Whole-brain activity mapping onto a zebrafish brain atlas

Abstract

In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open-source atlas containing molecular labels and definitions of anatomical regions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated extracellular signal–regulated kinase (ERK) as a readout of neural activity, we have developed a system to create and contextualize whole-brain maps of stimulus- and behavior-dependent neural activity. This mitogen-activated protein kinase (MAP)-mapping assay is technically simple, and data analysis is completely automated. Because MAP-mapping is performed on freely swimming fish, it is applicable to studies of nearly any stimulus or behavior. Here we demonstrate our high-throughput approach using pharmacological, visual and noxious stimuli, as well as hunting and feeding. The resultant maps outline hundreds of areas associated with behaviors.

Figure 1. Analysis pipeline: creating the zebrafish reference brain atlas (Z-Brain) and whole-brain activity maps (MAP-Maps)

A) tERK Confocal stacks registered to a reference brain. Shown are three fish (cyan, magenta, yellow) as maximum intensity projections. B) Registrations are applied to an anatomical label (reticulospinal backfills). C) The mean across all registered fish is calculated. Shown are Z and X maximum intensity projections D) Mean-stacks from a total of 29 transgenic, antigenic, or dye labels were generated (Supplementary Table 1). Shown are maximum intensity Z and X projections of 16 different labels (left), and a color Z and X random color projection image for 21 labels (right) E) Z and X mean projections of the outlines of the segmented Z-Brain regions, drawn in colors biased towards green = Telencephalon, cyan = Diencephalon, yellow = Mesencephalon, red = Rhombencephalon, magenta = Spinal Cord, F) To create a MAP-Map, pERK/tERK confocal stacks are acquired for ~10–30 fish per condition. G) Stacks are registered to Z-Brain, and the pERK level statistics are calculated at each voxel. H) Voxels found exhibiting significantly higher (green) and lower (magenta) pERK levels in the stimulus group are localized to create a MAP-Map (Online Methods). Shown are mean Z and X projections for heat exposure (Fig. 4c). I, J) The MAP-Map is then analyzed using the Z-Brain (see Online Methods). Shown is the Z and X maximum intensity projections depicting the mean signal in the Z-Brain regions. Scale bars represent 50um. R = Right, L = Left, A = Anterior, P = Posterior, D = Dorsal, V = Ventral.

Figure 2. pERK is a neural activity sensor in zebrafish neurons

A) A confocal slice of a fish stained for phosphorylated-ERK (pERK, yellow) and total-ERK (tERK, magenta), from which we calculate the normalized ‘pERK level’ (pERK/tERK). Te = Telencephalon, Me = Mesencephalon, Rh = Rhombencephalon. Boxes depict the approximate x/y positions of neurons shown in panels B and E, but in a different z-plane. B) ChR2-YFP was driven in multiple neuron types in Tg(−6.7FRhcrtR:gal4VP16);Tg(14xUAS-E1b:hChR2(H134R)-EYFP); atoh7^th241/th241; Tg(atoh7:GAP-RFP) larvae. Shown are neurons of the tangential and median vestibular nucleus (tVN and mVN), stimulated with either blue or green light. C) ChR2 activation significantly increased pERK levels (p = 8.03×10–35, ranksum test, n =1056 neurons from 20 larvae). In the boxplots, red line = median, blue box = 25^th and 75^th quartiles, whiskers extend to the most extreme non-outliers, red crosses mark points considered outliers. D) The total number of seconds an ROI was active during Ca²⁺ imaging plotted against the pERK level after fixation, revealing a significant correlation (p=1.1 × 10⁻⁶⁶, R = 0.40, Pearson’s correlation, n=1771 ROIs). Plotted are all of the ROIs (grey), the mean in 7-second x-bins (blue), and the linear best-fit line (red). E) Cells exhibiting Ca²⁺ activity localized by correlation (Online Methods and), compared their pERK level (green) reveals many instances of good signal correspondence (arrows), as well as potential false positives (asterisks) and false negatives (arrowheads). F) MAP-Map from exposure to 8.25mM pentylenetetrazol (PTZ) for 15 min. n=12/12 (PTZ/ no drug controls). Subpallium (SP), hypothalamus (Hy) and area postrema (AP). Smaller brain insets depict the mean signal within each Z-Brain region in this and other MAP-Map panels G) MAP-Map from exposure to 15mM MS-222 for 1hr. Telencephalon (Te) and preoptic area (PO), olfactory epithelium (OE), caudal hindbrain (cHB) and hypothalamus (Hy) n=12/12 (MS-222/no drug controls). H) MAP-Map from a 10 second light pulse delivered 30 seconds before fixation. n=15/16 (light pulse/darkness controls). Subpallium (SP), tectal neuropil (TN), cerebellum (CB) and hindbrain (HB). I) Mean pERK level (+/− SEM) in retinal arborization fields (AFs, 1–10) for dark adapted larvae (red), and at different chase-times after delivery of a 10 second light pulse (red), and J) resultant MAP-Maps. n = 8/13/12/13/13/13/13 (10sec/1min/2min/5min/10min/30min/Darkness). Scale bars represent 50um.

