Expression.

Gonadal hormone 17ß-estradiol (E2) and its receptors are key regulators of gene transcription by binding to estrogen responsive elements in the genome. Besides the classical genomic action, E2 regulates gene transcription via the modification of epigenetic marks on DNA and histone proteins. Depending on the reaction partner, liganded estrogen receptor (ER) promotes DNA methylation at the promoter or enhancer regions. In addition, ERs are important regulators of passive and active DNA demethylation. Furthermore, ERs cooperating with different histone modifying enzymes and chromatin remodeling complexes alter gene transcription. In this review, we survey the basic mechanisms and interactions between estrogen receptors and DNA methylation, demethylation and histone modification processes as well as chromatin remodeling complexes. The particular relevance of these mechanisms to physiological processes in memory formation, embryonic development, spermatogenesis and aging as well as in pathophysiological changes in carcinogenesis is also discussed.

Effect of Inflammation on Female Gonadotropin-Releasing Hormone (GnRH) Neurons: Mechanisms and Consequences.

: Inflammation has a well-known suppressive effect on fertility. The function of gonadotropin-releasing hormone (GnRH) neurons, the central regulator of fertility is substantially altered during inflammation in females. In our review we discuss the latest results on how the function of GnRH neurons is modified by inflammation in females. We first address the various effects of inflammation on GnRH neurons and their functional consequences. Second, we survey the possible mechanisms underlying the inflammation-induced actions on GnRH neurons. The role of several factors will be discerned in transmitting inflammatory signals to the GnRH neurons: cytokines, kisspeptin, RFamide-related peptides, estradiol and the anti-inflammatory cholinergic pathway. Since aging and obesity are both characterized by reproductive decline our review also focuses on the mechanisms and pathophysiological consequences of the impact of inflammation on GnRH neurons in aging and obesity.

Melatonin Suppresses the Kainate Receptor-Mediated Excitation on Gonadotropin-Releasing Hormone Neurons in Female and Male Prepubertal Mice.

Melatonin, a pineal gland secretion, is an amphiphilic neurohormone involved in the biological and physiologic regulation of bodily functions. Numerous studies have shown the effects of melatonin on the release of gonadotropins and their actions at one or several levels of the hypothalamic-pituitary-gonadal axis. However, direct melatonin action on gonadotropin-releasing hormone (GnRH) neurons and its mechanism of action remain unclear. Here, plasma melatonin levels were measured and the effect of melatonin on GnRH neurons was assessed using brain slice patch clamp techniques. The plasma melatonin levels in prepubertal mice were higher than those in the adults. Melatonin itself did not change the firing activity of GnRH neurons. Interestingly, the kainate receptor-mediated responses but not the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and N-methyl-D-aspartic acid (NMDA)-induced responses were suppressed by melatonin in both the voltage clamp and current clamp modes. The inhibitory effects of the kainate-induced response by melatonin tended to increase with higher melatonin concentrations and persisted in the presence of tetrodotoxin, a voltage-sensitive Na+ channel blocker, or luzindole, a non-selective melatonin receptor antagonist. However, the response was completely abolished by pretreatment with pertussis toxin. These results suggest that melatonin can regulate GnRH neuronal activities in prepubertal mice by partially suppressing the excitatory signaling mediated by kainate receptors through pertussis toxin-sensitive G-protein-coupled receptors.

Characterization of Neurons Expressing the Novel Analgesic Drug Target Somatostatin Receptor 4 in Mouse and Human Brains.

Somatostatin is an important mood and pain-regulating neuropeptide, which exerts analgesic, anti-inflammatory, and antidepressant effects via its Gi protein-coupled receptor subtype 4 (SST4) without endocrine actions. SST4 is suggested to be a unique novel drug target for chronic neuropathic pain, and depression, as a common comorbidity. However, its neuronal expression and cellular mechanism are poorly understood. Therefore, our goals were (i) to elucidate the expression pattern of Sstr4/SSTR4 mRNA, (ii) to characterize neurochemically, and (iii) electrophysiologically the Sstr4/SSTR4-expressing neuronal populations in the mouse and human brains. Here, we describe SST4 expression pattern in the nuclei of the mouse nociceptive and anti-nociceptive pathways as well as in human brain regions, and provide neurochemical and electrophysiological characterization of the SST4-expressing neurons. Intense or moderate SST4 expression was demonstrated predominantly in glutamatergic neurons in the major components of the pain matrix mostly also involved in mood regulation. The SST4 agonist J-2156 significantly decreased the firing rate of layer V pyramidal neurons by augmenting the depolarization-activated, non-inactivating K+ current (M-current) leading to remarkable inhibition. These are the first translational results explaining the mechanisms of action of SST4 agonists as novel analgesic and antidepressant candidates.

