Hyena (Crocuta Crocuta)
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Original Paper Brain Behav Evol 2003;62:182–192 Received: April 28, 2003 Returned for revision: June 11, 2003 DOI: 10.1159/000073270 Accepted after revision: July 1, 2003 Topography of Photoreceptors and Retinal Ganglion Cells in the Spotted Hyena (Crocuta crocuta) Jack B. Calderone Benjamin E. Reese Gerald H. Jacobs Neuroscience Research Institute and Department of Psychology, University of California, Santa Barbara, Calif., USA Key Words distribution defines a prominent visual streak with high- Mammals W Hyena W Crocuta crocuta W Photoreceptors W est spatial packing (approx. 4,200/mm2) in an area cen- Ganglion cells W Cones W Spatial acuity W Retinal tralis that is located in the temporal retina. The total num- topography ber of ganglion cells is estimated at about 260,000. Using standard assumptions the maximum spatial resolution of the spotted hyena is calculated to be about 8.4 cycles/ Abstract degree, a value similar to estimates obtained for other The spatial distributions of photoreceptors and retinal terrestrial carnivores. ganglion cells were examined in the spotted hyena (Cro- Copyright © 2003 S. Karger AG, Basel cuta crocuta). Two populations of cones were identified by immunocytochemical labeling. The hyena retina con- tains approximately 2.3 million middle- to long-wave- Introduction length sensitive (M/L) cones that reach peak densities of about 7,500/mm2 in the vicinity of the optic nerve head. A Carnivores comprise a diverse group of animals vary- sparser population of short-wavelength sensitive (S) ing greatly in size, natural habitat, and photic rhythmicity cones, totaling about 0.3 million, was also detected. [Macdonald, 2001]. Many of them survive as predators There is a striking disparity in the spatial distributions of and for that they must rely on a range of keen sensory the two cone types with S cones achieving peak density capacities – olfaction, audition and vision – to locate, in a region located well below the optic nerve head. The stalk and capture prey. Although natural observations differences in the spatial distributions of the two cone make clear that vision is an important component of this types have implications both for visual sensitivity and for sensory armamentarium, there are only modest numbers color vision. Hyena rods outnumber cones by about of direct studies of vision in carnivores, and these mostly 100:1 with rod density falling off modestly along a cen- involve laboratory investigations of domesticated species, tral-peripheral gradient. Ganglion cells were identified in principally cats and dogs. An alternative means to under- retinal wholemounts by Nissl staining patterns. Their standing species that might be impractical to test directly © 2003 S. Karger AG, Basel Gerald H. Jacobs ABC Neuroscience Research Institute, University of California Fax + 41 61 306 12 34 Santa Barbara, CA 93106 (USA) E-Mail karger@karger.ch Accessible online at: Tel. +1 805 893 2446, Fax +1 805 893 2005 www.karger.com www.karger.com/bbe E-Mail jacobs@psych.ucsb.edu under controlled conditions is to examine various fea- nized for reasons unrelated to the present investigation. This proce- tures of visual anatomy and, from these, draw inferences dure was carried out in accordance with institutional animal care and about vision using as a baseline the wealth of information use guidelines and with NIH Principles of Animal Care. These ani- mals were perfused transcardially with 4% paraformaldehyde. The about how such features generally correlate with visual cornea, lens, and vitreous were rapidly removed from the eyes and capacities in species where both have been studied the eyecups were postfixed in 4% paraformaldehyde for an addition- [Hughes, 1977]. Although this strategy is generally less al period of 4–8 h. Prior to the removal of the retina, spatial orienta- satisfactory than direct studies of vision, these anatomical tion was marked using the prominent tapetum as a reference. For immunocytochemistry, the retinas from the perfused animals indices do yield useful insights. We use this approach to were dissected from the eyecups and rinsed in phosphate-buffered examine some features of vision in a large carnivore, the saline (PBS). Following 5–13 days incubation in a 20% sucrose solu- spotted hyena. tion, the retinas were shock-frozen in liquid nitrogen and rinsed in Spotted hyenas are native across a broad range of Sub- PBS (3 ! 