Zona incerta subpopulations differentially encode and modulate anxiety

Abstract

Despite recent clinical observations linking the zona incerta (ZI) to anxiety, little is known about whether and how the ZI processes anxiety. Here, we subject mice to anxious experiences and observe an increase in ZI c-fos–labeled neurons and single-cell calcium activity as well as an efficient effect of ZI infusion of diazepam, a classical anxiolytic drug. We further identify that somatostatin (SOM)–, calretinin (CR)–, and vesicular glutamate transporter-2 (Vglut2)–expressing cells display unique electrophysiological profiles; however, they similarly respond to anxiety-provoking stimuli and to diazepam. Optogenetic manipulations reveal that each of these ZI neuronal populations triggers specific anxiety-related behavioral phenotypes. Activation of SOM-expressing neurons induced anxiety, while photoactivation of CR-positive cells and photoinhibition of Vglut2-expressing neurons produce anxiolysis. Furthermore, activation of CR- and Vglut2-positive cells provokes rearing and jumps, respectively. Our findings provide the first experimental evidence that ZI subpopulations encode and modulate different components of anxiety.

Fig. 1.. Anxious experience enhances ZI neuronal activation.

(A) Experimental setup where mice were placed on a narrow elevated rod. (B) Schematic of the control home cage condition. (C) Example confocal image of the immunohistochemical (IHC) staining against c-fos (green) in the ZI-containing coronal brain slices from mice that were exposed to the anxious elevated rod experience. Scale bar, 100 μm. (D) Example confocal image of the IHC labeling of c-fos in the ZI-containing coronal brain slices of mice that were left in the control home cage condition. Scale bar, 100 μm. (E) Bar graphs reporting the relative number of c-fos-labeled cells (normalized to the control mice) in the ZI for both experimental groups. Data are presented with means ± SEM. ****P < 0.0001.

Fig. 2.. ZI neurons are sensitive to anxiety-related cues.

(A) Surgical setup to express and visualize GCaMP6m in the ZI of wild-type (WT) C57BL/6 mice. (B) Example confocal image of a partial coronal section containing the GRIN lens tract and the GCaMP6m-expressing (green) cells in the ZI. Scale bars, 500 and 50 μm. (C) Schematics of the elevated-platform design with the compartments (edge, transition, and center) annotated. (D) Bar graph reporting the percentage of time mice explored the center (C) and edges (E). (E) Raw frame extracted from a ZI calcium recording (left) and maximum intensity projection visualization of identified cells (right) with example cells color coded. (F) Track plot of a single mouse (top) along the elevated platform (different compartments highlighted). Time-locked heatmap of calcium activity (represented in z scores) of all recorded cells (middle) and traces from example cells [(bottom; corresponding to (E)]. (G) Scatterplots of example recorded cells with calcium activity positively (red), negatively (blue), and not (black) correlated to the distance from the center in the recorded mouse. (H) Pie chart depicting the proportion of cells whose calcium activity correlate to the center distance. (I) Bar graphs reporting the differences in calcium activity while mice were in the edges versus the center. (J) Transient changes in activity as mice entered the edge. Schematic of the positional change (top). Line plot (left) depicting the average activity 2.5 s before and after the positional change. Heatmap (right) of the activity of all cells with a vertical bar depicting the type of correlation. Bar graphs (right) comparing the change in calcium activity between following the positional change. (K) Transient changes in activity as mice exited the edge. (L) Transient changes in activity as mice exited the center. (M) Transient changes in activity as mice entered the center. Data are presented with means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.. Local ZI diazepam infusion reduces anxiety without affecting movement.

(A) Surgical setup to place cannulas above the ZI. (B) Example confocal image of a partial coronal section containing the cannula tract with the red RetroBeads in the ZI to show the extent of spread. Scale bars, 500 and 250 μm. (C) Schematic of the setup to directly infuse diazepam (1 mg/kg) into the ZI or vehicle 30 min before testing the mouse on the EOM. (D) Track plot of a single example mouse on the EOM (dashed lines denote the open arms, while the filled lines indicate the closed arms) after vehicle (left) and diazepam (right) infusion with the speed color coded. (E) Bar graph of time the mice spent in the open arms after vehicle and diazepam infusions. (F) Bar graph depicting the number of times mice entered the open arm after vehicle and diazepam infusions. (G) Track plot of an example mouse in the OFT after vehicle (left) and diazepam (right) infusion with the speed color coded. (H) Bar graph depicting the distance mice traveled after vehicle or diazepam infusions. (I) Bar graph showing the time mice spent immobile after vehicle or diazepam infusions. Data are presented with means ± SEM. *P < 0.05.

Fig. 4.. SOM-, CR-, and Vglut2-expressing ZI neurons display unique electrophysiological properties.

