Evidence that Astroglia Influence Dendrite Morphogenesis and Synaptogenesis Independently in the Vertebrate Central Nervous System

In the absence of external spatial cues, dendritic arbors of neurons grown in vitro approximate those observed in situ. Absent, however, from these culture models is patterned orientation of dendritic trunks, and variation of branch geometry that provide identifiable characteristics of the cytoarchitecture of the intact brain. Although astroglia are present during key stages of dendritic development in vivo, little is known about whether local interactions with glia shape dendritic growth. Astroglial cells are good candidates for this kind of regulation because they can exert control over the formation of synapses, an event correlated with the maturational state of the dendrite. The present review highlights some key findings from vertebrate model systems offering evidence that astroglia can contribute to the shape, and growth, of the dendritic arbor. Drawing from our recent work using a co-culture system composed of neurons growing in differential contact with astroglia, we discuss findings that suggest: 1) growth of dendrites, and addition of synapses, can be independent; further, while astroglia promote synapse formation, they inhibit dendritic growth; 2) astroglia mediate dendrite growth through both paracrine, and contact-dependent mechanisms; and 3) astroglia appear to impose pattern by constraining the growth of dendrites within their zones of influence

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

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

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

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

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

<div><p>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 <i>in vitro</i> 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.</p></div

Local and physical interactions between dendrites and astroglia can alter the shape and growth of the dendritic arbor.

<p>Dendrites not in contact with astroglia are significantly longer than those in contact. Dendrites, and associated filopodia that are in close proximity to astroglia orient toward the glial cell which, upon contact, restricts growth.</p