Skip to main content
. 2019 Oct 18;8:e43775. doi: 10.7554/eLife.43775

Figure 3. Caudal and lateral hypothalamic responses to prey sensory cues are anti-correlated over short timescales.

(a) Top: Transgenic fish (2 hr food-deprived) with GCaMP6s expressed in cH and LH neurons were paralyzed, tethered in agarose with their eyes and nostrils freed and exposed to live paramecia (prey), as described in Materials and methods. Top image: GCaMP expression in the cH and LH driven by two transgenic lines, Tg(116A:Gal4) and Tg(76A:Gal4) respectively. Bottom image: Downsampled image stack used for analysis in (f). (b) Top: Mean calcium activity (Δf/f) from respective hypothalamic ROIs (shown in (a)) from four individual fish during a baseline food-deprived period (Dep.), exposure to water alone (Water), and a dense water drop of paramecia (Para). Traces from left and right hypothalamic lobes of the same animal are overlain, revealing a high degree of correlated activity on opposite sides of the midline. Paramecia presentation increases activity in the LH and reduces activity in the cH, revealing opposing activity on short timescales. Bottom: Δf/f traces within area marked by gray box (top), displayed at higher magnification. An increase in LH activity and corresponding reduction in cH activity is observable within seconds of paramecia presentation, except for fish D in which maximal responses only occur after a few minutes (beyond the displayed time window). (c) Average Δf/f triggered on lLH calcium spikes (left and right lobes averaged) shows a mean corresponding reduction in cH activity (n = 159 lLH spikes extracted from mean Δf/f traces from 14 fish across the entire duration of the experiment). (d) Raster plots showing mean calcium activity from the hypothalamic lobes (left and right lobes averaged) of 14 fish before and after presentation of water alone and water with paramecia. (e) Quantification of integrated fluorescence (sum Δf/f %), calcium spike frequency (spikes/min) and calcium spike amplitude (Δf/f %) per fish across experimental epochs (300 s food-deprived baseline (D), 300 s after water (W) delivery or 600 s after paramecia delivery (P)). Each colored line represents data from an individual fish (left and right lobes averaged). Water alone was sufficient to significantly reduce cH integrated fluorescence (p = 6.1×10−5) and spike frequency (p = 0.0127) but not spike amplitude (p = 0.9324). Water alone was similarly sufficient to increase lLH integrated fluorescence (p = 0.029) and spike frequency (p = 0.0098) but not spike amplitude (p = 0.13). Conversely, water alone was not sufficient to significantly modulate mLH integrated fluorescence (p = 0.48) or spike frequency (p = 0.20), but was sufficient to increase spike amplitude (p = 0.039). Paramecia delivery significantly increased mLH and lLH integrated fluorescence (mLH, p = 1.2×10−4; lLH, p = 0.045) and spike frequency (mLH, p = 6.1×10−5; lLH, 6.1 × 10−4), while only significantly increasing mLH spike amplitude (mLH, p = 0.045; lLH, p = 0.43), relative to water delivery. In contrast, paramecia delivery significantly reduced cH integrated fluorescence relative to water delivery alone (p = 3.1×10−4), but not spike frequency (p = 0.52) nor spike amplitude (p = 0.85). Asterisks denote p<0.05, one-tailed Wilcoxon signed-rank test. (f) Top: Cross-correlogram of hypothalamic cell-sized voxels (cells and/or neuropil from downsampled image stacks, see (a)) from four fish. The cH and LH voxels were mostly anti-correlated, whereas voxels within each cluster displayed correlated activity. Black arrowheads indicate region of lLH that appears to be most anti-correlated with the cH. Bottom: Correlation coefficients of other hypothalamic voxels relative to a selected voxel with the cH, mLH or lLH. See color key for numerical translation of color maps. (g) Summary of data from 14 fish, showing the probability of the nth most anti-correlated voxel belonging to each of the other regions (cH, mLH or lLH), normalized to chance probability (gray line) of belonging to each region (i.e. the fraction of all voxels occupied by each region). For example, if we consider all the voxels within the cH, there is a four-fold probability relative to chance of their most anti-correlated voxels (Rank = 1) being part of the lLH.

Figure 3.

Figure 3—figure supplement 1. Characterization of the 116A:Gal4 line.

Figure 3—figure supplement 1.

