Computational geometry analysis of dendritic spines by structured illumination microscopy

We are currently short of methods that can extract objective parameters of dendritic spines useful for their categorization. Authors present in this study an automatic analytical pipeline for spine geometry using 3D-structured illumination microscopy, which can effectively extract many geometrical parameters of dendritic spines without bias and automatically categorize spine population based on their morphological feature

Visual Search of Neuropil-Enriched RNAs from Brain In Situ Hybridization Data through the Image Analysis Pipeline Hippo-ATESC

International audienceMotivation: RNA molecules specifically enriched in the neuropil of neuronal cells and in particular in dendritic spines are of great interest for neurobiology in virtue of their involvement in synaptic structure and plasticity. The systematic recognition of such molecules is therefore a very important task. High resolution images of RNA in situ hybridization experiments contained in the Allen Brain Atlas (ABA) represent a very rich resource to identify them and have been so far exploited for this task through human-expert analysis. However, software tools that may automatically address the same objective are not very well developed. Results: In this study we describe an automatic method for exploring in situ hybridization data and discover neuropil-enriched RNAs in the mouse hippocampus. We called it Hippo-ATESC (Automatic Texture Extraction from the Hippocampal region using Soft Computing). Bioinformatic validation showed that the Hippo-ATESC is very efficient in the recognition of RNAs which are manually identified by expert curators as neuropil-enriched on the same image series. Moreover, we show that our method can also highlight genes revealed by microdissection-based methods but missed by human visual inspection. We experimentally validated our approach by identifying a non-coding transcript enriched in mouse synaptosomes. The code is freely available on the web at http://ibislab.ce.unipr.it/software/hippo/

Structural and functional alterations of cortical neurons in Alzheimer’s disease transgenic mice assessed by two-photon in vivo imaging

Alzheimer’s disease (AD), the most common form of dementia, has been proposed to result from the degeneration of synapses, putatively caused by assemblies of the amyloid-β peptide (Aβ). The spatiotemporal dynamics of this synaptopathy, its potential reversibility as well as its consequences on the function of single neurons and neuronal circuits, however, are not fully understood to date. In order to address these questions, I assessed structural and functional alterations of neurons in the neocortex in a transgenic mouse model of Alzheimer’s disease, namely APP/PS1 (APPswe, PS1L166P) mice, using in vivo two-photon imaging. Chronic imaging of dendrites and axons over the course of four weeks revealed not only a reduction in dendritic spine density close to amyloid plaques (proteinaceous extracellular deposits typical of AD), but I also identified synaptic instability as a main aspect contributing to AD pathology. Importantly, while synapse loss was confined to the immediate plaque vicinity (up to 15µm from the histological plaque border), synaptic instability was evident in a much larger region surrounding plaques (50 µm) and affected both, pre- and postsynaptic compartments. As the prevailing hypothesis in AD holds that Aβ conveys these detrimental effects on synapses one therapeutic approach is based on the pharmacological inhibition of Aβ generation. I thus assessed the impact of a novel selective γ-secretase inhibitor (GSI), a compound that prevents the last cleavage step necessary for the release of Aβ from the longer transmembrane amyloid precursor protein (APP). Notably, the GSI used here primarily interferes with the processing of APP and still allows for processing of other γ-secretase substrates, and hence should largely reduce side effects seen with earlier generations of GSIs before. Daily treatment with the GSI reduced the deposition of Aβ as evidenced by the initial reduction in the number of new plaques and a sustained decrease in the growth of these newly deposited plaques. Importantly, it also ameliorated the plaque-associated synaptic instability, without displaying overt adverse effects on dendritic spines in WT mice. These data represent the first in vivo evidence that selective pharmacological inhibition of the γ-secretase mediated APP cleavage can have beneficial effects on synaptic pathology in AD. Given the widespread impact of Aβ assemblies on neuronal structures, I then asked to which extent these structural alterations affect the function of neurons. To address this question, I recorded neuronal response properties in the primary visual cortex of behaving APP/PS1 mice, employing in vivo two-photon calcium imaging using the genetically encoded calcium indicator GCaMP6m. In order to probe the impact of AD related pathology on specific aspects of information processing, which rely on multiple neuronal circuits, I characterized visually driven and motor-related activity, as well as signals based on mismatches between actual and expected visual input. My data reveal a massive reduction in responsiveness under almost all conditions tested, which is line with the profound impact on neuronal structure. Stimulus selectivity, like orientation or direction tuning, were not altered in APP/PS1 mice, indicating that the main effect is caused by a change in response gain. Along with the massive decrease in feedforward signals, I observed an increase in spontaneous, hence uncorrelated neuronal activity in AD transgenic mice. Both features jointly affected the coding accuracy of the network, and I propose that this combination may represent a common characteristic leading to impaired information processing in AD. Surprisingly, I found that responses elicited after a discordance of actual and expected visual flow during running, i.e. a visuomotor mismatch, were selectively spared in APP/PS1 mice, suggesting a particular resilience of this very signal. Together, both studies demonstrate that global widespread structural changes of neurons in the AD brain are accompanied by a severe impact on information processing, most prominently seen in a strong reduction of feedforward signals. My data, thus, provide a correlate of impaired cognition in AD at the level of single neurons and neural circuits

