Comparison of dipsogenic responses of adult offspring as a function of different perinatal programming models

The perinatal environment interacts with the genotype of the developing organism resulting in a unique phenotype through a developmental or perinatal programming phenomenon. However, it remains unclear how this phenomenon differentially affects particular targets expressing specific drinking responses depending on the perinatal conditions. The main goal of the present study was to compare the dipsogenic responses induced by different thirst models as a function of two perinatal manipulation models, defined by the maternal free access to hypertonic sodium solution and a partial aortic ligation (PAL-W/Na) or a sham-ligation (Sham-W/Na). The programmed adult offspring of both perinatal manipulated models responded similarly when was challenged by overnight water dehydration or after a sodium depletion showing a reduced water intake in comparison to the non-programmed animals. However, when animals were evaluated after a body sodium overload, only adult Sham-W/Na offspring showed drinking differences compared to PAL and control offspring. By analyzing the central neurobiological substrates involved, a significant increase in the number of Fos + cells was found after sodium depletion in the subfornical organ of both programmed groups and an increase in the number of Fos + cells in the dorsal raphe nucleus was only observed in adult depleted PAL-W/Na. Our results suggest that perinatal programming is a phenomenon that differentially affects particular targets which induce specific dipsogenic responses depending on matching between perinatal programming conditions and the osmotic challenge in the latter environment. Probably, each programmed-drinking phenotype has a particular set point to elicit specific repertoires of mechanisms to reestablish fluid balance.

Impact of neurite alignment on organelle motion.

Intracellular transport is pivotal for cell growth and survival. Malfunctions in this process have been associated with devastating neurodegenerative diseases, posing a need for deeper understanding of the involved mechanisms. Here, we used an experimental methodology that lead neurites of differentiated PC12 cells in either of two configurations: an one-dimensional, where the neurites align along lines, or a two-dimensional configuration, where the neurites adopt a random orientation and shape on a flat substrate. We subsequently monitored the motion of functional organelles, the lysosomes, inside the neurites. Implementing a time-resolved analysis of the mean-squared displacement, we quantitatively characterized distinct motion modes of the lysosomes. Our results indicate that neurite alignment gives rise to faster diffusive and super-diffusive lysosomal motion in comparison to the situation where the neurites are randomly oriented. After inducing lysosome swelling through an osmotic challenge by sucrose, we confirmed the predicted slowdown in diffusive mobility. Surprisingly we found that the swelling-induced mobility change affected each of the (sub-/super-) diffusive motion modes differently and depended on the alignment configuration of the neurites. Our findings imply that intracellular transport is significantly and robustly dependent on cell morphology, which might be in part controlled by the extracellular matrix.

RNAi by Soaking Aedes aegypti Pupae in dsRNA

RNA-interference (RNAi) is a standard technique for functional genomics in adult mosquitoes. However, RNAi in immature, aquatic mosquito stages has been challenging. Several studies have shown successful larval RNAi, usually in combination with a carrier molecule. Except for one study in malaria mosquito, Anopheles gambiae, none of the previous studies has explored RNAi in mosquito pupae. Even in the study that used RNAi in pupae, double stranded RNA (dsRNA) was introduced by microinjection. Here, we describe a successful method by soaking pupae in water containing dsRNA without any carrier or osmotic challenge. The knockdown persisted into adulthood. We expect that this simple procedure will be useful in the functional analysis of genes that highly express in pupae or newly emerged adults.

Super-resolution shadow imaging reveals local remodeling of astrocytic microstructures and brain extracellular space after osmotic challenge

The extracellular space (ECS) plays a central role for brain physiology, shaping the time course and spread of neurochemicals, ions and nutrients that ensure proper brain homeostasis and neuronal communication. Astrocytes are the most abundant type of glia cell in the brain, whose processes densely infiltrate the brain’s parenchyma. As astrocytes are highly sensitive to changes in osmotic pressure, they are capable of exerting a potent physiological influence on the ECS.However, little is known about the spatial distribution and temporal dynamics of the ECS that surrounds astrocytes, owing mostly to a lack of appropriate techniques to visualize the ECS in live brain tissue. Mitigating this technical limitation, we applied the recent SUper-resolution SHadow Imaging technique (SUSHI) to astrocyte-labeled organotypic hippocampal brain slices, which allowed us to concurrently image the complex morphology of astrocytes and the ECS with nanoscale resolution in a live experimental setting.Focusing on ring-like astrocytic microstructures in the spongiform domain, we found them to enclose sizable pools of interstitial fluid and cellular structures like dendritic spines. Upon an experimental osmotic challenge, these microstructures remodeled and swelled up at the expense of the pools, effectively increasing the physical contact between astrocytic and cellular structures.Our study reveals novel facets of the dynamic microanatomical relationships between astrocytes, neuropil and the ECS in living brain tissue, which could be of functional relevance for neuronglia communication in a variety of (patho)physiological settings, e.g. LTP induction, epileptic seizures or acute ischemic stroke, where osmotic disturbances are known to occur.

