Fiber photometry in striatum reflects primarily nonsomatic changes in calcium.

All experimental procedures were approved by the Washington University Animal Care and Use Committee and the Northwestern University Animal Care and Use Committee. Subjects. The animals used in this study were 14 wild-type C57BL6 mice (8 males, 6 females), 8 Drd1-Cre mice (GENSAT line EY217, 6 males, 2 females), 6 A2a-Cre mice (GENSAT line KG139, 5 males, 1 female) and 4 Drd1-Cre crossed to Allen Institute reporter line Ai14 on a C57BL6/J background (4 females). Cre mice were obtained from the GENSAT project (The Gene Expression Nervous System Atlas (GENSAT) Project, NINDS Contracts N01NS02331 & HHSN271200723701C to The Rockefeller University (New York, NY)). Animals were housed in either the Washington University in St. Louis animal facilities in standard vivarium cages with ad libitum food and water and a nonreversed 12-h dark/light cycle or the Northwestern University animal facility with a reversed 12-h light/dark cycle. Viral transduction. Anesthesia was induced with 3–5% isoflurane and maintained at 0.5–1.5% isoflurane during stereotactic surgery. Ear bars and a mouth holder were used to keep the mouse head in place while the skin was shaved and disinfected with a povidone/iodine solution. The skull was exposed and 1-mm-diameter craniotomy was made with a microdrill mounted to the stereotaxic manipulator. Injections were performed with a glass pipette mounted in a Nanoject 3 infusion system (Drummond Scientific). Then 500 nl of virus AAV1-Syn-GCaMP6s or AAV2/9-CaMKII-GCaMP6s (1.2 × 10 12 GC ml −1 ) virus was infused over 10 min into either the dorsal striatum (AP = +0.5 mm, ML = +1.5 mm, DV = −2.8 mm) or ventral striatum (AP = +0.5 mm, ML = +1.2 mm, DV = −4.5 mm). The injector was left in place for 5 or 10 min before removal. Optical guide implantation and head bar placement. We used a 1.4-mm-diameter drill bit to create another craniotomy (AP = +1.0 mm, ML = +1.5 mm) for implantation of the optical guide tube. We fabricated this guide tube by using ultraviolet (UV) liquid adhesive (Norland no. 81) to fix a 2.5-mm-diameter disc of no. 0 glass to the tip of a 3.8-mm-long, extra-thin 18-gauge stainless steel tube (McMaster-Carr). We ground off any excess glass using a polishing wheel (Ultratec). Using a 27-gauge blunt-end needle, we aspirated the cortex down to DV = −2.1 mm from the dura and implanted the exterior glass face of the optical guide tube at DV = −2.35 mm. After stereotaxic placement of these components, we attached a head bar to the entire assembly using Metabond (Parkell) and dental acrylic. For mice used for two-photon imaging, we used additional dental acrylic to construct a reservoir for holding water for the water-immersion objective lens. Mice recovered for 3–4 weeks before two-photon imaging experiments or mounting of the miniature microscope. GRIN lens implantation and mounting of miniature microscope. After 3–4 weeks, we inserted a GRIN lens (1 mm diameter; 4.12 mm length; 0.46 numerical aperture; 0.45 pitch; GRINTECH GmbH or Inscopix Inc.) into the optical guide tube. In mice with uniform indicator expression, we secured the GRIN lens in the guide tube with UV-light-curable epoxy (Loctite 4305). For miniscope imaging, after affixing the GRIN lens, we lowered a miniature microscope (nVistaHD, Inscopix Inc.) toward the GRIN lens until the fluorescent tissue was in focus. To secure the miniature microscope to the cranium, we created a base on the cranium around the GRIN lens using blue-light-curable resin (Flow-It ALC; Pentron). We attached the base plate of the miniature microscope to the resin base using UV-light-curable epoxy (Loctite 4305). After affixing its base plate, we released the microscope and attached a base plate cover (Inscopix Inc.). We coated the resin with black nail polish (Black Onyx, OPI) to make it opaque. Implantation of electrode arrays. Following viral infusion, a combined electrophysiology/fiber photometry device was implanted. Fiber optic cannulae (200 μm diameter, 0.50 numerical aperture) with 1.25 mm ceramic ferrules were purchased from Thorlabs and cut to 6 mm long. These cannulae were mounted in a custom electrode array with 32 Teflon-coated tungsten microwires (35 μm diameter; Innovative Neurophysiology) that positioned the wires in a semicircle surrounding a central gap where the photometry fiber was mounted. This combined photometry/electrical recording device was implanted into the right DMS (AP = +0.5 mm, ML = +1.5 mm, DV = −2.8 mm). The device was secured to the skull with a thin layer of adhesive dental cement (C&B Metabond, Parkell) followed by a larger layer of acrylic dental cement (Lang Dental). Once the cement had fully cured, animals were placed back in their home-cage on a pre-heated pad at 37 °C. After recovery, animals received a subcutaneous injection of meloxicam (10 mg kg −1 ) and were again returned to their home-cages for recovery. Mice recovered for at least 2 weeks to allow for viral expression before recording. Electrophysiological recordings. Neurophysiological signals were recorded by a multichannel neurophysiology system (Plexon Omniplex, Plexon Inc.). Spike channels were acquired at 40 kHz and bandpass filtered from 150 Hz to 3 kHz before spike sorting. Recordings were performed in a 9″ × 12″ clear plastic box and lasted between 1 