Prolactin blood concentration relies on the scalability of the TIDA neurons' network efficiency in vivo.

Our understanding and management of reproductive health and related disorders such as infertility, menstrual irregularities, and pituitary disorders depend on understanding the intricate sex-specific mechanisms governing prolactin secretion. Using ex vivo experiments in acute slices, in parallel with in vivo calcium imaging (GRIN lens technology), we found that dopamine neurons inhibiting PRL secretion (TIDA), organize as functional networks both in and ex vivo. We defined an index of efficiency of networking (Ieff) using the duration of calcium events and the ability to form plastic economic networks. It determined TIDA neurons' ability to inhibit PRL secretion in vivo. Ieff variations in both sexes demonstrated TIDA neurons' adaptability to physiological changes. A variation in the number of active neurons contributing to the network explains the sexual dimorphism in basal [PRL]blood secretion patterns. These sex-specific differences in neuronal activity and network organization contribute to the understanding of hormone regulation.

Acquiring social safety engages oxytocin neurons in the supraoptic nucleus - role of Magel2 deficiency.

Introduction Exposure to social trauma may alter engagement with both fear-related and unrelated social stimuli long after. Intriguingly, how simultaneous discrimination of social fear and safety is affected in neurodevelopmental conditions remains underexplored. The role of the neuropeptide oxytocin is established in social behaviors, and yet unexplored during such a challenge post-social trauma. Methods Using Magel2 knockout mice, an animal model of Prader Willi Syndrome (PWS) and Schaaf-Yang Syndrome (SYS), we tested memory of social fear and safety after a modified social fear conditioning task. Additionally, we tracked the activity of oxytocin neurons in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus by fibre photometry, as animals were simultaneously presented with a choice between a fear and safe social cue during recall. Results Male Magel2 KO mice trained to fear females with electrical footshocks avoided both unfamiliar females and males during recalls, lasting even a week post-conditioning. On the contrary, trained Magel2 WT avoided only females during recalls, lasting days rather than a week post-conditioning. Inability to overcome social fear and avoidance of social safety in Magel2 KO mice were associated with reduced engagement of oxytocin neurons in the SON, but not the PVN. Conclusion In a preclinical model of PWS/SYS, we demonstrated region-specific deficit in oxytocin neuron activity associated with behavioral generalization of social fear to social safety. Insights from this study add to our understanding of oxytocin action in the brain at the intersection of social trauma and PWS/SYS.

TSH Pulses Finely Tune Thyroid Hormone Release and TSH Receptor Transduction.

Detection of circulating TSH is a first-line test of thyroid dysfunction, a major health problem (affecting about 5% of the population) that, if untreated, can lead to a significant deterioration of quality of life and adverse effects on multiple organ systems. Human TSH levels display both pulsatile and (nonpulsatile) basal TSH secretion patterns; however, the importance of these in regulating thyroid function and their decoding by the thyroid is unknown. Here, we developed a novel ultra-sensitive ELISA that allows precise detection of TSH secretion patterns with minute resolution in mouse models of health and disease. We characterized the patterns of ultradian TSH pulses in healthy, freely behaving mice over the day-night cycle. Challenge of the thyroid axis with primary hypothyroidism because of iodine deficiency, a major cause of thyroid dysfunction worldwide, results in alterations of TSH pulsatility. Induction in mouse models of sequential TSH pulses that mimic ultradian TSH profiles in periods of minutes were more efficient than sustained rises in basal TSH levels at increasing both thyroid follicle cAMP levels, as monitored with a genetically encoded cAMP sensor, and circulating thyroid hormone. Hence, this mouse TSH assay provides a powerful tool to decipher how ultradian TSH pulses encode thyroid outcomes and to uncover hidden parameters in the TSH-thyroid hormone set-point in health and disease.

Loss of glucocorticoid receptor phosphorylation contributes to cognitive and neurocentric damages of the amyloid-ß pathway.

Aberrant cortisol and activation of the glucocorticoid receptor (GR) play an essential role in age-related progression of Alzheimer's disease (AD). However, the GR pathways required for influencing the pathobiology of AD dementia remain unknown. To address this, we studied an early phase of AD-like progression in the well-established APP/PS1 mouse model combined with targeted mutations in the BDNF-dependent GR phosphorylation sites (serines 134/267) using molecular, behavioral and neuroimaging approaches. We found that disrupting GR phosphorylation (S134A/S267A) in mice exacerbated the deleterious effects of the APP/PS1 genotype on mortality, neuroplasticity and cognition, without affecting either amyloid-ß deposition or vascular pathology. The dynamics, maturation and retention of task-induced new dendritic spines of cortical excitatory neurons required GR phosphorylation at the BDNF-dependent sites that amyloid-ß compromised. Parallel studies in postmortem human prefrontal cortex revealed AD subjects had downregulated BDNF signaling and concomitant upregulated cortisol pathway activation, which correlated with cognitive decline. These results provide key evidence that the loss of neurotrophin-mediated GR phosphorylation pathway promotes the detrimental effects of the brain cortisol response that contributes to the onset and/or progression of AD dementia. These findings have important translational implications as they provide a novel approach to treating AD dementia by identifying drugs that increase GR phosphorylation selectively at the neurotrophic sites to improve memory and cognition.

