Research ArticleNEUROSCIENCE

Cavefish brain atlases reveal functional and anatomical convergence across independently evolved populations

See allHide authors and affiliations

Science Advances  16 Sep 2020:
Vol. 6, no. 38, eaba3126
DOI: 10.1126/sciadv.aba3126
  • Fig. 1 Behavioral and neuroanatomical evolution in larval A. mexicanus.

    (A) Image of 6–dpf (days postfertilization) fish. Scale bar, 500 μm. (B) Total sleep over 24 hours in 6-dpf A. mexicanus [one-way analysis of variance (ANOVA), F = 51.53, P < 0.0001; Dunnett’s to surface: Molino, P < 0.001; Pachòn, P < 0.001; and Tinaja, P < 0.001). (C) Feeding angle orienting to prey (0 ms) and then immediately after strike (20 ms) (Kruskal-Wallis test = 13.39, P = 0.003; Dunnett’s to surface: Molino, P > 0.56; Pachón, P < 0.01; and Tinaja, P < 0.03). ns, not significant. (D) Standard brain with labels registered to the template brain (left). Segmentations applied to the template brain (middle). Merge of standard brain with anatomical segmentations (right). (E) Registrations of Huc:GCaMP6s reveals overlap of tERK with transgenic label. (F) Projection of anatomical overlap between Huc:GCaMP (green) and tERK (magenta) of optic neuropil. (G) Segmentation of developmental regions of brains using tERK staining: telencephalon (green), diencephalon (magenta), mesencephalon (cyan), rhombencephalon (red), and spine (blue). Scale bar, 300 μm. (H) Developmental regions relative to whole-brain size. Two-way ANOVA: genotype, F = 13.91, P < 0.001; brain region, F = 3134, P < 0.001; interaction, F = 0.65, P = 0.58. Dunnett’s: rhombencephalon, Molino, P < 0.01; Pachòn, P < 0.001; and Tinaja, P < 0.001. Mesencephalon, Molino, P < 0.001; Pachòn, P < 0.001; and Tinaja, P < 0.001. Diencephalon, Molino, P < 0.001; Pachòn, P < 0.001; and Tinaja, P < 0.01. Telencephalon, Molino, P = 0.74; Pachòn, P = 0.68; Tinaja, P > 0.89. (I) Volumetric projections of nuclei within the rhombencephalon: cerebellum (red) and locus coeruleus (green). Scale bar, 200 μm. (J) Nuclei within the mesencephalon. Optic tectum (blue) and optic neuropil (light green). Scale bar, 200 μm. (K) Nuclei in the diencephalon: pineal gland (light red), habenula (pink), preoptic hypothalamus (purple), rostral zone of the hypothalamus (green), intermediate zone of the hypothalamus (blue), diffuse nucleus of the hypothalamus (yellow), and pituitary complex (dark blue). Scale bar, 100 μm. (L) Nuclei within telencephalon: subpallium (orange) and pallium (light green). Scale bar, 100 μm. (M) Quantifications of (F) to (I) normalized to surface fish. *P ≤ 0.05, **P < 0.01, and ***P < 0.005 for indicated comparisons in all statistical tests.

  • Fig. 2 Whole-brain imaging of circuits associated with sleep and feeding.

