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512-Channel and 13-Region Simultaneous Recordings Coupled With Optogenetic Manipulation in Freely Behaving Mice

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512-Channel and 13-Region Simultaneous Recordings Coupled With Optogenetic Manipulation in Freely Behaving Mice

Kun Xie et al. Front Syst Neurosci.

Abstract

The development of technologies capable of recording both single-unit activity and local field potentials (LFPs) over a wide range of brain circuits in freely behaving animals is the key to constructing brain activity maps. Although mice are the most popular mammalian genetic model, in vivo neural recording has been traditionally limited to smaller channel count and fewer brain structures because of the mouse's small size and thin skull. Here, we describe a 512-channel tetrode system that allows us to record simultaneously over a dozen cortical and subcortical structures in behaving mice. This new technique offers two major advantages - namely, the ultra-low cost and the do-it-yourself flexibility for targeting any combination of many brain areas. We show the successful recordings of both single units and LFPs from 13 distinct neural circuits of the mouse brain, including subregions of the anterior cingulate cortices, retrosplenial cortices, somatosensory cortices, secondary auditory cortex, hippocampal CA1, dentate gyrus, subiculum, lateral entorhinal cortex, perirhinal cortex, and prelimbic cortex. This 512-channel system can also be combined with Cre-lox neurogenetics and optogenetics to further examine interactions between genes, cell types, and circuit dynamics across a wide range of brain structures. Finally, we demonstrate that complex stimuli - such as an earthquake and fear-inducing foot-shock - trigger firing changes in all of the 13 brain regions recorded, supporting the notion that neural code is highly distributed. In addition, we show that localized optogenetic manipulation in any given brain region could disrupt network oscillations and caused changes in single-unit firing patterns in a brain-wide manner, thereby raising the cautionary note of the interpretation of optogenetically manipulated behaviors.

Keywords: BRAIN project; earthquakes; fear conditioning; functional connectivity; large-scale recordings; learning and memory; neural code; optogenetics.

