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Relationship between the fluorescence intensity (normalized values) and the depth within the tissue. A: Cross sectional profiles showing those molecules in which the detected signal is optimal at the surface of the sections. B: Cross sectional profiles showing those molecules in which the detected fluorescence intensity remains at optimal values through the section.
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The quantification of the expression of different molecules is a key question in both basic and applied sciences. While protein quantification through molecular techniques leads to the loss of spatial information and resolution, immunohistochemistry is usually associated with time-consuming image analysis and human bias. In addition, the scarce aut...
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... were acquired using a confocal microscope (Leica TCS SPE): we obtained z- stacks from every layer, strata or region of interest covering the whole thickness of the tissue. As previously mentioned, all the acquisition parameters were kept the same at all times: including a pinhole of 1 AU, the laser intensity, the gain of the photomultiplier, the offset of the histogram, and the image magnification. After analyzing the expression of the molecules of interest, we selected a single plane at the depth where the antibody detection was optimal. This selection depended on each antibody: generally synaptic proteins, such as the vesicular glutamate trans- porter 1 or synaptophysin do not penetrate more than a few microns on the surface of the sections, while other molecules, such as FosB or calcium binding proteins penetrate easily throughout the tissue (see Fig. 1A and ...
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... carry out all the analysis within an optimal range of antibody detection. As com- mented above, the optimal depth is different for each antibody and we need to test the histogram for each molecule for optimal results (see Fig. 1). The main idea underlying this methodology is that all processing of the tissue has been, as much as possible, identical: same perfusion protocol, same incubation times, same laser intensity and acquisition settings such as the pinhole the gain and offset, to name a few. We use a macro that identifies a threshold separating the top % brightest part of the histogram from the rest, and therefore adjusting to the signal-to-noise ratio. Depending on the specific molecule to be analyzed, we use between 1 and 5%, but that can be easily adapted to other circumstances (see macro 3; in lines 15 and 46 we can select the percentage to be discarded after the binarization for each channel i.e. a value of 95 means we are selecting the top ...
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... antibody detection was optimal. This selection depended on each antibody: generally synaptic proteins, such as the vesicular glutamate trans- porter 1 or synaptophysin do not penetrate more than a few microns on the surface of the sections, while other molecules, such as FosB or calcium binding proteins penetrate easily throughout the tissue (see Fig. 1A and ...
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... carry out all the analysis within an optimal range of antibody detection. As com- mented above, the optimal depth is different for each antibody and we need to test the histogram for each molecule for optimal results (see Fig. 1). The main idea underlying this methodology is that all processing of the tissue has been, as much as possible, identical: same perfusion protocol, same incubation times, same laser intensity and acquisition settings such as the pinhole the gain and offset, to name a few. We use a macro that identifies a threshold separating the top % ...
Citations
... The perimeter of the cell somata was calculated. The density of VGLUT1 perisomatic puncta was calculated by dividing the numbers of puncta by perimeter [38,39]. For VGLUT1/PSD95 analysis, each image was threshold-adjusted using the default autothreshold and then converted into a binary image. ...
Non-invasive brain stimulation therapy for autism spectrum disorder (ASD) has shown beneficial effects. Recently, we and others demonstrated that visual sensory stimulation using rhythmic 40 Hz light flicker effectively improved cognitive deficits in mouse models of Alzheimer’s disease and stroke. However, whether rhythmic visual 40 Hz light flicker stimulation can ameliorate behavioral deficits in ASD remains unknown. Here, we show that 16p11.2 deletion female mice exhibit a strong social novelty deficit, which was ameliorated by treatment with a long-term 40 Hz light stimulation. The elevated power of local-field potential (LFP) in the prefrontal cortex (PFC) of 16p11.2 deletion female mice was also effectively reduced by 40 Hz light treatment. Importantly, the 40 Hz light flicker reversed the excessive excitatory neurotransmission of PFC pyramidal neurons without altering the firing rate and the number of resident PFC neurons. Mechanistically, 40 Hz light flicker evoked adenosine release in the PFC to modulate excessive excitatory neurotransmission of 16p11.2 deletion female mice. Elevated adenosine functioned through its cognate A1 receptor (A1R) to suppress excessive excitatory neurotransmission and to alleviate social novelty deficits. Indeed, either blocking the A1R using a specific antagonist DPCPX or knocking down the A1R in the PFC using a shRNA completely ablated the beneficial effects of 40 Hz light flicker. Thus, this study identified adenosine as a novel neurochemical mediator for ameliorating social novelty deficit by reducing excitatory neurotransmission during 40 Hz light flicker treatment. The 40 Hz light stimulation warrants further development as a non-invasive ASD therapeutics.
