Imaging pheromone sensing in a mouse vomeronasal acute tissue slice preparation.

Peter Karlson and Martin Lüscher used the term pheromone for the first time in 1959 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules secreted and/or contained in biological fluids, such as urine, a liquid known to be a main source of pheromones. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions, hierarchical organisations and sexual interactions and are consequently directly correlated with the survival of a given species. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO), a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs). Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize. Two main vomeronasal receptor families, V1Rs and V2Rs, are composed respectively by 240 and 120 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs) and formyl peptide receptors (FPRs) are also expressed in VSNs. Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons TRPC2 independent transduction channels have been suggested. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex. Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy

Ultrasound-guided spermatic cord block for scrotal surgery

Background Performing spermatic cord block for scrotal surgery avoids the potential risks of neuraxial and general anaesthesia and provides long-lasting postoperative analgesia. A blindly performed block is often inefficient and bears its own potential risks (intravascular injection of local anaesthetics, haematoma formation and perforation of the deferent duct). The use of ultrasound may help to overcome these disadvantages. The aim of this study was to test the feasibility and monitor the success rate of a new ultrasound-guided spermatic cord block. Methods Twenty consecutive patients undergoing urologic surgery (subcapsular orchiectomy or vaso-vasostomy) were included in this prospective study. Using a linear ultrasound probe, the spermatic cord was identified by locating the spermatic artery and the deferent duct. A 23 G Microlance needle was advanced close to the deferent duct by avoiding vessel perforation, and local anaesthetic was deposited around the deferent duct under direct visualization. The primary outcome was the success rate of the block which was defined as surgery without any substitution of opioids, additional local anaesthetics, or sedatives. Results In 20 patients, 40 blocks were performed with a success rate of 95% (n=38). The failure rate was 5% (n=2) and no conversion to general anaesthesia was needed. The mean duration of the block was 14.1 h (sd 6.9). Conclusions The use of ultrasound guidance to perform spermatic cord block is feasible and has a high success rate. Our new approach may become a suitable alternative to neuraxial or general anaesthesia especially in the ambulatory surgical setting. Registry: International Standard Randomised Controlled Trial Number Register; www.controlled-trials.com; Registry Nr.: ISRCTN4464781

Different Learning Curves for Axillary Brachial Plexus Block: Ultrasound Guidance versus Nerve Stimulation

Little is known about the learning of the skills needed to perform ultrasound- or nerve stimulator-guided peripheral nerve blocks. The aim of this study was to compare the learning curves of residents trained in ultrasound guidance versus residents trained in nerve stimulation for axillary brachial plexus block. Ten residents with no previous experience with using ultrasound received ultrasound training and another ten residents with no previous experience with using nerve stimulation received nerve stimulation training. The novices' learning curves were generated by retrospective data analysis out of our electronic anaesthesia database. Individual success rates were pooled, and the institutional learning curve was calculated using a bootstrapping technique in combination with a Monte Carlo simulation procedure. The skills required to perform successful ultrasound-guided axillary brachial plexus block can be learnt faster and lead to a higher final success rate compared to nerve stimulator-guided axillary brachial plexus block

Ultrasound-guided thoracic paravertebral puncture and placement of catheters in human cadavers: where do catheters go?†

Background Paravertebral regional anaesthesia is used to treat pain after several surgical procedures. This study aimed to improve on our first published ultrasound-guided approach to the paravertebral space (PVS) and to investigate a possible discrepancy between the needle, catheter, and contrast dye position. Methods In 10 cadavers, we conducted 26 ultrasound-guided paravertebral approaches combined with loss of resistance (LOR) and after an interim analysis performed 36 novel, pure ultrasound-guided (PUSG) paravertebral approaches. Needle-tip position was controlled by a first computed tomography (CT) scan. After placement of the catheters, the tips were assessed by a second CT and the spread of injected contrast dye was assessed by further CT scans. The part of the PVS near the intervertebral foramen was defined as the primary target to reach. Results The first CT scans assessing 62 needle tips revealed that: 13 (50%) of LOR and 34 (94%) of PUSG approaches were at the target; and two (8%) LOR and no PUSG approaches were outside the PVS. With the second CT scans 60 catheter-tip positions were analysed: three (12%) of LOR and five (14%) of PUSG approaches were at the target, three (12%) of LOR and two (6%) of PUSG approaches were outside the PVS. No catheters were detected in the epidural space. In two cases, insertion of the catheter was not possible. In cases with major epidural contrast, the widest contrast dye spread was 7.7 (3.5) [mean (sd)] vertebral segments. Conclusions Our new PUSG technique has a high success rate for paravertebral needle placement. Although needles were correctly positioned, catheters were usually found distant from the needle-tip positio

