Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model.

The etiology of necrotizing enterocolitis (NEC) is not known. Alterations in gut microbiome, mucosal barrier function, immune cell activation, and blood flow are characterized events in its development, with stress as a contributing factor. The hormone corticotropin-releasing factor (CRF) is a key mediator of stress responses and influences these aforementioned processes. CRF signaling is modulated by NEC's main risk factors of prematurity and formula feeding. Using an established neonatal rat model of NEC, we tested hypotheses that: (i) increased CRF levels-as seen during stress-promote NEC in formula-fed (FF) newborn rats, and (ii) antagonism of CRF action ameliorates NEC. Newborn pups were formula-fed to initiate gut inflammation and randomized to: no stress, no stress with subcutaneous CRF administration, stress (acute hypoxia followed by cold exposure-NEC model), or stress after pretreatment with the CRF peptide antagonist Astressin. Dam-fed unstressed and stressed littermates served as controls. NEC incidence and severity in the terminal ileum were determined using a histologic scoring system. Changes in CRF, CRF receptor (CRFRs), and toll-like receptor 4 (TLR4) expression levels were determined by immunofluorescence and immunoblotting, respectively. Stress exposure in FF neonates resulted in 40.0% NEC incidence, whereas exogenous CRF administration resulted in 51.7% NEC incidence compared to 8.7% in FF non-stressed neonates (p<0.001). Astressin prevented development of NEC in FF-stressed neonates (7.7% vs. 40.0%; p = 0.003). CRF and CRFR immunoreactivity increased in the ileum of neonates with NEC compared to dam-fed controls or FF unstressed pups. Immunoblotting confirmed increased TLR4 protein levels in FF stressed (NEC model) animals vs. controls, and Astressin treatment restored TLR4 to control levels. Peripheral CRF may serve as specific pharmacologic target for the prevention and treatment of NEC.

Transient lamellipodia predict sites of dendritic branch formation in hippocampal neurons.

Extensive branching creates the complex dendritic arbor of mammalian CNS neurons but capturing the complete process of branch formation with time-lapse recordings has been challenging. Here, we report that application of BMP7 to cultured hippocampal neurons accelerated dendritic growth sufficiently to document branches forming in less than 20 h via frequent time-lapse imaging (10-min intervals). In these recordings, most branches emerged as collateral sprouts from the shaft of a parent branch. Analysis of the recordings showed that filopodia were abundant and formed transiently throughout the length of dendrites but among these, only a small subset occurred at sites where branches later emerged. Conversely, formation of lamellipodia was rare and coincided with sites where collateral branches emerged. This pattern suggests that lamellipodial structures act as an important intermediate form of cytoskeletal remodeling related to a cellular commitment to branch, whereas filopodia appear to be related to events prior to such commitment.

Interactions with Astroglia Influence the Shape of the Developing Dendritic Arbor and Restrict Dendrite Growth Independent of Promoting Synaptic Contacts.

Astroglia play key roles in the development of neurons, ranging from regulating neuron survival to promoting synapse formation, yet basic questions remain about whether astrocytes might be involved in forming the dendritic arbor. Here, we used cultured hippocampal neurons as a simple in vitro model that allowed dendritic growth and geometry to be analyzed quantitatively under conditions where the extent of interactions between neurons and astrocytes varied. When astroglia were proximal to neurons, dendrites and dendritic filopodia oriented toward them, but the general presence of astroglia significantly reduced overall dendrite growth. Further, dendritic arbors in partial physical contact with astroglia developed a pronounced pattern of asymmetrical growth, because the dendrites in direct contact were significantly smaller than the portion of the arbor not in contact. Notably, thrombospondin, the astroglial factor shown previously to promote synapse formation, did not inhibit dendritic growth. Thus, while astroglia promoted the formation of presynaptic contacts onto dendrites, dendritic growth was constrained locally within a developing arbor at sites where dendrites contacted astroglia. Taken together, these observations reveal influences on spatial orientation of growth as well as influences on morphogenesis of the dendritic arbor that have not been previously identified.

Evidence that angiogenesis lags behind neuron and astrocyte growth in experience-dependent plasticity.