Rapid non-classical effects of steroids on the membrane receptor dynamics and downstream signaling in neurons.

Contribution to Special Issue on Fast effects of steroids. Although rapid effects of steroid hormones on membrane receptors and intracellular signaling molecules have been extensively studied in neurons, we are only beginning to understand the molecular mechanisms behind these non-classical steroid actions. Single molecule tracking (SMT) studies on live cells demonstrated that surface trafficking of membrane receptors determines their ligand binding properties and downstream signaling events. Recent findings suggest that one of the underlying mechanisms of non-classical steroid actions is the alteration of receptor movements on the membrane surface. In order to highlight this novel aspect of steroid effects, we first address the types of receptor movements in the plasma membrane and the role of cortical actin dynamics in receptor movement. We then discuss how single molecules and the surface movements of receptors can be detected in live cells. Next, we review the fundamental processes, which determine the effect of steroids on the plasma membrane: steroid movement through the lipid bilayer and the role of steroid membrane receptors. Using glutamate and neurotrophin receptors (NTRs) as examples, we demonstrate the features of receptor dynamics in the membrane. In addition, we survey the available data of rapid steroid actions on membrane receptor trafficking: we discuss how glucocorticoids act on the surface diffusion of glutamate receptor molecules and how estradiol acts on NTRs and gamma-aminobutyric acid type A receptors (GABAARs) and their related signaling events as well as on cortical actin. Finally, we address the physiological relevance of rapid steroid action on membrane receptors dynamics.

Non-classical effects of estradiol on cAMP responsive element binding protein phosphorylation in gonadotropin-releasing hormone neurons: mechanisms and role.

Gonadotropin-releasing hormone (GnRH) is produced by a heterogenous neuronal population in the hypothalamus to control pituitary gonadotropin production and reproductive function in all mammalian species. Estradiol is a critical component for the communication between the gonads and the central nervous system. Resolving the mechanisms by which estradiol modulates GnRH neurons is critical for the understanding of how fertility is regulated. Extensive studies during the past decades have provided compelling evidence that estradiol has the potential to alter the intracellular signal transduction mechanisms. The common target of many signaling pathways is the phosphorylation of a key transcription factor, the cAMP response element binding protein (CREB). This review first addresses the aspects of estradiol action on CREB phosphorylation (pCREB) in GnRH neurons. Secondly, this review considers the receptors and signaling network that regulates estradiol's action on pCREB within GnRH neurons and finally it summarizes the physiological significance of CREB to estrogen feedback.

The role of cAMP response element-binding protein in estrogen negative feedback control of gonadotropin-releasing hormone neurons.

The mechanisms through which estradiol (E2) regulates gonadotropin-releasing hormone (GnRH) neurons to control fertility are unclear. Previous studies have demonstrated that E2 rapidly phosphorylates cAMP response element-binding protein (CREB) in GnRH neurons in vivo. In the present study, we used GnRH neuron-specific CREB-deleted mutant mice [GnRH-CREB knock-outs (KOs)] with and without global cAMP response element modulator (CREM) deletion (global-CREM KOs) to investigate the role of CREB in estrogen negative feedback on GnRH neurons. Evaluation of GnRH-CREB KO mice with and without global CREM deletion revealed normal puberty onset. Although estrus cycle length in adults was the same in controls and knock-out mice, cycles in mutant mice consisted of significantly longer periods of diestrus and less estrus. In GnRH-CREB KO mice, basal levels of luteinizing hormone (LH) and the postovariectomy increment in LH were normal, but the ability of E2 to rapidly suppress LH was significantly blunted. In contrast, basal and postovariectomy LH levels were abnormal in GnRH-CREB KO/global-CREM KO mice. Fecundity studies showed that GnRH-CREB KO with and without global CREM deletion were normal up to ∼9 months of age, at which time they became prematurely reproductively senescent. Morphological analysis of GnRH neurons revealed a significant reduction (p < 0.01) in GnRH somatic spine density of GnRH-CREB KO mice compared to control females. These observations implicate CREB within the GnRH neuron as an important target for E2's negative feedback actions. They also indicate that the rapid modulation of CREB by E2 is of physiological significance in the CNS.

Maximum likelihood-based estimation of diffusion coefficient is quick and reliable method for analyzing estradiol actions on surface receptor movements.

