Md498

Fiber photometry was performed using a previously described system (Zhou et al., 2017 (link); Sun et al., 2019 ; Wang et al., 2019 (link), 2020 (link); Wu et al., 2020 (link)). To record fluorescent signals, the beam from a 488 nm laser (OBIS 488LS, Coherent) was reflected by a dichroic mirror (MD498, Thorlabs), focused by an objective lens (10×, NA: 0.3; Olympus), and then coupled to an optical commutator (Doric Lenses). An optical fiber (200 mm o.d., NA: 0.37, 1.5 m long) coupled the light between the commutator and the implanted optical fiber. GCaMP6s fluorescence was collected by the same fiber and objective, then bandpass-filtered (MF525–39, Thorlabs) and detected by a photomultiplier tube (R3896, Hamamatsu). An amplifier (C7319, Hamamatsu) converted the photomultiplier tube current output to a voltage signal, which was further filtered through a low-pass filter (35 Hz cut-off; Brownlee, 440). The analog voltage signals were digitized at 500 Hz and recorded by fiber photometry software (Thinkerbiotech, Nanjing, China) for the duration of each behavioral session.

For fluorescence Ca²⁺ recordings, light from a 473-nm LED was reflected by a dichroic mirror (MD498, Thorlabs). The emission signals collected through the implanted optical fiber in V1 were filtered by a bandpass filter (MF525-39, Thorlabs) and detected by a photomultiplier tube (PMT, R3896, Hamamatsu). The light at the tip of the optical fiber was adjusted to 10–30 μW to minimize bleaching. An amplifier converted the output of the PMT to voltage signals, which were digitized using a data acquisition card (USB6009, National Instrument) at 200 Hz with custom-written programs.

Following AAV-DIO-GCaMP6m virus injection, an optical fibre (230 μm O.D., 0.37 numerical aperture (NA); Shanghai Fiblaser) was placed in a ceramic ferrule and inserted towards the DRN through the craniotomy. The ceramic ferrule was supported with a skull-penetrating M1 screw and dental acrylic. Mice were individually housed for at least 1 week to recover.
To record fluorescence signals, laser beam from a 488-nm laser (OBIS 488LS; Coherent) was reflected by a dichroic mirror (MD498; Thorlabs), focused by a × 10 objective lens (NA=0.3; Olympus) and then coupled to an optical commutator (Doric Lenses). An optical fibre (230 μm O.D., NA=0.37, 2-m long) guided the light between the commutator and the implanted optical fibre. The laser power was adjusted at the tip of optical fibre to the low level of 0.01–0.02 mW, to minimize bleaching. The GCaMP fluorescence was bandpass filtered (MF525-39, Thorlabs) and collected by a photomultiplier tube (R3896, Hamamatsu). An amplifier (C7319, Hamamatsu) was used to convert the photomultiplier tube current output to voltage signals, which was further filtered through a low-pass filter (40 Hz cut-off; Brownlee 440). The analogue voltage signals were digitalized at 500 Hz and recorded by a Power 1401 digitizer and Spike2 software (CED, Cambridge, UK).

Details for the fiber photometric system are described previously^{81 (link)}. The excitation source is a 488 nm semiconductor laser (Coherent, Inc. OBIS 488 LS, tunable power up to 60 mW). A dichroic mirror with a 452–490 nm reflection band and a 505–800 nm transmission band (Thorlabs, Inc. MD498) is employed for wavelength selection. A multimode fiber (Thorlabs, Inc., 200 μm in diameter and 0.39 in numerical aperture) coupled to an objective lens (Olympus, ×10, NA 0.3) is used for optical transmission. The fluorescence signals are collected with a photomultiplier tube (PMT) (Hamamatsu, Inc. R3896) after filtering by a GFP bandpass filter (Thorlabs, MF525-39). An amplifier (C7319, Hamamatsu) is used to convert the current output from the PMT to voltage signals, which are further filtered through a lowpass filter (40 Hz cutoff; Brownlee 440, USA). The fluorescence signals are digitalized at 100 Hz and recorded by a Power 1401 digitizer and Spike2 software (CED, UK). Photometric data are exported and analyzed with MATLAB. The fluorescence signals (ΔF/F₀) are processed by averaging the baseline signal F₀ over a 4.5-s long control time window and presented as heatmaps or per-event plots. AUC (area under the curve) of the DA signal is used for statistical analysis. AUC is the integral of a curve that describes the variation of DA signals during light stimulation.

Following AAV-EF1α-DIO-GCaMP6f virus injection, an optical fiber (125 µm O.D., 0.37 numerical aperture (NA); Newdoon, Shanghai) was placed in a ceramic ferrule and inserted towards the NAc. Fiber photometry^{53 (link)} uses the same fiber to both excite and record from GCaMP in real time. To record fluorescence signals, laser beam was passed through a 488 nm laser (OBIS 488LS; Coherent), reflected off a dichroic mirror (MD498; Thorlabs), focused by objective lens (Olympus), and coupled through a fiber collimation package (F240FC-A, Thorlabs) into a patch cable connected to the ferrule of the upright optic fiber implanted in the mouse via a ceramic sleeve (125 µm O.D.; Newdoon, Shanghai). GCaMP6 fluorescence was bandpass filtered (MF525–39, Thorlabs) and collected by a photomultiplier tube (R3896, Hamamatsu). An amplifier (C7319, Hamamatsu) was used to convert the photomultiplier tube current output to voltage signals, which was further filtered through a low-pass filter (40 Hz cut-off; Brownlee 440). The photometry voltage traces were downsampled using interpolation to match the EEG/EMG sampling rate of 512 Hz by using a Power1401 digitizer and Spike2 software (CED, Cambridge, UK). Analysis of the resulting signal was performed with custom written MATLAB software.

Customized fiber photometry experiments were performed as previously described (62 (link)). Excitation from a 488 nm laser (OBIS 488LS; Coherent) produced fluorescence signals that were reflected by a dichroic mirror (MD498; Thorlabs). The light was transmitted through an optical fiber (200 mm OD, NA = 0.37, 1 m long) between the rotary joint and the implanted fiber. The analog voltage signals were digitized at 50 Hz. The fiber photometry experiment was conducted using a fiber photometry system purchased from ThinkerTech Nanjing BioSicence Inc. For the NORT, mice were allowed to visit objects as described above, along with video recording and fiber photometry acquisition to synchronously record behaviors and calcium signals. All the GCaMP6s signals were subtracted from the background and segmented based on behavioral events in individual trials. We calculated the values of fluorescence change as follows: (ΔF/F) by (F – F₀)/F₀, where F₀ was defined as the average fluorescence signal from 2 seconds preceding event onset. The ΔF/F data were assessed with an average line with a shaded area indicating SEM.

To record fluorescence signals for the GCaMP6f and GRAB_DA sensors, a laser beam from a 488 nm laser (OBIS 488LS; Coherent) was reflected by a dichroic mirror (MD498; Thorlabs, Newton, NJ, USA) (Li et al., 2016 ) (Thinkertech Nanjing Bioscience Inc, Co., Ltd.).