Reversible developmental stasis in response to nutrient availability in the Xenopus laevis central nervous system.

Many organisms confront intermittent nutrient restriction (NR), but the mechanisms to cope with nutrient fluctuations during development are not well understood. This is particularly true of the brain, the development and function of which is energy intensive. Here we examine the effects of nutrient availability on visual system development in Xenopus laevis tadpoles. During the first week of development, tadpoles draw nutrients from maternally provided yolk. Upon yolk depletion, animals forage for food. By altering access to external nutrients after yolk depletion, we identified a period of reversible stasis during tadpole development. We demonstrate that NR results in developmental stasis characterized by a decrease in overall growth of the animals, a failure to progress through developmental stages, and a decrease in volume of the optic tectum. During NR, neural progenitors virtually cease proliferation, but tadpoles swim and behave normally. Introducing food after temporary NR increased neural progenitor cell proliferation more than 10-fold relative to NR tadpoles, and cell proliferation was comparable to that of fed counterparts 1 week after delayed feeding. Delayed feeding also rescued NR-induced body length and tectal volume deficits and partially rescued developmental progression defects. Tadpoles recover from developmental stasis if food is provided within the first 9 days of NR, after which access to food fails to increase cell proliferation. These results show that early stages of tadpole brain development are acutely sensitive to fluctuations in nutrient availability and that NR induces developmental stasis from which animals can recover if food becomes available within a critical window.

Influence of advancing season on dietary composition, intake, site of digestion, and microbial efficiency in beef steers grazing season-long or twice-over rotation native range pastures in western North Dakota.

Eight ruminally and duodenally cannulated beef steers (374 +/- 11 kg) were used to evaluate effects of advancing season and grazing treatment (season-long; SL or twice-over rotation; TOR) on dietary composition (Exp. 1 and 2), intake, site and extent of digestion, and microbial efficiency (Exp. 2) of native range. In Exp. 1, six 11-d sample collections were conducted from early June to mid-November 2000 and 2001. In vitro OM disappearance decreased (P < or = 0.04) for both years and both treatments with advancing season. Dietary N declined (P < or = 0.07), whereas fiber content increased (P < or = 0.05) during both years in both treatments, with the exception of NDF (P = 0.55) during yr 2 (YR2) on the TOR, as season advanced. In Exp. 2, three 11-d sample collections were conducted from late July to mid-September 2000 on SL and TOR. Organic matter intake (g/kg of BW) was not altered (P = 0.28) by grazing treatments or advancing season. Total tract OM and apparent ruminal OM digestion were not different (P > or = 0.12) between treatment and decreased (P < or = 0.04) with advancing season. Grazing treatment x season interactions (P = 0.06) were present for true ruminal OM digestibility with TOR being greater (P < or = 0.10) than SL in late August and mid-September but not late July. Microbial efficiency was greater (P = 0.07; 15.1 vs. 10.8 +/- 1.6 g of microbial N/kg of OM truly fermented) in SL than TOR, respectively. Degradable intake protein (g/d) was less (P = 0.05) in TOR than SL during late July to early August and not affected by treatment in late August or mid-September. However, undegradable intake protein was unchanged (P > or = 0.54) between treatment and across season. These data suggest that mixed-grass range forage consumed by cattle after late September is deficient in N, particularly degradable intake protein, and that forage intake may be insufficient to support adequate performance in lactating cows independent of grazing management strategies evaluated. Knowledge of diet quality and forage intake should aid producers in meeting the nutritional needs of livestock grazing these forages.

Influence of advancing season on dietary composition, intake, site of digestion, and microbial efficiency in beef steers grazing a native range in western North Dakota.

Four ruminally and duodenally cannulated beef steers (388 +/- 12 kg) were used to evaluate effects of advancing season on forage quality, intake, site of digestion, and microbial efficiency while grazing mixed-grass prairie in western North Dakota. Five 11-d sample collections were conducted from late June to mid-November. Chromic oxide (8 g) was dosed twice daily at 0700 and 1900 h via gelatin capsule from d 2 to 11 of each collection period, and duodenal and fecal collections were performed on d 7 to 11. Masticate samples were collected for each sampling period. Dietary N declined linearly (P = 0.01), from 1.95% in June to 1.15% in November, whereas NDF increased linearly (P = 0.01), 72.4% in June to 85.1% in November. Total OM intake (g/kg of BW) decreased linearly (P

The molecular basis of CaMKII function in synaptic and behavioural memory.