The rapid effects of estradiol on membrane receptors are in the focus of the estradiol research field, however, the molecular mechanisms of these non-classical estradiol actions are poorly understood. Since the lateral diffusion of membrane receptors is an important indicator of their function, a deeper understanding of the underlying mechanisms of non-classical estradiol actions can be achieved by investigating receptor dynamics. Diffusion coefficient is a crucial and widely used parameter to characterize the movement of receptors in the cell membrane. The aim of this study was to investigate the differences between maximum likelihood-based estimation (MLE) and mean square displacement (MSD) based calculation of diffusion coefficients. In this work we applied both MSD and MLE to calculate diffusion coefficients. Single particle trajectories were extracted from simulation as well as from α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor tracking in live estradiol-treated differentiated PC12 (dPC12) cells. The comparison of the obtained diffusion coefficients revealed the superiority of MLE over the generally used MSD analysis. Our results suggest the use of the MLE of diffusion coefficients because as it has a better performance, especially for large localization errors or slow receptor movements.

Stereology of gonadotropin-releasing hormone and kisspeptin neurons in PACAP gene-deficient female mice.

The hypothalamic gonadotropin-releasing hormone (GnRH)-kisspeptin neuronal network regulates fertility in all mammals. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide isolated from the hypothalamus that is involved in the regulation of several releasing hormones and trop hormones. It is well-known that PACAP influences fertility at central and peripheral levels. However, the effects of PACAP on GnRH and kisspeptin neurons are not well understood. The present study investigated the integrity of the estrous cycle in PACAP-knockout (KO) mice. The number and immunoreactivity of GnRH (GnRH-ir) neurons in wild-type (WT) and PACAP KO female mice were determined using immunohistochemistry. In addition, the number of kisspeptin neurons was measured by counting kisspeptin mRNA-positive cells in the rostral periventricular region of the third ventricle (RP3V) and arcuate nucleus (ARC) using the RNAscope technique. Finally, the mRNA and protein expression of estrogen receptor alpha (ERα) was also examined. Our data showed that the number of complete cycles decreased, and the length of each cycle was longer in PACAP KO mice. Furthermore, the PACAP KO mice experienced longer periods of diestrus and spent significantly less time in estrus. There was no difference in GnRH-ir or number of GnRH neurons. In contrast, the number of kisspeptin neurons was decreased in the ARC, but not in the R3PV, in PACAP KO mice compared to WT littermates. Furthermore, ERα mRNA and protein expression was decreased in the ARC, whereas in the R3PV region, ERα mRNA levels were elevated. Our results demonstrate that embryonic deletion of PACAP significantly changes the structure and presumably the function of the GnRH-kisspeptin neuronal network, influencing fertility.

17ß-estradiol does not have a direct effect on the function of striatal cholinergic interneurons in adult mice in vitro.

The striatum is an essential component of the basal ganglia that is involved in motor control, action selection and motor learning. The pathophysiological changes of the striatum are present in several neurological and psychiatric disorder including Parkinson's and Huntington's diseases. The striatal cholinergic neurons are the main regulators of striatal microcircuitry. It has been demonstrated that estrogen exerts various effects on neuronal functions in dopaminergic and medium spiny neurons (MSN), however little is known about how the activity of cholinergic interneurons are influenced by estrogens. In this study we examined the acute effect of 17ß-estradiol on the function of striatal cholinergic neurons in adult mice in vitro. We also tested the effect of estrus cycle and sex on the spontaneous activity of cholinergic interneurons in the striatum. Our RNAscope experiments showed that ERα, ERß, and GPER1 receptor mRNAs are expressed in some striatal cholinergic neurons at a very low level. In cell-attached patch clamp experiments, we found that a high dose of 17ß-estradiol (100 nM) affected the spontaneous firing rate of these neurons only in old males. Our findings did not demonstrate any acute effect of a low concentration of 17ß-estradiol (100 pM) or show any association of estrus cycle or sex with the activity of striatal cholinergic neurons. Although estrogen did not induce changes in the intrinsic properties of neurons, indirect effects via modulation of the synaptic inputs of striatal cholinergic interneurons cannot be excluded.

Effects of estrogen on beta-amyloid-induced cholinergic cell death in the nucleus basalis magnocellularis.

