Name: neurovascular network explorer 2.0: a simple tool for exploring and sharing a database of optogenetically-evoked vasomotion in mouse cortex in vivo
Uploaded: 2018-05-04T13:00:00
Description: Watch this Scientific Journal Video about Neurovascular Network Explorer 2.0: A Simple Tool for Exploring and Sharing a Database of Optogenetically-evoked Vasomotion in Mouse Cortex In Vivo at JoVE.co

We gratefully acknowledge support from the NIH (NS057198, EB00790, MH111359, and S10RR029050) and the Ministry of Education, Youth and Sports of the Czech Republic (CEITEC 2020, LQ1601). KK was supported by postdoctoral fellowships from the International Headache Society in 2014 and The Scientific and Technological Research Council of Turkey in 2015. MT was supported by postdoctoral fellowship from the German Research Foundation (DFG TH 2031/1).

1. Installation of NNE 2.0
<ol>
	<li>Go to UCSD Neurovascular Imaging Laboratory website1 and left-click at &#8216;NNE 2.0 HDbase v1.0&#8217; to download the zipped program files to the desired location on your PC. 
	NOTE: NNE 2.0 requires a Windows-operating system of versions 7-10, at least 2.8 GB of free space to download the zipped file and 6.9 GB to install the program.</li>
	<li>Unzip &#8216;NNE2_HDbase_v1.0.zip&#8217;. 
	NOTE: The unzipped folder NNE2 contains 10 files: &#8216;h...