Figure 3. Neural activity underling the optomotor response

A) Larvae were presented with gratings moving to the right (green) or left (magenta). B) This induces the optomotor response (OMR) and turning in the direction of motion (n = 18 fish per group, mean +/− SEM). C) MAP-Map highlighting the differential activity of fish presented in B, revealing increased activity for rightward (green) and leftward (magenta) motion. Pretectum (PT) and anterior hindbrain (aHB). (n = 17/18, right/left). D) Two-photon GCaMP5G Ca²⁺ imaging data from 17 fish stimulated with moving gratings were registered into the Z-Brain, and compared to the MAP-Map. Shown are Z and X projections. E) Virtual colocalization analysis to Z-Brain labels, comparing the OMR-induced activity in the medial-aHB (m-aHB) and lateral-aHB (l-aHB) to the Tg(Gad1b:GFP) label. The MAP-Map activity patterns are shown as outlines of the activated areas. F) Tg(Gad1B:GFP) fish were presented with gratings moving to the right, to compare the pERK level within the Gad1B-positive cells in the m-aHB and l-aHB. Shown are pERK level probability histograms, revealing significantly increased pERK levels on the right side of the brain (p=4.3×10⁻²⁵, and p=8.2×10⁻¹⁵ for the m-aHB and l-aHB respectively, ranksum test, n=8 fish). The inset shows the results for non-GFP labeled cells, which do not show such a strong (although still significant) shift in distribution (p=6.9×10⁻⁴, and p=3.6×10⁻³ for the m-aHB and l-aHB, respectively). Scale bars represent 50um.

Figure 4. Activity induced by aversive stimuli and hunting/feeding

Fish were exposed to aversive stimuli for 15 minutes, and MAP-Mapped A) 10uM Mustard oil vs. DMSO controls (n = 19/18 fish) B) Dish-taps vs. no taps controls (n = 28/29 fish) C) 37°C Heat vs. room temperature controls (n = 23/21 fish) D) Electric shocks vs. controls (n = 21/21 fish) E) The intersection of the MAP-Maps in A–D. Subpallium (SP), preoptic area (PO), caudal hypothalamic neural cluster (cHY). F) Z-Brain virtual colocalization reveals co-activation of the locus coeruleus (LC) labeled by Et(Vmat2:GFP), and G) cells labeled by the Tg(−6.7FRhcrtR:gal4VP16;UAS:Kaede) line in the caudal hindbrain H) 2-photon Ca2+ imaging of Tg(−6.7FRhcrtR:gal4VP16);Tg(UAS:GCaMP5G) transgenic larvae stimulated with dish-taps and electric shocks. Functional images depict fluorescence correlation with the stimuli. i) MAP-Map revealing the activity induced by a 1hr of paramecia exposure, mapped relative to a non-fed control group (n = 28/23). Tectal neuropil (TN), hypothalamus (Hy), area postrema (AP), subpallium (Sp), ventral hindbrain (vHB), inferior olive (IO), cerebellum (CB), dorsal-caudal hindbrain (dcHB). Scale bars represent 50um

Figure 5. Spatial independent component analysis across fish as a method to localize functional brain networks

A) The pERK level stack is reshaped into a vector, and the vectors from 820 fish are then combined into an array for independent component analysis (ICA) (see Online Methods) B–D) Voxels for each recovered independent component (IC) are painted with their intensity proportional to the z-score of the loadings of the ICA signal linearly mapped between z = 1–4, and are shown as maximum Z and X projections. B) IC #4 highlights a putative motor network, which associates regions overlapping with reticulospinal neurons (RS), the nucleus of the medial longitudinal fascicle (nucMLF), and the spinal cord. Overlap with the spinal backfill Z-Brain label is shown as an X-projection over the boxed area (right). C) IC #17 highlights a putative visual response network. i) This IC overlaps with areas of the retinal arborization fields (AF) 4, 8 and 9, and the tectal neuropil (TN) labeled by Tg(Isl2b:Gal4);Tg(uas:Dendra) in the Z-Brain. ii) Prominent signals are also observed in the left habenula (L-Hab) and interpeduncular nucleus (IPN). Dashed lines represent the position of the resliced views in ii and iii. D) IC #21 highlights a putative octavolateral network, since it contains prominent signals in the torus semicircularis (TS). Foci of signal in the rostral hypothalamus overlap with the cell bodies of Tg(Qrfp:GFP) labeled neurons (right panel, arrows), which send projections to the TS (arrowheads), implicating these cells in the network.