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The suprachiasmatic nuclei (SCN) of the anterior hypothalamus host the circadian pacemaker that synchronizes mammalian rhythms with the day-night cycle. SCN neurons are intrinsically rhythmic, thanks to a conserved cell-autonomous clock mechanism. In addition, circuit-level emergent properties confer a unique degree of precision and robustness to SCN neuronal rhythmicity. However, the multicellular functional organization of the SCN is not yet fully understood. Although SCN neurons are well coordinated, experimental evidences indicate that some neurons oscillate out of phase in SCN explants, and possibly to a larger extent in vivo. Here, we used microendoscopic Ca2+i imaging to investigate SCN rhythmicity at a single cell resolution in free-behaving mice. We found that SCN neurons in vivo exhibited fast Ca2+i spikes superimposed upon slow changes in baseline Ca2+i levels. Both spikes and baseline followed a time-of-day modulation in many neurons, but independently from each other. Daily rhythms in basal Ca2+i were well coordinated, while spike activity from the same neurons peaked at multiple times of the light cycle, and unveiled clock-independent interactions at the multicellular level. Hence, fast Ca2+i spikes and slow changes in baseline Ca2+i levels highlighted how diverse activity patterns could articulate within the temporal network unity of the SCN in vivo, and provided support for a multiplex neuronal code in the circadian pacemaker.

Key points Mouse chromaffin cells in acute adrenal slices exhibit two distinct spiking patterns, a repetitive mode and a bursting mode. A sodium background conductance operates at rest as demonstrated by the membrane hyperpolarization evoked by a low Na+ -containing extracellular saline. This sodium background current is insensitive to TTX, is not blocked by Cs+ ions and displays a linear I-V relationship at potentials close to chromaffin cell resting potential. Its properties are reminiscent of those of the sodium leak channel NALCN. In the adrenal gland, Nalcn mRNA is selectively expressed in chromaffin cells. The study fosters our understanding of how the spiking pattern of chromaffin cells is regulated and adds a sodium background conductance to the list of players involved in the stimulus-secretion coupling of the adrenomedullary tissue. Abstract Chromaffin cells (CCs) are the master neuroendocrine units for the secretory function of the adrenal medulla and a finely-tuned regulation of their electrical activity is required for appropriate catecholamine secretion in response to the organismal demand. Here, we aim at deciphering how the spiking pattern of mouse CCs is regulated by the ion conductances operating near the resting membrane potential (RMP). At RMP, mouse CCs display a composite firing pattern, alternating between active periods composed of action potentials spiking with a regular or a bursting mode, and silent periods. RMP is sensitive to changes in extracellular sodium concentration, and a low Na+ -containing saline hyperpolarizes the membrane, regardless of the discharge pattern. This RMP drive reflects the contribution of a depolarizing conductance, which is (i) not blocked by tetrodotoxin or caesium, (ii) displays a linear I-V relationship between -110 and -40 mV, and (iii) is carried by cations with a conductance sequence gNa > gK > gCs . These biophysical attributes, together with the expression of the sodium-leak channel Nalcn transcript in CCs, state credible the contribution of NALCN. This inaugural report opens new research routes in the field of CC stimulus-secretion coupling, and extends the inventory of tissues in which NALCN is expressed to neuroendocrine glands.

Migration of GnRH neurons, a critical step for vertebrate reproduction, depends on circuit-intrinsic synaptic communication., Migration of gonadotropin-releasing hormone (GnRH) neurons from their birthplace in the nasal placode to their hypothalamic destination is critical for vertebrate reproduction and species persistence. While their migration mode as individual GnRH neurons has been extensively studied, the role of GnRH-GnRH cell communication during migration remains largely unexplored. Here, we show in awake zebrafish larvae that migrating GnRH neurons pause at the nasal-forebrain junction and form clusters that act as interhemisphere neuronal ensembles. Within the ensembles, GnRH neurons create an isolated, spontaneously active circuit that is internally wired through monosynaptic glutamatergic synapses into which newborn GnRH neurons integrate before entering the brain. This initial phase of integration drives a phenotypic switch, which is essential for GnRH neurons to properly migrate toward their hypothalamic destination. Together, these experiments reveal a critical step for reproduction, which depends on synaptic communication between migrating GnRH neurons.

International audience; Functional imaging in behaving animals is essential to explore brain functions. Real-time optical imaging of brain functions is limited by light scattering, skull distortion, timing resolution and subcellular precision that altogether, make challenging the rapid acquisition of uncorrupted functional data of cells integrated de novo in the neurogliovascular unit. We report multimodal transcranial in vivo optical imaging for the fast and direct visualization of microcirculation in the perfusion domain where new cells incorporated in the neurogliovascular unit during the progression of a seizure disorder and its treatment. Using this methodology, we explored the performance improvement of cells integrated de novo in the neurogliovascular unit. We report fast transcranial imaging of blood microcirculation at sites of pericyte turnover in the epileptic brain and after treatment with a trophic factor that revealed key features of the regenerating neurogliovascular unit.