Nested calcium dynamics support daily cell unity and diversity in the suprachiasmatic nuclei of free-behaving mice.

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. Indeed, 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, to tackle this issue we used microendoscopic Ca2+ i imaging and investigated 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 highly coordinated, while spike activity from the same neurons peaked at multiple times of the light cycle, and unveiled clock-independent coactivity in neuron subsets. Hence, fast Ca2+ i spikes and slow changes in baseline Ca2+ i levels highlighted how multiple individual activity patterns could articulate within the temporal unity of the SCN cell network in vivo, and provided support for a multiplex neuronal code in the circadian pacemaker.

Dual imaging of dendritic spines and mitochondria in vivo reveals hotspots of plasticity and metabolic adaptation to stress.

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.

A sodium background conductance controls the spiking pattern of mouse adrenal chromaffin cells in situ.

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.

Regeneration of the neurogliovascular unit visualized in vivo by transcranial live-cell imaging.

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.

IGSF1 Deficiency Results in Human and Murine Somatotrope Neurosecretory Hyperfunction.

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.

Imaging and Manipulating Pituitary Function in the Awake Mouse.

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.

Integrin ß1 Optimizes Diabetogenic T Cell Migration and Function in the Pancreas.

T cell search behavior is dictated by their need to encounter their specific antigen to eliminate target cells. However, mechanisms controlling effector T cell motility are highly tissue-dependent. Specifically, how diabetogenic T cells encounter their target beta cells in dispersed islets throughout the pancreas (PA) during autoimmune diabetes remains unclear. Using intra-vital 2-photon microscopy in a mouse model of diabetes, we found that CXCR3 chemokine downregulated CD8+ T cell motility specifically within islets, promoting effector cell confinement to their target sites. By contrast, T cell velocity and directionality in the exocrine tissue were enhanced along blood vessels and extracellular matrix fibers. This guided migration implicated integrin-dependent interactions, since integrin blockade impaired exocrine T cell motility. In addition, integrin ß1 blockade decreased CD4+ T cell effector phenotype specifically in the PA. Thus, we unveil an important role for integrins in the PA during autoimmune diabetes that may have important implications for the design of new therapies.

Topographic Reorganization of Cerebrovascular Mural Cells under Seizure Conditions.

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.

CD4+ T Helper Cells Play a Key Role in Maintaining Diabetogenic CD8+ T Cell Function in the Pancreas.

Autoreactive CD8+ and CD4+ T cells have been assigned independent key roles in the destruction of insulin-producing beta cells resulting in type 1 diabetes. Although CD4 help for the generation of efficient CD8+ T cell responses in lymphoid tissue has been extensively described, whether these two cell populations cooperate in islet destruction in situ remains unclear. By using intravital 2-photon microscopy in a mouse model of diabetes, we visualized both effector T cell populations in the pancreas during disease onset. CD4+ T helper cells displayed a much higher arrest in the exocrine tissue than islet-specific CD8+ T cells. This increased arrest was major histocompatibility complex (MHC) class II-dependent and locally correlated with antigen-presenting cell recruitment. CD8+ T cells deprived of continued CD4 help specifically in the pancreas, through blocking MHC class II recognition, failed to maintain optimal effector functions, which contributed to hamper diabetes progression. Thus, we provide novel insight in the cellular mechanisms regulating effector T cell functionality in peripheral tissues with important implications for immunotherapies.

Phasic and Tonic mGlu7 Receptor Activity Modulates the Thalamocortical Network.

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.

Somatostatin triggers rhythmic electrical firing in hypothalamic GHRH neurons.

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.

Evidence for Status Epilepticus and Pro-Inflammatory Changes after Intranasal Kainic Acid Administration in Mice.

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 (>90%). 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.

Metabolism Regulates Exposure of Pancreatic Islets to Circulating Molecules In Vivo.

Pancreatic ß-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 ß-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 ß-cell expansion or islet blood flow velocity. Together, these data support a role for islet vascular permeability in shaping ß-cell adaptive responses to metabolic demand by modulating the access and sensing of circulating molecules.

Long-chain acylcarnitines regulate the hERG channel.

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.

Existence of long-lasting experience-dependent plasticity in endocrine cell networks.

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.