    (A) Whole-brain volumetric reconstructions of confocal imaging with anti-tERK (white) and anti-TH (green) for surface, Molino, Pachón, and Tinaja cavefish, with dorsal (left) and sagittal (right) views. Scale bar, 250 μm. (B to F) Numbers of cells expressing TH in distinct regions of the brain (B). TH+ cell quantification in the locus coeruleus (one-way ANOVA, F = 0.509, P > 0.68; Molino, P > 0.64; Pachón, P > 0.54; and Tinaja, P > 0.83). (C) TH+ cell number in the telencephalon (one-way ANOVA, F = 18.87; P < 0.001; Molino, P > 0.66; Pachón, P < 0.001; and Tinaja, P < 0.001). (D) Quantification of pretectal TH+ neuron cells (one-way ANOVA, F = 35.19, P < 0.001; Molino, P < 0.001; Pachón, P < 0.001; and Tinaja, P < 0.001). (E) Number of TH+ cells in the hypothalamus (one-way ANOVA, F = 20.16, P < 0.001; Molino, P < 0.001; Pachón, P < 0.001; and Tinaja, P < 0.001). (F) TH+ cell number in the medial octavolateralis nucleus (one-way ANOVA, F = 9.532, P < 0.001; Molino, P < 0.001; Pachón, P < 0.002; and Tinaja, P > 0.44). (G) Whole-brain volumetric reconstructions of confocal imaging with anti-tERK (white) and anti–α-MSH (green) for four populations of A. mexicanus. Scale bar, 300 μm. (H) Single-plane view of the α-MSH (green) cell cluster in the pituitary complex in surface fish and Molino, Pachón, and Tinaja cavefish. Scale bar, 50 μm. (I) Total number of cells expressing α-MSH in the pituitary complex. (J) Mean fluorescence intensity of α-MSH+ individual cells from (I). All comparisons were carried with n > 8, and all post hoc tests compared cavefish to surface fish. N > 8 for all measurements. *P ≤ 0.05, **P < 0.01, and ***P < 0.005 for indicated comparisons in all statistical tests.

  • Fig. 3 Whole-brain pERK neural activity imaging reveals altered landscape of brain activity.

    (A) Average pERK activity maps overlaid onto standard brains with segmentations in major brain subdivisions in the indicated population of A. mexicanus. Scale bar, 200 μm. (B) Scree plot for principal components analysis (PCA). Together, principal component 1 (PC1) and PC2 explain 44.78% of the variation in the PCA; further components do not meet the Kaiser criterion for further analysis. (C) Loading plot for PCA analysis showing the correlations between regions and the amount each region contributes to the PCs. Vectors that form small angles show correlated neural activity. (D) PCA of whole-brain neural activity in the brain of free-swimming fish. PC1 (one-way ANOVA, F = 8.019, P < 0.001; Dunnett’s post hoc: Molino, P < 0.003; Pachón, P < 0.001; and Tinaja, P > 0.95). PC2 (one-way ANOVA, F = 8.786, P = 0.0001; Dunnett’s post hoc: Molino, P < 0.001; Pachón, P < 0.05; and Tinaja, P = 0.001). Percentages indicate the amount of variance in neural activity explained by each PC. (E) Maximum-intensity projections of mean pERK signal in the rostral zone of the hypothalamus. Scale bar, 100 μm. (F) Quantification of pERK signal in rostral zone of the hypothalamus (one-way ANOVA, F = 4.69, P < 0.01; Molino, P < 0.01; Pachón, P < 0.04; and Tinaja, P ≥ 0.95). (G) Maximum-intensity projections of pERK signal in habenula. Scale bar, 100 um. (H) Quantification of pERK activity in the habenula (one-way ANOVA, F = 4.16, P = 0.012; Molino, P = 0.99; Pachón, P = 0.018; and Tinaja, P < 0.02). (I) Maximum-intensity projections of pERK signal in the pallium. Scale bar, 100 μm. (J) Quantification of pallial neural activity (one-way ANOVA, F = 6.18, P = 0.001; Molino, P < 0.001; Pachón, P < 0.03; and Tinaja, P < 0.04). N > 10 for all pERK activity mapping. *P ≤ 0.05, **P < 0.01, and ***P < 0.005 for indicated comparisons in all statistical tests.

  • Fig. 4 pERK neural activity during feeding reveals sensory transformation in cavefish and convergence on hypothalamic circuitry.

