Abnormal development of dendritic spines in FMR1 knock-out mice

Fragile X syndrome is caused by a mutation in the FMR1 gene leading to absence of the fragile X mental retardation protein (FMRP). Reports that patients and adult FMR1 knock-out mice have abnormally long dendritic spines of increased density suggested that the disorder might involve abnormal spine development. Because spine length, density, and motility change dramatically in the first postnatal weeks, we analyzed these properties in mutant mice and littermate controls at 1, 2, and 4 weeks of age. To label neurons, a viral vector carrying the enhanced green fluorescent protein gene was injected into the barrel cortex. Layer V neurons were imaged on a two-photon laser scanning microscope in fixed tissue sections. Analysis of >16,000 spines showed clear developmental patterns. Between 1 and 4 weeks of age, spine density increased 2.5-fold, and mean spine length decreased by 17% in normal animals. Early during cortical synaptogenesis, pyramidal cells in mutant mice had longer spines than controls. At 1 week, spine length was 28% greater in mutants than in controls. At 2 weeks, this difference was 10%, and at 4 weeks only 3%. Similarly, spine density was 33% greater in mutants than in controls at 1 week of age. At 2 or 4 weeks of age, differences were not detectable. The spine abnormality was not detected in neocortical organotypic cultures. The transient nature of the spine abnormality in the intact animal suggests that FMRP might play a role in the normal process of dendritic spine growth in coordination with the experience-dependent development of cortical circuits

The number of glutamate receptors opened by synaptic stimulation in single hippocampal spines

The number of receptors opening after glutamate release is critical for understanding the sources of noise and the dynamic range of synaptic transmission. We imaged [Ca2+] transients mediated by synaptically activated NMDA receptors (NMDA-Rs) in individual spines in rat brain slices. We show that Ca2+ influx through single NMDA-Rs can be reliably detected, allowing us to estimate the number of receptors opening after synaptic transmission. This number is small: at the peak of the synaptic response, less than one NMDA-R is open, on average. Therefore, stochastic interactions between transmitter and receptor contribute substantially to synaptic noise, and glutamate occupies a small fraction of receptors. The number of receptors opening did not scale with spine volume, and smaller spines experience larger [Ca2+] transients during synaptic transmission. Our measurements further demonstrate that optical recordings can be used to study single receptors in intact systems

Imaging high-resolution structure of GFP-expressing neurons in neocortex in vivo

To detect subtle changes in neuronal morphology in response to changes in experience, one must image neurons at high resolution in vivo over time scales of minutes to days. We accomplished this by infecting postmitotic neurons in rat and mouse barrel cortex with a Sindbis virus carrying the gene for enhanced green fluorescent protein. Visualized with 2-photon excitation laser scanning microscopy, infected neurons showed bright fluorescence that was distributed homogeneously throughout the cell, including axonal and dendritic arbors. Single dendritic spines could routinely be resolved and their morphological dynamics visualized. Viral infection and imaging were achieved throughout postnatal development up to early adulthood (P 8-30), although the viral efficiency of infection decreased with age. This relatively noninvasive method for fluorescent labeling and imaging of neurons allows the study of morphological dynamics of neocortical neurons and their circuits in vivo

Boosting of Synaptic Potentials and Spine Ca Transients by the Peptide Toxin SNX-482 Requires Alpha-1E-Encoded Voltage-Gated Ca Channels

The majority of glutamatergic synapses formed onto principal neurons of the mammalian central nervous system are associated with dendritic spines. Spines are tiny protuberances that house the proteins that mediate the response of the postsynaptic cell to the presynaptic release of glutamate. Postsynaptic signals are regulated by an ion channel signaling cascade that is active in individual dendritic spines and involves voltage-gated calcium (Ca) channels, small conductance (SK)-type Ca-activated potassium channels, and NMDA-type glutamate receptors. Pharmacological studies using the toxin SNX-482 indicated that the voltage-gated Ca channels that signal within spines to open SK channels belong to the class CaV2.3, which is encoded by the Alpha-1E pore-forming subunit. In order to specifically test this conclusion, we examined the effects of SNX-482 on synaptic signals in acute hippocampal slices from knock-out mice lacking the Alpha-1E gene. We find that in these mice, application of SNX-482 has no effect on glutamate-uncaging evoked synaptic potentials and Ca influx, indicating that that SNX-482 indeed acts via the Alpha-1E-encoded CaV2.3 channel

Cetaceans have complex brains for complex cognition

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Glutamate Induces the Elongation of Early Dendritic Protrusions via mGluRs in Wild Type Mice, but Not in Fragile X Mice

