Synaptic activity and the formation and maintenance of neuronal circuits

One of the most fundamental features of neurons is their polarized organization with two types of neurites extending from the cell body, axons and dendrites that are both functionally and morphologically distinct. During development, both axons and dendrites possess highly dynamic and actin-rich growth cones and filopodia extending from their shafts, which are subsequently replace by fundamentally stable axonal varicosities and dendritic spines. Together they form the basic elements of mature synapses. To mimic in vivo neuronal development, I have used organotypic cultures of brain tissue from transgenic mice expressing either green fluorescent protein (GFP) bearing a surface membrane localization signal or actin-GFP in combination with live cell imaging system. This approach provided me with high-resolution images of developing neurons’ fine structure in organized tissue. Co-cultures of fluorescent and non-fluorescent hippocampal slices enabled me then to examine simultaneously dendrite differentiation in the fluorescent slice and to track the fate of fluorescent axons growing into the non-fluorescent slice. Together this granted me a powerful tool to study neuronal network formation and developmental maturation of axons and dendrites. Co-cultures of embryonic tissue showed a sustained cross-innervation of axonal projections. Over time neurons in these co-cultures formed a dense axonal network with numerous axonal varicosities along their shaft. This axonal plexus remained present beyond 2 months in vitro. Dendrites in these embryonic co-cultures subsequently switched from producing labile filopodia to fundamentally stable dendritic spines. These mature dendritic spines had morphologies similar to those reported from studies of adult brain. Both axons and dendrites exhibited a successive focalisation of actin-based dynamics to the site of the synaptic junction. The observed changes in shape of mature axonal varicosities and dendritic spines together with the rapidly extension and retraction of actin-rich protrusions from the top of varicosities and spine heads suggest a retained capacity for experience-dependent fine-tuning e.g. during either periods of learning and memory or during brain damage resulting in an altered connectivity for both pre- and postsynaptic compartments in the mature mammalian central nervous system. The observed morphological dynamics suggest a high degree of preservation of morphological plasticity at the synapse in mature neuronal networks. Co-cultures of postnatal brain slices showed intensive invasion of axonal projections during the first two weeks in culture, followed by dramatic axonal regression and resulting in a near complete absence of cross-innervating axons after 1 month in vitro. In contrast, dendrite development in each of these postnatal cultures was fundamentally normal and occurred similar to that observed in embryonic co-cultures. I then co-cultured embryonic and postnatal slices to investigate whether the difference in capacity to cross-innervate between postnatal co-cultures and embryonic co-cultures were the result of tissue maturation. We found that the postnatal slice degenerated so that after 1 month in culture it had almost disappeared whereas the neighbouring embryonic slice had matured without noticeable problems. Staining these co-cultures of embryonic and postnatal slices showed a massive invasion of microglial cells into the dying postnatal slice. The difference between embryonic and postnatal neurons in their capacity to maintain cross-innervating synaptic connection suggests the existence of a developmental switch resulting in the inability of sustained afferent cross-innervation between postnatal brain slices. At the same time, in heterochronic co-cultures it causes miscommunication between postnatal and embryonic cells leading to profound degeneration of postnatal tissue. The thick layer of microglia surrounding postnatal tissue suggests their involvement in neuronal degeneration similar to that observed in axotomy-induced neuronal death and various neurodegenerative conditions such as Alzheimer’s disease. The earlier suggested preservation of morphological plasticity at the synapse in mature neuronal networks was illustrated by cooling mature hippocampal slices, either acutely cut brain slices or organotypic cultures, to room temperature. Dendritic spines are highly sensitive to reduced temperature with rapid loss of actin-based motility followed by disappearance of the entire spine structure within 12 hours. However, rewarming these cooled slices to 37˚C resulted in the rapid extension of filopodia from the surface of dendrites and re-establishment of dendritic spines within several of hours. These data underline the high degree of plasticity retained by neuronal connections in the mature CNS and suggest a link between dendritic spine structure and global brain function

Munc18 and Munc13 regulate early neurite outgrowth

Background information. During development, growth cones of outgrowing neurons express proteins involved in vesicular secretion, such as SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins, Munc13 and Munc18. Vesicles are known to fuse in growth cones prior to synapse formation, which may contribute to outgrowth

Cre-Dependent Expression of Multiple Transgenes in Isolated Neurons of the Adult Forebrain

Background: Transgenic mice with mosaic, Golgi-staining-like expression of enhanced green fluorescent protein (EGFP) have been very useful in studying the dynamics of neuronal structure and function. In order to further investigate the molecular events regulating structural plasticity, it would be useful to express multiple proteins in the same sparse neurons, allowing co-expression of functional proteins or co-labeling of subcellular compartments with other fluorescent proteins. However, it has been difficult to obtain reproducible expression in the same subset of neurons for direct comparison of neurons expressing different functional proteins. Principal Findings: Here we describe a Cre-transgenic line that allows reproducible expression of transgenic proteins of choice in a small number of neurons of the adult cortex, hippocampus, striatum, olfactory bulb, subiculum, hypothalamus, superior colliculus and amygdala. We show that using these Cre-transgenic mice, multiple Cre-dependent transgenes can be expressed together in the same isolated neurons. We also describe a Cre-dependent transgenic line expressing a membrane associated EGFP (EGFP-F). Crossed with the Cre-transgenic line, EGFP-F expression starts in the adolescent forebrain, is present in dendrites, dendritic protrusions, axons and boutons and is strong enough for acute or chronic in vivo imaging. Significance: This triple transgenic approach will aid the morphological and functional characterization of neurons in various Cre-dependent transgenic mice