EphA4 expression promotes network activity and spine maturation in cortical neuronal cultures

<p>Abstract</p> <p>Background</p> <p>Neurons form specific connections with targets via synapses and patterns of synaptic connectivity dictate neural function. During development, intrinsic neuronal specification and environmental factors guide both initial formation of synapses and strength of resulting connections. Once synapses form, non-evoked, spontaneous activity serves to modulate connections, strengthening some and eliminating others. Molecules that mediate intercellular communication are particularly important in synaptic refinement. Here, we characterize the influences of EphA4, a transmembrane signaling molecule, on neural connectivity.</p> <p>Results</p> <p>Using multi-electrode array analysis on <it>in vitro </it>cultures, we confirmed that cortical neurons mature and generate spontaneous circuit activity as cells differentiate, with activity growing both stronger and more patterned over time. When EphA4 was over-expressed in a subset of neurons in these cultures, network activity was enhanced: bursts were longer and were composed of more spikes than in control-transfected cultures. To characterize the cellular basis of this effect, dendritic spines, the major excitatory input site on neurons, were examined on transfected neurons <it>in vitro</it>. Strikingly, while spine number and density were similar between conditions, cortical neurons with elevated levels of EphA4 had significantly more mature spines, fewer immature spines, and elevated colocalization with a mature synaptic marker.</p> <p>Conclusions</p> <p>These results demonstrate that experimental elevation of EphA4 promotes network activity <it>in vitro</it>, supporting spine maturation, producing more functional synaptic pairings, and promoting more active circuitry.</p

Dynamic FoxP2 levels in male zebra finches are linked to morphology of adult-born Area X medium spiny neurons

The transcription factor FOXP2 is crucial for the formation and function of cortico-striatal circuits. FOXP2 mutations are associated with specific speech and language impairments. In songbirds, experimentally altered FoxP2 expression levels in the striatal song nucleus Area X impair vocal learning and song production. Overall FoxP2 protein levels in Area X are low in adult zebra finches and decrease further with singing. However, some Area X medium spiny neurons (MSNs) express FoxP2 at high levels (FoxP2(high) MSNs) and singing does not change this. Because Area X receives many new neurons throughout adulthood, we hypothesized that the FoxP2(high) MSNs are newly recruited neurons, not yet integrated into the local Area X circuitry and thus not active during singing. Contrary to our expectation, FoxP2 protein levels did not predict whether new MSNs were active during singing, assayed via immediate early gene expression. However, new FoxP2(high) MSNs had more complex dendrites, higher spine density and more mushroom spines than new FoxP2(low) MSNs. In addition, FoxP2 expression levels correlated positively with nucleus size of new MSNs. Together, our data suggest that dynamic FoxP2 levels in new MSNs shape their morphology during maturation and their incorporation into a neural circuit that enables the maintenance and social modulation of adult birdsong