Functional MRI in the Nile crocodile: a new avenue for evolutionary neurobiology

Abstract

Crocodilians are important for understanding the evolutionary history of amniote neural systems as they are the nearest extant relatives of modern birds and share a stem amniote ancestor with mammals. Although the crocodilian brain has been investigated anatomically, functional studies are rare. Here, we employed functional magnetic resonance imaging (fMRI), never tested in poikilotherms, to investigate crocodilian telencephalic sensory processing. Juvenile Crocodylus niloticus were placed in a 7 T MRI scanner to record blood oxygenation level-dependent (BOLD) signal changes during the presentation of visual and auditory stimuli. Visual stimulation increased BOLD signals in rostral to mid-caudal portions of the dorso-lateral anterior dorsal ventricular ridge (ADVR). Simple auditory stimuli led to signal increase in the rostromedial and caudocentral ADVR. These activation patterns are in line with previously described projection fields of diencephalic sensory fibres. Furthermore, complex auditory stimuli activated additional regions of the caudomedial ADVR. The recruitment of these additional, presumably higher-order, sensory areas reflects observations made in birds and mammals. Our results indicate that structural and functional aspects of sensory processing have been likely conserved during the evolution of sauropsids. In addition, our study shows that fMRI can be used to investigate neural processing in poikilotherms, providing a new avenue for neurobiological research in these critical species.

Keywords: audio-visual stimulation; dorsal ventricular ridge; hierarchical processing; poikilotherms; reptile.

Figure 1.

Experimental set-up and stimulation procedure. (a) Custom-made restrainer. Mildly sedated crocodiles were placed in an MRI compatible tube. To immobilize the animal in Y and Z directions, the snout, tail and back of the animal were taped to the tube. To control motion artefacts in X direction, the jaw and head were secured by blocks of Plasticine. (b) Timing of visual stimuli. The visual stimuli consisted of binocular visual stimulation using two different colours (green and red), with a flickering frequency at 2, 5 or 8 Hz. Each stimulus was presented in 20 repeated blocks consisting of 24 s light followed by 36 s darkness. (c) Timing of auditory stimuli. Three different stimuli were used to stimulate the auditory system. Random chords centred around 1000 or 3000 Hz were used as simple stimuli, while classical music (by Johann Sebastian Bach) was used as a complex stimulus. Stimuli were played using a speaker in front of the animals. Stimulation was done in 40 repeated blocks with each block consisting of 12 s sound followed by 18 s silence. As an example, the spectrogram of the classical music stimulus is shown.

Figure 2.

Activation of the Nile crocodile forebrain after binocular visual stimulation. (a) 3D view and two axial slices illustrating the spatial pattern of significant signal changes during binocular visual stimulation with red light flickering at 8 Hz. Significant activations were found in the thalamic nucleus rotundus (Rt, left slice) and a forebrain area strongly resembling the avian entopallium (cVs_I, right slice) (group results, mixed-effect model, FLAME1 + 2, p < 0.05, N = 5). (b–e) Mean BOLD response of all five crocodiles in cVs_I and Rt measured after stimulation with red or green light at 2, 5 or 8 Hz flickering frequency. As the shape of BOLD responses did not differ between left and right hemisphere, the average of both hemispheres is plotted. The larger variation of BOLD responses in Rt in comparison with cVs_I were caused by the higher distance between the surface coil and Rt, causing an increase in the noise level. Stimulation intervals are marked in grey. Error bars represent s.e.m.

Figure 3.

Auditory activation in the Nile crocodile brain in response to different stimuli. (a) 3D dorsal view representing the location of significant BOLD activations after stimulation. While auditory areas cAu_I and cAu_II responded to all stimuli, cAu_III was only activated in response to the complex stimulus (music by Bach). Mean BOLD response of all four crocodiles in areas cAu_I (b), cAu_II (c), cAu_III (d) measured after stimulation with random chords, centred around 1000 (black line) or 3000 Hz (blue line) and classical music (red line). As the shape of BOLD activations did not differ between left and right hemisphere, the average of both hemispheres is plotted. Stimulation intervals are marked in grey. Error bars represent the standard error of the mean (s.e.m.). Z-score maps were acquired during classical music presentation (group results, mixed-effect model, FLAME1 + 2, p < 0.05, N = 4).