    (A) Average whole-brain pERK activity patterns registered to standard brains (white) in nonfeeding fish (green) and fish undergoing a 10-min feeding assay (magenta). Scale bar, 200 μm. (B) PCA of whole-brain activity (reflected by pERK signal) in fish undergoing 10-min feeding assay PC1 explained 27.53% of the variability of the brain activity (one-way ANOVA, F = 6.652, P = 0.001; Molino, P > 0.40; Pachón, P < 0.03; and Tinaja, P < 0.001). There were no statistical differences between populations along PC2, which explained 18.80% of the variability. (C) Maximum-intensity projection of pERK neural activity in medial octavolateralis nucleus (MON) activity for nonfed (green) and fed (magenta) fish. Scale bar, 50 μm. (D) Quantitation of the change in pERK activity in the MON during feeding (one-way ANOVA, F = 22.14, P < 0.001; Molino, Pachón, and Tinaja, P < 0.001). (E) Maximum-intensity projection of pERK activity in the optic tectum of nonfed (green) and feeding (magenta) fish. Scale bar, 200 μm. (F) Quantification of change in pERK activity in the optic tectum during feeding (one-way ANOVA, F = 6.13, P = 0.002; Molino, P > 0.02; Pachón, P < 0.001; and Tinaja, P > 0.04). (G) Maximum-intensity projection of pERK activity in the diffuse nucleus of the hypothalamus of nonfed (green) and feeding (magenta) fish. Scale bar, 100 μm. (H) Quantification of change in pERK activity in the MON during feeding (one-way ANOVA, F = 1.43, P = 0.24; Molino, P > 0.91; Pachón, P > 0.33; and Tinaja, P > 0.8). N > 10 for all feeding pERK neural activity.

  • Fig. 5 Whole-brain activity imaging of sleep-like state reveals heterogeneous neural signatures.

    (A) A 24-hour sleep quantification with control dimethyl sulfoxide (DMSO; green), EMPA (magenta), and propranolol (yellow) treatments (two-way ANOVA, genotype, F = 13.18, P < 0.01; drug treatment, F = 34.50, P < 0.001; genotype-phenotype, F = 2.13, P = 0.234; Dunn’s analysis: surface EMPA, P = 0.894; surface propranolol, P < 0.42; Molino EMPA, P < 0.02; Molino propranolol, P < 0.01; Pachòn EMPA, P = 0.011; Pachòn propranolol, P < 0.001; Tinaja EMPA, P < 0.001; and Tinaja propranolol, P = 0.034). (B) Whole-brain activity in DMSO (green), EMPA (magenta), or propranolol (yellow). Scale bar, 200 μm. (C) PCA of neural activity in fish treated with EMPA. PC1 explains 29.17% of the PCA variance (one-way ANOVA, F = 16.75, P < 0.001; Molino, P > 0.56; Pachón, P > 0.96; and Tinaja, P < 0.001). PC2 explains 22.78% of the variance (one-way ANOVA, F = 0.849, P > 0.47; Molino, P > 0.84; Pachòn, P > 0.74; and Tinaja, P > 0.90). (D) PCA of neural activity in sleeping fish treated with propranolol PC1 explains 31.94% of the variation (one-way ANOVA, F = 7.475, P < 0.001; Molino, P < 0.02; Pachón, P < 0.05; and Tinaja, P > 0.61). PC2 explains 18.60% of the neural activity variation (one-way ANOVA, F = 2.03, P = 0.131; Molino, P = 0.435; Pachòn, P = 0.579; and Tinaja, P = 0.921). (E) Average neural activity in preoptic area of the hypothalamus in awake DMSO (green) and sleeping EMPA (magenta) and sleeping propranolol (yellow). Scale bar, 100 μm. (F) Quantification of the change in pERK neural activity in the preoptic area of the hypothalamus in sleeping versus waking fish (two-way ANOVA, F = 6.959, P < 0.001; for EMPA treatment: Molino, P > 0.34; Pachón, P < 0.001; and Tinaja, P > 0.95; propranolol treatment: Molino, P < 0.02; Pachón, P < 0.001; and Tinaja, P < 0.001). (G) Hindbrain area containing TH+ locus coeruleus neurons (white circles) with average neural activity of awake DMSO (green) and sleeping EMPA-treated (magenta) or EMPA-propranolol–treated fish (yellow). Scale bar, 50 μm (H) Quantification of the change in pERK signal (two-way ANOVA, drug treatment, F = 1.71, P = 0.124; EMPA: Molino, P = 0.1; Pachòn, P = 0.743; and Tinaja, P = 0.727; for propranolol treatment: Molino, P > 0.97; Pachón, P > 0.99; and Tinaja, P > 0.99). *P ≤ 0.05, **P < 0.01, and ***P < 0.005 for indicated comparisons in all statistical tests.

Supplementary Materials

Stay Connected to Science Advances

Navigate This Article