Minimally invasive, wide-field two-photon imaging of the brainstem at cellular resolution

Abstract

Brain-viscera communication is crucial for regulating mental health, with the vagus nerve being a key structure mediating this interaction. Clinically, artificial vagus nerve stimulation (VNS) is used to treat various neuropsychiatric disorders, highlighting the importance of vagal afferent fibers in emotion regulation. The nucleus tractus solitarii (NTS) is a brainstem structure proposed to receive signals from vagal afferents and relay them to brain networks for emotion regulation. However, due to the anatomical complexity and difficulty in accessing the deep-brain NTS region in vivo, its underlying mechanisms remain unclear. Here, we developed a wide-field and deep-brain two-photon imaging method using a double-prism optical interface. This approach enables cellular-resolution imaging to specifically detect NTS neural activity while largely preserving the overlying cerebellum, a region also implicated in emotion regulation. We evaluated NTS neuronal responses to VNS and a gastrointestinal hormone, demonstrating the method's utility for investigating the vagus-NTS pathway in vivo.

Keywords: CP: Imaging; CP: Neuroscience; brain-viscera communication; brainstem; emotion; in vivo two-photon calcium imaging; nucleus tractus solitarii; vagus nerve stimulation.

Graphical abstract

Figure 1

In vivo NTS imaging through a double prism (A) AAV injection for GCaMP expression in the mouse NTS neurons (sagittal view). (B) GCaMP expression in the NTS (coronal view). (Left) Schematic illustration around the NTS. (Right) GCaMP expression (green) in the NTS, exclusive to the 10N neurons (marked by ChAT in magenta). Blue, DAPI. (C) Double-prism schematic with example picture (bottom right). The letter “m” below the double prism appears through it. (D) Vacuum-type holder for double-prism implantation. Black arrows, directions of the double-prism insertion; thick blue arrows, vacuum directions for holding; thin blue arrow, protrusion to push the double prism during the implantation. (E) Imaging NTS through the double prism inserted between the cerebellum and brainstem from the posterior edge of the cerebellum. (F) Head plate attachment after the double-prism implantation. (G) Example of in vivo brainstem imaging. (Left) Representative two-photon image showing GCaMP fluorescence. Each number indicates a region of interest (ROI), corresponding to a putative single neuron. Baseline fluorescence (green-fire-blue) was estimated from median fluorescence over time. (Middle) Magnified view of the left image. (Right) GCaMP traces from selected ROIs. Gray lines, VNS timing. Zp4, 4th z-stacked plane (see also Figure 4). D, dorsal; R, right; P, posterior; A, anterior.

Figure 2

Evaluating spatial resolution through the double prism (A) Two-photon imaging of yellow-green fluorescent beads through the cover glass alone (left) and the double prism attached to the cover glass (right). (B) Representative horizontal (xy) images of fluorescent beads in agarose gel captured through the cover glass (left) and double prism (right) at 50 μm depth from the surface (i.e., the bottom end of the cover glass or double prism). (C) Representative vertical (xz) sections of agarose gel captured through the cover glass (left) or double prism (right) and reconstructed from 3D scanned image data. (D) Representative horizontal images of the fluorescent beads in the brain-mimetic gel, captured as in (B). (E) Representative vertical sections of brain-mimetic gel, captured as in (C). (F) Definition of “center” and “peripheral” regions for spatial resolution analysis through the double prism. Center: 500 × 500 μm field of view (FOV) at the center of the 2 × 2 mm prism window. Peripheral: FOV located 500 μm left and 500 μm upward from the center. (G) FWHM values were analyzed in horizontal (xy) and vertical (xz) sections using bead images isolated from 3D volume data. Representative images in agarose gel (left) and corresponding fluorescence intensity line profiles (right) are shown for each condition. (H) Summary of FWHM values (μm), comparing resolution between the cover glass and double prism. Agar, agarose gel; BrainM, brain-mimetic gel; D-prism (center), data at the center of the double prism. (I) Summary of FWHM values (μm), comparing the resolution between center and peripheral regions through the double prism, with data acquired via z scanning at each position. Brightness variations across experiments were normalized in (B)–(E) and (G) to ensure consistent brightness at the surface (0.0 μm). Boxes in (H) and (I): 25th–75th percentile; red bars in (H) and (I): median.

Figure 3

Histological examination of GCaMP expression in the nodose ganglion and NTS (A) Schematic of afferent vagal projections to the NTS. (B) Representative image of a nodose ganglia from an animal expressing GCaMP in NTS neurons. We performed immunohistochemical staining of substance P, a marker protein of the mouse nodose ganglion. GCaMP signals in fixed tissues were detected using identical optical settings (see STAR Methods for details) for both nodose ganglia (this image) and NTS (Figure 1B), revealing much lower signal levels in the nodose ganglion. Images are maximum intensity projections of a z series of confocal images. Blue, DAPI; magenta, substance P. (C) Single z-plane images (not maximum projections) of the NTS area expressing GCaMP, captured with a higher-NA objective lens for precise visualization of the density of GCaMP-expressing NTS neurons.