Long-term potentiation (LTP) in the CA1 region of the hippocampus has been the primary model by which to study the cellular and molecular basis of memory. Calcium/calmodulin-dependent protein kinase II (CaMKII) is necessary for LTP induction, is persistently activated by stimuli that elicit LTP, and can, by itself, enhance the efficacy of synaptic transmission. The analysis of CaMKII autophosphorylation and dephosphorylation indicates that this kinase could serve as a molecular switch that is capable of long-term memory storage. Consistent with such a role, mutations that prevent persistent activation of CaMKII block LTP, experience-dependent plasticity and behavioural memory. These results make CaMKII a leading candidate in the search for the molecular basis of memory.

Design of a logarithmic k-space spiral trajectory.

Spiral acquisitions are used in fast cardiac imaging because they traverse k-space efficiently and minimize flow artifacts. A variable pitch logarithmic spiral trajectory is designed to critically sample the low-frequency region in k-space and gradually undersample the high-frequency region. An approximate analytical expression for the trajectory provides a fast means to calculate the gradient waveforms and the sampled data points. A numerical method is introduced based on the trajectory curvature and the rate of change in the gradient magnitude with time for the composite Archimedean-logarithmic trajectory. The pulse sequence is implemented and images are acquired on phantoms and human hearts. The images show improved image resolution and some improvement in image quality as a result of increased extent in k-space and reduction in aliasing artifacts, respectively.

Nitric oxide is an essential negative regulator of cell proliferation in Xenopus brain.

Mechanisms controlling the transition of a neural precursor cell from proliferation to differentiation during brain development determine the distinct anatomical features of the brain. Nitric oxide (NO) may mediate such a transition, because it can suppress DNA synthesis and cell proliferation. We cloned the gene encoding the neuronal isoform of Xenopus NO synthase (XNOS) and found that in the developing brain of Xenopus tadpoles, a zone of XNOS-expressing cells lies adjacent to the zone of dividing neuronal precursors. Exogenous NO, supplied to the tadpole brain in vivo, decreased the number of proliferating cells and the total number of cells in the optic tectum. Conversely, inhibition of NOS activity in vivo increased the number of proliferating cells and the total number of cells in the optic tectum. NOS inhibition yielded larger brains with grossly perturbed organization. Our results indicate that NO is an essential negative regulator of neuronal precursor proliferation during vertebrate brain development.

Developmental regulation of CPG15 expression in Xenopus.

Mechanisms controlling dendritic arbor formation affect the establishment of neuronal circuits. Candidate plasticity gene 15 (CPG15) is a glycosylphosphatidyl inositol (GPI)-linked activity-induced protein that has been shown to function as an intercellular signaling molecule that can promote the morphological and physiological development of the Xenopus retinotectal system. A thorough understanding of CPG15 function requires knowledge of the spatiotemporal expression of the endogenous protein. We therefore cloned Xenopus cpg15 and used RNA in situ hybridization and immunohistochemistry to determine the pattern of CPG15 expression. cpg15 mRNA and CPG15 protein are first detectable in the developing spinal cord and become widespread as development proceeds. CPG15 is expressed in sensory regions of the brain, including the visual, auditory, and olfactory systems. Within the retina, CPG15 is only expressed in retinal ganglion cells. CPG15 protein is concentrated in axon tracts, including retinal axons. These data support a model in which CPG15 expressed in retinal ganglion cells is trafficked to retinal axons, where it modulates postsynaptic dendritic arbor elaboration, and synaptic maturation.

Single-cell electroporation for gene transfer in vivo.

We report an electroporation technique for targeting gene transfer to individual cells in intact tissue. Electrical stimulation through a micropipette filled with DNA or other macromolecules electroporates a single cell at the tip of the micropipette. Electroporation of a plasmid encoding enhanced green fluorescent protein (GFP) into the brain of intact Xenopus tadpoles or rat hippocampal slices resulted in GFP expression in single neurons and glia. In vivo imaging showed morphologies, dendritic arbor dynamics, and growth rates characteristic of healthy cells. Coelectroporation of two plasmids resulted in expression of both proteins, while electroporation of fluorescent dextrans allowed direct visualization of transfer of molecules into cells. This technique will allow unprecedented spatial and temporal control over gene delivery and protein expression.