Context The X-linked immunoglobulin superfamily, member 1 (IGSF1), gene is highly expressed in the hypothalamus and in pituitary cells of the POU1F1 lineage. Human loss-of-function mutations in IGSF1 cause central hypothyroidism, hypoprolactinemia, and macroorchidism. Additionally, most affected adults exhibit higher than average IGF-1 levels and anecdotal reports describe acromegaloid features in older subjects. However, somatotrope function has not yet been formally evaluated in this condition. Objective We aimed to evaluate the role of IGSF1 in human and murine somatotrope function. Patients, Design, and Setting We evaluated 21 adult males harboring hemizygous IGSF1 loss-of-function mutations for features of GH excess, in an academic clinical setting. Main Outcome Measures We compared biochemical and tissue markers of GH excess in patients and controls, including 24-hour GH profile studies in 7 patients. Parallel studies were undertaken in male Igsf1-deficient mice and wild-type littermates. Results IGSF1-deficient adult male patients demonstrated acromegaloid facial features with increased head circumference as well as increased finger soft-tissue thickness. Median serum IGF-1 concentrations were elevated, and 24-hour GH profile studies confirmed 2- to 3-fold increased median basal, pulsatile, and total GH secretion. Male Igsf1-deficient mice also demonstrated features of GH excess with increased lean mass, organ size, and skeletal dimensions and elevated mean circulating IGF-1 and pituitary GH levels. Conclusions We demonstrate somatotrope neurosecretory hyperfunction in IGSF1-deficient humans and mice. These observations define a hitherto uncharacterized role for IGSF1 in somatotropes and indicate that patients with IGSF1 mutations should be evaluated for long-term consequences of increased GH exposure.

Summary: Reorganization of the neurovascular unit has been suggested in the epileptic brain, although the dynamics and functional significance remain unclear. Here, we tracked the in vivo dynamics of perivascular mural cells as a function of electroencephalogram (EEG) activity following status epilepticus. We segmented the cortical vascular bed to provide a size- and type-specific analysis of mural cell plasticity topologically. We find that mural cells are added and removed from veins, arterioles, and capillaries after seizure induction. Loss of mural cells is proportional to seizure severity and vascular pathology (e.g., rigidity, perfusion, and permeability). Treatment with platelet-derived growth factor subunits BB (PDGF-BB) reduced mural cell loss, vascular pathology, and epileptiform EEG activity. We propose that perivascular mural cells play a pivotal role in seizures and are potential targets for reducing pathophysiology. : Arango-Lievano et al. follow how status epilepticus changes the dynamics of mural cell turnover at the cortical vasculature, causing vessel damage. PDGF-BB, a growth factor promoting the assembly of mural cells at the vascular unit, ameliorates vessel function and reduces spontaneous epileptiform activity. Keywords: neurovascular, epilepsy, pericyte, blood flow, PDGF-BB, PDGFR-B, in vivo microscopy

Metabolic adaptation is a critical feature of synaptic plasticity. Indeed, synaptic plasticity requires the utilization and resupply of metabolites, in particular when the turnover is high and fast such as in stress conditions. What accounts for the localized energy burden of the post-synaptic compartment to the build up of chronic stress is currently not understood. We used in vivo microscopy of genetically encoded fluorescent probes to track changes of mitochondria, dendritic spines, ATP and H2O2 levels in pyramidal neurons of cortex before and after chronic unpredictable mild stress. Data revealed hotspots of postsynaptic mitochondria and dendritic spine turnover. Pharmacogenetic approach to force expression of the metabolic stress gene NR4A1 caused the fragmentation of postsynaptic mitochondria and loss of proximal dendritic spine clusters, whereas a dominant-negative mutant counteracted the effect of chronic stress. When fragmented, dendritic mitochondria produced lesser ATP at resting state and more on acute demand. This corresponded with significant production of mitochondrial H2O2 oxidative species in the dendritic compartment. Together, data indicate that pyramidal neurons adjust proximal dendritic spine turnover and mitochondria functions in keeping with synaptic demands., Highlights • Addition of dendritic spine clusters match with more proximal mitochondria coverage. • Loss of dendritic spine clusters match with less proximal mitochondria coverage. • Dendrites alter spine dynamics, ATP and H202 production in keeping with excitation. • In excess, the transcription factor NR4A1 promotes cross-clustering losses. • Blocking NR4A1 prevents net cross-clustering losses mediated by chronic stress.

Hypothyroidism refers to the pathological state of thyroid hormone (TH) deficiency. It affects between 0.3% and 3.7% of the general population in the USA and between 0.2% and 5.3% in Europe. Hypothyroid patients suffer from extreme fatigue, altered heart rate, depression, weight gain, and difficulty in conceiving. Also, maternal hypothyroidism, which affects 0.5% of all pregnant women, is the leading preventable cause of mental retardation in offspring. Importantly, diagnosis of hypothyroidism is based on a single thyroid-stimulating-hormone (TSH) measurement. TSH secretion by pituitary thyrotrophs is under the positive control of hypothalamic thyrotropin-releasing-hormone (TRH) and negative feedback exerted by THs. Despite the undeniable implication of thyrotrophs in the hypothalamus-pituitary-thyroid (HPT) axis regulation, little is known about the mechanisms underlying adaptive TSH secretion. Being the smallest endocrine cell population of the pituitary (2-4%), thyrotrophs display high plasticity in cell shape, number and network topology throughout development and in disease. Moreover, enzymatically-dispersed thyrotrophs lose their ability to express and secrete TSH in both basal and TRH-stimulated conditions. Altogether, these findings led us to the hypothesis that thyrotrophs form a very finely regulated and highly plastic functionally-organized cell population capable of adapting to physiological (i.e. pregnancy) and pathological (i.e. hypothyroidism) demand. The aim of this study was to investigate how thyrotrophs function in their native environment. Using gradient-index (GRIN) lenses implanted at the pituitary level and a 2g head-mounted miniscope, multicellular calcium activities of the thyrotroph population were monitored in freely-moving TSHβ-crexR26fl-flGCaMP6f mice. Strikingly, thyrotrophs displayed both short-lived calcium spikes of high amplitude, and slowly-evolving calcium waves propagating among the thyrotroph population (n=3). Such calcium waves recurred every 80min during episodes lasting 3min and were interspaced by short-lived activity/silent periods. As TSH displays a 2.722min half-life (n=6), repetitive bouts of calcium-dependent TSH exocytosis would lead to cumulative increase in circulating TSH levels. Hence, the thyrotroph population functions in vivo as a robust generator of repetitive calcium waves which would orchestrate the generation of ultradian TSH fluctuations. Such signaling events could be monitored during longitudinal studies (weeks to months) in which individual animals could be their own controls.