... On these planes, 16 small squares of the neuropil (336 μm 2 ) per layer and animal were selected for analysis to avoid blood vessels and cell somata. Images were processed using FIJI/ImageJ software as described before [62,63]: the background was subtracted with rolling value of 50, converted to 8-bit deep images and binarized using a determined threshold value. This value depended on the marker and the area analyzed and was kept the same for all images with the same marker and area. ...
Severe psychiatric illnesses, for instance schizophrenia, and affective diseases or autism spectrum disorders, have been associated with cognitive impairment and perturbed excitatory-inhibitory balance in the brain. Effects in juvenile mice can elucidate how erythropoietin (EPO) might aid in rectifying hippocampal transcriptional networks and synaptic structures of pyramidal lineages, conceivably explaining mitigation of neuropsychiatric diseases. An imminent conundrum is how EPO restores synapses by involving interneurons. By analyzing ~12,000 single-nuclei transcriptomic data, we generated a comprehensive molecular atlas of hippocampal interneurons, resolved into 15 interneuron subtypes. Next, we studied molecular alterations upon recombinant human (rh)EPO and saw that gene expression changes relate to synaptic structure, trans-synaptic signaling and intracellular catabolic pathways. Putative ligand-receptor interactions between pyramidal and inhibitory neurons, regulating synaptogenesis, are altered upon rhEPO. An array of in/ex vivo experiments confirms that specific interneuronal populations exhibit reduced dendritic complexity, synaptic connectivity, and changes in plasticity-related molecules. Metabolism and inhibitory potential of interneuron subgroups are compromised, leading to greater excitability of pyramidal neurons. To conclude, improvement by rhEPO of neuropsychiatric phenotypes may partly owe to restrictive control over interneurons, facilitating re-connectivity and synapse development.
... Various software for digital image processing are available [5,22], and the most widely used is ImageJ, an intensely robust open-source tool that allows the analysis of numerous parameters in a single image [23]. In this context, IF images represent highly informative systems, as they allow to obtain valuable information about the localisation and the expression levels of cellular proteins [24,25]. The development of automated computer systems capable of identifying and quantifying IF signals is a particularly daring challenge, as it could not only remove the interpretation bias introduced by the user but also ensure objective, standardised and reproducible analysis, even on large image sets. ...
In recent years, optical imaging and efficient computational approaches have improved the ability to analyse and understand biological phenomena. Immunofluorescence (IF) is a widely used immunochemical technique that provides information about protein localisation and expression levels. However, the manual analysis of IF images can present important limitations, such as operator workload and interpretative bias. Thus, the development of automated tools for IF signal computation is crucial. Several software programs have been proposed to address this challenge, but there is still a need for more accurate and reliable systems. In this work, we present Q-IF, a software for automatically measuring cellular IF signals with an intuitive and easy-to-use interface. We describe the software and validate its results in different biological scenarios using SH-SY5Y neuroblastoma cells, human fibroblasts, and rat brain sections. The Q-IF system automatically carries out the entire process, from IF signal quantification to statistical analysis, thus evading operator biases and speeding up the analysis workflow. Our results demonstrate the accuracy and reliability of the Q-IF system, highlighting its potential as a valuable tool for IF analysis in biological research.
... We analysed the density of the puncta expressing synaptic proteins using immunofluorescent labelling for synaptophysin, vGLUT1 and GAD67, in the different subregions of the hippocampus, and to perform the quantification we used a previously described methodology (Guirado et al., 2018). Sections of hippocampus (Bregma between − 2.06 and − 2.30 mm) for each group were examined with a Leica SPE confocal microscope using the same parameters, and they were digitalized. ...
