Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion

Oxytocin (OT) is a prominent regulator of many aspects of mammalian social behavior and stored in large dense-cored vesicles (LDCVs) in hypothalamic neurons. It is released in response to activity-dependent Ca²⁺ influx, but is also dependent on Ca²⁺ release from intracellular stores, which primes LDCVs for exocytosis. Despite its importance, critical aspects of the Ca²⁺-dependent mechanisms of its secretion remain to be identified. Here we show that lysosomes surround dendritic LDCVs, and that the direct activation of endolysosomal two-pore channels (TPCs) provides the critical Ca²⁺ signals to prime OT release by increasing the releasable LDCV pool without directly stimulating exocytosis. We observed a dramatic reduction in plasma OT levels in TPC knockout mice, and impaired secretion of OT from the hypothalamus demonstrating the importance of priming of neuropeptide vesicles for activity-dependent release. Furthermore, we show that activation of type 1 metabotropic glutamate receptors sustains somatodendritic OT release by recruiting TPCs. The priming effect could be mimicked by a direct application of nicotinic acid adenine dinucleotide phosphate, the endogenous messenger regulating TPCs, or a selective TPC2 agonist, TPC2-A1-N, or blocked by the antagonist Ned-19. Mice lacking TPCs exhibit impaired maternal and social behavior, which is restored by direct OT administration. This study demonstrates an unexpected role for lysosomes and TPCs in controlling neuropeptide secretion, and in regulating social behavior.

Oxytocin (OT) was initially described as a hormone regulating parturition (1, 2) and lactation (3), but it is now also well-recognized as a prosocial hormone regulating maternal behavior (4, 5), social recognition (6–8), and social interactions (9–14). OT is stored in large dense-core vesicles (LDCV) in the magno- and parvocellular secretory neurons of the paraventricular nuclei (PVN) and the supraoptic nuclei (SON) of the hypothalamus. This neuropeptide is released from hypothalamic neurons into different brain structures, and via the pituitary gland into the systemic bloodstream. OT neurons are multifunctional multisensory cells that respond to a wide variety of stimuli to fulfil their numerous roles described above (for review see ref. 15). However, the regulatory mechanisms underlying OT secretion are still unclear. Although OT secretion is known to be independently regulated in different neuronal compartments such as axons, soma, and dendrites (8, 16–20), the intracellular signaling pathways underlying secretion in these complex multitask neurons are still unclear.OT release from both somatodendritic sites (16) and neurohypophysial axonal terminals (21) have been proposed to be dependent on Ca²⁺ release from intracellular stores in addition to Ca²⁺ influx via voltage-dependent Ca²⁺ channels. Indeed release of OT from pituitary axonal terminals and hypothalamus was shown to be highly dependent on the expression of cluster of differentiation 38 (CD38) (21, 22), a transmembrane multifunctional enzyme that catalyzes the synthesis of the Ca²⁺ mobilizing messenger, cyclic ADP-ribose (cADPR). cADPR which promotes Ca²⁺ release via ryanodine receptors (RyRs) from the endoplasmic reticulum (ER) (23) caused a large increase in OT release from isolated oxytocinergic nerve endings (21). However, CD38 also may catalyze the synthesis of another major Ca²⁺ mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), which specifically mobilizes Ca²⁺ from acidic organelles such as lysosomes (24, 25) via two-pore channels (TPCs) (26). However, in contrast to cADPR, NAADP was found not to directly stimulate OT release (21).Somatodendritic OT secretion has also been shown to exhibit the important property of priming, which greatly increases the extent of exocytosis of OT-containing LCDVs (16). Priming in neuroendocrine cells is the phenomenon whereby an initial signal prepares cells for an anticipated subsequent trigger, sometimes involving the autocrine action of the peptide released (8, 16, 27). It is of fundamental significance in the neuroendocrine system where such a mechanism mediates such phenomena as the OT-controlled milk-ejection reflex and the LH surge at ovulation (28). In hypothalamic neurons, this initial priming signal can be OT itself (self-priming) or other hormones or neurotransmitters which activate metabotropic cell surface receptors to mobilize Ca²⁺ from intracellular stores. This results in a substantial augmentation of the secretory response to subsequent cell activation and is thought to occur by recruiting a reserve pool of LCDVs to the plasma membrane, increasing their probability of release. It had been previously suggested that Ca²⁺ release from Ca²⁺ storage organelles primes vesicles for sustained OT release since pharmacological release of Ca²⁺ from the ER by the sarcoendoplasmic reticulum Ca²⁺ ATPase (SERCA) inhibitor thapsigargin and consequential Ca²⁺ entry promoted priming effects (16, 29, 30).Lysosomes are recognized as important organelles in autophagic macromolecular degradation and membrane repair, but they also contain a high concentration of Ca²⁺ that could be mobilized for cell signaling (24, 25, 31, 32). Electron microscopy has shown the presence of lysosomes in axon endings, dendrites, cell-bodies, and Herring bodies of OT neurons located in areas rich in LDCVs (33–35), but so far, their role in hypothalamic neurons as a source of Ca²⁺ has remained unexplored. In various cell types, endosomes and lysosomes release Ca²⁺ through the activation of the endolysosomal TPCs, modulated by NAADP (26, 36–39) and phosphatidylinositol-3, 5-bisphosphate (PI(3, 5)P₂) (40, 41). TPCs belong to an ancient cation channel family present in numerous species with three known isoforms (TPC1, TPC2, and TPC3). In humans and rodents, only TPC1 and TPC2 are expressed, displaying different ionic selectivity in part depending on how they are activated: NAADP favoring Ca²⁺ permeation (42, 43), while PI(3, 5)P₂ favors Na⁺ permeation (40, 43–45). TPCs have been implicated in many aspects of cell signaling, autophagy, and vesicular trafficking in various cell types (42, 46, 47), but despite the growing interest in these channels, their roles in the central nervous system remain largely unexplored (for review, see ref. 22).Here on the basis that CD38 is a major regulator of OT secretion (21), we have examined the role of endolysosomal TPCs as the principal targets of the NAADP branch of the CD38 signaling pathway and found that they play critical roles in the regulation of OT secretion and thus control social behavior.     