<ol>
	<li>. Use Our Data Available from: <a target="_blank" href="https://neurosciences.ucsd.edu/research/labs/nil/Pages/UseOurData.aspx">https://neurosciences.ucsd.edu/research/labs/nil/Pages/UseOurData.aspx</a> (2017)</li><li>Craddock, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+human+connectomes+at+the+macroscale.">Imaging human connectomes at the macroscale.</a> Nat Methods. 10 (6), 524-539 (2013).</li><li>Devor, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frontiers+in+optical+imaging+of+cerebral+blood+flow+and+metabolism.">Frontiers in optical imaging of cerebral blood flow and metabolism.</a> J Cereb Blood Flow Metab. 32 (7), 1259-1276 (2012).</li><li>Ji, N., Freeman, J., Smith, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technologies+for+imaging+neural+activity+in+large+volumes.">Technologies for imaging neural activity in large volumes.</a> Nat Neurosci. 19 (9), 1154-1164 (2016).</li><li>Maze, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+tools+and+current+challenges+in+the+modern+era+of+neuroepigenomics.">Analytical tools and current challenges in the modern era of neuroepigenomics.</a> Nat Neurosci. 17 (11), 1476-1490 (2014).</li><li>Medland, S. E., Jahanshad, N., Neale, B. M., Thompson, P. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+challenges+in+probing+the+human+brain.">Progress and challenges in probing the human brain.</a> Nature. 526 (7573), 371-379 (2015).</li><li>Kotter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroscience+databases:+tools+for+exploring+brain+structure-function+relationships.">Neuroscience databases: tools for exploring brain structure-function relationships.</a> Philos Trans R Soc Lond B Biol Sci. 356 (1412), 1111-1120 (2001).</li><li>Uhlirova, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roadmap+for+estimation+of+cell-type-specific+neuronal+activity+from+non-invasive+measurements.">The roadmap for estimation of cell-type-specific neuronal activity from non-invasive measurements.</a> Philos Trans R Soc Lond B Biol Sci. 371 (1705), (2016).</li><li>Aine, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+Neuroimaging+in+Schizophrenia:+Description+and+Dissemination.">Multimodal Neuroimaging in Schizophrenia: Description and Dissemination.</a> Neuroinformatics. , (2017).</li><li>Amunts, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BigBrain:+an+ultrahigh-resolution+3D+human+brain+model.">BigBrain: an ultrahigh-resolution 3D human brain model.</a> Science. 340 (6139), 1472-1475 (2013).</li><li>Laird, A. R., Lancaster, J. L., Fox, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrainMap:+the+social+evolution+of+a+human+brain+mapping+database.">BrainMap: the social evolution of a human brain mapping database.</a> Neuroinformatics. 3 (1), 65-78 (2005).</li><li>Lein, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+atlas+of+gene+expression+in+the+adult+mouse+brain.">Genome-wide atlas of gene expression in the adult mouse brain.</a> Nature. 445 (7124), 168-176 (2007).</li><li>Shin, D. D., Ozyurt, I. B., Liu, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cerebral+Blood+Flow+Biomedical+Informatics+Research+Network+(CBFBIRN)+database+and+analysis+pipeline+for+arterial+spin+labeling+MRI+data.">The Cerebral Blood Flow Biomedical Informatics Research Network (CBFBIRN) database and analysis pipeline for arterial spin labeling MRI data.</a> Front Neuroinform. 7, 21 (2013).</li><li>Uhlirova, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurovascular+Network+Explorer+2.0:+A+Database+of+2-Photon+Single-Vessel+Diameter+Measurements+from+Mouse+SI+Cortex+in+Response+To+Optogenetic+Stimulation.">Neurovascular Network Explorer 2.0: A Database of 2-Photon Single-Vessel Diameter Measurements from Mouse SI Cortex in Response To Optogenetic Stimulation.</a> Front Neuroinform. 11, 4 (2017).</li><li>Sridhar, V. B., Tian, P., Dale, A. M., Devor, A., Saisan, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurovascular+Network+Explorer+1.0:+a+database+of+2-photon+single-vessel+diameter+measurements+with+MATLAB((R))+graphical+user+interface.">Neurovascular Network Explorer 1.0: a database of 2-photon single-vessel diameter measurements with MATLAB((R)) graphical user interface.</a> Front Neuroinform. 8, 56 (2014).</li><li>Tian, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+depth-specific+microvascular+dilation+underlies+laminar+differences+in+blood+oxygenation+level-dependent+functional+MRI+signal.">Cortical depth-specific microvascular dilation underlies laminar differences in blood oxygenation level-dependent functional MRI signal.</a> Proc Natl Acad Sci U S A. 107 (34), 15246-15251 (2010).