Down syndrome (DS) is the most common genetic disorder associated with intellectual disability. To study this syndrome, several mouse models have been developed. Among the most common is the Ts65Dn model, which mimics most of the alterations observed in DS. Ts65Dn mice, as humans with DS, show defects in the structure, density, and distribution of dendritic spines in the cerebral cortex and hippocampus. Fasudil is a potent inhibitor of the RhoA kinase pathway, which is involved in the formation and stabilization of dendritic spines. Our study analysed the effect of early chronic fasudil treatment on the alterations observed in the hippocampus of the Ts65Dn model. We observed that treating Ts65Dn mice with fasudil induced an increase in neural plasticity in the hippocampus: there was an increment in the expression of PSA-NCAM and BDNF, in the dendritic branching and spine density of granule neurons, as well as in cell proliferation and neurogenesis in the subgranular zone. Finally, the treatment reduced the unbalance between excitation and inhibition present in this model. Overall, early chronic treatment with fasudil increases cell plasticity and eliminates differences with euploid animals.
... files. The images were analyzed with NIH ImageJ software by following step-by-step workflow according to the published protocols (78,79). Quantification of the molecules of interest, i.e. neutrophils, NETs, cytokines, or C3, and CXCR4 were based on an automatic threshold analysis of immunofluorescence images to automatically identify the top brightest structures of each image as described in our published work (15,16). ...
Sunlight triggers lupus flares causing both local skin and systemic inflammation, including lupus nephritis, through poorly understood mechanisms. To address this knowledge gap, we found that UVB irradiation of asymptomatic, young female lupus-prone mice induced skin and kidney inflammation with proteinuria, accompanied by neutrophil infiltration and neutrophil extracellular trap (NET) formation. Furthermore, UVB irradiation induced co-expression of CXCR4 and cytokines or C3 by neutrophils in vitro and in vivo, in the skin and kidneys of lupus-prone mice, indicating their transmigratory and pro-inflammatory potentials. A causality study demonstrated that inhibiting CXCR4 attenuated renal neutrophil infiltration, accumulation of NETs, NET-associated cytokines or C3, and proteinuria in UVB-irradiated lupus-prone mice. Moreover, controlling NETosis through a novel strategy targeting nuclear envelope regulation reduced deposition of NET-associated cytokines or C3 in skin and kidneys, attenuating proteinuria in UVB-irradiated MRL-lpr-lmnB1Tg mice. Our investigation unveils a new mechanism by which neutrophil NETosis drives the early onset of lupus flares triggered by UVB-irradiation. Targeting neutrophil transmigration and NETosis could be promising therapeutic strategies.
... On these planes, 16 small squares of the neuropil (336 μm 2 ) per layer and animal were selected for analysis to avoid blood vessels and cell somata. Images were processed using FIJI/ImageJ software as described before 102,103 : the background was subtracted with rolling value of 50, converted to 8-bit deep images and binarized using a determined threshold value. This value depended on the marker and the area analyzed and was kept the same for all images with the same marker and area. ...
Erythropoietin (EPO) aids in rectifying hippocampal transcriptional networks and synaptic structures of pyramidal lineages, thereby mitigating mood and cognition-associated disorders. An imminent conundrum is how EPO restores synapses by involving interneurons. By analyzing ∼ 12,000 single-nuclei transcriptomic data, we generated a comprehensive molecular atlas of hippocampal interneurons, resolved into 15 interneuron subtypes. Next, we studied molecular alterations upon recombinant human (rh)EPO and saw that gene expression changes relate to synaptic structure, trans-synaptic signaling and intracellular catabolic pathways. Putative ligand-receptor interactions between pyramidal and inhibitory neurons, regulating synaptogenesis, are altered upon rhEPO. An array of in/ex vivo experiments confirms that specific interneuronal populations exhibit reduced dendritic complexity, synaptic connectivity, and changes in plasticity-related molecules. Metabolism and inhibitory potential of interneuron subgroups are compromised, leading to greater excitability of pyramidal neurons. To conclude, improvement by rhEPO of neuropsychiatric phenotypes may partly owe to restrictive control over interneurons, facilitating re-connectivity and synapse development.