Extensive efforts have been made to explore how the activities of multiple brain cells combine to alter physiology through imaging and cell-specific manipulation in different animal models. However, the temporal regulation of peripheral organs by the neuroendocrine factors released by the brain is poorly understood. We have established a suite of adaptable methodologies to interrogate in vivo the relationship of hypothalamic regulation with the secretory output of the pituitary gland, which has complex functional networks of multiple cell types intermingled with the vasculature. These allow imaging and optogenetic manipulation of cell activities in the pituitary gland in awake mouse models, in which both neuronal regulatory activity and hormonal output are preserved. These methodologies are now readily applicable for longitudinal studies of short-lived events (e.g., calcium signals controlling hormone exocytosis) and slowly evolving processes such as tissue remodeling in health and disease over a period of days to weeks.

International audience; Pancreatic β-cells modulate insulin secretion through rapid sensing of blood glucose and integration of gut-derived signals. Increased insulin demand during pregnancy and obesity alters islet function and mass and leads to gestational diabetes mellitus and type 2 diabetes in predisposed individuals. However, it is unclear how blood-borne factors dynamically access the islets of Langerhans. Thus, understanding the changes in circulating molecule distribution that accompany compensatory β-cell expansion may be key to developing novel antidiabetic therapies. Here, using two-photon microscopy in vivo in mice, we demonstrate that islets are almost instantly exposed to peaks of circulating molecules, which rapidly pervade the tissue before clearance. In addition, both gestation and short-term high-fat-diet feeding decrease molecule extravasation and uptake rates in vivo in islets, independently of β-cell expansion or islet blood flow velocity. Together, these data support a role for islet vascular permeability in shaping β-cell adaptive responses to metabolic demand by modulating the access and sensing of circulating molecules.

OBJECTIVE: The L-type Ca(2+) current (I(Ca,L)) contributes to the generation and modulation of the pacemaker action potential (AP). We investigated facilitation of I(Ca,L) in sino-atrial cells. METHODS: Facilitation was studied in regularly-beating cells isolated enzymatically from young albino rabbits (0.8-1 kg). We used the whole-cell patch-clamp technique to vary the frequency of the test depolarizations evoked at -10 mV or the conditioning diastolic membrane potential prior to the test pulse. RESULTS: High frequencies (range 0.2-3.5 Hz) slowed the decay kinetics of I(Ca,L) evoked from a holding potential (HP) of -80 mV in 68% of cells resulting in a larger Ca(2+) influx during the test pulse. The amount of facilitation increased progressively between 0.2 and 3.0 Hz. When the frequency was changed from 0.1 to 1 Hz, the averaged increase in the time integral of I(Ca,L) was 27+/-7% (n=22). Application of conditioning voltages between -80 and -50 mV induced similar facilitation of I(Ca,L) in 73% of cells. The maximal increase of Ca(2+) entry occurred between -60 and -50 mV, and was on average 38+/-14% for conditioning prepulses of 5 s in duration (n=15). Numerical simulations of the pacemaker activity showed that facilitation of I(Ca,L) promotes stability of sino-atrial rate by enhancing Ca(2+) entry, thus establishing a negative feedback control against excessive heart rate slowing. CONCLUSION: Facilitation of I(Ca,L) is present in rabbit sino-atrial cells. The underlying mechanism reflects modulation of I(Ca,L) decay kinetics by diastolic membrane potential and frequency of depolarization. This phenomenon may provide an important regulatory mechanism of sino-atrial automaticity.

Mutation of the metabotropic glutamate receptor type 7 (mGlu7) induces absence-like epileptic seizures, but its precise role in the somatosensory thalamocortical network remains unknown. By combining electrophysiological recordings, optogenetics, and pharmacology, we dissected the contribution of the mGlu7 receptor at mouse thalamic synapses. We found that mGlu7 is functionally expressed at both glutamatergic and GABAergic synapses, where it can inhibit neurotransmission and regulate short-term plasticity. These effects depend on the PDZ-ligand of the receptor, as they are lost in mutant mice. Interestingly, the very low affinity of mGlu7 receptors for glutamate raises the question of how it can be activated, namely at GABAergic synapses and in basal conditions. Inactivation of the receptor activity with the mGlu7 negative allosteric modulator (NAM), ADX71743, enhances thalamic synaptic transmission. In vivo administration of the NAM induces a lethargic state with spindle and/or spike-and-wave discharges accompanied by a behavioral arrest typical of absence epileptic seizures. This provides evidence for mGlu7 receptor-mediated tonic modulation of a physiological function in vivo preventing synchronous and potentially pathological oscillations.