</li><li>. Use Our Data Available from: <a target="_blank" href="https://neurosciences.ucsd.edu/research/labs/nil/Pages/UseOurData.aspx">https://neurosciences.ucsd.edu/research/labs/nil/Pages/UseOurData.aspx</a> (2017)</li><li>Uhlirova, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type+specificity+of+neurovascular+coupling+in+cerebral+cortex.">Cell type specificity of neurovascular coupling in cerebral cortex.</a> Elife. 5, (2016).</li><li>Gagnon, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+microvascular+origin+of+BOLD-fMRI+from+first+principles+with+two-photon+microscopy+and+an+oxygen-sensitive+nanoprobe.">Quantifying the microvascular origin of BOLD-fMRI from first principles with two-photon microscopy and an oxygen-sensitive nanoprobe.</a> J Neurosci. 35 (8), 3663-3675 (2015).</li><li>Sakadzic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+high-resolution+measurement+of+partial+pressure+of+oxygen+in+cerebral+vasculature+and+tissue.">Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue.</a> Nat Methods. 7 (9), 755-759 (2010).</li><li>Nizar, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+stimulus-induced+vasodilation+occurs+without+IP3+receptor+activation+and+may+precede+astrocytic+calcium+increase.">In vivo stimulus-induced vasodilation occurs without IP3 receptor activation and may precede astrocytic calcium increase.</a> J Neurosci. 33 (19), 8411-8422 (2013).</li><li>Reznichenko, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+alterations+in+calcium+buffering+capacity+in+transgenic+mouse+model+of+synucleinopathy.">In vivo alterations in calcium buffering capacity in transgenic mouse model of synucleinopathy.</a> J Neurosci. 32 (29), 9992-9998 (2012).</li><li>Langer, J., Rose, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptically+induced+sodium+signals+in+hippocampal+astrocytes+in+situ.">Synaptically induced sodium signals in hippocampal astrocytes in situ.</a> J Physiol. 587 (Pt 24), 5859-5877 (2009).</li><li>Gong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+recording+of+neural+spikes+in+awake+mice+and+flies+with+a+fluorescent+voltage+sensor.">High-speed recording of neural spikes in awake mice and flies with a fluorescent voltage sensor.</a> Science. 350 (6266), 1361-1366 (2015).</li><li>Tantama, M., Hung, Y. P., Yellen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+reporters:+Fluorescent+protein-based+genetically+encoded+indicators+of+signaling+and+metabolism+in+the+brain.">Optogenetic reporters: Fluorescent protein-based genetically encoded indicators of signaling and metabolism in the brain.</a> Prog Brain Res. 196, 235-263 (2012).</li><li>Devor, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Overshoot"+of+O(2)+is+required+to+maintain+baseline+tissue+oxygenation+at+locations+distal+to+blood+vessels.">"Overshoot" of O(2) is required to maintain baseline tissue oxygenation at locations distal to blood vessels.</a> J Neurosci. 31 (38), 13676-13681 (2011).</li><li>Devor, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-induced+changes+in+blood+flow+and+2-deoxyglucose+uptake+dissociate+in+ipsilateral+somatosensory+cortex.">Stimulus-induced changes in blood flow and 2-deoxyglucose uptake dissociate in ipsilateral somatosensory cortex.</a> J Neurosci. 28 (53), 14347-14357 (2008).</li><li>Rauch, A., Rainer, G., Logothetis, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+a+serotonin-induced+dissociation+between+spiking+and+perisynaptic+activity+on+BOLD+functional+MRI.">The effect of a serotonin-induced dissociation between spiking and perisynaptic activity on BOLD functional MRI.</a> Proc Natl Acad Sci U S A. 105 (18), 6759-6764 (2008).</li><li>Lemmon, V. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimum+information+about+a+spinal+cord+injury+experiment:+a+proposed+reporting+standard+for+spinal+cord+injury+experiments.">Minimum information about a spinal cord injury experiment: a proposed reporting standard for spinal cord injury experiments.</a> J Neurotrauma. 31 (15), 1354-1361 (2014).</li><li>Ascoli, G. A., Donohue, D. 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NNE 2.0 was written in order to share the vascular imaging data of a specific study20 but with the intention of developing a simple tool for sharing and exploring data of similar kind by other users. Researchers interested in inspecting the associated database of vascular data may use the GUI to browse the data, select subsets of data, compare them to their own experimental results or process them further using their own computational procedures. Users familiar with MATLAB can utilize directly the...