(A) Example confocal images of ZI-containing coronal brain slices with the FISH labeling of the mRNAs for Vglut2 (green) and Vgat (cyan) and the merged image. Scale bars, 100 and 25 μm. (B) Pie chart quantification of the proportion of cells in ZI expressing Vglut2, Vgat, or both (3390 cells total, three mice). (C) Example confocal images of the ZI-containing coronal brain sections with the FISH labeling of the mRNAs for SOM (red), CR (green), PV (cyan), and the merged. Scale bars, 100 and 25 μm. (D) Pie chart representation of the percentage of expressing the mRNAs for SOM, CR, PV, or any combination of the three (1684 cells total, three mice). (E) Schematic of the experimental procedure. (F) Example bright-field (top) and fluorescent (bottom) images of patched cells: SOM (cyan), CR (magenta), and Vglut2 (maroon). (G) Bar graphs reporting the capacitance from SOM-, CR-, and Vglut2-expressing ZI cells. (H) Bar graphs depicting the resting membrane potential (Vm) from SOM-, CR-, and Vglut2-expressing ZI cells. (I) Bar graphs of the action potential width from SOM-, CR-, and Vglut2-expressing ZI cells. (J) Bar graphs presenting the rheobase from SOM-, CR-, and Vglut2-expressing ZI cells. (K) Bar graphs summarizing the maximum firing frequency from SOM-, CR-, and Vglut2-expressing ZI cells. (L) Example current clamp recordings from SOM-, CR-, and Vglut2-expressing ZI cells upon various current step injections. (M) Summary input/output curve for SOM-, CR-, and Vglut2-positive cells. Data are presented with means ± SEM. **P < 0.01, ****P < 0.0001.

Fig. 5.. Diazepam potentiates inhibitory drive onto SOM-, CR-, and Vglut2-expressing ZI neurons.

(A) Schematic of the experimental setup. (B) Example IPSC from representative SOM-expressing (cyan), CR-expressing (magenta), and Vglut2-expressing (maroon) ZI cells during baseline (base) and following subsequent bath application of diazepam (Dz) and PTX. Scale bars, 100 pA (vertical) and 25 ms (horizontal). Bar graphs report the amplitude of the IPSCs and the paired-pulse ratio (PPR) of both conditions. (C) Example sIPSCs (spontaneous inhibitory post-synaptic currents) recorded from representative SOM-, CR-, and Vglut2-expressing ZI cells during baseline (base) and during bath application of diazepam (Dz) and PTX. Scale bars, 30 pA (vertical) and 500 ms (horizontal). Bar graphs of the amplitude and the frequency of sIPSCs in the baseline and following diazepam application are shown. Data are presented with means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.. Optogenetic manipulation of SOM-, CR-, and Vglut2-expressing ZI neurons triggers distinct anxiety-related traits.

(A) Schematic of the task. (B) Example confocal images of coronal brain slices with the expression of ChR2 in SOM-, CR-, and Vglut2-expressing ZI cells and optic fiber tract above (high-magnification inserts below the fiber). Scale bars, 25 μm. Laser stimulation protocols for ChR2 (bottom left) and Arch (bottom right). (C) Example track plots of mice on the EOM (dashed lines denote the open arms; filled lines represent the closed arms) during laser OFF (top) and ON (bottom, shaded) periods with the speed color coded. (D) Bar graph depicting the delta (ON-OFF) open arm time in mice expressing EYFP, ChR2, and Arch in SOM-expressing ZI cells. (E) Bar graph depicting the delta body entries into the open arms [corresponding to (D)]. (F) Bar graph depicting the delta open arm time in CR-CRE mice. (G) Bar graph depicting the delta body entries [corresponding to (F)]. (H) Bar graph depicting the delta open arm time in Vglut2-CRE mice. (I) Bar graph depicting the delta body entries [corresponding to (H)]. (J) Schematic of the experimental procedure. (K) Schematic of setup (top, extracted from movie S3). Laser stimulation protocol for ChR2 (bottom). (L) Example minimum intensity projection of 0.5-s and 1-min windows before and after laser stimulation (blue shaded) in mice expressing ChR2 in the Vglut2-expressing ZI cells. (M) Bar graph presenting the cumulative jumps made by mice expressing ChR2 in the Vglut2-expressing ZI cells. (N) Example minimum intensity projection of 15-s and 1-min windows in the stimulation off and on period (blue shaded) in mice expressing ChR2 in the CR-expressing ZI cells. (O) Bar graph presenting the cumulative rears made by mice expressing ChR2 in the CR-expressing ZI cells. Data are presented with means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Photo credit: Zhuoliang Li, University of Basel.