The Intervisions cliniques continuing medical education program: A forum for exchange and mutual knowledge development between general practitioners and psychiatrists

Objective This article presents the Intervisions cliniques program, a continuing Medical Education (CME) initiative for psychiatrists and general practitioners which uses case discussion to foster the acquisition of mental health care knowledge. Method This project is a case study, using qualitative methods. An operational model of Intervisions Cliniques was drawn for analysis. Data mainly come from the program-related literature, direct observation and interviews with 9 key actors, assembled in 2 groups of promoters and participants of the program. Findings The findings help to define the context and vision in which the Intervisions cliniques program was set up and implemented, to identify the perceived benefits and disappointments with regard to the program. Set up in the Canadian public managed care system and university-affiliated hospital, the Intervisions cliniques program helps to create lines of communication and privileged exchanges between general practitioners and psychiatrists as well as to foster mutual knowledge and a crosscutting and interactive view within a shared clinical territory. Several benefits noted by the participants and the organizing committee demonstrate the importance and multimodal effects of this type of initiative. The case-discussion format used as a CME method meets the training needs of general practitioners and is widely appreciated. However, the pressing needs expressed by the general practitioners with regard to improving shared mental health care still pose many challenges. Conclusions Making the link with the duties of responding psychiatrists, who are now funded in Québec, seems to be part of the solution. Maintaining and disseminating the Intervisions Cliniques program will require provincial-wide system implementation strategies to train psychiatrist, support them and regional managers to monitor the quality of this innovative practice within a public managed care system. Clinical implications • In the context of Continuing Medical Education (CME), case discussions are valued because they help to address the full complexity of clinical situations. • In the development of shared mental health care, direct contacts between general practitioners and psychiatrists are an optimal way to foster mutual knowledge and a crosscutting and interactive view within a shared clinical territory. • The Intervisions cliniques program contributes to shared mental health care and is a CME strategy that should be reproduced. Limitations • The operational model presented cannot identify all the links which unite the structures, processes and effects of the Intervisions cliniques program. • Interviews with local and regional managers would have helped to better determine all the issues involved in maintaining and generalizing the Intervisions cliniques program. • Clinical examples illustrating experiences of shared mental health care following participation in the Intervisions cliniques program would have helped to better determine what contribution the Intervisions cliniques program has made to shared mental health care

Role of SNX16 in the Dynamics of Tubulo-Cisternal Membrane Domains of Late Endosomes

In this paper, we report that the PX domain-containing protein SNX16, a member of the sorting nexin family, is associated with late endosome membranes. We find that SNX16 is selectively enriched on tubulo-cisternal elements of this membrane system, whose highly dynamic properties and formation depend on intact microtubules. By contrast, SNX16 was not found on vacuolar elements that typically contain LBPA, and thus presumably correspond to multivesicular endosomes. We conclude that SNX16, together with its partner phosphoinositide, define a highly dynamic subset of late endosomal membranes, supporting the notion that late endosomes are organized in distinct morphological and functional regions. Our data also indicate that SNX16 is involved in tubule formation and cholesterol transport as well as trafficking of the tetraspanin CD81, suggesting that the protein plays a role in the regulation of late endosome membrane dynamics

Hrs and SNX3 Functions in Sorting and Membrane Invagination within Multivesicular Bodies

After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs—an adaptor-like protein that binds membrane PtdIns3P via a FYVE motif—and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis

Late Endosomal Cholesterol Accumulation Leads to Impaired Intra-Endosomal Trafficking

Background Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. Methodology/Principal Findings Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2–3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. Conclusions/Significance These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation

SNX12 Role in Endosome Membrane Transport

In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes

The Sodium/Proton Exchanger NHE8 Regulates Late Endosomal Morphology and Function

The pH and lumenal environment of intracellular organelles is considered essential for protein sorting and trafficking through the cell. We provide the first evidence that a mammalian sodium (potassium)/proton exchanger, NHE8, plays a key role in the control of protein trafficking and endosome morphology