Contrasting strategies of osmotic and ionic regulation in freshwater crabs and shrimps: gene expression of gill ion transporters

Owing to their extraordinary niche diversity, the Crustacea are ideal for comprehending the evolution of osmoregulation. The processes that effect systemic hydro-electrolytic homeostasis maintain hemolymph ionic composition via membrane transporters located in highly specialized gill ionocytes. We evaluated physiological and molecular hyper- and hypo-osmoregulatory mechanisms in two phylogenetically distant, freshwater crustaceans, the crab Dilocarcinus pagei and the shrimp Macrobrachium jelskii, when osmotically challenged for up to 10 days. When in distilled water, D. pagei survived without mortality, hemolymph osmolality and [Cl−] increased briefly, stabilizing at initial values, while [Na+] decreased continually. Gill V(H+)-ATPase, Na+/K+-ATPase and Na+/K+/2Cl− gene expressions were unchanged. In M. jelskii, hemolymph osmolality, [Cl−] and [Na+] decreased continually for 12 h, the shrimps surviving only around 15 to 24 h exposure. Gill transporter gene expressions increased 2- to 5-fold. After 10-days exposure to brackish water (25 ‰S), D. pagei was isosmotic, iso-chloremic and iso-natriuremic. Gill V(H+)-ATPase expression decreased while Na+/K+-ATPase and Na+/K+/2Cl− expressions were unchanged. In M. jelskii (20 ‰S), hemolymph was hypo-regulated, particularly [Cl−]. Transporter expressions initially increased 3- to 12-fold, declining to control values. Gill V(H+)-ATPase expression underlies the ability of D. pagei to survive in fresh water while V(H+)- and Na+/K+-ATPase and Na+/K+/2Cl− expressions enable M. jelskii to confront hyper/hypo-osmotic challenge. These findings reveal divergent responses in two unrelated crustaceans inhabiting a similar osmotic niche. While D. pagei does not secrete salt, tolerating elevated cellular isosmoticity, M. jelskii exhibits clear hypo-osmoregulatory ability. Each species has evolved distinct strategies at the transcriptional and systemic levels during its adaptation to fresh water.

Hog1 Controls Lipids Homeostasis Upon Osmotic Stress in Candida albicans

As opportunistic pathogen, Candida albicans adapts to different environmental conditions and its corresponding stress. The Hog1 MAPK (Mitogen Activated Protein Kinase) was identified as the main MAPK involved in the response to osmotic stress. It was later shown that this MAPK is also involved in the response to a variety of stresses and therefore, its role in virulence, survival to phagocytes and establishment as commensal in the mouse gastrointestinal tract was reported. In this work, the role of Hog1 in osmotic stress is further analyzed, showing that this MAPK is involved in lipid homeostasis. The hog1 mutant accumulates lipid droplets when exposed to osmotic stress, leading to an increase in cell permeability and delaying the endocytic trafficking routes. Cek1, a MAPK also implicated in the response to osmotic challenge, did not play a role in lipid homeostasis indicating that Hog1 is the main MAP kinase in this response. The alteration on lipid metabolism observed in hog1 mutants is proposed to contribute to the sensitivity to osmotic stress.

Substrate Stiffness-Dependent Regulatory Volume Decrease and Calcium Signaling in Chondrocytes

The pericellular matrix stiffness is strongly associated with its biochemical and structural changes During the aging and osteoarthritis progresses of articular cartilage. However, how substrate stiffness modulates the chondrocyte regulatory volume decrease (RVD) and calcium signaling remains unknown. This study aims to investigate the effects of substrate stiffness on the chondrocyte RVD and calcium signaling by recapitulating the physiologically relevant substrate stiffness. Our results showed that substrate stiffness induced completely different dynamical deformation between the cell swelling and recovering progresses. Chondrocytes swelled faster on the soft substrate but recovered slower than the stiff substrate during the RVD response induced by the hypo-osmotic challenge. We found that stiff substrate enhanced the cytosolic Ca2+ oscillation of chondrocytes in the iso-osmotic medium. More importantly, chondrocytes exhibited a distinctive cytosolic Ca2+ oscillation during the RVD response. Soft substrate significantly improved the Ca2+ oscillation during the cell swelling whereas stiff substrate enhanced the cytosolic Ca2+ oscillation during the cell recovering. Our work also suggests that TRPV4 channel are involved in the chondrocyte sensing substrate stiffness and RVD response by mediating Ca2+ signaling in a stiffness-dependent manner. It helps to understand a previously unidentified relationship between substrate stiffness and RVD response under the hypo-osmotic challenge.

Osmokinetics: Defining the Characteristics of Osmotic Challenge to the Ocular Surface

The association of severe dry eye disease with elevated osmolarity in the tear film is a subject of ongoing discussions. As the absolute value of osmolarity in tear film is highly variable, the daily variation in osmolarity (DVO) has recently been proposed to further identify the osmotic stress at the ocular surface. However, the DVO alone does not explain the variations in the available published data or allow their use in diagnostic testing or therapy. We therefore investigated and evaluated further details of osmokinetics and their importance for ocular surface disease on the basis of the available literature. Additionally, osmolarity was measured in the tear samples of volunteers in the morning hours between 8 – 10 a. m., midday noon–2 p. m., and afternoon between 3 – 5 p. m., i.e., during normal office hours. The results were compared with available published data which suggested that within the DVO, the daily maximal amplitude of osmotic variation (M-DVO) and the frequency of osmotic cycles (defined as daily osmolarity cycles, DOC) could be the main factors that further characterize osmokinetics. In addition, a decisive role could be the level of osmolarity at which the variation does occurs (L-DVO). The possible effects of these characteristics on ocular surface pathophysiology are discussed, along with their relationship to topical therapy with hypo-osmolar solutions, and the model of the osmotic roller coaster is introduced.