Determination of transmitter function by neuronal activity

The role of neuronal activity in the determination of transmitter function was studied in cultures of dissociated sympathetic neurons from newborn rat superior cervical ganglia. Cholinergic and adrenergic differentiation were assayed by incubating the cultures with radioactive choline and tyrosine and determining the rate of synthesis and accumulation of labelled acetylcholine and catecholamines. As in previous studies, pure neuronal cultures grown in control medium displayed much lower ratios of acetylcholine synthesis to catecholamine synthesis than did sister cultures grown in medium previously conditioned by incubation on appropriate nonneuronal cells (conditioned medium). However, here we report that neurons treated with the depolarizing agents elevated K+ or veratridine, or stimulated directly with electrical current, either before or during application of conditioned medium, displayed up to 300-fold lower acetylcholine/catecholamine ratios than they would have without depolarization, and thus remained primarily adrenergic. Elevated K+ and veratridine produced this effect on cholinergic differentiation without significantly altering neuronal survival. Because depolarization causes Ca2+ entry in a number of cell types, the effects of several Ca2+ agonists and antagonists were investigated. In the presence of the Ca2+ antagonists D600 or Mg2+, K+ did not prevent the induction of cholinergic properties by conditioned medium. Thus depolarization, either steady or accompanying activity, is one of the factors determining whether cultured sympathetic neurons become adrenergic or cholinergic, and this effect may be mediated by Ca2+

Effects of high hydrostatic pressure on neuromuscular transmission in shallow-living and deep-living crustaceans

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution November, 1975The effects of high hydrostatic pressure on excitatory neuromuscular transmission in shallow- and deep-living crustaceans were compared. Pressure caused depression of the amplitude of excitatory junctional potentials (e.j.p.s) at the neuromuscular junction in the shallow-living crab, Libinia emarginata. A pressure of 100 atm depressed the e.j.p. amplitude by about one-half. In the deep- sea crab, Geryon quinquedens, which ranges to a depth of 2000 m (or 200 atm pressure), adaptations to high pressure were observed in two different types of muscle fibers: 1) In fibers with e.j.p.s that showed high levels of facilitation, the magnitude of pressure-induced depression decreased with increasing frequency of nerve stimulation; i. e., there was a pressure-induced increase in facilitation. Also a pressure-induced increase in the duration of the falling phase of the e.j.p. was observed which served to increase the level of depolarization resulting from summation of the e.j.p.s at high frequencies of nerve stimulation. In these highly facilitating fibers the physiologically significant frequencies that cause appreciable contraction are probably high. At high frequencies the pressure-induced increases in facilitation and summation together served to completely counteract the depressive effect of pressure, and the net depolarization attained during a train of nerve stimulation was relatively unaffected by pressures up to at least 200 atm. 2) Fibers with e.j.p.s showing low levels of facilitation may undergo significant contraction at low frequencies of nerve impulses where neither facilitation nor summation play a significant role. The amplitude of e.j.p.s recorded from this fiber-type in the deep-sea crab were, on the average, unaffected by pressures to 200 atm. The e.j.p.s of some of these fibers showed depression, but others were amplified under pressure. The results of experiments with the lobster, Homarus americanus, which ranges to a depth intermediate between Libinia and Geryon were in many respects intermediate between the results obtained with the two species of crab. Studies of the effect of pressure on isometric tension developed by whole muscles in Homarus and Geryon were consistent with the results of the studies of the e.j.p.; pressure depressed the rate of rise of tension in Homarus and had little effect in Geryon. The results of this work provides a physiological basis for the observation that shallow-living animals are generally immobilized by pressures in excess of 200 atm. Experiments were performed in an attempt to elucidate the mechanism underlying the pressure-induced depression of e.j.p. amplitude. Results were suggestive that the depression of e.j.p. amplitude reflects a pressure-induced decrease in the number of quanta of transmitter substance released by the nerve endings

Visualization of an Alphaherpesvirus Membrane Protein That Is Essential for Anterograde Axonal Spread of Infection in Neurons