Bill Greenough's work on the cell biology of information storage suggests that we cannot understand the mechanism of long-term memory without understanding the series of cellular transactions that drive coordinated structural changes in neurons, glia, and blood vessels. Here, we show that after 4 days of differential housing, neuropil of EC cortex has expanded significantly, but the vasculature has not, resulting in a dilution of the blood supply. Significant growth of neurons and astrocytes has been reported within this time period, suggesting expression of synaptic plasticity might involve temporally coordinated genomic responses by both neurons and glia. Given that astrocytes appear to couple neuronal and vascular growth during development, we hypothesize that they may also mediate the onset of angiogenesis in response to neural demand in the EC brain. Further, these results may imply that a neuron's capacity for plasticity could be constrained by the rate of vascular expansion.

Synapses on demand require dendrites at the ready: how defining stages of dendritic development in vitro could inform studies of behaviorally driven information storage in the brain.

Bill Greenough's work provides a framework for thinking about synaptogenesis not only as a key step in the initial wiring of neural systems according to a species typical plan (i.e., experience-expectant development), but also as a mechanism for storing information based an individual's unique experience over its lifetime (i.e., experience-dependent plasticity). Analysis of synaptic development in vitro brings a new opportunity to test the limits of expectant-expectant development at the level of the individual neuron. We analyzed dendritic growth, synapse formation, and the development of specialized cytoplasmic microdomains during development in cultured hippocampal neurons, to determine if the timing of each of these events is correlated. Taken together, the findings reported here support the hypotheses that (1) dendritic development is rate limiting in synapse formation and (2) synaptic circuits are assembled in a step-wise fashion consistent with a stage-specific shift from genomically pre-programmed to activity-dependent mechanisms.

Dendrites differ from axons in patterns of microtubule stability and polymerization during development.

BACKGROUND: Dendrites differ from axons in patterns of growth and development, as well as in morphology. Given that microtubules are key structural elements in cells, we assessed patterns of microtubule stability and polymerization during hippocampal neuron development in vitro to determine if these aspects of microtubule organization could distinguish axons from dendrites. RESULTS: Quantitative ratiometric immunocytochemistry identified significant differences in microtubule stability between axons and dendrites. Most notably, regardless of developmental stage, there were high levels of dynamic microtubules throughout the dendritic arbor, whereas dynamic microtubules were predominantly concentrated in the distal end of axons. Analysis of microtubule polymerization using green fluorescent protein-tagged EB1 showed both developmental and regional differences in microtubule polymerization between axons and dendrites. Early in development (for example, 1 to 2 days in vitro), polymerization events were distributed equally in both the anterograde and retrograde directions throughout the length of both axons and dendrites. As development progressed, however, polymerization became biased, with a greater number of polymerization events in distal than in proximal and middle regions. While polymerization occurred almost exclusively in the anterograde direction for axons, both anterograde and retrograde polymerization was observed in dendrites. This is in agreement with predicted differences in microtubule polarity within these compartments, although fewer retrograde events were observed in dendrites than expected. CONCLUSION: Both immunocytochemical and live imaging analyses showed that newly formed microtubules predominated at the distal end of axons and dendrites, suggesting a common mechanism that incorporates increased microtubule polymerization at growing process tips. Dendrites had more immature, dynamic microtubules throughout the entire arbor than did axons, however. Identifying these differences in microtubule stability and polymerization is a necessary first step toward understanding how they are developmentally regulated, and may reveal novel mechanisms underlying neuron maturation and dendritic plasticity that extend beyond the initial specification of polarity.

Experience-dependent plasticity in the mushroom bodies of the solitary bee Osmia lignaria (Megachilidae).

All members of the solitary bee species Osmia lignaria (the orchard bee) forage upon emergence from their natal nest cell. Conversely, in the honey bee, days-to-weeks of socially regulated behavioral development precede the onset of foraging. The social honey bee's behavioral transition to foraging is accompanied by neuroanatomical changes in the mushroom bodies, a region of the insect brain implicated in learning. If these changes were general adaptations to foraging, they should also occur in the solitary orchard bee. Using unbiased stereological methods, we estimated the volume of the major compartments of the mushroom bodies, the neuropil and Kenyon cell body region, in adult orchard bees. We compared the mushroom bodies of recently emerged bees with mature bees that had extensive foraging experience. To separate effects of general maturation from field foraging, some orchard bees were confined to a cage indoors. The mushroom body neuropil of experienced field foragers was significantly greater than that of both recently emerged and mature caged orchard bees, suggesting that, like the honey bee, this increase is driven by outdoor foraging experience. Unlike the honey bee, where increases in the ratio of neuropil to Kenyon cell region occur in the worker after emerging from the hive cell, the orchard bee emerged from the natal nest cell with a ratio that did not change with maturation and was comparable to honey-bee foragers. These results suggest that a common developmental endpoint may be reached via different development paths in social and solitary species of foraging bees.