Hypothesis of interference to superinfection between bovine spastic paresis and bovine spongiform encephalopathy; suggestions for experimentation, theoretical and practical interest

Sub-acute transmissible spongiform encephalopathies (TSEs) or prion diseases are diseases of little known etiology. The origin of these diseases would appear to be an abnormal protease-resistant prion protein (PrP(res)) which would be infectious by directly inducing its defective conformation to the normal native protein (PrP(C)). This hypothesis does not account for certain aspects of TSEs, such as interference to superinfection: in laboratory animals, inoculation by means of an attenuated strain with a long incubation period protects against later infection by a very virulent strain with a short incubation period. The hypothesis is put forward that there exists a possibility of interference to superinfection between neurodegenerative diseases of unknown origin, thought to be similar to TSEs, and a later infection by a TSE. The study of this interference between bovine spastic paresis (BSP) and bovine spongiform encephalopathy (BSE) could be used as a model for this hypothesis. BSP is a very rare disease among cattle, of unknown etiology; it is curable, in the very early stages, by using tryptophan and especially lithium, potentiated by copper and manganese. An etiology close to that of TSEs has been suggested on several occasions. If interference could be demonstrated between BSP and BSE, interesting data would be provided concerning the etiology, the pathogenesis and possibly the treatment and prevention of these diseases. Notably, such data could lead to the development of a treatment and a prevention with lithium and amino acids precursors of neuromediators (tryptophan, tyrosine, glutamic acid, etc.), as well as the developing of a vaccine to combat TSEs, especially BSE and scrapie.     