The scaffold protein, Homer1b/c, regulates axon pathfinding in the central nervous system in vivo.

Homer proteins are a family of multidomain cytosolic proteins that have been postulated to serve as scaffold proteins that affect responses to extracellular signals by regulating protein-protein interactions. We tested whether Homer proteins are involved in axon pathfinding in vivo, by expressing both wild-type and mutant isoforms of Homer in Xenopus optic tectal neurons. Time-lapse imaging demonstrated that interfering with the ability of endogenous Homer to form protein-protein interactions resulted in axon pathfinding errors at stereotypical choice points. These data demonstrate a function for scaffold proteins such as Homer in axon guidance. Homer may facilitate signal transduction from cell-surface receptors to intracellular proteins that govern the establishment of axon trajectories.

Dendritic arbor development and synaptogenesis.

In vivo studies indicate that synaptic activity promotes dendritic arbor elaboration at early stages of brain development. At later stages of development, synaptic activity stabilizes dendritic structure. The different roles of synaptic activity with respect to structural plasticity probably reflect the regulated spatiotemporal expression of key components within signaling pathways.

Postsynaptic CPG15 promotes synaptic maturation and presynaptic axon arbor elaboration in vivo.

The formation of CNS circuits is characterized by the coordinated development of neuronal structure and synaptic function. The activity-regulated candidate plasticity gene 15 (cpg15) encodes a glycosylphosphatidylinositol (GPI)-linked protein whose in vivo expression increases the dendritic arbor growth rate of Xenopus optic tectal cells. We now demonstrate that tectal cell expression of CPG15 significantly increases the elaboration of presynaptic retinal axons by decreasing rates of branch retractions. Whole-cell recordings from optic tectal neurons indicate that CPG15 expression promotes retinotectal synapse maturation by recruiting functional AMPA receptors to synapses. Expression of truncated CPG15, lacking its GPI anchor, does not promote axon arbor growth and blocks synaptic maturation. These results suggest that CPG15 coordinately increases the growth of pre- and postsynaptic structures and the number and strength of their synaptic contacts.

Synapse formation: if it looks like a duck and quacks like a duck ....

Neuroligin and neurexin form an intercellular adhesion complex sufficient to trigger formation of functional presynaptic elements in vitro. This single molecular interaction appears to initiate clustering of synaptic vesicles, assembly of vesicle-release machinery and morphological changes at the presynaptic membrane.

Combined connectivity and a gray-level morphological filter in magnetic resonance coronary angiography.

A connectivity algorithm combined with a new gray-level morphological filter dramatically improves the segmentation of tortuous coronary arteries from 3D MRI. Small coronary arteries are segmented from the larger ventricles with a new filter. These blood vessels are segmented from the noise background with connectivity. Coronary angiograms were computed in nine datasets acquired on volunteers with 3D stack of spirals and contrast-enhanced navigator sequences by both a maximum intensity projection and surface rendering. Surface images provided depth information needed to distinguish branching arteries from crossing veins. Magn Reson Med 43:892-895, 2000.

Rho GTPases regulate distinct aspects of dendritic arbor growth in Xenopus central neurons in vivo.

The development and structural plasticity of dendritic arbors are governed by several factors, including synaptic activity, neurotrophins and other growth-regulating molecules. The signal transduction pathways leading to dendritic structural changes are unknown, but likely include cytoskeleton regulatory components. To test whether GTPases regulate dendritic arbor development, we collected time-lapse images of single optic tectal neurons in albino Xenopus tadpoles expressing dominant negative or constitutively active forms of Rac, Cdc42 or RhoA. Analysis of images collected at two-hour intervals over eight hours indicated that enhanced Rac activity selectively increased branch additions and retractions, as did Cdc42 to a lesser extent. Activation of endogenous RhoA decreased branch extension without affecting branch additions and retractions, whereas dominant-negative RhoA increased branch extension. Finally, we provide data suggesting that RhoA mediates the promotion of normal dendritic arbor development by NMDA receptor activation.