Figures

FIGURE 1
FIGURE 1
The design of 512-channel tetrode arrays for brain-wide neural recording and optogenetic probing in mice. (A) An eight-tetrode bundle with optical fiber in the center. The 3 × 3 array square formation was used to arrange eight tetrodes at the outline, surrounding the optical fiber at the center. The left one shows the optrode bundle with green laser-light off, whereas the right photo shows the optrode bundle with green laser-light on. (B) An example of a tetrode array consisting of two rows of eight evenly spaced tetrodes. The tetrode was cut into a specific length depending on the recording depth. The polyimide tubing used for holding the wires together remained above the cortical surface. (C) Schematic drawing for the 512-channel tetrode device for recording in a total of 13 different brain regions in mice. Two tetrode arrays targeting the subiculum (S) and secondary auditory cortex (AuV) also contained optical fibers for opsin-based manipulation of neural activity. The scales are marked by black bars.
FIGURE 2
FIGURE 2
Construction and assembly of 512-channel tetrodes and headstage. (A) Two different lengths of tetrode wires. (B) A total of sixteen 32-channel connector pins were used to make a 512-channel headstage. (C) An assembled PrL/ACC (Cg1 and Cg2) module consisting of three 32-connector pins with 96-channel arrays (see the detailed arrangement in C:5, ACC/PrL module). The copper bar was used for temporally holding the assembled polyimide tubes and connector pins during the construction of the module and surgery. The tips of tetrodes protruding from the polyimide tubes are inserted into the brain. Two grounding wires were used for each module; one is to be attached to the skull and the other to the copper-mesh cage wrapped around the finished headstage after surgery. (D) Specifications of various tetrode arrays used for constructing seven different brain region modules. (E) Seven assembled tetrode array modules prior to surgical implantation. (F) The planar fiberglass used to secure four 128-channel connector pins (each consisting of four rows of 36 pin-connector arrays) on each arm. The copper bar in the middle of the fiberglass was used as a pole for holding the headstage to the dental-cement base in the skull and also for tethering to a helium balloon.
FIGURE 3
FIGURE 3
Visualization and verification of representative tetrode tracks in the mouse brain. (A) Tetrode arrays in the somatosensory cortex (S1Tr) and the auditory cortex (AuV). (B) A tetrode track in the S1HL. (C) A tetrode track in the prefrontal cortex (ACC). (D) A tetrode track in the prelimbic cortex (PrL). (E) Two tetrode tracks in the hippocampal dentate gyrus (DG). The square box is enlarged at a higher magnification.
FIGURE 4
FIGURE 4
Mice implanted with 512-channel headstage for recording from 13 brain structures. (A) A mouse with a 512-channel headstage, which also contained two optical fibers for light stimulation of the AuV and subiculum (S), respectively. The mouse is an order of magnitude smaller than a Long Evans rat which is typically used for in vivo neural recording. (B) The 512-channel headstage was connected through the ultra-thin cables to the 512-channel Plexon Neural Data Acquisition System. (C) A helium balloon was used to alleviate the weight of cables and headstage, enabling the mouse to perform behavioral tasks.
FIGURE 5
FIGURE 5
Mice implanted with the 512-channel headstage can still move freely during behavioral tests. The video track shows the movement trajectories inside a novel chamber during behavioral exploration. A total of 10 min were plotted. The object contour tracking mode in CinePlex Studio Application Version 3.2 (Plexon Inc.) was used to track the movement trajectories.
FIGURE 6
FIGURE 6
Large-scale local field potentials (LFP) simultaneously recorded from 13 different brain regions. An LFP signal was collected from one out of every four channels for each tetrode. This provided a total of 128 channels of LFP signals. A 5-s segment of LFP is used as an example. LFPs from different tetrodes within each region are more similar than those of different regions. Please note LFP in the AuV seemed to be more coherent with LFP in the DG (see large oscillatory signals at the time period from 2.1 to 2.7 s). Thirteen brain regions are labeled on the right with different colors.
FIGURE 7
FIGURE 7
Stable recordings of single units in mice. The top three panels show four well-isolated units in the PCA plot used in the MClust recorded from the same tetrode. These units remained stable over a period of a week (from Day 1 to Day 7). The L-ratio and isolation distance corresponding to these four units are listed beneath the panel, the table provides quantitative measures for the separation quality for these units.
FIGURE 8
FIGURE 8
Earthquake event triggered robust changes in firing rates in some of the recorded units across all 13 brain regions. Peri-event spike raster and histograms illustrated two example units recorded from the same tetrode from each of the 13 brain regions, placed in top and bottom rows. The left side of subplots shows the units with firing changes (peri-event spike raster in orange color, whereas peri-event spike histogram in light blue), the right side of subplots showing another representative unit (in gray) recorded from the same tetrode did not show significant firing change. Please note that only units of the Cg1 (but not Cg2) of the ACC are shown here.
FIGURE 9
FIGURE 9
Foot-shock triggered robust changes in firing rates in some of the recorded units across all 13 brain regions. Peri-event spike raster and histograms illustrated two example units recorded from the same tetrode from each of the 13 brain regions, placed in top and bottom rows. The left side of subplots shows the units with firing changes (peri-event spike raster in orange color, whereas peri-event spike histogram in light blue), the right side of subplots showing another representative unit (in gray) from the same tetrode did not show significant firing change. Please note that only units in the Cg1 of the ACC are shown here.
FIGURE 10
FIGURE 10
Optogenetic stimulation in the auditory cortex triggered dynamic changes in LFP across the 13 brain regions. Green-laser light stimulation (10 Hz) in the AuV of the CaMKII-Cre/Arch transgenic mice showed suppressed neural oscillation at lower frequencies during the 2-s light-stimulation, but caused apparent rebound theta oscillation across nearly all 13 brain areas after the termination of light stimulation at the AuV. Green bars at the top of the LFP power spectrum indicate the light stimulation duration. LFP color maps were averaged over 10 trials.
FIGURE 11
FIGURE 11
Optogenetic stimulation in the subiculum triggered dynamic changes in LFP across the 13 brain regions. Green-laser light stimulation (10 Hz) in the subiculum (S) of the CaMKII-Cre/Arch transgenic mice showed suppressed neural oscillation at lower frequencies during the 2-s light stimulation, but caused strong rebound theta oscillation across nearly all 13 brain areas after the termination of light stimulation at the subiculum. Green bars at the top of the LFP power spectrum indicate the 2-s light stimulation duration in the subiculum. LFP color maps were averaged over 10 trials.
FIGURE 12
FIGURE 12
Single-unit responses to green light stimulation. (A) Peri-event spike raster (in orange color) and spike histogram (in light blue) show that a responsive unit in the AuV suppressed its firing in time-locked manner during 2-s light stimulation (left spike raster). Another unit recorded from the same tetrode did not change its firing (second left panel, in gray). Some neurons in the other brain regions also responded to the AuV light stimulation. Two units from the ACC were plotted, one unit exhibited increased firing changes (in color), and another unit (in gray) from the same tetrode did not change its firing rate. (B) Peri-event spike raster and histogram show that a unit in the subiculum reduced its firing during 2-s light stimulation (left spike rasters in color). Another unit recorded from the same tetrode did not change its firing (in gray). Two units from the LEnt were plotted here, one unit exhibited increased firing changes (in color), and another unit from the same tetrode did not change its firing (in gray).

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