Hypothalamic growth hormone-releasing hormone (GHRH) neurons orchestrate body growth/maturation and have been implicated in feeding responses and ageing. However, the electrical patterns that dictate GHRH neuron functions have remained elusive. Since the inhibitory neuropeptide somatostatin (SST) is considered to be a primary oscillator of the GH axis, we examined its acute effects on GHRH neurons in brain slices from male and female GHRH-GFP mice. At the cellular level, SST irregularly suppressed GHRH neuron electrical activity, leading to slow oscillations at the population level. This resulted from an initial inhibitory action at the GHRH neuron level via K+ channel activation, followed by a delayed, sst1/sst2 receptor-dependent unbalancing of glutamatergic and GABAergic synaptic inputs. The oscillation patterns induced by SST were sexually dimorphic and could be explained by differential actions of SST on both GABAergic and glutamatergic currents. Thus, a tripartite neuronal circuit involving a fast hyperpolarization and a dual regulation of synaptic inputs appeared sufficient in pacing the activity of the GHRH neuronal population. These “feed-forward loops” may represent basic building blocks involved in the regulation of GHRH release and its downstream sexual specific functions.

Both endocrine and non-endocrine cells of the pituitary gland are organized into structural and functional networks which are formed during embryonic development but which may be modified throughout life. Structural mapping of the various endocrine cell types has highlighted the existence of distinct network motifs and relationships with the vasculature which may relate to temporal differences in their output. Functional characterization of the network activity of growth hormone and prolactin cells has revealed a role for cell organization in gene regulation, the plasticity of pituitary hormone output and remarkably the ability to memorize altered demand. As such, the description of these endocrine cell networks alters the concept of the pituitary from a gland which simply responds to external regulation to that of an oscillator which may memorize information and constantly adapt its coordinated networks’ responses to the flow of hypothalamic inputs.

Background— Reperfusion during acute myocardial infarction remains the best treatment for reducing infarct size. Postconditioning, applied at the onset of reperfusion, reduces myocardial infarction both in animals and humans. The objective of this study was to identify the time delay to apply postconditioning at reperfusion, allowing preservation of cardioprotection in the mouse myocardium. This is a major issue in the management of acute myocardial infarction patients. Methods and Results— Mice were subjected to 40 minutes of ischemia and 60 minutes of reperfusion (IR 60′ ). Postconditioning protocols corresponding to repetitive ischemia (3 cycles of 1 minute of ischemia and 1 minute of reperfusion) were applied during early reperfusion at various time durations (Δt) after reopening of the coronary artery (Δt=10 seconds, 1, 5, 10, 15, 20, 30, and 45 minutes; PostC Δt ). Infarct size/area at risk was reduced by 71% in PostC Δ1 compared with IR 60′ mice ( P =5×10 −6 ). There was a linear correlation ( r 2 =0.91) between infarct size and Δt, indicating that the cardioprotective effect of delayed postconditioning was progressively attenuated when Δt time increased. The protective effect of PostC Δ1 and PostC Δ15 was still effective when the duration of reperfusion was prolonged to 24 hours (IR 24 hours ; PostC Δ1 and PostC Δ15 versus IR 24 hours , P =0.001). Similar results were obtained for internucleosomal DNA fragmentation and lactate dehydrogenase release. Conclusions— This study in our in vivo mouse model of myocardial IR shows for the first time that delaying the intervention of postconditioning to 30 minutes does not abrogate the cardioprotective effect of postconditioning. This finding provides evidence that the time window of protection afforded by postconditioning may be larger than initially reported.

It is well established that Pacemaker activity of the sino-atrial node (SAN) initiates the heartbeat. However, the atrioventricular node (AVN) can generate viable pacemaker activity in case of SAN failure, but we have limited knowledge of the ionic bases of AVN automaticity. We characterized pacemaker activity and ionic currents in automatic myocytes of the mouse AVN. Pacemaking of AVN cells (AVNCs) was lower than that of SAN pacemaker cells (SANCs), both in control conditions and upon perfusion of isoproterenol (ISO). Block of I(Na) by tetrodotoxin (TTX) or of I(Ca,L) by isradipine abolished AVNCs pacemaker activity. TTX-resistant (I(Nar)) and TTX-sensitive (I(Nas)) Na(+) currents were recorded in mouse AVNCs, as well as T-(I(Ca,T)) and L-type (I(Ca,L)) Ca(2+) currents I(Ca,L) density was lower than in SANCs (51%). The density of the hyperpolarization-activated current, (I(f)) and that of the fast component of the delayed rectifier current (I(Kr)) were, respectively, lower (52%) and higher (53%) in AVNCs than in SANCs. Pharmacological inhibition of I(f) by 3 µM ZD-7228 reduced pacemaker activity by 16%, suggesting a relevant role for I(f) in AVNCs automaticity. Some AVNCs expressed also moderate densities of the transient outward K(+) current (I(to)). In contrast, no detectable slow component of the delayed rectifier current (I(Ks)) could be recorded in AVNCs. The lower densities of I(f) and I(Ca,L), as well as higher expression of I(Kr) in AVNCs than in SANCs may contribute to the intrinsically slower AVNCs pacemaking than that of SANCs.

There are well-recognized sex differences in many pituitary endocrine axes, usually thought to be generated by gonadal steroid imprinting of the neuroendocrine hypothalamus. However, the recognition that growth hormone (GH) cells are arranged in functionally organized networks raises the possibility that the responses of the network are different in males and females. We studied this by directly monitoring the calcium responses to an identical GH-releasing hormone (GHRH) stimulus in populations of individual GH cells in slices taken from male and female murine GH-eGFP pituitary glands. We found that the GH cell network responses are sexually dimorphic, with a higher proportion of responding cells in males than in females, correlated with greater GH release from male slices. Repetitive waves of calcium spiking activity were triggered by GHRH in some males, but were never observed in females. This was not due to a permanent difference in the network architecture between male and female mice; rather, the sex difference in the proportions of GH cells responding to GHRH were switched by postpubertal gonadectomy and reversed with hormone replacements, suggesting that the network responses are dynamically regulated in adulthood by gonadal steroids. Thus, the pituitary gland contributes to the sexually dimorphic patterns of GH secretion that play an important role in differences in growth and metabolism between the sexes.