Fragile X syndrome (FXS), the most common inherited from of autism and mental impairment, is caused by transcriptional silencing of the Fmr1 gene, resulting in the loss of the RNA-binding protein FMRP. Dendritic spines of cortical pyramidal neurons in affected individuals are abnormally immature and in Fmr1 knockout (KO) mice they are also abnormally unstable. This could result in defects in synaptogenesis, because spine dynamics are critical for synapse formation. We have previously shown that the earliest dendritic protrusions, which are highly dynamic and might serve an exploratory role to reach out for axons, elongate in response to glutamate. Here, we tested the hypothesis that this process is mediated by metabotropic glutamate receptors (mGluRs) and that it is defective in Fmr1 KO mice. Using time-lapse imaging with two-photon microscopy in acute brain slices from early postnatal mice, we find that early dendritic protrusions in layer 2/3 neurons become longer in response to application of glutamate or DHPG, a Group 1 mGluR agonist. Blockade of mGluR5 signaling, which reverses some adult phenotypes of KO mice, prevented the glutamate-mediated elongation of early protrusions. In contrast, dendritic protrusions from KO mice failed to respond to glutamate. Thus, absence of FMRP may impair the ability of cortical pyramidal neurons to respond to glutamate released from nearby pre-synaptic terminals, which may be a critical step to initiate synaptogenesis and stabilize spines

The long journey to a Systems Biology of neuronal function

Computational neurobiology was born over half a century ago, and has since been consistently at the forefront of modelling in biology. The recent progress of computing power and distributed computing allows the building of models spanning several scales, from the synapse to the brain. Initially focused on electrical processes, the simulation of neuronal function now encompasses signalling pathways and ion diffusion. The flow of quantitative data generated by the "omics" approaches, alongside the progress of live imaging, allows the development of models that will also include gene regulatory networks, protein movements and cellular remodelling. A systems biology of brain functions and disorders can now be envisioned. As it did for the last half century, neuroscience can drive forward the field of systems biology

Distinct Mammalian Precursors Are Committed to Generate Neurons with Defined Dendritic Projection Patterns

The mechanisms that regulate how dendrites target different neurons to establish connections with specific cell types remain largely unknown. In particular, the formation of cell-type–specific connectivity during postnatal neurogenesis could be either determined by the local environment of the mature neuronal circuit or by cell-autonomous properties of the immature neurons, already determined by their precursors. Using retroviral fate mapping, we studied the lamina-specific dendritic targeting of one neuronal type as defined by its morphology and intrinsic somatic electrical properties in neonatal and adult neurogenesis. Fate mapping revealed the existence of two separate populations of neuronal precursors that gave rise to the same neuronal type with two distinct patterns of dendritic targeting—innervating either a deep or superficial lamina, where they connect to different types of principal neurons. Furthermore, heterochronic and heterotopic transplantation demonstrated that these precursors were largely restricted to generate neurons with a predetermined pattern of dendritic targeting that was independent of the host environment. Our results demonstrate that, at least in the neonatal and adult mammalian brain, the pattern of dendritic targeting of a given neuron is a cell-autonomous property of their precursors

Proteomics, ultrastructure, and physiology of hippocampal synapses in a fragile X syndrome mouse model reveal presynaptic phenotype

Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc

Prenatal stress and subsequent exposure to chronic mild stress influence dendritic spine density and morphology in the rat medial prefrontal cortex

<p>Abstract</p> <p>Background</p> <p>Both prenatal stress (PS) and postnatal chronic mild stress (CMS) are associated with behavioral and mood disturbances in humans and rodents. The aim of this study was to reveal putative PS- and/or CMS-related changes in basal spine morphology and density of pyramidal neurons in the rat medial prefrontal cortex (mPFC).</p> <p>Results</p> <p>We show that rats exposed to PS and/or CMS display changes in the morphology and number of basal spines on pyramidal neurons in the mPFC. CMS had a negative effect on spine densities, particularly on spines of the mushroom type, which are considered to form stronger and more stable synapses than other spine types. PS alone did not affect spine densities, but had a negative effect on the ratio of mushroom spines. In addition, PS seemed to make rats less responsive to some of the negative effects of CMS, which supports the notion that PS represents a predictive adaptive response.</p> <p>Conclusion</p> <p>The observed changes may represent a morphological basis of PS- and CMS-related disturbances, and future studies in the field should not only consider total spine densities, but also separate between different spine types.</p