Combined anterior segment OCT and wavefront-based autorefractor using a shared beam

We have combined an anterior segment (AS) optical coherence tomography (OCT) system and a wavefront-based aberrometer with an approach that senses ocular wavefront aberrations using the OCT beam. Temporal interlacing of the OCT and aberrometer channels allows for OCT images and refractive error measurements to be acquired continuously and in real-time. The system measures refractive error with accuracy and precision comparable to that of clinical autorefractors. The proposed approach provides a compact modular design that is suitable for integrating OCT and wavefront-based autorefraction within the optical head of the ophthalmic surgical microscope for guiding cataract surgery or table-top devices for simultaneous autorefraction and ocular biometry.     

Povidone-iodine enema as a preoperative bowel preparation for colorectal surgery

To evaluate the effects of povidone-iodine (PI) enema on the bacterial flora of colorectal mucosa, the authors studied 113 patients who were candidates for colorectal surgery. The study of the rectum included 72 patients. Total bacterial concentrations after a PI enema (N=44) were significantly lower than after a simple water enema (N=12,P<0.001), or than after a water enema associated with intravenous metronidazole (N=16,P<0.01). The study of the colon included 41 patients. Total bacterial concentrations did not differ after a PI enema (N=24) than after a water enema (N=11); both groups were associated with intravenous metronidazole. In contrast, both preparations significantly reduced bacterial concentrations when compared with oral administration of polyethyleneglycol (N=6,P<0.01). Similar results were observed in rectal and colonic studies, when analysis was restricted to the anaerobic flora. PI is an antiseptic that, when administered alone in an enema or in association with metronidazole, significantly reduces bacterial concentrations in the mucosa of the colon and rectum. It may be proposed as a simple preoperative preparation for colorectal surgery.     

Brief report: benchmarking human pluripotent stem cell markers during differentiation into the three germ layers unveils a striking heterogeneity: all markers are not equal

Pluripotent stem cells (PSC) are functionally characterized by their capacity to differentiate into all the cell types from the three germ layers. A wide range of markers, the expression of which is associated with pluripotency, has been used as surrogate evidence of PSC pluripotency, but their respective relevance is poorly documented. Here, we compared by polychromatic flow cytometry the kinetics of loss of expression of eight widely used pluripotency markers (SSEA3, SSEA4, TRA-1-60, TRA-1-81, CD24, OCT4, NANOG, and alkaline phosphatase [AP]) at days 0, 5, 7, and 9 after induction of PSC differentiation into cells representative of the three germ layers. Strikingly, each marker showed a different and specific kinetics of disappearance that was similar in all the PSC lines used and for all the induced differentiation pathways. OCT4, SSEA3, and TRA-1-60 were repeatedly the first markers to be downregulated, and their expression was completely lost at day 9. By contrast, AP activity, CD24, and NANOG proteins were still detectable at day 9. In addition, we show that differentiation markers are coexpressed with pluripotency markers before the latter begin to disappear. These results suggest that OCT4, SSEA3, and TRA-1-60 might be better to trace in vitro the emergence of pluripotent cells during reprogramming.     

Intra-individual comparison of sentinel lymph node scintigraphy on the day of injection and on the following day in breast cancer

OBJECTIVES: To compare, intra-individually, the detection rates of sentinel node on lymphoscintigraphy performed on the day of injection (D0) and on the following day (D1) in breast carcinoma. We also compared 2-day and 1-day protocols in the two groups of patients. METHODS: The 2-day and 1-day protocols included 76 patients in group 1 and 23 patients in group 2. Patients from group 1 underwent lymphoscintigraphy twice--at 2 h (lymphoscintigraphy 1) and 18 h (lymphoscintigraphy 2) post-injection at four sites periareolar using 99mTc sulfur colloid. Patients from group 2 underwent lymphoscintigraphy only at 2 h post-injection. The detection rates and the number of sentinel nodes were compared in the two lymphoscintigraphy examinations for group 2. RESULTS: The detection rate on lymphoscintigraphy in group 1 was 92% at D0 and 96% at D1. The overall agreement between lymphoscintigraphy 1 and lymphoscintigraphy 2 was 69/76 (91%). In 2/76 women, the sentinel node disappeared at D1 on lymphoscintigraphy, but remained detectable during surgery, and in 5/76 women, the sentinel node appeared at D1 on lymphoscintigraphy. The mean number of sentinel nodes detected on lymphoscintigraphy was 2.05+/-0.14 at D0 and 1.76+/-0.11 at D1 (P=0.004) in group 2, the detection rate of the sentinel node was 20/23 (87%). CONCLUSION: Our study demonstrated that for patients undergoing the 2-day protocol for sentinel node procedure in early stage breast cancer, the optimal imaging time would be to perform lymphoscintigraphy 1 h after injection, with the possibility of imaging patients the following day in cases where lymphoscintigraphy was negative.     

