The Retinal Wholemount Technique: A Window to Understanding the Brain and Behaviour

Abstract

The accessibility of the vertebrate retina has provided the opportunity to assess various parameters of the visual abilities of a range of species. This thin but complex extension of the brain achieves a large proportion of the necessary visual processing of an optical image before information is delivered to the brain as neural impulses. Studies of the retina as a wholemount or a flattened sheet of neural tissue are abundant due to the large amount of information that can be analysed, as follows: the level of summation or convergence; the coverage, stratification and potential sites of synaptic connections; the spatial resolving power; the arrangement of neuronal arrays or mosaics; electrophysiological access for the recording of responses to visual stimuli; the spatial arrangement of cell dendritic fields; location of retinal ‘blind spots’ (optic nerve, falciform process and pecten); topographic differences in retinal cell sampling; spectral filters, and reflective structures. The present study examines all aspects of the wholemount technique, including enucleation, fixation, retinal extraction, flattening, staining, visualization of labelled cells and stereological mapping of cell density. Uniquely, it highlights the crucial technical and often species-specific differences encountered when examining a range of vertebrate taxa (fishes, reptiles, birds and mammals). This broad comparative approach will enable future studies to overcome technical difficulties, thus permitting larger conceptual questions to be posed regarding the diversity of visual tasks across phylogenetic boundaries.

Comparison of functional and anatomical estimations of visual acuity in two species of coral reef fish

“…The standard length of each fish was recorded to the nearest millimetre. Once enucleated, the diameter of the eye was measured to the nearest 0.1 mm along the dorsal-ventral, axial (corneal-scleral) and equatorial (anterior-posterior) axis (Ullmann et al, 2012). The technique for dissecting the retinal layer from the eye and subsequent mounting was largely derived from Stone (1981) and only variations to the technique are described below.…”

Disruptive colouration in reef fish: does matching the background reduce predation risk?

“…Retinal whole-mounts were generated by removing the retina from the eye, fixing the tissue and then mounting the entire retina on a slide with the photoreceptors pointing towards the viewer. Photoreceptors were then counted using specialised software on a modified microscope, using design-based stereology methods similar to those previously published elsewhere (Coimbra et al, 2012;de Busserolles et al, 2014a;Ullmann et al, 2012) and described in detail in the Appendix.…”

“…Retinas from each fish were extracted and fixed in 4% paraformaldehyde immediately using standard methods (Coimbra et al, 2012;Coimbra et al, 2009;Ullmann et al, 2012). For optimal photoreceptor viewing and counting, all retinas required bleaching to remove the densely pigmented retinal pigment epithelium (RPE) that covered large parts of the retina.…”

“…For optimal photoreceptor viewing and counting, all retinas required bleaching to remove the densely pigmented retinal pigment epithelium (RPE) that covered large parts of the retina. Bleaching followed similar protocols for other fish and cephalopods (Talbot and Marshall, 2011;Ullmann et al, 2012), in which retinas were submerged for up to 2 h in a 6% hydrogen peroxide solution (in PBS, pH 10) followed by several rinses in fresh, chilled PBS to remove all traces of the oxidative solution (Ullmann et al, 2012). Any remaining vitreous was removed and relaxation slits were cut around the periphery to allow the tissue to lie flat.…”

“…To understand the design and success of various camouflage strategies, we must consider how colour patterns are viewed by relevant signal receivers (Endler, 1983). Therefore, we first measured the visual acuity of two reef fish predators using information on the anatomy of their eyes and the density of photoreceptors in the area of the eye most likely used for focussing a clear image (Collin and Pettigrew, 1989;Ullmann et al, 2012). This information was combined to apply relevant blurring to images used in behavioural assays and natural scenes, so that they represented a predator's-eye-view of a scene.…”