... Colour signals were taken by digital camera and measured by ImageJ freeware. ImageJ is a popular freeware which can be used to quantify or extract colour intensity from photos or images [28][29][30]. ...
Microfluidic paper-based channels play an important role in microfluidic paper-based analytical devices ( μ PADs). There are some fabrication methods which could be utilised to fabricate microfluidic channels on paper substrate. Among these methods, inkjet printing process is considered as a promising fabrication method with many advantages such as low-cost, material saving, high precision, etc. The aim of this work is to apply inkjet printing technology to fabricate paper channels of μ PADs. A new design of μ PAD was proposed in this paper to demonstrate how to fabricate inkjet-printed hydrophobic lines to make paper-based biosensor. Biological target of our μ PADs is human chOrionic gonadotropin (hCG). Colorimetric signals from μ PADs were captured by digital camera and measured by ImageJ software, which showed that these μ PADs can determine hCG in the range from 1,000 to 10,000 ng ml ⁻¹ . These results showed that piezoelectric inkjet printing technology can fabricate 250 μ m-width hydrophobic lines on paper substrate, helping in fabricating μ PADs in next applications.
... For neuropil analysis, we randomly selected 4 rectangular ROIs (100μm 2 ) per image and used the aforementioned procedure to measure the fluorescent intensity and the number of synaptic puncta. Additionally, analysis of images from Ncan KO mice was performed as previously described (Schindelin et al., 2012;Guirado et al., 2018). Briefly, after applying the subtract background tool (the rolling value = 50) and Gaussian blur (s value = 1) filters, the original outline was expanded 1 μm from the cell body edge and the region of interest (ROI) was defined as the area between both outlines. ...
The condensed form of neural extracellular matrix (ECM), perineuronal nets (PNNs), is predominantly associated with parvalbumin-expressing (PV+) interneurons in the cortex and hippocampus. PNNs are enriched in several lecticans, including neurocan (Ncan). A polymorphism in the human Ncan gene has been associated with alterations in hippocampus-dependent memory function, variation of prefrontal cortex structure, and a higher risk for schizophrenia or bipolar disorder. Ncan knockout (KO) mice show related behavioral abnormalities, such as hyperactivity. Here we focused on studying how dysregulation of Ncan specifically in the mPFC may affect cognitive and synaptic functions. Intracortical adeno-associated virus (AAV) delivery was used to express shRNA against Ncan. Analysis of PNNs in Ncan shRNA-injected mice revealed a reduction in PNNs labelling by Wisteria floribunda agglutinin (WFA) around PV+ interneurons. Reduced Ncan expression resulted in a loss of the mPFC-dependent temporal order recognition and impairment of reversal spatial learning in a labyrinth (dry maze) task. As a potential synaptic substrate of these cognitive abnormalities, we report a robust reduction in the perisomatic GABAergic innervation of PV+ cells in Ncan KO and Ncan shRNA-injected mice. We also observed an increase in the density of vGLUT1-immunopositive synaptic puncta in the neuropil of Ncan shRNA-injected mice, which was, however, compensated in Ncan KO mice. Thus, our findings highlight a functional role of Ncan in supporting perisomatic GABAergic inhibition, temporal order recognition memory and cognitive flexibility, as one of the important cognitive resources depleted in neuropsychiatric disorders.
Contribution to the field
In this study, we asked if the extracellular matrix proteoglycan neurocan (Ncan) plays a functional role in the prefrontal cortex (PFC) of mice. Using viral delivery and expression of shRNA to knock down the expression of Ncan in the PFC, we provide evidence that neuronal Ncan is essential for the maintenance of perineuronal nets enveloping perisomatic interneurons by influencing the expression of glycoepitopes stained with Wisteria floribunda agglutinin and by modulating mRNA expression levels of other PNNs constituents. At the behavioral level, the knockdown of Ncan in mPFC impaired the temporal order recognition memory and consolidation/retrieval of spatial memories after reversal learning in the dry maze task. At the synaptic level, we found that Ncan knockdown reduced perisomatic GABAergic innervation of perisomatic interneurons and increased the density of vGLUT1+ excitatory presynaptic terminals in the neuropil of the PFC. Moreover, knockdown of Ncan changed the expression levels of several genes involved in activity-dependent synaptic remodeling. In summary, we conclude that neuronal Ncan is essential for multiple cognitive flexibility-related synaptic and cognitive functions in the PFC.