Endocrine cells in the mammalian pituitary are arranged into three-dimensional homotypic networks that wire the gland and act to optimise hormone output by allowing the transmission of information between cell ensembles in a temporally precise manner. Despite this, the structure-function relationships that allow cells belonging to these networks to display coordinated activity remain relatively uncharacterised. This review discusses the recent technological advances that have allowed endocrine cell network structure and function to be probed and the mathematical models that can be used to analyse and present the resulting data. In particular, we focus on the mechanisms that allow endocrine cells to dynamically function as a population to drive hormone release as well as the experimental and theoretical methods that are used to track and model information flow through the network.

Although growth hormone (GH)- and prolactin (PRL)-secreting pituitary adenomas are considered benign, in many patients, tumour growth and/or invasion constitute a particular challenge. In other tumours, progression relies in part on dysfunction of intercellular adhesion mediated by the large family of cadherins. In the present study, we have explored the contribution of cadherins in GH and PRL adenoma pathogenesis, and evaluated whether this class of adherence molecules was related to tumour invasiveness. We have first established, by quantitative polymerase chain reaction and immunohistochemistry, the expression profile of classical cadherins in the normal human pituitary gland. We show that the cadherin repertoire is restricted and cell-type specific. Somatotrophs and lactotrophs express mainly E-cadherin and cadherin 18, whereas N-cadherin is present in the other endocrine cell types. This repertoire undergoes major differential modification in GH and PRL tumours: E-cadherin is significantly reduced in invasive GH adenomas, and this loss is associated with a cytoplasmic relocalisation of cadherin 18 and catenins. In invasive prolactinomas, E-cadherin distribution is altered and is accompanied by a mislocalisation of cadherin 18, β-catenin and p120 catenin. Strikingly, de novo expression of N-cadherin is present in a subset of adenomas and cells exhibit a mesenchymal phenotype exclusively in invasive tumours. Binary tree analysis, performed by combining the cadherin repertoire with the expression of a subset of known molecular markers, shows that cadherin/catenin complexes play a significant role in discrimination of tumour invasion.

Pituitary growth hormone (GH)-secreting cells regulate growth and metabolism in animals and humans. To secrete highly ordered GH pulses (up to 1,000-fold rise in hormone levels in vivo ), the pituitary GH cell population needs to mount coordinated responses to GH secretagogues, yet GH cells display an apparently heterogeneous scattered distribution in 2D histological studies. To address this paradox, we analyzed in 3D both positioning and signaling of GH cells using reconstructive, two-photon excitation microscopy to image the entire pituitary in GH-EGFP transgenic mice. Our results unveiled a homologous continuum of GH cells connected by adherens junctions that wired the whole gland and exhibited the three primary features of biological networks: robustness of architecture across lifespan, modularity correlated with pituitary GH contents and body growth, and connectivity with spatially stereotyped motifs of cell synchronization coordinating cell activity. These findings change our view of GH cells, from a collection of dispersed cells to a geometrically connected homotypic network of cells whose local morphology and connectivity can vary, to alter the timing of cellular responses to promote more coordinated pulsatile secretion. This large-scale 3D view of cell functioning provides a powerful approach to identify and understand other networks of endocrine cells that are thought to be scattered in situ . Many dispersed endocrine systems exhibit pulsatile outputs. We suggest that cell positioning and associated cell-cell connection mechanisms will be critical parameters that determine how well such systems can deliver a coordinated secretory pulse of hormone to their target tissues.

FK-506, a widely used immunosuppressant, has caused a few clinical cases with QT prolongation and torsades de pointe at high blood concentration. The proarrhytmogenic potential of FK-506 was investigated in single rat ventricular cells using the whole cell clamp method to record action potentials (APs) and ionic currents. Fluorescence measurements of Ca2+ transients were performed with indo-1 AM using a multiphotonic microscope. FK-506 (25 μmol/l) hyperpolarized the resting membrane potential (RMP; −3 mV) and prolonged APs (AP duration at 90% repolarization increased by 21%) at 0.1 Hz. Prolongation was enhanced by threefold at 3.3 Hz, and early afterdepolarizations (EADs) occurred in 59% of cells. EADs were prevented by stronger intracellular Ca2+ buffering (EGTA: 10 vs. 0.5 mmol/l in the patch pipette) or replacement of extracellular Na+ by Li+, which abolishes Na+/Ca2+ exchange [Na+/Ca2+ exchanger current ( INaCa)]. In indo-1-loaded cells, FK-506 generated doublets of Ca2+ transients associated with increased diastolic Ca2+ in one-half of the cells. FK-506 reversibly decreased the L-type Ca2+ current ( ICaL) by 25%, although high-frequency-dependent facilitation of ICaL persisted, and decreased three distinct K+ currents: delayed rectifier K+ current ( IK; >80%), transient outward K+ current (+ current ( IK1; >40%). A shift in the reversal potential of IK1 (−5 mV) accounted for RMP hyperpolarization. Numerical simulations, reproducing all experimental effects of FK-506, and the use of nifedipine showed that frequency-dependent facilitation of ICaL plays a role in the occurrence of EADs. In conclusion, the effects of FK-506 on the cardiac AP are more complex than previously reported and include inhibitions of IK1 and ICaL. Alterations in Ca2+ release and INaCa may contribute to FK-506-induced AP prolongation and EADs in addition to the permissive role of ICaL facilitation at high rates of stimulation.