Alzheimer disease is characterized by accumulation of ß-amyloid (Aß) and cognitive dysfunctions linked to early loss of cholinergic neurons. As estrogen-based hormone replacement therapy has beneficial effects on cognition of demented patients, and it may prevent memory impairments, we investigated the effect of estrogen-pretreatment on Aß-induced cholinergic neurodegeneration in the nucleus basalis magnocellularis (NBM). We tested which Aß species induces the more pronounced cholinotoxic effect in vivo. We injected different Aß assemblies in the NBM of mice, and measured cholinergic cell and cortical fiber loss. Spherical Aß oligomers had the most toxic effect. Pretreatment of ovariectomized mice with estrogen before Aß injection decreased cholinergic neuron loss and partly prevented fiber degeneration. By using proteomics, we searched for proteins involved in estrogen-mediated protection and in Aß toxicity 24 h following injection. The change in expression of, e.g., DJ-1, NADH ubiquinone oxidoreductase, ATP synthase, phosphatidylethanolamine-binding protein 1, protein phosphatase 2A and dimethylarginine dimethylaminohydrolase 1 support our hypothesis that Aß induces mitochondrial dysfunction, decreases MAPK signaling, and increases NOS activation in NBM. On the other hand, altered expression of, e.g., MAP kinase kinase 1 and 2, protein phosphatase 1 and 2A by Aß might increase MAPK suppression and NOS signaling in the cortical target area. Estrogen pretreatment reversed most of the changes in the proteome in both areas. Our experiments suggest that regulation of the MAPK pathway, mitochondrial pH and NO production may all contribute to Aß toxicity, and their regulation can be prevented partly by estrogen pretreatment.

Estrogen augments the T cell-dependent but not the T-independent immune response.

Estrogen plays a critical regulatory role in the development and maintenance of immunity. Its role in the regulation of antibody synthesis in vivo is still not completely clear. Here, we have compared the effect of estrogen on T cell-dependent (TD) and T cell-independent type 2 (TI-2) antibody responses. The results provide the first evidence that estrogen enhances the TD but not the TI-2 response. Ovariectomy significantly decreased, while estrogen re-administration increased the number of hapten-specific IgM- and IgG-producing cells in response to TD antigen. In vitro experiments also show that estrogen may have a direct impact on B and T cells by inducing rapid signaling events, such as Erk and AKT phosphorylation, cell-specific Ca(2+) signal, and NFkappaB activation. These non-transcriptional effects are mediated by classical estrogen receptors and partly by an as yet unidentified plasma membrane estrogen receptor. Such receptor- mediated rapid signals may modulate the in vivo T cell-dependent immune response.

Super-Resolution Imaging With Lanthanide Luminescent Nanocrystals: Progress and Prospect.

Stimulated emission depletion (STED) nanoscopy has overcome a serious diffraction barrier on the optical resolution and facilitated new discoveries on detailed nanostructures in cell biology. Traditional fluorescence probes employed in the super-resolution imaging approach include organic dyes and fluorescent proteins. However, some limitations of these probes, such as photobleaching, short emission wavelengths, and high saturation intensity, still hamper the promotion of optical resolution and bio-applications. Recently, lanthanide luminescent probes with unique optical properties of non-photobleaching and sharp emissions have been applied in super-resolution imaging. In this mini-review, we will introduce several different mechanisms for lanthanide ions to achieve super-resolution imaging based on an STED-like setup. Then, several lanthanide ions used in super-resolution imaging will be described in detail and discussed. Last but not least, we will emphasize the future challenges and outlooks in hope of advancing the next-generation lanthanide fluorescent probes for super-resolution optical imaging.

Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons.

Gonadal steroid 17ß-estradiol (E2) exerts rapid, non-genomic effects on neurons and strictly regulates learning and memory through altering glutamatergic neurotransmission and synaptic plasticity. However, its non-genomic effects on AMPARs are not well understood. Here, we analyzed the rapid effect of E2 on AMPARs using single-molecule tracking and super-resolution imaging techniques. We found that E2 rapidly decreased the surface movement of AMPAR via membrane G protein-coupled estrogen receptor 1 (GPER1) in neurites in a dose-dependent manner. The cortical actin network played a pivotal role in the GPER1 mediated effects of E2 on the surface mobility of AMPAR. E2 also decreased the surface movement of AMPAR both in synaptic and extrasynaptic regions on neurites and increased the synaptic dwell time of AMPARs. Our results provide evidence for understanding E2 action on neuronal plasticity and glutamatergic neurotransmission at the molecular level.

Sex differences in rapid nonclassical action of 17ß-oestradiol on intracellular signalling and oestrogen receptor α expression in basal forebrain cholinergic neurones in mouse.