Inappropriate use of dialysis for some elderly patients: nephrology nurses' perceptions and concerns.

The purpose of this study was to determine what percentage of elderly patients (age 65 years and older) nephrology nurses (NN) believe should not be dialyzed and to identify NN percep tions and concerns about the inappropriate use of dialysis for these patients. The design was descriptive using a cross-sectional survey instrument. Data was collected in 1996 from 393 NN (44% response rate), with a mean age of 41 years and 9 years of dialysis experience. The sample was 1,000 NN randomly selected from 7,000 members of the American Nephrology Nurses'Association (ANNA) who selected hemodialysis as a special area of interest. Quantitative analysis of respondents revealed that NN felt that 15% of elderly dialysis patients (EDP) should not be dialyzed, and 80% were troubled by decisions to start dialysis for some elderly patients. Content analysis of qualitative responses supported the NN concerns revealed in the quantitative analysis. Five themes with interrater reliability of > 98% were identified: qualiy of life (QoL), patient/family inadequate knowledge, patient influences on decision making age, and other (cost/legal reasons). QoL issues accounted for 75% of NN responses. Statistical analyses including an ANOVA, t-tests, and chi square failed to identify relationships between themes and demographic factors, such as age, gender, years of experience, education level, unit profit status, and type of staff position. The researchers conclude that NN believe dialysis is inappropriate for a significant percentage of EDP, these perceptions and concerns are widespread, and QoL issues are key factors in decisions to withhold or withdraw dialysis. NN may decrease the inappropriate use of dialysis by identifying QoL factors for EDP, facilitating discussions with family members and nephrologists, and continuing to advocate for the rights of EDP with poor QoL.

Postsynaptic calcium/calmodulin-dependent protein kinase II is required to limit elaboration of presynaptic and postsynaptic neuronal arbors.

Neuronal dendritic and axonal arbors grow to a characteristic size and then stabilize their structures. Activity-dependent stop-growing signals may limit neuronal process elaboration. We tested whether endogenous calcium/calmodulin-dependent protein kinase II (CaMKII) activity in postsynaptic optic tectal cells is required to restrict the elaboration of neuronal processes in the Xenopus tadpole retinotectal projection. Optic tectal cells were infected with vaccinia viruses that express CaMKII-specific inhibitory peptides. In vivo time-lapse imaging revealed that expression of CaMKII inhibitors blocked the growth restriction that normally occurs during maturation of tectal cell dendritic arbors. Postsynaptic CaMKII inhibition also increased the growth of presynaptic retinotectal axon arbors. The results indicate that endogenous postsynaptic CaMKII activity is required to limit the growth of presynaptic and postsynaptic arbor structures in vivo.

Dendritic dynamics in vivo change during neuronal maturation.

In vivo imaging of optic tectal neurons in the intact Xenopus tadpole permits direct observation of the structural dynamics that occur during dendritic arbor formation. Based on images of single DiI-labeled neurons collected at daily intervals over a period of 6 d, we divided tectal cell development into three phases according to the total length of the dendritic arbor. During phase 1, the cell differentiates from a neuroepithelial cell type and extends an axon out of the tectum. The total dendritic branch length (TDBL) is <100 micrometers. During phase 2, when TDBL is 100-400 micrometers, the dendritic arbor grows rapidly. During phase 3, when TDBL is >400 micrometers, the dendritic arbor grows slowly and appears stable. Neurons at different positions along the rostrocaudal developmental axis of the tectum were imaged at 2 hr intervals over 6 hr and at 24 hr intervals over several days. Images collected at 2 hr intervals were analyzed to determine rates of branch additions and retractions. Morphologically complex, phase 3 neurons show half the rate of branch additions and retractions as phase 2 neurons. Therefore, rapidly growing neurons have dynamic dendritic arbors, and slower-growing neurons are structurally stable. The change in growth rate and dendritic arbor dynamics from phase 2 to phase 3 correlates with the developmental increase in synaptic strength in neurons located along the rostrocaudal tectal axis. The data are consistent with the idea that strong synaptic inputs stabilize dendritic arbor structures and that weaker synaptic inputs are permissive for a greater degree of dynamic rearrangements and a faster growth rate in the dendritic arbor.