Effects of substrate geometry on growth cone behavior and axon branching.

At the leading edge of a growing axon, the growth cone determines the path the axon takes and also plays a role in the formation of branches, decisions that are regulated by a complex array of chemical signals. Here, we used microfabrication technology to determine whether differences in substrate geometry, independent of changes in substrate chemistry, can modulate growth cone motility and branching, by patterning a polylysine grid of narrow (2 or 5 microm wide) intersecting lines. The shape of the intersections varied from circular nodes 15 microm in diameter to simple crossed lines (nodeless intersections). Time-lapse recordings of cultured hippocampal neurons showed that simple variations in substrate geometry changed growth cone shape, and altered the rate of growth and the probability of branching. When crossing onto a node intersection the growth cone paused, often for hours, and microtubules appeared to defasciculate. Once beyond the node, filopodia and lamellipodia persisted at that site, sometimes forming a collateral branch. At nodeless intersections, the growth cone passed through with minimal hesitation, often becoming divided into separate areas of motility that led to the growth of separate branches. When several lines intersected at a common point, growth cones sometimes split into several subdivisions, resulting in the emergence of as many as five branches. Such experiments revealed an intrinsic preference for branches to form at angles less than 90 degrees . These data show that simple changes in the geometry of a chemically homogeneous substrate are detected by the growth cone and can regulate axonal growth and the formation of branches.

New ways to print living cells promise breakthroughs for engineering complex tissues in vitro.

The ability to control the placement of cells and the assembly of networks in vitro has tremendous potential for understanding the regulation of development as well as for generating artificial tissues. To date, most engineering tools that can place materials with precision are not compatible with the requirements of living cells, and so approaches to tissue engineering have focused on patterning substrates as a way of controlling cell growth rather than patterning cells directly. In this issue of Biochemical Journal, however, Eagles et al. adapt electrohydrodynamic printing technology to 'print' living cells from a neuronal cell line on to a substrate. The importance of this approach is that it has the potential for unprecedented control over the position of cells in culture by directly placing them, thus allowing for the systematic assembly of cell networks.

Diminished experience-dependent neuroanatomical plasticity: evidence for an improved biomarker of subtle neurotoxic damage to the developing rat brain.

Millions of children are exposed to low levels of environmental neurotoxicants as their brains are developing. Conventional laboratory methods of neurotoxicology can detect maldevelopment of brain structure but are not designed to detect maldevelopment of the brain's capacity for plasticity that could impair learning throughout life. The environmental complexity (EC) paradigm has become classic for demonstrating the modifications in brain structure that occur in response to experience and thus provides a set of indices for plasticity in the healthy brain. In this study, we have tested the hypothesis that if degradation of experience-dependent cortical plasticity is used as a biomarker, then developmental neurotoxic effects will be detected at doses below those that alter cortical morphogenesis overtly. Pregnant Long-Evans hooded rats received a single injection of either saline vehicle or 1, 5, 10, or 25 mg/kg of the well-characterized developmental neurotoxicant methylazoxymethanol acetate (MAM) on the 16th or 17th day of gestation. On postnatal days 35-39, male offspring were assigned to either a complex environment (EC) or an individual cage (IC) for 28 days to stimulate neuroanatomical plasticity. This response was measured as the difference between the thickness of visual cortex of IC and EC littermates at a given dose. The threshold dose for significant reduction of cortical thickness was 25 mg/kg, but the threshold dose for failure of plasticity was much lower and could be detected at 1 mg/kg, the lowest dose used. No other method of assessment has detected lasting effects of prenatal exposure to MAM at such a low dose. These data suggest that this simple test of plasticity could be an efficient way to detect subtle neurotoxic damage to the developing brain.