Figure 4

Volumetric time-lapse imaging of brainstem neural activity (A) Schematic of two-photon time-lapse imaging in vivo at various depths through the implanted double prism. Colored circles illustrate regions of interest (ROIs) at Zp1–Zp3. (B) Examples of in vivo volumetric imaging of the wide brainstem area, including the NTS. Results from the same mouse are shown in Figures 1G and 5C and Video S4. Here, seven z-stacked planes (termed Zp) were acquired at each time point, and Zp1–Zp5 are shown (Zp1, -2, and -4 correspond to Figure 5C). Baseline fluorescence (green-fire-blue) was estimated from median fluorescence for each Zp. See STAR Methods for details. D, dorsal; P, posterior; A, anterior; R, right.

Figure 5

In vivo imaging of NTS activity during repeated VNS (A) VNS during in vivo NTS imaging was conducted through a bipolar platinum electrode on the cervical vagal trunk, secured with the insulating silicone adhesive. To create an anodal block for efferent direction, the cathode (black) and anode (red) were placed on the rostral and caudal sites of the vagus nerve, respectively. (B) Schematic of VNS protocol. This example represents a session at 1.0 mA and 10 Hz. (C) Example of in vivo NTS imaging during VNS at 2.0 mA and 10 Hz. AAVdj/Syn.GCaMP7f was used. (Left) Zp1, Zp2, and Zp4, which contain automatically detected ROIs in this mouse, are shown (Zp4 is the same data as Figure 1G). Baseline fluorescence (green-fire-blue) was estimated from median fluorescence. (Right) Detection of spontaneous and/or VNS-evoked neural activities (ΔF/F0) from 21 example neurons outside (red) and inside (orange) the NTS. See also Video S4. (D) Higher-magnification imaging provides clear single-cell precision. NTS imaging was performed during VNS at 0.5 mA and 10 Hz. AAV1/Syn.GCaMP6f was used. (Left) Zp1 and Zp5 from this mouse are shown. Baseline GCaMP fluorescence is shown in grayscale to enhance ROI visibility, with each ROI individually color coded for clear separation. (Right) Eleven ROIs, each corresponding to a putative single neuron, are sorted by response magnitude to VNS. See also Video S5. Zp, z-stacked horizontal plane. Gray in activity trace panels indicates VNS timing. A, anterior; R, right.

Figure 6

Variable responsiveness of NTS neurons under various VNS conditions (A) Schematic of VNS protocol. As in this example, each experiment in this figure includes a session with three 10-s VNS trials, separated by 50-s intervals. (B) Average signal (0–10, 0–20, and 10–20 s from VNS onset), peak time, and amplitude of neural response (ΔF/F0) to VNS (gray area) were calculated to assess neural responses. Horizontal axis represents time from VNS onset. (C) Responses of an example neuron to VNS at a fixed 10-Hz frequency, with various intensities. Colored lines indicate responses to the first, second, and third VNSs. Dashed lines indicate the mean responses across trials. (D) Responses of simultaneously recorded neurons (n = 8) in a mouse to VNS at 10 Hz with various stimulus intensities. The mean ΔF/F0 of each neuron for 10 s from VNS onset at different intensities is summarized. (E) Summary of neuronal responses to VNS at 10 Hz with varying stimulus intensities. Mean values across three trials for five parameters (defined in B) were calculated per neuron (gray) and for all neurons (black). (F) Responses of the same neuron as in (C) to VNS at a fixed 1.0 mA intensity, with various frequencies. (G) Responses of simultaneously recorded neurons (n = 8) to VNS at 1.0 mA with various frequencies, summarized as in (D). (H) Summary of neuronal responses to VNS at 1.0 mA with varying stimulus frequencies, summarized as in (E). AAV1/Syn.GCaMP6s was used. Gray shaded area indicates VNS timing. Boxes in (D) and (G): 25th–75th percentile; red bars in (D) and (G): median of all neurons; error bars in (E) and (H): standard deviation.

Figure 7

Imaging NTS neural activity in response to intravenous CCK administration (A) Schematic of in vivo NTS imaging during saline or CCK administration via the tail vein of an anesthetized mouse (co-administered with red fluorescent dye, SR101). (B) Averaged GCaMP fluorescence during pre- and post-administration (60 s) of saline and CCK. (Top) Baseline-subtracted GCaMP signal (green) overlaid on the baseline image (gray). (Middle) Baseline image. (Bottom) Baseline-subtracted signal. Dashed circles indicates the NTS area. See also Videos S7 and S8. (C) Traces of GCaMP fluorescence from simultaneously recorded NTS neurons (same neurons on the left and right). Gray dotted lines indicate the timings of the injections (saline, left; CCK, right). Traces representing more than 5 min after the injection are expanded to highlight the increased frequency of spontaneous activity (i.e., frequent calcium transients) observed uniquely after CCK administration. (D) Mean ΔF/F0 for each 60-s bin was calculated over time for saline (left) and CCK (right) administration. Each data point represents the mean ΔF/F0 for each neuron in each bin. (E) Mean ΔF/F0 for 60 s immediately after saline or CCK administration. Horizontal red bars: median across all neurons. ∗∗p < 0.01, two-tailed Wilcoxon signed-rank test (p = 0.0011). (F) Cumulative distribution of calcium-transient intervals from 60 to 240 s after saline or CCK administration. Neural activation frequency was assessed by identifying calcium-transient timings and measuring interval lengths (Figure S6; see also STAR Methods). Data from all neurons (6 neurons shown in C) were pooled for each group for statistical comparison. ∗∗p < 0.01, Kolmogorov-Smirnov test (p = 0.0075). AAVdj/Syn.GCaMP7f was used.