Tobramycin disposition in ICU patients receiving a once daily regimen: population approach and dosage simulations

AIM

The aim of this study was to evaluate the disposition of tobramycin (TOB) in critically ill patients (ICU) by a population pharmacokinetic approach, to determine the covariates involved, and to simulate tobramycin dosage regimens.

METHODS

Forty-nine adult ICU patients received TOB (5 mg kg⁻¹) once daily. NonMem modelling was performed on 32 patients. The 17 other patients were used for the qualification process by normalized prediction distribution error. Then Monte Carlo simulations (MCS) were performed.

RESULTS

A two-compartment model with a proportional error best fitted the data. TOB total clearance (CL_TOB) was significantly correlated with Cockcroft creatinine clearance (COCK) and height. TOB clearance was 4.8 ± 1.9 l h⁻¹ (range 1.22–8.95), the volume of distribution of the central compartment was 24.7 ± 3.7 l (range 17.34–32.83) and that of the peripheral compartment and the inter-compartmental clearance were 30.6 l and 4.74 l h⁻¹, respectively. Only 29% of the patients presented a target AUC between 80 and 125 mg l^–1 h and 61% were lower than 80 mg l⁻¹ h. After considering COCK and height, MCS showed that only 50% of the population could achieve the target AUC for the 375 and 400 mg dosages.

CONCLUSION

Even after taking into account COCK and height, for strains with an MIC ≤ 1 mg l⁻¹, MCS doses evidenced that peak and AUC pharmacodynamic targets could not be reached simultaneously in more than 45% of the ICU patient population. Combination therapy in addition to drug monitoring are required to manage efficacy and toxicity.     

In vitro push-out strength of seven luting agents to dentin

PURPOSE: New luting agents, described as resin-modified glass-ionomer cements and compomers, have been developed during the last decade to improve the retention of cemented restorations. The aims of this study were to (1) compare the push-out strength of these new luting materials against both conventional cements and bonding luting agents, and (2) evaluate the influence of dentin surface treatment on both glass-ionomer cement and 4-META adhesive resin push-out strength. MATERIALS AND METHODS: Conical standardized cavities were drilled in the center of coronal dentin disks. Ninety sandblasted Ni-Cr inlays, divided into nine batches, were luted into the cavities according to the surface treatment and the nature of the following luting agents: zinc phosphate cement, zinc polycarboxylate cement, type 1 glass-ionomer +/- polyacrylic acid, resin-modified glass-ionomer, polyacid-modified composite resin, filled bis-GMA phosphate ester resin, and 4-META adhesive resin +/- application of activated monomer. Each specimen was placed in a holding device, and a steel rod was used to apply a force on the inlay until rupture occurred. The push-out strength was calculated, and the failure mode was controlled. RESULTS: There were significant differences between some of the groups. The highest push-out strength was achieved by the 4-META adhesive resin after application of activated monomer. The lowest value was attained with zinc phosphate and polycarboxylate cements. CONCLUSION: Both resin-modified glass-ionomer and polyacid-modified composite resin luting materials exhibited a push-out strength similar to resin-based materials. Specific dentin surface treatments significantly enhanced the push-out strengths of glass-ionomer cement and 4-META adhesive resin.