International audience; Kainic acid (KA) is routinely used to elicit status epilepticus (SE) and epileptogenesis. Among the available KA administration protocols, intranasal instillation (IN) remains understudied. Dosages of KA were instilled IN in mice. Racine Scale and Video-EEG were used to assess and quantify SE onset. Time spent in SE and spike activity was quantified for each animal and confirmed by power spectrum analysis. Immunohistochemistry and qPCR were performed to define brain inflammation occurring after SE, including activated microglial phenotypes. Long term video-EEG recording was also performed. Titration of IN KA showed that a dose of 30 mg/kg was associated with low mortality while eliciting SE. IN KA provoked at least one behavioral and electrographic SE in the majority of the mice (\textgreater90%). Behavioral and EEG SE were accompanied by a rapid and persistent microglial-astrocytic cell activation and hippocampal neurodegeneration. Specifically, microglial modifications involved both pro- (M1) and anti-inflammatory (M2) genes. Our initial long-term video-EEG exploration conducted using a small cohort of mice indicated the appearance of spike activity or SE. Our study demonstrated that induction of SE is attainable using IN KA in mice. Typical pro-inflammatory brain changes were observed in this model after SE, supporting disease pathophysiology. Our results are in favor of the further development of IN KA as a means to study seizure disorders. A possibility for tailoring this model to drug testing or to study mechanisms of disease is offered.

International audience; BACKGROUND AND PURPOSE: In some pathological conditions carnitine concentration is high while in others it is low. In both cases,cardiac arrhythmias can occur and lead to sudden cardiac death. It has been proposed that in ischaemia, acylcarnitine (acyl-CAR), but not carnitine, is involved in arrhythmias through modulation of ionic currents. We studied the effects of acyl-CARs on hERG, K(IR)2.1 and K(v)7.1/minK channels (channels responsible for I(KR), I(K1) and I(KS) respectively). EXPERIMENTAL APPROACH: HEK293 cells stably expressing hERG, K(IR)2.1 or Kv7.1/minK were studied using the patch clamp technique. Free carnitine (CAR) and acyl-CAR derivatives from medium- (C8 and C10) and long-chain (C16 and C18:1) fatty acids were applied intra- and extracellularly at different concentrations. For studies on hERG, C16 and C18:1 free fatty acid were also used. KEY RESULTS: Extracellular long-chain (LCAC), but not medium-chain, acyl-CAR,induced an increase of I(hERG) amplitude associated with a dose-dependent speeding of deactivation kinetics. They had no effect on K(IR)2.1 or Kv7.1/minK currents.Computer simulations of these effects were consistent with changes in action potential profile. CONCLUSIONS AND APPLICATIONS: Extracellular LCAC tonically regulates I(hERG) amplitude and kinetics under physiological conditions. This modulation may contribute to the changes in action potential duration that precede cardiac arrhythmias in ischaemia, diabetes and primary systemic carnitine deficiency.

The pulsatile secretion of hormones from the mammalian pituitary gland drives a wide range of homeostatic responses by dynamically altering the functional set-point of effector tissues. To accomplish this, endocrine cell populations residing within the intact pituitary display large-scale changes in coordinated calcium-spiking activity in response to various hypothalamic and peripheral inputs. Although the pituitary gland is structurally compartmentalized into specific and intermingled endocrine cell networks, providing a clear morphological basis for such coordinated activity, the mechanisms which facilitate the timely propagation of information between cells in situ remain largely unexplored. Therefore, the aim of the current review is to highlight the range of signalling modalities known to be employed by endocrine cells to coordinate intracellular calcium rises, and discuss how these mechanisms are integrated at the population level to orchestrate cell function and tissue output.

The atrioventricular node controls cardiac impulse conduction and generates pacemaker activity in case of failure of the sino-atrial node. Understanding the mechanisms of atrioventricular automaticity is important for managing human pathologies of heart rate and conduction. However, the physiology of atrioventricular automaticity is still poorly understood. We have investigated the role of three key ion channel-mediated pacemaker mechanisms namely, Ca(v)1.3, Ca(v)3.1 and HCN channels in automaticity of atrioventricular node cells (AVNCs). We studied atrioventricular conduction and pacemaking of AVNCs in wild-type mice and mice lacking Ca(v)3.1 (Ca(v)3.1(-/-)), Ca(v)1.3 (Ca(v)1.3(-/-)), channels or both (Ca(v)1.3(-/-)/Ca(v)3.1(-/-)). The role of HCN channels in the modulation of atrioventricular cells pacemaking was studied by conditional expression of dominant-negative HCN4 channels lacking cAMP sensitivity. Inactivation of Ca(v)3.1 channels impaired AVNCs pacemaker activity by favoring sporadic block of automaticity leading to cellular arrhythmia. Furthermore, Ca(v)3.1 channels were critical for AVNCs to reach high pacemaking rates under isoproterenol. Unexpectedly, Ca(v)1.3 channels were required for spontaneous automaticity, because Ca(v)1.3(-/-) and Ca(v)1.3(-/-)/Ca(v)3.1(-/-) AVNCs were completely silent under physiological conditions. Abolition of the cAMP sensitivity of HCN channels reduced automaticity under basal conditions, but maximal rates of AVNCs could be restored to that of control mice by isoproterenol. In conclusion, while Ca(v)1.3 channels are required for automaticity, Ca(v)3.1 channels are important for maximal pacing rates of mouse AVNCs. HCN channels are important for basal AVNCs automaticity but do not appear to be determinant for β-adrenergic regulation.