and 3 hr. Video and tracking data was also recorded in real time with the Plexon Cineplex system. Fiber photometry recordings. Fiber photometry acquisition was performed with a Neurophotometrics fiber photometry system (FP3001, Neurphotometrics LTD). Briefly, this system utilizes a 470-nm blue-light LED, which was left on continuously at 40–100 μW to excite GCaMP, and a fluorescence light path that includes a dichroic mirror to pass emitted green fluorescence to a complementary metal-oxide semiconductor camera (FLIR BlackFly). Fluorescence signals from the camera are processed with Bonsai ( https://bonsai-rx.org/docs ) and transmitted as a voltage signal to the Plexon Omniplex for simultaneous digitizing with the electrophysiological data. Behavior. Open field recordings were performed after 2 weeks of viral injection in GCaMP8f mice, and 1–3 months in GCaMP6s mice. The operant feeding, air puffs and foot shock behaviors were tested 6–7 weeks after viral injections. Operant field task. Mice were placed in an open field chamber and spontaneous activity was recorded for 1.5 h. Operant feeding task. In two overnight (16-hour) sessions, mice were trained to hold an isometric lever for at least 200 ms, which dispensed a pellet into the chamber from a FED3 device 23 . Following training, mice were fasted for 6 hours and simultaneous spiking and photometry data was recorded as they completed this same task for 3 hours during the daytime. Air puffs. Mice were placed in an open field chamber and left to acclimate for 15 min. Fifteen air puffs were manually delivered, with a duration of approximately 500 ms each, at pseudo-random intervals between 1 and 3 min. Foot shocks. Mice were placed in a shock box chamber and left to acclimate for 15 min. Foot shocks were delivered at pseudo-random intervals of 1 to 3 min. Foot shocks of 0.7 mA and three different lengths were delivered (100 ms, 250 ms and 500 ms, in that order, 12–15 shocks per intensity). In total, between 40 and 45 shocks were delivered per mouse. Electrophysiology/fiber photometry data analysis. Single units and multiunits were manually discriminated using principal component analysis (Offline Sorter; Plexon), using multivariate analysis of covariance analyses to determine if single-unit clusters were statistically distinct from multiunit clusters. Where single-unit isolation did not reach statistical significance, spike clusters were combined into multiunits. Data analysis was performed using a custom Python pipeline (code available at: https://osf.io/8j7g2/ ), described below. Photometry signal preprocessing. The fiber photometry signal was processed using a custom Python pipeline (code available at: https://osf.io/8j7g2/ ). We first applied both a low-pass (6 Hz) and a high-pass filter (0.0005 Hz) to the photometry signal to correct for high-frequency noise and photo bleaching, respectively, and the signal was down-sampled to 20 Hz. Time derivative of photometry. To obtain the time derivative of the photometry signal, a discrete derivative was done using the np.diff() function. Photometry deconvolution. To deconvolve the photometry signal, we used a one-dimensional constrained deconvolution algorithm, which assumes that the sensor fluorescence follows a second-degree autoregressive process. We used the CaImAn implementation of this algorithm 3 . Population spiking preprocessing. For analyzing the population spiking response around behavioral events, we created peri-event histograms of each unit. To obtain the population spiking, all the trials from all the neurons were concatenated and averaged, resulting in one average time series that reflected the average population spiking. Photometry transient and spiking burst detection. Photometry transients were detected using the scipy.find_peaks() function, setting a minimum prominence of 2. For detecting population spiking bursts, all spikes from all the neurons were concatenated and a firing rate histogram was created with bins of 0.05 (or 20 Hz) to create a signal analogous to the photometry signal. A Gaussian filter with a sigma value of 0.5 was applied. Population spiking bursts were detected in the same way to photometry transients, by using the scipy.find_peaks() function with the prominence parameter set to a minimum of 2. Normalization of signals. In all peri-event histograms, all the signals were z -scored to a baseline period. One-photon endoscopic calcium imaging recordings. Brain imaging in freely moving mice occurred in a circular arena (31 cm in diameter). To habituate mice to this arena, mice explored it for 1 h on each of three sequential days before any calcium ion (Ca 2+ ) imaging. Before each imaging session, we head-fixed each mouse to a metal frame by its implanted head bar and allowed the mouse to walk or run on a running wheel. We then attached the miniature microscope and adjusted the focal setting to optimize the field of view. After securing the microscope to the head of the mouse, we detached the mouse from its head restraint and allowed it to freely explore the circular arena. After allowing ≥10 min for the mouse to habituate to the arena, fluorescence Ca 2+ imaging commenced using 50–200 μW of illumination power at the specimen plane and a 20-Hz frame acquisition rate. Endoscopic calcium imaging analysis. Cropping. To better match the surface area of the most