RESEARCH ARTICLE Retinal Photoreceptor Arrangement, SWS1 and LWS Opsin Sequence, and Electroretinography in the South American Marsupial Thylamys elegans (Waterhouse, 1839) Adria´n G. Palacios,1* Francisco Bozinovic,2 Alex Vielma,1 Catherine A. Arrese,3 David M. Hunt,4 and Leo Peichl5 1Centro de Neurociencia de Valparaı´so, Facultad de Ciencias, Universidad de Valparaı´so, Valparaı´so 2370006, Chile 2Centro de Estudios Avanzados en Ecologı´a & Biodiversidad, Departamento de Ecologı´a, Facultad de Ciencias Biolo´gicas, PUC, Santiago 6513677, Chile 3School of Animal Biology, University of Western Australia, Crawley, Western Australia 6009, Australia 4UCL Institute of Ophthalmology, London EC1V 9EL, United Kingdom 5Max Planck Institute for Brain Research, 60528 Frankfurt am Main, Germany ABSTRACT vision. Cone densities peaked in a horizontally elongated We studied the retinal photoreceptors in the mouse opos- region ventral to the optic nerve head. In ventral—but not sum Thylamys elegans, a nocturnal South American mar- dorsal—retina, roughly 40% of the LWS opsin-expressing supial. A variety of photoreceptor properties and color vi- cones occurred as close pairs (double cones), and one sion capabilities have been documented in Australian member of each double cone contained a colorless oil marsupials, and we were interested to establish what sim- droplet. The corneal electroretinogram (ERG) showed a ilarities and differences this American marsupial showed. high scotopic sensitivity with a rod peak sensitivity at 505 Thylamys opsin gene sequencing revealed two cone nm. At mesopic light levels, the spectral ERG revealed the opsins, a longwave-sensitive (LWS) opsin and a shortwave- contributions of a UV-sensitive SWS1 cone mechanism sensitive (SWS1) opsin with deduced peak sensitivities at and an LWS cone mechanism with peak sensitivities at 365 560 nm and 360 nm (ultraviolet), respectively. Immunocy- nm and 555 nm, respectively, confirming the tuning pre- tochemistry located these opsins to separate cone popu- dictions from the cone opsin sequences. The two spectral lations, a majority of LWS cones (density range 1,600– cone types provide the basis for dichromatic color vision, 5,600/mm2) and a minority of SWS1 cones (density range or trichromacy if the rods contribute to color processing at 100–690/mm2). With rod densities of 440,000– mesopic light levels. J. Comp. Neurol. 518:1589–1602, 590,000/mm2, the cones constituted 0.4–1.2% of the 2010. photoreceptors. This is a suitable adaptation to nocturnal © 2009 Wiley-Liss, Inc. INDEXING TERMS: retina; electroretinogram; cone opsin; photoreceptors; UV vision; marsupials Among Marsupials, the order of Didelphimorphia (com- considerable variation in number and retinal topography mon opossums) is one of the most diverse in phylogenetic across species, correlating with the predominant diel ac- and geographic habitat specialization (Eduardo Palma et tivity pattern, whether diurnal, crepuscular, or nocturnal al., 2002). American marsupials are primitive metatherian (Ahnelt and Kolb, 2000; Peichl, 2005). The typical mamma- mammals that separated from eutherian mammals around 125 Myr ago during the Cretaceous, and from the Austra- Grant sponsor: Comisio´n Nacional de Investigacio´n Cientı´fica y Tecno- lian marsupial lineage about 60 Myr ago in the Eocene/ lo´gica (CONICYT); Grant number: PBCT-ACT45 (to A.G.P.); Grant sponsor: Australian Research Council; Grant number: Discovery grant DP0662985 Paleocene when Australia separated from Antarctica. (to D.M.H./C.A.A.); Grant sponsor: Leverhulme Trust; Grant number: Mammals have a “duplex” retina with rod photorecep- F/07134 (to D.M.H.). tors for scotopic vision and cone photoreceptors for pho- *CORRESPONDENCE TO: Adrian G. Palacios, Ph. D., Centro de Neuro- ciencia de Valparaı´so, Universidad de Valparaı´so, Facultad de Ciencias, topic vision and color vision. The photoreceptors show P.O. Box 5030, Valparaı´so, Chile. E-mail: [email protected] Received 14 August 2009; Revised 1 October 2009; Accepted 19 November 2009 DOI 10.1002/cne.22292 Published online December 8, 2009 in Wiley InterScience (www.interscience. © 2009 Wiley-Liss, Inc. wiley.com). The Journal of Comparative Neurology ͦ Research in Systems Neuroscience 518:1589–1602 (2010) 1589 Palacios et al. