Evolution of a central dopamine circuit underlies adaptation of light-evoked sensorimotor response in the blind cavefish, Astyanax mexicanus

Abstract

Adaptive behaviors emerge in novel environments through functional changes in neural circuits. While relationships between circuit function and behavior have been well studied, how evolution shapes those circuits and leads to behavioral adpation is poorly understood. The Mexican cavefish, Astyanax mexicanus, provides a unique genetically amendable model system, equipped with above ground eyed surface fish and multiple evolutionarily divergent populations of blind cavefish that have evolved in complete darkness. These differences in environment and vision provide an opprotunity to examine how a neural circuit is functionally influenced by the presence of light. Here, we examine differences in the detection, and behavioral response induced by non visual light reception. Both populations exhibit photokinetic behavior, with surface fish becoming hyperactive following sudden darkness and cavefish becoming hyperactive following sudden illumination. To define these photokinetic neural circuits, we integrated whole brain functional imaging with our Astyanax brain atlas for surface and cavefish responding to light changes. We identified the caudal posterior tuberculum as the central modulator for both light or dark stimulated photokinesis. To unconver how spatiotemporal neuronal activity differed between surface fish and cavefish, we used stable pan-neuronal GCaMP Astyanax transgenics to show that a subpopulation of darkness sensitve neurons in surface fish are now light senstive in cavefish. Further functional analysis revealed that this integrative switch is dependent on dopmane signaling, suggesting a key role for dopamine and a highly conserved dopamine circuit in modulating the evolution of a circuit driving an essential behavior. Together, these data shed light into how neural circuits evolved to adapte to novel settings, and reveal the power of Astyanax as a model to elucidate mechanistic ingiths underlying sensory adaptation.

Figure 1.

Cavefish exhibit a negative photokinesis index in comparison to a positive photokinesis index in surface fish. a Diagram illustrating eyed fish behavior and the light cycles used in the photokinesis experiments. Surface fish exhibit hyperactive behavior in dark cycles. Larvae were dark adapted before 5 minute alternating cycles of light on (yellow) to light off (black). b Line graph showing the inverse behavioral patterns of hyperactive behavior for cavefish (lights on=yellow bars) and surface fish (lights-off=black bars). c Photokinesis index box plots quantifying relationship between increased activity and light cycle (positive = light off, negative = light on). d. Average velocity of 30 second binned light on to light off transitions. Sample sizes, surface fish (n=42) and cavefish (n=34). Statistical significance represents output from a Tukey multiple comparisons corrected one-way ANOVA. ** = p<0.01, **** = p<0.0001.

Figure 2.

Brain mapping identifies overlap and variation of neural activity in the ventral forebrain between surface fish and cavefish. The first column displays a sagittal maximum projection through the middle of the brain, with directional arrows representing dorsal (D), ventral (V), rostral (R) and caudal (C). The second column provides an optical section through the Astyanax brain atlas and tERK reference brain. The last two columns contain pERK neural activity maps displaying regions of increased pERK immunostaining during lights off (magenta) or lights on (green) in surface fish and cavefish. Rows represent dorsal to ventral optical sections through the (a) pineal (P) and optic tectum (TeO), (b&c) thalamus (Th) and tegmentum (T), and finally (d&e) the subpallium (SP), preoptic region (PO), posterior tuberculum (PT) and intermediate hypothalamus (Int Hyp). Sample sizes, surface fish (n=18) and cavefish (n=20).

Figure 3.

Photokinesis behavior in F₂ hybrids is highly variable and photokinesis indices are negatively correlated to brain region size. a Diagram illustrating the crossing scheme for producing F₂ hybrid larvae to correlate behavior to biological traits. b Scatter plot displaying the relationship between light-on and light-off transition states. Surface fish are grouped along the y-axis, cavefish along the x-axis, and F₂ hybrids are scattered across both grandparental populations. c Photokinesis index violin plots exhibiting the range of behaviors of F₂ hybrids, from positive to negative photokinesis values. Scatter plot of brain region volume against photokinesis behavior for (d) tegmentum, (e) hypothalamus and (f) posterior tuberculum Sample sizes, surface fish (n=42), cavefish (n=34), surface to cave F₂ hybrids (n=199).

Figure 4.

Clusters of neurons in the posterior tuberculum of cavefish exhibit light-on tuning in comparison to light-off tuning in surface fish. a. Diagram illustrating the experimental setup of light stimulated agarose embedded surface and cavefish GCaMP6s larvae. Projection inset shows the segmented region analyzed in the live imaging dataset. Surface fish (b) and cavefish (c) timeseries clusters based on stimulus tuning, dark cycles (grey stripes) or light cycles (yellow stripes). Light condition peaks are noted by black arrows for cluster 4 (red) and cluster 5 (green). Coronal projections represent analyzed regions and display color coded neuronal clusters. d Box plots comparing average delta f over f for lights-off and lights-on conditions of clusters 4 and 5 in the caudal posterior tuberculum. Sample size, surface fish (n=4) and cavefish (n=5). Statistical significance represents t-test comparisons between populations for each cluster according to light conditions, ** = p<0.01, *** = p<0.001, **** = p<0.0001

Figure 5.

Light tuned PT neurons are dopaminergic and photokinesis is extinguished by dopamine antagonism or physical ablation. a pipeline for live to fix imaging; neural activity is recorded, larvae are fixed in agarose and stained with RNA probes. b Stained larvae are then imaged and registered to a maximum projection of the live stack. c-e Medial projection of the posterior tuberculum with colored dots and arrows corresponding to neural activity traces for surface fish (d) and cavefish (e). Images resolutions are (b&c) 512 × 512, zoom 1.2, and (d&e) 512 × 128, zoom 2.4. f Violin plot showing photokinesis indices for surface fish, cavefish, surface exposed to haloperidol and exposed to haloperidol. Sample sizes, surface fish (n=18), cavefish (n=10), surface fish + haloperidol (n=15) and cavefish + haloperidol (n=12). g Examples of 2-photon sham, posterior tuberculum (PT) and tyrosine hydroxylase (th) positive ablations. h Photokinesis indices following ablation experiments. Sample sizes, cavefish (n=14), cavefish sham ablations (n=6), cavefish PT ablations (n=7) and cavefish PT +th ablations (n=3). Scale bars, 512 × 512 = 50 μm and 512 × 128 = 20 μm. Statistical significance represents a Tukey multiple comparisons corrected one-way ANOVA. *** = p<0.001, **** = p<0.0001