commonly used fiber photometry fiber, we cropped each 1-mm GRIN lens image into six 200-μm 2 regions with the FIJI distribution of ImageJ 21 . Somatic activity extraction. We used the CaImAn 3 cell body extraction Jupyter notebook pipeline to extract somatic activity from miniscope videos. Briefly, this pipeline implements motion correction and the CNMF-E algorithm 5 in an online notebook, returning quality metrics and images of extracted somatic signals for subsequent analysis. We then averaged the activity trace of somatic signals. Nonsomatic activity extraction. ROIs covering cell bodies were extracted with the CaImAn pipeline and were used to mask out regions of the video that contained somatic activity. The fluorescence in the remaining pixels was averaged for each frame of the video to create an average nonsomatic signal. Extraction of photometry signals. The proxy for photometry signal was obtained by averaging the intensity of the entire field for each frame of the raw video. Two-photon calcium imaging recordings. Drd1-Cre; Ai14 mice injected with AAV2/9-CaMKII-GCaMP6s were used for two-photon calcium imaging. GCaMP6s was constitutively expressed in both MSN types, whereas tdTomato expression was restricted to D1-MSNs. After habituating mice to head-fixation on a running wheel, we used a two-photon microscope with a piezoelectric actuator (Bruker) to acquire videos of Ca 2+ activity and tdTomato expression at three imaging planes separated by 20 μm in the dorsal striatum of head-fixed mice during wheel running. We used a tunable laser (Insight X3, Spectra Physics) and a 16x/0.8NA objective (Nikon) to acquire 512 × 512-pixel videos of each plane at a 30-Hz frame acquisition rate (effectively 6 Hz per plane). We used 920 nm excitation light to simultaneously excite tdTomato and GCaMP6s fluorescence, which we detected using gallium arsenide phoshide photomultiplier tubes and bandpass filters (520/40 for GCaMP6s and 595/50 for tdTomato). Two-photon calcium imaging processing. We used an exponential fit to normalize slow variations in green and red fluorescence intensity that were assumed to be due to photo bleaching. We then motion corrected the tdTomato video using NormCorre 22 . We then applied the tdTomato motion correction transformations to the video frames of the green GCaMP6s fluorescence video. We then corrected for fluctuations in background fluorescence intensity in the GCaMP6s video by applying a Gaussian low-pass filter to each image, then dividing each image frame by its low-pass filtered version. We then down-sampled the GCaMP6s video by a factor of 2 via linear interpolation. Two-photon calcium imaging analysis. Data analysis was performed using a custom Python pipeline (code available at: https://osf.io/8j7g2/ ), described below. Fast Fourier Transformations (FFTs). All FFTs analyses were done using the numpy.fft2 module of numpy. To obtain the signal coming from different spatial frequencies, an FFT was applied to each frame, and then a bandpass filter was applied through a circular mask that selected for specific spatial frequencies. Finally, an inverse FFT was performed to recreate a filtered image that contained only the spatial frequencies allowed by the filter. Nonsomatic activity extraction. ROIs containing cell bodies were identified with the EXTRACT algorithm and were used to mask out areas of the movie containing somatic signals. Fluorescence from the remaining pixels was averaged for each frame of the video to obtain a nonsomatic signal. Histology. At the end of the experiments, we performed histological verification of implant placements. Animals were anesthetized with isoflurane and decapitated, and their brains were quickly removed and placed in 10% formalin solution, in which they were incubated overnight. The brains were then moved to 30% sucrose solution until sectioning. Coronal slices containing the striatum were prepared using a freezing microtome (Leica SM2010R). Slices were mounted on microscope slides with a mounting media and imaged with an epifluorescence microscope (Zeiss). For in vivo electrophysiology, electrode placement was assessed via observation of implant tracts or electric lesions that were made under anesthesia before decapitation (performed with a 5-s long pulse of 10 mA; Ugo Basile Lesion Making Device). Statistics and reproducibility. No statistical methods were used to pre-determine sample sizes, but our sample sizes are similar to those reported in previous publications 4 , 12 . Confirmation of viral expression and optic fiber/electrode implantation in each mouse was done through histology (Histology), resulting in similar expression and implant localization to the representative example in Fig. 1b . The experimenters were not blinded to the allocation of groups, since both photometry and spiking activity, and calcium imaging signal was acquired from every mouse and all comparisons were paired. No data were excluded. Data distribution was assumed to be normal, but this was not formally tested. There were no experimental groups in this study, and thus no randomization, since photometry and spiking signal ( Figs. 1 and 2 and Extended Data Figs. 1 – 4 ) and miniscope-based calcium signals ( Fig. 3 and Extended Data Figs. 5 and 6 ) were collected from all the animals in the experiment.

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