(a) Z-projection images of whole mount Tg(116A:Gal4;UAS:GFP) fish at low (left) and high (right) intensities. Scale bar = 100 μm. (b) Overlap of Tg(116A:Gal4;UAS:GFP) (green) with anti-5-HT (magenta) immunostaining is seen in all layers of the caudal hypothalamus, as well as the anterior and posterior paraventricular organ (aPVO and pPVO). Each row shows a different z-plane, moving from dorsal to ventral. Scale bar = 50 μm. (c) Higher magnification images of the cH, aPVO and pPVO from left side of image in (c). (d) Minimal overlap of Tg(116A:Gal4;UAS:nfsb-mCherry) (magenta) with dopaminergic neurons labeled by Tg(TH2:GCaMP5) (green). Note that the Tg(116A:Gal4;UAS:nfsb-mCherry) transgenic, which is used in ablation experiments, shows sparser labeling than with Tg(UAS:GFP). In this fish, 2 out of 17 (11.8%) of Tg(116A:Gal4;UAS:nfsb-mCherry) cells overlapped with Tg(TH2:GCaMP5) expression. Scale bar = 50 μm. (e) Quantification of 5-HT overlap with Tg(116A:Gal4;UAS:GFP) in the cH, aPVO and pPVO.
Figure 3—figure supplement 2. Overlap of 116A:Gal4 and 76A:Gal4 driven reporter expression with hypothalamic activity under conditions of food deprivation and feeding.

Figure 3—figure supplement 2.

(a) mLH and lLH activity in voraciously-feeding (food-deprived 2 hr + 15 min paramecia) fish overlaps with Tg(76A:Gal4;UAS:GCaMP6s) expression (green, dissected brains). All visible pERK-positive neurons (magenta) were also co-labeled with GCaMP6s. Tg(116A:Gal4) is also expressed (green). Scale bar = 50 μm. (b) mLH and lLH activity in voraciously-feeding fish overlaps with Tg(76A:Gal4;UAS:GCaMP6s) expression (whole-mount). All visible pERK-positive neurons were also co-labeled with GFP. Note that more dorsally and anteriorly (as visible in the third panel of (i), and the z-projection in (ii)) other neurons beyond the LH are labeled by Tg(76A:Gal4;UAS:GCaMP6s). Scale bar = 50 μm. (c) pERK-positive cells (magenta) in 2 hr food-deprived fish overlap partially with Tg(116A:Gal4) expression (green, dissected brains). (i) Overlap with Tg(116A:Gal4;UAS:GFP) (ii) Overlap with Tg(116A:Gal4;UAS:nfsb-mCherry). Scale bar = 20 μm.
Figure 3—figure supplement 3. Calcium imaging of the cH and LH over food deprivation.

Figure 3—figure supplement 3.

Note that fish were imaged ~20 min after embedding, thus initial food deprivation time is already 20 min. Hence, the initial reduction in LH active cell count, which occurs within 30 min (Figure 2) may not be observable using this imaging method. (a) Fish 1 and 2 were imaged using volumetric imaging for 115 min, whereas fish 3 and 4 were imaged only at a single plane, and for a slightly shorter time period of 90 min (see images in (b)) (i): Mean Δf/f across the entire (both lobes) of the cH, mLH and lLH (i.e. raw) show increases in baseline fluorescence over time. (ii) Mean Δf/f with baseline subtracted (i.e. detrended). Since a rising baseline over long imaging periods is difficult to interpret (see text for discussion), we also display detrended traces. (b) (i): Average intensity projection images showing imaged regions with ROIs outlined. (ii) Spike-triggered averages based on extracted lLH calcium spikes (from detrended traces) usually reveal an accompanying reduction in cH calcium fluorescence (Δf/f). (c) Calcium spike frequency (spikes/min, left) and calcium spike amplitude (Δf/f %, right) for each ROI averaged over 5 min bins throughout the imaging session for the above four fish. Colored lines are the means, shaded areas reflects SEM. (d) Over the entire imaging period, calcium spike frequency (left) was significantly higher in the cH as compared to the mLH (p = 0.014) and lLH (p = 0.014). Calcium spike amplitude (right) was also significantly higher in the cH as compared to the mLH (p = 0.014), but not the lLH (p = 0.057), asterisks denote p<0.05, one-tailed Wilcoxon rank-sum test.