International audience; The mammalian circadian system is composed of multiple peripheral clocks that are synchronized by a central pacemaker in the suprachiasmatic nuclei of the hypothalamus. This system keeps track of the external world rhythms through entrainment by various time cues, such as the light-dark cycle and the feeding schedule. Alterations of photoperiod and meal time modulate the phase coupling between central and peripheral oscillators. In this study, we used real-time quantitative PCR to assess circadian clock gene expression in the liver and pituitary gland from mice raised under various photoperiods, or under a temporal restricted feeding protocol. Our results revealed unexpected differences between both organs. Whereas the liver oscillator always tracked meal time, the pituitary circadian clockwork showed an intermediate response, in between entrainment by the light regimen and the feeding-fasting rhythm. The same composite response was also observed in the pituitary gland from adrenalectomized mice under daytime restricted feeding, suggesting that circulating glucocorticoids do not inhibit full entrainment of the pituitary clockwork by meal time. Altogether our results reveal further aspects in the complexity of phase entrainment in the circadian system, and suggest that the pituitary may host oscillators able to integrate multiple time cues.

Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Here we have developed in vivo approaches to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Our findings suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation.

Background Ghrelin targets the arcuate nucleus, from where growth hormone releasing hormone (GHRH) neurones trigger GH secretion. This hypothalamic nucleus also contains neuropeptide Y (NPY) neurons which play a master role in the effect of ghrelin on feeding. Interestingly, connections between NPY and GHRH neurons have been reported, leading to the hypothesis that the GH axis and the feeding circuits might be co-regulated by ghrelin. Principal Findings Here, we show that ghrelin stimulates the firing rate of identified GHRH neurons, in transgenic GHRH-GFP mice. This stimulation is prevented by growth hormone secretagogue receptor-1 antagonism as well as by U-73122, a phospholipase C inhibitor and by calcium channels blockers. The effect of ghrelin does not require synaptic transmission, as it is not antagonized by γ-aminobutyric acid, glutamate and NPY receptor antagonists. In addition, this hypothalamic effect of ghrelin is independent of somatostatin, the inhibitor of the GH axis, since it is also found in somatostatin knockout mice. Indeed, ghrelin does not modify synaptic currents of GHRH neurons. However, ghrelin exerts a strong and direct depolarizing effect on GHRH neurons, which supports their increased firing rate. Conclusion Thus, GHRH neurons are a specific target for ghrelin within the brain, and not activated secondary to altered activity in feeding circuits. These results support the view that ghrelin related therapeutic approaches could be directed separately towards GH deficiency or feeding disorders.

Absence epilepsy is a neurological disorder that causes a recurrent loss of consciousness and generalized spike-and-wave discharges on an electroencephalogram (EEG). The role of metabotropic glutamate receptors (mGluRs) and associated scaffolding proteins in absence epilepsy has been unclear to date. We investigated a possible role for these proteins in absence epilepsy, focusing on the mGluR7a receptor and its PDZ-interacting protein, protein interacting with C kinase 1 (PICK1), in rats and mice. Injection of a cell-permeant dominant-negative peptide or targeted mutation of the mGluR7a C terminus, both of which disrupt the interaction between the receptor and PDZ proteins, caused behavioral symptoms and EEG discharges that are characteristic of absence epilepsy. Inactivation of the Pick1 gene also facilitated pharmacological induction of the absence epilepsy phenotype. The cortex and thalamus, which are known to participate in absence epilepsy, were involved, but the hippocampus was not. Our results indicate that disruption of the mGluR7a-PICK1 complex is sufficient to induce absence epilepsy-like seizures in rats and mice, thus providing, to the best of our knowledge, the first animal model of metabotropic glutamate receptor-PDZ protein interaction in absence epilepsy.

International audience; An increase in circulating catecholamine levels represents one of the mechanisms whereby organisms cope with stress. In the periphery, catecholamines mainly originate from the sympathoadrenal system. As we reported, in addition to the central control through cholinergic innervation, a local gap junction-delineated route between adrenal chromaffin cells contributes to catecholamine exocytosis. Here, we investigated whether this intercellular communication is modified when the hormonal demand is increased as observed during cold stress. Our results show that in cold exposed rats, gap-junctional communication undergoes a functional plasticity, as evidenced by an increased number of dye-coupled cells. Of a physiological interest is that this upregulation of gap-junctional coupling results in the appearance of a robust electrical coupling between chromaffin cells that allows the transmission of action potentials between coupled cells. This enhancement of gap-junctional communication parallels an increase in expression levels of connexin36 (Cx36) and connexin43 (Cx43) proteins. Both transcriptional and posttranslational mechanisms are involved because Cx36 transcripts are increased in stressed rats and the expression of the scaffolding protein zonula occludens-1, known to interact with both Cx36 and Cx43, is also upregulated. Consistent with an upregulated coupling extent in stressed rats, the cytosolic Ca(2+) concentration rises triggered in a single cell by an iontophoretic application of nicotine occur simultaneously in several neighboring cells. These results describe for the first time a functional plasticity of junctional coupling between adult chromaffin cells that should be crucial for adaptation to stress or sensitization to subsequent stressors.