Pseudorabies virus (PRV), an alphaherpesvirus with a broad host range, replicates and spreads in chains of synaptically connected neurons. The PRV protein Us9 is a small membrane protein that is highly conserved among alphaherpesviruses and is essential for anterograde axonal spread in neurons. Specifically, the Us9 protein is required for the sorting of newly assembled PRV particles into axons. However, the molecular details underlying the function of Us9 are poorly understood. Here we constructed PRV strains that express functional green fluorescent protein (GFP)-Us9 fusion proteins in order to visualize axonal transport of viral particles in infected rat superior cervical ganglion neurons. We show that GFP-Us9-labeled structures are transported exclusively in the anterograde direction within axons. Additionally, the vast majority of anterograde-directed capsids (labeled with VP26-monomeric red fluorescent protein) and a viral membrane protein (labeled with glycoprotein M fused to mCherry) are cotransported with GFP-Us9 in the anterograde direction. In contrast, during infection with PRV strains that express nonfunctional mutant GFP-Us9 proteins, cotransport of mutant GFP-Us9 with capsids in axons is abolished. These findings show that axonal sorting of progeny viral particles is dependent upon the association of viral structures with membranes that contain functional Us9 proteins. This association is required for anterograde spread of infection in neurons

The effects of changing climate on faunal depth distributions determine winners and losers

Changing climate is predicted to impact all depths of the global oceans, yet projections of range shifts in marine faunal distributions in response to changing climate seldom evaluate potential shifts in depth distribution. Marine ectotherms’ thermal tolerance is limited by their ability to maintain aerobic metabolism (oxygen- and capacity-limited tolerance), and is functionally associated with their hypoxia tolerance. Shallow-water (<200 m depth) marine invertebrates and fishes demonstrate limited tolerance of increasing hydrostatic pressure (pressure exerted by the overlying mass of water), and hyperbaric (increased pressure) tolerance is proposed to depend on the ability to maintain aerobic metabolism, too. Here, we report significant correlation between the hypoxia thresholds and the hyperbaric thresholds of taxonomic groups of shallow-water fauna, suggesting that pressure tolerance is indeed oxygen-limited. Consequently, it appears that the combined effects of temperature, pressure, and oxygen concentration constrain the fundamental ecological niches (FENs) of marine invertebrates and fishes. Including depth in a conceptual model of oxygen- and capacity-limited FENs’ responses to ocean warming and deoxygenation confirms previous predictions made based solely on consideration of the latitudinal effects of ocean warming (e.g. Cheung et al., 2009), that polar taxa are most vulnerable to the effects of climate change, with Arctic fauna experiencing the greatest FEN contraction. In contrast, the inclusion of depth in the conceptual model reveals for the first time that temperate fauna as well as tropical fauna may experience substantial FEN expansion with ocean warming and deoxygenation, rather than FEN maintenance or contraction suggested by solely considering latitudinal range shifts

One at a time, live tracking of NGF axonal transport using quantum dots

Retrograde axonal transport of nerve growth factor (NGF) signals is critical for the survival, differentiation, and maintenance of peripheral sympathetic and sensory neurons and basal forebrain cholinergic neurons. However, the mechanisms by which the NGF signal is propagated from the axon terminal to the cell body are yet to be fully elucidated. To gain insight into the mechanisms, we used quantum dot-labeled NGF (QD-NGF) to track the movement of NGF in real time in compartmentalized culture of rat dorsal root ganglion (DRG) neurons. Our studies showed that active transport of NGF within the axons was characterized by rapid, unidirectional movements interrupted by frequent pauses. Almost all movements were retrograde, but short-distance anterograde movements were occasionally observed. Surprisingly, quantitative analysis at the single molecule level demonstrated that the majority of NGF-containing endosomes contained only a single NGF dimer. Electron microscopic analysis of axonal vesicles carrying QD-NGF confirmed this finding. The majority of QD-NGF was found to localize in vesicles 50–150 nm in diameter with a single lumen and no visible intralumenal membranous components. Our findings point to the possibility that a single NGF dimer is sufficient to sustain signaling during retrograde axonal transport to the cell body