... Minor changes in spine morphology can modulate synaptic transmission, increase synaptic strength, and increase the number and size of dendritic spines [164][165][166][167][168]. IHC enables the differentiation of excitatory and inhibitory synapses, with antibodies selectively targeting either proteins predominantly expressed in excitatory synapses (e.g., PSD-95) or in inhibitory synapses (e.g., gephyrin) [169][170][171][172]. Most synaptic proteins (e.g., SVA2) are expressed in excitatory and inhibitory synapses, enabling a global brain mapping and quantifying synaptic density [171,173,174]. ...
Early cognitive decline in patients with Alzheimer’s (AD) is associated with quantifiable structural and functional connectivity changes in the brain. AD dysregulation of Aβ and tau metabolism progressively disrupt normal synaptic function, leading to loss of synapses, decreased hippocampal synaptic density and early hippocampal atrophy. Advances in brain imaging techniques in living patients have enabled the transition from clinical signs and symptoms-based AD diagnosis to biomarkers-based diagnosis, with functional brain imaging techniques, quantitative EEG, and body fluids sampling. The hippocampus has a central role in semantic and episodic memory processing. This cognitive function is critically dependent on normal intrahippocampal connections and normal hippocampal functional connectivity with many cortical regions, including the perirhinal and the entorhinal cortex, parahippocampal cortex, association regions in the temporal and parietal lobes, and prefrontal cortex. Therefore, decreased hippocampal synaptic density is reflected in the altered functional connectivity of intrinsic brain networks (aka large-scale networks), including the parietal memory, default mode, and salience networks. This narrative review discusses recent critical issues related to detecting AD-associated early cognitive decline with brain synaptic structural and functional markers in high-risk or neuropsychologically diagnosed patients with subjective cognitive impairment or mild cognitive impairment.
... We show that our method can work on both, without specializing on either. In immunohistochemistry, measuring fluorescence signal in confocal microscopy images has largely relied on manual annotation or traditional image intensity thresholding techniques such as histogram adaptation or clustering, with ImageJ-based Fiji being a widely used software for this purpose [40,41]. The small areas, granular distribution, unclear signal, noise, and background borders of the fluorescence signal, and the variability in the image quality, acquisition protocols, and staining and sample quality present inherent challenges for both approaches, with the latter often employing semi-automated techniques for annotation quality control [42]. ...
Identification of small objects in fluorescence microscopy is a non-trivial task burdened by parameter-sensitive algorithms, for which there is a clear need for an approach that adapts dynamically to changing imaging conditions. Here, we introduce an adaptive object detection method that, given a microscopy image and an image level label, uses kurtosis-based matching of the distribution of the image differential to express operator intent in terms of recall or precision. We show how a theoretical upper bound of the statistical distance in feature space enables application of belief theory to obtain statistical support for each detected object, capturing those aspects of the image that support the label, and to what extent. We validate our method on 2 datasets: distinguishing sub-diffraction limit caveolae and scaffold by stimulated emission depletion (STED) super-resolution microscopy; and detecting amyloid- β deposits in confocal microscopy retinal cross-sections of neuropathologically confirmed Alzheimer’s disease donor tissue. Our results are consistent with biological ground truth and with previous subcellular object classification results, and add insight into more nuanced class transition dynamics. We illustrate the novel application of belief theory to object detection in heterogeneous microscopy datasets and the quantification of conflict of evidence in a joint belief function. By applying our method successfully to diffraction-limited confocal imaging of tissue sections and super-resolution microscopy of subcellular structures, we demonstrate multi-scale applicability.