The organization of the peptidergic neurons of the hypothalamic arcuate nucleus is not fully understood. These include growth hormone-releasing hormone (GHRH) neurons involved in growth and metabolism. We studied identified GHRH neurons of GHRH–green fluorescent protein transgenic mice using patch-clamp methods and focused on gender differences, which govern the physiological patterns of GHRH release. Both the spontaneous firing rates and the intrinsic properties of GHRH neurons were similar in males and females, although higher glutamatergic currents were noticed in females. Surprisingly, marked gender differences in GHRH neuronal activity were observed in response to the muscarinic agonist carbachol (CCh). In females, CCh enhanced action potential firing in all GHRH neurons. In males, CCh enhanced action potential firing in two-thirds of GHRH neurons, whereas it decreased firing in the remainders. M1agonist McN-A343 (10 μm) mimicked, and M1antagonist pirenzepine (3 μm) blocked the effects of CCh. In both genders, CCh did not change the intrinsic properties of GHRH neurons, although it strongly increased the frequency of glutamatergic currents, in the presence or absence of tetrodotoxin. In males only, CCh enhanced the frequency of GABAergic currents, and this modulation was antagonized by tetrodotoxin. Thus, the muscarinic regulation involved differential control of afferent inputs at short and long distances in male and female mice. The dual-level control could be a mechanism whereby the selective modulation of the GHRH system (short-distance control) is adjusted to the integrated regulation of arcuate nucleus activity (long-distance control).

We have previously shown that oxytocin neurons located in the four hypothalamic magnocellular nuclei display synchronous bursts of action potentials before each milk ejection. The mechanisms involved in such a synchronization have, however, not yet been elucidated. In this study, we test the hypothesis of an extranuclear synchronization arising from a common extrahypothalamic input innervating bilateral magnocellular nuclei. First, two different retrograde tracers were injected into the right and left supraoptic nuclei of rats that were fixed 5-7 days later. Each tracer labelled numerous neurons in various brain regions ipsilateral or contralateral to the injection site, but colocalization of the two tracers within the same cell body could only be detected bilaterally in neurons in the ventromedial regions of the medulla oblongata. The axonal projections of these medullary neurons were then visualized by the unilateral microinjection of an anterograde tracer (BDA) within the ventromedial medulla oblongata. BDA-labelled axons afferent to the hypothalamus were found to branch towards both supraoptic nuclei through medial portions of the optic chiasma. Finally, in anaesthetized lactating rats, surgical lesions were placed medially through the optic chiasma and the electrical activity of oxytocin neurons in bilateral supraoptic nuclei was pair-recorded during suckling. The incidence of synchronous bursts in oxytocin neurons located within bilateral supraoptic nuclei were dramatically altered only when the medial portions of the optic chiasma were totally lesioned. Taken together, these data suggest that medullary neurons afferent to bilateral supraoptic nuclei are involved in the recruitment and synchronization of bursting in oxytocin neurons during suckling.

Endogenous production of SRIH and GHRH was analyzed in human breast tissue. SRIH precursor (pro-SRIH) was identified after Sephadex G-50 filtration of acetic acid extracts of normal and tumoral human breast samples. SRIH-(1–14) or -(1–28) could not be detected in breast tissue, whereas the immunoreactive SRIH released in vitro was characterized as SRIH-(1–28). Endogenous production of GHRH was assessed by identification of GHRH messenger ribonucleic acid by PCR followed by sequencing of the amplified complementary DNA and by high performance liquid chromatographic characterization of immunoreactive GHRH contained in the tissue and released in vitro. There were no differences in pro-SRIH or GHRH-(1–44) tissue contents between normal and tumoral samples. The release of both peptides was evidenced in perifusion and static incubation. Perifusion of normal breast tissue (n = 3) showed pulsatile release of SRIH and GHRH. Perifusion of tumors (n = 4) showed SRIH release in 50% of the cases. SRIH release was pulsatile in one case. GHRH release was observed in the four tumoral samples analyzed, but was pulsatile in only one case. In static incubation, tumors (n = 6) secreted 13 times more GHRH than did normal samples (n = 3; 383 ± 92 vs. 29.6 ± 4.6 fmol/mg protein; P < 0.05). Stimulation of GHRH release by exogenous SRIH was observed only with the normal tissue.Together these data provide evidence for the existence of local production of SRIH and GHRH by human breast. Hypersecretion of GHRH by breast tumors indicates that this peptide could play a role in maintaining epithelial cell proliferation as is the case for other peptides produced locally.

Experience-dependent plasticity of cell and tissue function is critical for survival by allowing organisms to dynamically adjust physiological processes in response to changing or harsh environmental conditions. Despite the conferred evolutionary advantage, it remains unknown whether emergent experience-dependent properties are present in cell populations organized as networks within endocrine tissues involved in regulating body-wide homeostasis. Here we show, using lactation to repeatedly activate a specific endocrine cell network in situ in the mammalian pituitary, that templates of prior demand are permanently stored through stimulus-evoked alterations to the extent and strength of cell–cell connectivity. Strikingly, following repeat stimulation, evolved population behaviour leads to improved tissue output. As such, long-lasting experience-dependent plasticity is an important feature of endocrine cell networks and underlies functional adaptation of hormone release., Experience-dependent plasticity and functional adaptation are thought to be restricted to the central nervous and immune systems. This study shows that long-lasting experience-dependent plasticity is a key feature of endocrine cell networks, allowing improved tissue function and hormone output following repeat demand.