Developmental axon pruning mediated by BDNF-p75NTR–dependent axon degeneration

The mechanisms that regulate the pruning of mammalian axons are just now being elucidated. Here, we describe a mechanism by which, during developmental sympathetic axon competition, winning axons secrete brain-derived neurotrophic factor (BDNF) in an activity-dependent fashion, which binds to the p75 neurotrophin receptor (p75NTR) on losing axons to cause their degeneration and, ultimately, axon pruning. Specifically, we found that pruning of rat and mouse sympathetic axons that project to the eye requires both activity-dependent BDNF and p75NTR. p75NTR and BDNF are also essential for activity-dependent axon pruning in culture, where they mediate pruning by directly causing axon degeneration. p75NTR, which is enriched in losing axons, causes axonal degeneration by suppressing TrkA-mediated signaling that is essential for axonal maintenance. These data provide a mechanism that explains how active axons can eliminate less-active, competing axons during developmental pruning by directly promoting p75NTR-mediated axonal degeneration

Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling

RhoA is activated from internalized Nogo-A to promote growth cone collapse and inhibit neurite outgrowth

Thick collagen-based 3D matrices including growth factors to induce neurite outgrowth

Designing synthetic microenvironments for cellular investigations is a very active area of research at the crossroads of cell biology and materials science. The present work describes the design and functionalization of a three-dimensional (3D) culture support dedicated to the study of neurite outgrowth from neural cells. It is based on a dense self-assembled collagen matrix stabilized by 100-nm wide interconnected native fibrils without chemical crosslinking. The matrices were made suitable for cell manipulation and direct observation in confocal microscopy by anchoring them to traditional glass supports with a calibrated thickness of ∼50 μm. The matrix composition can be readily adapted to specific neural cell types, notably by incorporating appropriate neurotrophic growth factors. Both PC-12 and SH-SY5Y lines respond to growth factors (nerve growth factor and brain-derived neurotrophic factor, respectively) impregnated and slowly released from the support. Significant neurite outgrowth is reported for a large proportion of cells, up to 66% for PC12 and 49% for SH-SY5Y. It is also shown that both growth factors can be chemically conjugated (EDC/NHS) throughout the matrix and yield similar proportions of cells with longer neurites (61% and 52%, respectively). Finally, neurite outgrowth was observed over several tens of microns within the 3D matrix, with both diffusing and immobilized growth factors

Unusual finding of endocervical-like mucinous epithelium in continuity with urothelium in endocervicosis of the urinary bladder

Endocervicosis in the urinary bladder is a rare benign condition. We present a case in a 37-year-old woman with classical clinical and pathological features of endocervicosis. The unusual observation of endocervical-like mucinous epithelium in continuity with the urothelium in addition to fully developed endocervicosis prompted immunohistochemical profiling of the case using antibodies to cytokeratins (AE1/AE3, CK19, CK7, CK5/6, CK20), HBME-1, estrogen receptor (ER) and progesterone receptor (PR) to assess the relationship of the surface mucinous and endocervicosis glandular epithelia. The surface mucinous epithelium, urothelium and endocervicosis glands were immunopositive for AE1/AE3, CK7 and CK19 while CK20 was only expressed by few urothelial umbrella cells. The surface mucinous epithelium was CK5/6 and HBME-1 immunonegative but showed presence of ER and PR. This was in contrast to the urothelium's expression of CK5/6 but not ER and PR. In comparison, endocervicosis glands expressed HBME-1, unlike the surface mucinous epithelium. The endocervicosis epithelium also demonstrated the expected presence of ER and PR and CK5/6 immunonegativity. The slightly differing immunohistochemical phenotypes of the surface mucinous and morphologically similar endocervicosis glandular epithelium is interesting and requires further clarification to its actual nature. The patient has remained well and without evidence of disease 18-months following transurethral resection of the lesion