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ lian retina contains two spectral cone types, a majority of fat-tailed dunnart, quenda, and quokka, the SWS1 cones middle-to-long-wave-sensitive (LWS) cones and a minority peak in the dorsal peripheral retina, with an additional of short-wave-sensitive (SWS) cones expressing either the ventral peak in the quenda; in contrast, in the honey pos- SWS2 pigment, as found in monotremes (Davies et al., sum, the highest SWS1 cone density is a ring around the 2007; Wakefield et al., 2008), or the SWS1 pigment, as retinal periphery (Arrese et al., 2003, 2005). Among the found in all other mammals (for review, see Jacobs, 1993; American marsupials, Didelphis marsupialis aurita has an Bowmaker and Hunt, 2006). Depending on species, the LWS cone peak in a temporally located area centralis, and LWS cones have their peak sensitivity in the green to yel- relatively high LWS cone densities in a ventrally located ␭ low part of the spectrum ( max range about 500–560 nm), horizontal streak; SWS1 cones are unevenly distributed ␭ the SWS1 cones in the blue to ultraviolet part ( max range across the retina, with highest densities in the dorsal pe- about 360–450 nm), and the SWS2 cones at 440 nm (Da- riphery (Ahnelt et al., 1995). In Didelphis virginiana, total vies et al., 2007). cone density also peaks in a temporal area centralis, but Whereas the basic pattern in eutherian mammals is falls off rather symmetrically toward the periphery without cone dichromacy with two types of cone visual pigment, showing a horizontal streak (Kolb and Wang, 1985; this there is recent evidence that some Australian marsupials study did not identify the spectral cone types). possess three spectral cone types and are potential Given these differences and the phylogenetic position of trichromats (Arrese et al., 2002, 2006a,b; Cowing et al., marsupials, further elucidation of the photoreceptor prop- 2008), although only two cone opsin genes have been erties of South American marsupials is crucial for under- found (Strachan et al., 2004; Cowing et al., 2008). How- standing the evolution of mammalian photoreceptor char- ever, a second rod opsin gene was found in the Australian acteristics. We have studied the elegant fat-tailed mouse fat-tailed dunnart, and if that were expressed in a subpopu- opossum Thylamys elegans (Didelphinae) from an as yet lation of cones, it could account for the trichromacy (Cow- unstudied genus by using a combination of molecular, im- ing et al., 2008). There are only a few studies on the pho- munohistochemical, and electrophysiological techniques. toreceptors of American marsupials. Walls (1939) Thylamys is a strictly nocturnal species (Meserve, 1981) provided their first description, by using the North Ameri- from central Chile, with a partly arboreal habit. It feeds can opossum Didelphis virginiana and the mouse opossum primarily on insects but occasionally on seeds and fruits. Marmosa mexicana. Kolb and Wang (1985) quantified rod and cone densities in Didelphis virginiana by conventional MATERIALS AND METHODS histology, and Ahnelt et al. (1995) analyzed the distribution of photoreceptors in the South American opossum Didel- Animals phis marsupialis aurita with SWS1 and LWS opsin-specific Adult male mouse opossums (Thylamys elegans) were antibodies. Recently Hunt et al. (2009) showed that two captured in the wild from central Chile, and brought to the nocturnal American opossum species (Monodelphis do- laboratory and individually maintained in wire cages in a mestica and Didelphis aurita, order Didelphimorphia, sub- standard animal facility at the Universidad de Valparaiso ␭ (Chile). Each cage contained a food dispenser and shelter, family Didelphinae) have SWS1 and LWS opsins with max around 360 nm (UV) and 550 nm, respectively. In addition, provided by cardboard tubes filled with cotton. Animals the Monodelphis genome possesses a single rod or Rh1 were acclimated for 1 week after capture to prevailing opsin gene. In contrast therefore to Australian marsupials, natural conditions of temperature (15–18°C) and photo- in which a second rod opsin gene has been found that may period, and fed ad libitum with commercial cat food (Whis- account for the trichromacy, this is not the case for South kas, Waltham, UK). All experiments were approved by the American marsupials, and the expectation would be that bioethics committee of the Universidad de Valparaiso and they are dichromats. complied with the international Guide for the Care and Use Cone topographies vary markedly across marsupials. of Laboratory Animals (National Academy Press, 1996). Among the Australian marsupials, the Tammar wallaby has Permission to work on collected specimens was under au- the highest LWS cone densities in a horizontal “visual thorization #3014 from the Chilean Servicio Agricola y Ga- streak” and in the ventral peripheral retina, whereas the nadero (SAG). To obtain retinae for the molecular and his- highest SWS1 cone densities occur in the dorsal periphery tological analysis, animals were euthanized by an (Hemmi and Gru¨nert, 1999). The fat-tailed