Rapid nonclassical effects of 17ß-oestradiol (E2 ) on intracellular signalling have been identified in the basal forebrain, although the extent to which these actions may be different in males and females is unknown. Previous work has shown that E2 rapidly phosphorylates cAMP responsive element binding protein (CREB) via ΕRα in female cholinergic neurones. Using this indicator, the present study examined whether nonclassical actions of E2 occur in a sexually dimorphic manner within basal forebrain cholinergic neurones in mice. In addition, we investigated the expression and intracellular distribution of oestrogen receptor (ΕR)α in cholinergic neurones in female and male mice. Animals were gonadectomised and treated 2 weeks later with E2 . The number of CREB-expressing cholinergic neurones was not altered in any of the brain regions after E2 treatment in both males and females. However, E2 treatment rapidly (< 15 minutes) increased (P < 0.05) the number of cholinergic neurones expressing phosphorylated CREB (pCREB) in the substantia innominata and medial septum but not in the striatum in female mice. By contrast, E2 did not change pCREB expression in cholinergic neurones in male mice at any time point (15 minutes, 1 hour, 4 hours), irrespective of the neuroanatomical location. We also observed that, in females, more cholinergic neurones expressed nuclear ΕRα in all regions, whereas males showed more cholinergic neurones with cytoplasmic or both nuclear and cytoplasmic expression of ΕRα. Taken together, these results demonstrate a marked sex difference in the E2 -induced nonclassical effect and intracellular distribution of ΕRα in basal forebrain cholinergic neurones in vivo.

Nonclassical estrogen modulation of presynaptic GABA terminals modulates calcium dynamics in gonadotropin-releasing hormone neurons.

There is increasing recognition that estrogen exerts multifaceted regulatory effects on GnRH neurons. The acute effects of estrogen on calcium dynamics in these cells were examined using a transgenic mouse line that allows real-time measurement of intracellular calcium concentration ([Ca2+]i) in GnRH neurons in the acute brain slice preparation. 17-beta-Estradiol (E2) at 100 pm-100 nm was found to activate [Ca2+]i transients in approximately 40% of GnRH neurons with an approximate 15-min latency. This effect was not replicated by E2-BSA, which limits E2 action to the membrane, 17-alpha-estradiol, the inactive isomer at classical estrogen receptors (ERs), or G-1 the GPR30 agonist. E2 continued to activate [Ca2+]i transients when transcription was blocked. An ER alpha-selective agonist was equally potent in activating [Ca2+]i transients, and E2 remained effective in ERbeta knockout x GnRH-Pericam mice. E2's activation of [Ca2+]i transients continued in the presence of tetrodotoxin, which blocks action potential-dependent transmission, but was abolished completely by the further addition of a gamma-aminobutyric acid (GABA)A receptor antagonist. Exogenous GABA was found to initiate [Ca2+]i transients in GnRH neurons. Whole cell, voltage-clamp recordings of GnRH-green fluorescence protein neurons revealed that E2 generated discrete bursts of miniature inhibitory postsynaptic currents with a latency of approximately 15 min. These observations provide evidence for a new mechanism of nonclassical estrogen action within the brain. Estrogen interacts with the classical ERalpha at the level of the GABAergic nerve terminal to regulate action potential-independent GABA release that, in turn, controls postsynaptic calcium dynamics.

The Role of Interleukin-10 in Mediating the Effect of Immune Challenge on Mouse Gonadotropin-Releasing Hormone Neurons In Vivo.

Immune challenge alters neural functioning via cytokine production. Inflammation has profound impact on the central regulation of fertility, but the mechanisms involved are not clearly defined. The anti-inflammatory cytokine interleukin (IL)-10 is responsible for balancing the immune response in the brain. To examine whether IL-10 has an effect on the function of the gonadotropin-releasing hormone (GnRH) neurons, we first examined the effect of immune responses with distinct cytokine profiles, such as the T cell-dependent (TD) and T cell-independent (TI) B-cell response. We investigated the effect of the TD and TI immune responses on ERK1/2 phosphorylation in GnRH neurons by administering fluorescein isothiocyanate/keyhole limpet hemocyanin (KLH-FITC) or dextran-FITC to female mice. Although dextran-FITC had no effect, KLH-FITC induced ERK1/2 phosphorylation in GnRH neurons after 6 d. KLH-FITC treatment increased the levels of IL-10 in the hypothalamus (HYP), but this treatment did not cause lymphocyte infiltration or an increase in the levels of proinflammatory cytokines. In IL-10 knock-out (KO) mice, KLH-FITC-induced ERK1/2 phosphorylation in the GnRH neurons was absent. We also showed that in IL-10 KO mice, the estrous cycle was disrupted. Perforated patch-clamp recordings from GnRH-GFP neurons, IL-10 immunohistochemistry, and in vitro experiments on acute brain slices revealed that IL-10 can directly alter GnRH neuron firing and induce ERK1/2 phosphorylation. These observations demonstrate that IL-10 plays a role in influencing signaling of GnRH neurons in the TD immune response. These results also provide the first evidence that IL-10 can directly alter the function of GnRH neurons and may help the maintenance of the integrity of the estrous cycle.