The brain is considered one of the most intricate organs and the desire to untangle its complex function is unflagging. It is being studied at different scales from the molecular to the behavioral level using a wide palette of tools2,3,4,5,6,7,8. The amount of non-homogeneous experimental data grows with unprecedented speed. The awareness of the need for experimental data sharing, organization and standardization grows with the amount of acquired data. It has become evident that neuroinformatics will play a critical role in integrating experimental data across scales into models of brain function and dysfunction9,10.
To this end some studies, especially larger-scale studies, were able to earmark resources to make their results available via extensive databases11,12,13,14,15. However, a vast amount of experimental data from individual studies and regular-sized laboratories never reached the wider research community. This is mainly for two reasons: first, more dedicated time is needed to build a database and create tools that would enable the user to interact with the database; and second, more money is needed to support these tasks. Motivated by these challenges, a MATLAB based graphical user interface (GUI) engine called the Neurovascular Network Explorer 2.0 (NNE 2.0)16 was developed as a simple and low-cost tool for databasing, sharing and exploring of vascular imaging data. This manuscript provides a manual for operation of NNE 2.0 and the associated database of experimental data.
NNE 2.0 is already a second-generation software engine. The first generation, called Neurovascular Network Explorer 1.0 (NNE 1.0)17 was built to interact with a database of sensory-evoked vasodilation in rat primary somatosensory cortex (SI) in vivo measured by 2-photon microscopy18. NNE 1.0, its source code as well as the associated database are freely downloadable as a zipped file called &#8216;NNE 1 Tian&#8217; from UCSD Neurovascular Imaging Laboratory website1. More information about NNE 1.0 and the associated database can be found in17.
The second generation, the NNE 2.0, interacts with a database of optogenetically-evoked dilation of individual vessels in mice SI in vivo measured by 2-photon microscopy20. The user can browse, select and visualize data based on selection categories such as cortical depth, branching order, vessel diameter, animal subject or a particular arteriolar tree. The GUI further performs simple mathematical operations such as averaging and peak-normalization in selected categories. NNE 2.0 enables to view and browse through images capturing 3D volumes of vasculature as well as identify the location of the functional measurement within the vascular trees. This feature can be used to reconstruct vascular morphologies in 3D and populate them with real single-vessel vaso-motion measurements. These reconstructions can in turn be incorporated into computational models of brain function21,22. NNE 2.0, its source code and the associated database are freely downloadable as a zipped file called &#8216;NNE 2.0 HDbase v1.0&#8217; from UCSD Neurovascular Imaging Laboratory website1.
NNE 2.0 works with a database called &#39;vdb.mat&#39;. This database is a matrix containing temporal profiles (time-courses) of single vessel diameter changes evoked by an optogenetic stimulus and measured at different locations of arteriolar trees. Each time-course was computed using custom-written software. It calculates the relative change of a vessel diameter from expansion of a fluorescent intensity profile acquired by scanning across the vessel. The fluorescent contrast was presented by intravascular injection of fluorescein isothiocyanate (FITC)-labeled dextran. For more information about the data and analysis procedures, please see20,23. The database has 305 time-courses (i.e. database entries) in total. In addition to the diameter change, each entry to the database holds an array of additional metadata which (1) quantify the time-course (2) describe the measured vessel and (3) identify the measurement location within a 3D volume of cortical vasculature. The metadata include the onset time, peak amplitude, peak amplitude time, cortical depth, branching order, vessel diameter at baseline, path to original reference images and 3D image stacks for each measurement and low-magnification maps of brain surface vasculature. Please see all parameters in the metadata listed and described in detail previously in Table 116.
NNE 2.0 interacts with reference images that are X-Y scans of a plane where the diameter measurement occurred. Each database entry has one corresponding reference image with a reference name displayed in the GUI. Each database entry also has an associated stack of images (3D stack) capturing a 3D volume of the vascular tree within which the measurement occurred. The GUI enables to choose a particular database entry and display the corresponding reference image as well as the 3D stack. It also guides the user to find the matching reference image and frame in the 3D stack (the same features can be found in both images). All stack and reference images in their full resolution (1024 pix x 1024 pix) are included in folders hana_stk and hana_refs, respectively. Low-magnification maps of brain vasculature are included in folder &#39;maps&#39;. All three folders as well as the database matrix &#8216;vdb.mat&#8217; are downloaded in the zipped file &#8216;NNE 2.0 HDbase v1.0&#8217; from the UCSD Neurovascular Imaging Laboratory website1 and saved to the root folder of NNE 2.0 during the installation process.
The GUI has been designed as a set of four panels (Panel 1 (Main Panel) &#8211; Panel 4) which open sequentially as the user explores the database and selects specific data based on selection categories. Each panel is divided into two main parts: (1) the right column provides the possibility to interact with the database by selecting parameters and categories of the data and displays important information from the metadata; (2) the left column displays the data in the form of time-courses (diameter change in time) and scatter-plots. There are four types of scatter plots displaying (1) dilation onset time (2) time of the dilation peak (3) maximum diameter change (peak amplitude) and (4) baseline diameter (diameter before stimulation) as function of cortical depth. The user has the possibility to display average time-courses and values for selected data grouped either by cortical depth or branching order. This is to highlight the feature of gradient diameter-change behavior with increasing depth and branching order20. NNE 2.0 allows the user to export the selected subset of data in the format of &#39;.xls&#39;, &#39;.csv&#39; or &#39;.mat&#39;.