10 min). The retinas were placed in 0.3% hydrogen perox- Saharan Africa where they live in complexly organized ide, again rinsed, and allowed to sit in a solution of 2% bovine serum albumin and 10% normal goat serum for 3 h. They were then exposed social groups [Kruuk, 1972; Holekamp et al., 1997; Wahaj to primary antibodies for 72 h. The primaries used were polyclonal et al., 2001]. Such associations require accurate inter-indi- antibodies either specific to mammalian short-wavelength (S) sensi- vidual recognition and, amongst other cues, spotted hye- tive pigments (JH455; 1:100,000 dilution) or to middle (M) to long nas use visual information to accomplish this task (L) wavelength sensitive pigments (JH492; 1:30,000 dilution; both [Kruuk, 1972], likely taking advantage of the prominent kindly provided by J. Nathans). This was followed by 24 h incubation in goat anti-rabbit biotinylated secondary antibody, a subsequent spot patterns that give the species its common name and 24-h exposure to avidin-biotin-peroxidase complex and a final reac- which are sufficiently unique among individuals that field tion with diaminobenzidine. The retinas were subsequently flat scientists conventionally use them to identify individuals. mounted and labeled cones were counted on average from 700 sites These animals are classified as nocturnal, but they are also taken at 1 mm intervals across the retina. At each site, counts were sporadically active during daylight hours; predominantly made across four adjacent regions each comprising 0.0081 mm2 in area and these counts were averaged to estimate the local cone densi- the 3–4 h after dawn and for a similar period preceding ty. Estimates of rod densities were also obtained by counting cells at nightfall [Kruuk, 1972]. Indeed, the majority of contem- 18 locations throughout two of the retinas. Rods were identified in porary studies of hyena behavior have involved observa- microscopic viewing of the flat mounts under oil immersion by using tions made during these crepuscular transition times. the identification criteria noted below. In deriving receptor density These daily activity patterns suggest that, like the majori- maps no corrections were made for possible tissue shrinkage. To obtain information about retinal ganglion cell distributions, a ty of other mammals, spotted hyenas would be expected retina was dissected from the eye of an adult hyena that had died of to have rod-dominated retinas, but with a sufficient cone natural causes. The retina was flat mounted with the ganglion cell population to allow reasonable vision under daylight con- layer upward and the wholemount was stained with 0.5 % cresyl vio- ditions [Jacobs, 1993]. That prospect receives qualitative let. To distinguish ganglion cell somata from other types of cells support from observations made by Kruuk who, in his present in the ganglion cell layer (principally displaced amacrine cells and glial cells) we used the structural criteria first developed by classic field studies, offered the summary opinion that Hughes [1981] and subsequently employed in quantitative studies of ‘hyenas could see in the daylight about as well as I could’ ganglion cells in a number of other carnivores [Henderson, 1985; [1972, p. 215]. To further examine these possibilities Peichl, 1992; Mass and Supin, 2000]. These criteria identify as gan- opsin antibody staining was used to obtain an indication glion cells those having relatively large cell bodies, a significant of the density and distribution of photoreceptors in the amount of Nissl substance, and a homogenous nucleus. Direct com- parisons of ganglion cell counts made using these structural criteria hyena retina and we have also charted the topographic and counts of ganglion cells identified by retrograde labeling follow- distribution of ganglion cells in retinal wholemounts. ing injection of retinorecipient nuclei in the brain have been shown From these results, inferences can be drawn about spec- to give very similar results in two mammalian species [Pettigrew et tral sensitivity and visual resolution as well as the pros- al., 1988]. Although application of these structural criteria necessari- pects for color vision. ly has a subjective component, their consistent use has been well accepted as a means of estimating ganglion cell density and distribu- tion in mammalian material. Using these cellular identification criteria, ganglion cell counts Materials and Methods were made at over 700 sites taken at 1 mm intervals across the retina. Because the retina was attached to the slide during processing it was Eyes were obtained from adult spotted hyenas that were members assumed, in accord with earlier observations [Wässle et al., 1981], of a colony maintained at the University of California, Berkeley. Sev- that there was no significant amount of tissue shrinkage. eral eyes were removed postmortem from animals that had died of natural causes and these were stored in formalin prior to our exami- nation. Three eyes were obtained from two animals that were eutha- Hyena Photoreceptors and Ganglion Cells Brain Behav Evol 2003;62:182–192 183 Results were similar for the two retinas examined, the maximum density was found to be consistently higher in one animal Gross Eye Characteristics with the result that the estimated total numbers of S cones Size measurements were obtained from 5 fixed eyes. for the two were, respectively, 379,300 and 200,600. The eye of the adult spotted hyena is nearly spherical These counts suggest that the hyena retina has, overall, an (fig. 1A) with a mean axial length of 25.4 mm (SD = M/L:S cone ratio of about 8:1.