The Biochemistry, Ultrastructure, and Subunit Assembly Mechanism of AMPA Receptors

The AMPA-type ionotropic glutamate receptors (AMPA-Rs) are tetrameric ligand-gated ion channels that play crucial roles in synaptic transmission and plasticity. Our knowledge about the ultrastructure and subunit assembly mechanisms of intact AMPA-Rs was very limited. However, the new studies using single particle EM and X-ray crystallography are revealing important insights. For example, the tetrameric crystal structure of the GluA2cryst construct provided the atomic view of the intact receptor. In addition, the single particle EM structures of the subunit assembly intermediates revealed the conformational requirement for the dimer-to-tetramer transition during the maturation of AMPA-Rs. These new data in the field provide new models and interpretations. In the brain, the native AMPA-R complexes contain auxiliary subunits that influence subunit assembly, gating, and trafficking of the AMPA-Rs. Understanding the mechanisms of the auxiliary subunits will become increasingly important to precisely describe the function of AMPA-Rs in the brain. The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex. Because the AMPA-Rs are important drug targets for treating various neurological and psychiatric diseases, it is likely that these new native complexes will require detailed mechanistic analysis in the future. The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing. Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity

Books and Research Evaluation @ SSP

My slides for "Seminar 4: Innovation in Scholarly Book Publishing: What Have We Achieved and What More Is Needed?" at #SSP201

Bookmetrix for APE

<div>EXPLORE THE IMPACT OF YOUR BOOKS</div><div>Bookmetrix brings together a collection of performance metrics, helping you to see how your books are being discussed, cited, and used around the world.</div

The Rigor and Transparency Index Quality Metric for Assessing Biological and Medical Science Methods.

The reproducibility crisis is a multifaceted problem involving ingrained practices within the scientific community. Fortunately, some causes are addressed by the author's adherence to rigor and reproducibility criteria, implemented via checklists at various journals. We developed an automated tool (SciScore) that evaluates research articles based on their adherence to key rigor criteria, including NIH criteria and RRIDs, at an unprecedented scale. We show that despite steady improvements, less than half of the scoring criteria, such as blinding or power analysis, are routinely addressed by authors; digging deeper, we examined the influence of specific checklists on average scores. The average score for a journal in a given year was named the Rigor and Transparency Index (RTI), a new journal quality metric. We compared the RTI with the Journal Impact Factor and found there was no correlation. The RTI can potentially serve as a proxy for methodological quality

Height-Independent Estimation of Glomerular Filtration Rate in Children: An Alternative to the Schwartz Equation

ObjectiveTo compare the diagnostic performance of 2 height-independent equations used to calculate estimated glomerular filtration rate (eGFR), those of Pottel (eGFR-Pottel) and the British Columbia Children's Hospital (BCCH) (eGFR-BCCH), with the commonly used Schwartz equation (eGFR-Schwartz).Study designWe externally validated eGFR-Pottel and eGFR-BCCH in a well-characterized pediatric patient population (n = 152) and compared their diagnostic performance with that of eGFR-Schwartz using Bland-Altman analysis. All patients underwent glomerular filtration rate measurement using the gold standard single-injection inulin clearance method (GFR-inulin).ResultsMedian GFR-inulin was 92.0 mL/min/1.73 m² (IQR, 76.1-107.4 mL/min/1.73 m²). Compared with GFR-inulin, the mean bias for eGFR-Schwartz was −10.1 mL/min/1.73 m2 (95% limits of agreement [LOA], −77.5 to 57.2 mL/min/1.73 m2), compared with −12.3 mL/min/1.73 m² (95% LOA, −72.6 to 47.9 mL/min/1.73 m2) for eGFR-Pottel and −22.1 mL/min/1.73 m² (95% LOA, −105.0 to 60.8 mL/min/1.73 m2) for eGFR-BCCH. eGFR-Pottel showed comparable accuracy to eGFR-Schwartz, with 77% and 76% of estimates within 30% of GFR-inulin, respectively. eGFR-BCCH was less accurate than eGFR-Schwartz (66% of estimates within 30% of GFR-inulin; P < .01).ConclusionThe performance of eGFR-Pottel is superior to that of eGFR-BCCH and comparable with that of eGFR-Schwartz. eGFR-Pottel is a valid alternative to eGFR-Schwartz in children and could be reported by the laboratory if height data are not available