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			<td height="40" style="height:40px;width:216px;">MATLAB</td>
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			<td style="width:167px;">program</td>
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			<td height="40" style="height:40px;">Winrar</td>
			<td>Rarlabs</td>
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			<td>program</td>
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neurovascular network explorer 2.0: a simple tool for exploring and sharing a database of optogenetically-evoked vasomotion in mouse cortex in vivo

The importance of sharing experimental data in neuroscience grows with the amount and complexity of data acquired and various techniques used to obtain and process these data. However, the majority of experimental data, especially from individual studies of regular-sized laboratories never reach wider research community. A graphical user interface (GUI) engine called Neurovascular Network Explorer 2.0 (NNE 2.0) has been created as a tool for simple and low-cost sharing and exploring of vascular imaging data. NNE 2.0 interacts with a database containing optogenetically-evoked dilation/constriction time-courses of individual vessels measured in mice somatosensory cortex in vivo by 2-photon microscopy. NNE 2.0 enables selection and display of the time-courses based on different criteria (subject, branching order, cortical depth, vessel diameter, arteriolar tree) as well as simple mathematical manipulation (e.g. averaging, peak-normalization) and data export. It supports visualization of the vascular network in 3D and enables localization of the individual functional vessel diameter measurements within vascular trees.
NNE 2.0, its source code, and the corresponding database are freely downloadable from UCSD Neurovascular Imaging Laboratory website1. The source code can be utilized by the users to explore the associated database or as a template for databasing and sharing their own experimental results provided the appropriate format.

NNE 2.0 and the associate database serve to browse and view the data of the database, sort out the data based on selection criteria, download the selected data, and find the vascular measurements within the corresponding vascular tree.
Panel 1 features selection of data based on categories: &#39;Cortical Depth&#39;, &#39;Branching Order&#39;, &#39;Baseline Diameter&#39; and &#39;Subjects&#39; &#8211; Figure ...

Watch this Scientific Journal Video about Neurovascular Network Explorer 2.0: A Simple Tool for Exploring and Sharing a Database of Optogenetically-evoked Vasomotion in Mouse Cortex In Vivo at JoVE.co

Neurovascular Network Explorer 2.0: A Simple Tool for Exploring and Sharing a Database of Optogenetically-evoked Vasomotion in Mouse Cortex In Vivo

A graphical user interface for exploring and sharing a database of optogenetically-induced vascular responses in mouse somatosensory cortex in vivo measured by 2-photon microscopy is presented. It allows browsing the data, criteria-based selection, averaging, localization of measurements within a 3D volume of vasculature and exporting the data.

The importance of sharing experimental data in neuroscience grows with the amount and complexity of data acquired and various techniques used to obtain ...

