DOI: 10.5061/dryad.85387h3
title: Data from: Stability of spontaneous, correlated activity in mouse auditory cortex

description: Neural systems can be modeled as complex networks in which neural elements
 are represented as nodes linked to one another through structural or
 functional connections. The resulting network can be analyzed using
 mathematical tools from network science and graph theory to quantify the
 system's topological organization and to better understand its
 function. Here, we used two-photon calcium imaging to record spontaneous
 activity from the same set of cells in mouse auditory cortex over the
 course of several weeks. We reconstruct functional networks in which cells
 are linked to one another by edges weighted according to the correlation
 of their fluorescence traces. We show that the networks exhibit modular
 structure across multiple topological scales and that these multi-scale
 modules unfold as part of a hierarchy. We also show that, on average,
 network architecture becomes increasingly dissimilar over time, with
 similarity decaying monotonically with the distance (in time) between
 sessions. Finally, we show that a small fraction of cells maintain
 strongly-correlated activity over multiple days, forming a stable temporal
 core surrounded by a fluctuating and variable periphery. Our work
 indicates a framework for studying spontaneous activity measured by
 two-photon calcium imaging using computational methods and graphical
 models from network science. The methods are flexible and easily extended
 to additional datasets, opening the possibility of studying cellular level
 network organization of neural systems and how that organization is
 modulated by stimuli or altered in models of disease.
description: The data contains
 fluorescent traces for the cells identified as described above. Please see
 the readme file for info.    
description: Two-photon microscopy Four mice were implanted
 with cranial windows over auditory cortex. Briefly, the mice were
 anaesthetized with 1.5-3\% isoflurane and a 3mm circular craniotomy was
 performed over auditory cortex (stereotaxic coordinates) using a 3mm
 biopsy punch. An adeno-associated virus (AAV) vector encoding the calcium
 indicator GCaMP6s (AAV1-SYN-GCAMP6s, UPENN vector core) was injected for
 expression in layer 2/3 neurons in left A1 within the window (750 nl, 1.89
 * 10-12 genome copies per ml) . After injection a glass circular 3mm
 coverslip (size 0, Warner Instruments) was placed in the craniotomy and
 fixed in place using a mix of Krazy glue and dental cement. A custom-made
 stainless steel head-plate (eMachine Shop) was fixed to the skull using
 C\&B Metabond dental cement (Parkell). All imaging sessions were
 carried out inside a single-walled acoustic isolation booth (Industrial
 Acoustics) as previously described. Mice were placed in the imaging setup,
 and the headpost was secured to a custom base (eMachine Shop) serving to
 immobilize the head. Mice were gradually habituated to the apparatus over
 3 days, 3-4 weeks after surgery.  Using two-photon
 microscopy (Ultima in vivo multiphoton microscope, Bruker) changes in
 fluorescence of GCaMP6s in transfected neurons caused by fluctuations in
 calcium activity were recorded in awake, head-fixed mice. We recorded from
 the same cells over many days in layer 2/3 of auditory cortex, using blood
 vessel architecture, depth from the surface, and the shape of cells to
 return to the same imaging site. Laser power at the brain surface was kept
 below 30 mW. Chronic imaging of the same field of view across days was
 carried out for the duration of the experiment. 
 Recordings were made at 512*512 pixels and 13-bit resolution at
 approximately 30 frames per second. Spontaneous activity was recorded for
 10 minutes in each session. Publicly available toolboxes were used to
 register the resulting images, select regions of interest, estimate
 neuropil contamination, and extract the changes in fluorescence from each
 cell. Upon conclusion of the imaging sessions, brains were extracted
 following perfusion in 0.01M phosphate buffer pH 7.4 (PBS) and 4\%
 paraformaldehyde (PFA), post-fixed in PFA overnight and cryopreserved in
 30\% sucrose solution for 2 days prior to slicing. The location and spread
 of GCaMP6s was confirmed through fluorescent imaging. These methods are
 consistent with the recommendations of the American Veterinary Medical
 Association (AVMA) Guidelines on Euthanasia. Cell
 tracking over days To identify ROIs from different
 imaging sessions that correspond to the same cell we performed a
 multi-step routine: (1) The mean fluorescence images from each day were
 registered by transforming the coordinates of landmarks present in both
 images in MATLAB (2017a) using the fitgeotrans function. The resulting
 transformation was used to transform the ROIs from the second imaging
 session to match the first; all sessions were aligned to the first imaging
 session. (2) We calculated the distance between all pairs of centroids
 across the two sessions. For each ROI from session 2, we computed the
 percentage overlap of the 10 cells with the smallest centroid distances
 from session 1. Cells that had more than 1 ROI with higher than 20\%
 overlap were manually inspected; the ROI that matched the current cell was
 selected from the overlapping ROIs or none were selected if it was unclear
 whether they were the same cell. A good match was determined by
 considering the percent overlap and the shape of the ROIs. All other cells
 were assigned the closest ROI as matching. (3) We manually inspected any
 cells that had duplicate matching ROIs; again considering the shape and
 the percent overlap of matching ROIs, we selected the ROI that matched
 that cell, or we decided that none was a good match. (4) To check for
 false positive matches, we manually inspected any matches where the
 centroid distances were greater than the mean + 1 std of all the matches,
 and any matches that had less than 30\% ROI overlap; the match was deemed
 good or not depending on the match criteria. Neurons that were not matched
 to any ROI were counted as different or new and assigned a new cell
 number. This process was repeated for subsequent sessions, registering the
 imaging field to the first session and comparing the ROIs to the
 cumulative ROIs from previous sessions. A final manual inspection of all
 the unique ROIs was performed after all the imaging sessions were
 registered; ROIs that overlapped were excluded from the dataset since it
 was unclear whether they were the same or different cells.