The presented software called Neurovascular Network Explorer 2.0 or NNE 2.0, is a MATLAB based graphical user interface which allows visualization, inspection, selection, and export of optogenetically-evoked vascular diameter changes measured by two photon microscopy. In any two supports localization of the diameter measurements within a 3D structure of vascular network which can be used in modern studies of brain function. And any two can be used to explore the associated database as well as a template to sharing and exploring users own data structured into database of similar format.Start the NNE 2.0 program by selecting NNE 2.execute. Images in the left column of the main panel show graphs of time courses and perimeters of all entries in the database vdb. mat Begin by selecting the range for cortical depth in the right column.A minimum depth of 40 microns and a maximum depth of 560 microns is used here. Then select branching order, left-click on the arrow and choose one of the options from the list shown. Next, select baseline diameter and type in the range.A minimum diameter of two microns and a maximum diameter of 45 microns is used here. Now, select the subjects to be analyzed in the right column of the panel. Left-click on the arrow and choose from the available options.Press submit to display and explore the selected data in panel two. Prior to exploring the selected subset of data, group average the data by left-clicking either average by cortical depth or average by branching order. The choice is highlighted in green below.Then select data based on vessel morphology or subject. Select all data for tree consists of the data for a single diving arterial and its branches. Next, left-click on submit to display selected data in graphs of individual time courses, group average time courses, and scatter plots of onset times, time-to-peak's peak amplitudes, and baseline diameters.Left-click on a trace in the graph of individual time courses. The selected time courses then highlighted in magenta and its onset time, time-to-peak, peak amplitude, and baseline diameter will be marked by red circles in the graphs below. Then right-click anywhere on panel two with a cross cursor to view and explore all traces for the selected subject ID or tree ID in panel three.Select a time course in the top graph of the left column by left-clicking on a trace. The selected trace will be highlighted in magenta in the graph and descriptive parameters of the database entry will be displayed on top of the graph. The corresponding onset time, time-to-peak, peak amplitude, and baseline diameter are displayed in the graphs below.The time course in thick black is the average of all displayed traces. Left-click on the export set button to save traces displayed in the top graph into the NNE 2.0 folder. Please make sure none of the exported files are open during the export action.If one of the files is open, a warning will appear. In this case, the user should close the exported file and restart NNE 2. To inspect all data for subject instead of tree, close panel three, restart NNE 2.0, then after repeating the selection of categories in panel one as before, select all data for subject in panel two.Right-click anywhere in panel three with a cross cursor to go to panel four to explore reference images and 3D image stacks for all traces in the top graph of panel three. Please know that panel four will open only if option all data for tree was selected in panel two. If all data for subject were selected instead, the user will be prompted to change the selection and direct it to panel one.Select a time course by left-clicking on it in the graph on top of the left column. At the bottom right of the left column, explore the corresponding reference image which in this case is loaded automatically from the hana_refs folder. At the bottom left of the left column, explore the corresponding 3D image stack loaded automatically from the hana_stk folder.Scroll through this stack using the arrows or slider below the figure. When the stack image reaches the level of the reference image, the stack image is highlighted and indicated as frame level. Click export set in the right column to export the highlighted time course into a file ref_stacks_trace.xls which is saved to the NNE 2 folder. Finally, close panel four to go back to panel one to start exploring a new set of data or close panel one to end the program. To use the functional measurements along with the 3D vascular structure, a corresponding image stack is located in folder hana_stk using its stack reference name displayed in panel four.To recognize the particular diving arterial, the user can refer to a low magnification map of surface vasculature with diving segments of measured arterial or trees labeled with corresponding numbers. This stack index number at frame level localizes the measurement within appropriate depth. The branching order along with the scan trajectory, localizes the measurement within the imaging plane.Taken together, the user is provided with 3D topography of vasculature populated by stimulus induced vasomotion at multiple locations within vascular trees. And NNE 2 was written to share the vascular imaging data of a specific study but with the intention of developing a simple tool for sharing and exploring data of similar kind by other users. In the prerequisite for using NNE 2 is a template, is a database in the format of a matrix And this could be achieved either by processing the data directly in MATLAB or using tools to build the matrix from other formats.In NNE 2 has the potential to help sharing experimental data across research communities. Facilitating further utilization of the data as well as the development for standards for data acquisition and processing.

neurovascular-network-explorer-20-simple-tool-for-exploring-sharing

Research

JoVE Journal

Neuroscience

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.