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ELECTRORETINOGRAMS AND RETINAL STRUCTURE OF THE EASTERN SCREECH (Otusasio) AND GREAT (Bubovirginianus)

STEVENJ. AULT

ABSTRACT-- Electroretinograms(ERGs) were recorded from 2 speciesof : (Otusasio) and Great Horned Owl (Bubovirginianus). Dark adaptationand flickerstimuli were used to determineretinal activityand to inferretinal structure. The darkadaptation results showed typical patterns associated with composed primarily of rods.This was indicated by the late regeneration of thescotopic b-wave. Flicker ERGs, however, also indicated a residual conecomponent. This wasindicated by the one-to-oneresponse at highluminance levels and high flickerfrequencies. The ERG dataconfirm existing histological observations of highrod numbersand few cones in the retinasof nocturnal owls.

Owl retinashave been examined histologically by mg/ml), Acepromazine(0.1 mg/ml), and Xylazine (1.0 mg/ml). a number of investigators(Bornshein and Tansley Dosagewas 1 ml/kgbody weight, administered IM. Averagedura- tion of anesthesiawas approximately I h. 1961; Hocking and Mitchell 1961; Oehme 1961; Eachsubject was placed into a light-tight,electrically grounded Fite 1973; Yew et al. 1977; Bowmaker and Martin box,and a cornealelectrode was placed on theeye. Space between 1978). All reported retinaswith high concentra- the cornea and electrodewas flooded with a saline(0.9% NaC1) tionsof rods, as would be expectedfor basically conductingsolution. A referenceelectrode was placed in the skin nocturnal . However, Fite (1973) and of the ear flap or in the skinof the ear canal.A groundelectrode wasinserted in the wing skin.A fiber opticlight guidewas hlaced a Oehme(1961) point out thatowl retinasdo possess few mm from the cornea. very smallconcentrations of cones,even in the most Electroretinograph.-- The light source was a 300 watt nocturnal species. tungsten-halogenlamp that coulddeliver steady, single-flash, or Fewelectroretinographic studies have been per- flickering stimuli. Flicker stimuli were produced by a motor- driven discthat interruptedthe light to giveequal time on andoff. formed on owls. Bornsheinand Tansley (1961) The light beamwas focused upon a fiber opticlight ,guidewhich obtained ERGs from Short-earedOwl (Asioflam- deliveredlight to the subject'seye in Maxwellianview (Armington meus)and compared responseof the to that 1974).The light beamwavelength and intensitywere adjusted by of the pigeon. These authorsalso correlated the theuse of variouscolor and neutral density filters. Unfiltered light intensityfrom thisapparatus was approximately 1.076 x 104mil- ERG resultswith histologicalpreparations of the lilamberts(mL)(1 mL = 0.001 lumens/cm2). owl and pigeon retinas. The ERGs and the his- The recordingelectrodes used were silver pedestalcorneal tologicalexamination revealed a retina composed contactlens systems. Reference and ,groundelectrodes were silver predominantly of rods in the Short-eared Owl. skin needle probes.Signals from the electrodeswere channeled Martin and Gordon (1975) recorded ERGs from through a Tektronix TM 504 pre-amplifier.The signalwas amplifiedand displayed on a Tektronix5103N dual-tracestorage the nocturnalTawny Owl (Strixaluco) to determine oscilloscope.Traces were permanently recorded by Polaroid its retinal spectralsensitivity. The ERG data sup- photography. ported earlier findings (Martin 1974; Martin and Procedure.-- Two testscommonly used in electroretinography, Gordon 1974) that the Tawny Owl possessesa re- dark adaptationand flicker stimuli,were used.Dark adaptation testswere used to observechanges in the ERG as the retina ad- tina with cone receptorsthat are presentin large justedto darkness.The eyewas first pre-adaptedto lightfor 5 min enough numbers to contribute to the visual re- to insurebleaching of the photopigments.Pre-adaptation retinal sponse. illuminancewas approximately1.076 x 10a mL for the Eastern The purposeof this investigationwas to record Screech Owl and 1.076 x 10 x mL for the Great Horned Owl. Different intensities were used for both owls to assess the effec- electroretinographicactivity of the EasternScreech tivenessof suchintensities for pre-adaptation,single-flash (20 Owl and Great Horned Owl, two speciesnot previ- msecduration) stimuli attenuated with a Kodak#2 neutraldensity ously investigated.Correlation of the ERG data filterand a Kodak#26 gelfilm red filter, weredelivered at widely with retinal structureof thesespecies was made in spacedintervals (see Figs. 1-3) to the eye to observethe retina's an attempt to better define the relativerole of the increasingsensitivity to darkness.After 20-30 min, full-intensity single-flashstimuli of red (Kodak #26 gel film), blue (Kodak#47, rods and conesin the visualprocess of theseowls. 47A, 47B gel film) and white (no filters)were givensuccessively to MATERIALS AND METHODS assessthe degreeof photopic(cone) and scotopic(rod) recovery. The secondtest utilized flickering stimuli of variousintensities Subjectsand Anesthesia.-- One EasternScreech Owl and one and flicker frequencies.Various neutral density filters,but no Great Horned Owl wereused for electroretinography.Both were color filters, were used. anesthetizedwith an anestheticmixture containingKetamine (10 Histology. -- The retinas of a Great Horned and Eastern

62 RAPTORRESEARCH 18(2):62-66 SUMMER 1984 OWL ELECTRORETINOGRAMS 63

15 O0

time (rain) 0 20 O0

0 30 25 O0

I 00 • • 30 00

3:00 • red

5:00 • blue

I0.00 •,-/-'-•• while

IO0,uV • 50msec

Figure 1. ERGsduring dark adaptationin the EasternScreech Owl. Time indicatesminutes into dark adaptation. Stimulus: 1.076x 104mL attenuatedwith #2 neutraldensity filter and #26 red filter; 20msecduration. a) Slight regenerationof photopicb-wave after 1 minute into dark adaptation.b) Slight regenerationof scotopicb-wave after 30 minutesinto dark adaptation. ScreechOwl wereexamined histologically. The Great Horned was adaptation) low-amplitude waveforms were ob- the same used in the ERG study. The subjectswere served(Figs. lb, 2b). The final red, blue, and white euthanizedwith lethalinjection of Ketamineand enucleated.The stimuli produced waveformsof low amplitude. posteriorportion of the eye was cut away and fixed in Bouin's solution.The tissuewas dehydrated in a gradedethanol series and Flicker Stimuli. -- A change in waveforms were clearedin cedarwoodoil. Portionsof peripheral retina were em- observed as the flickering stimuli were increased bedded in paraffin, sectionedmeridionally at 5 m on a rotary from low to high intensitiesand flicker frequencies; microtomeand stainedwith Hematoxylinand Eosin. this wasbest demonstratedby the Eastern Screech Owl. At low intensitiesand low flicker frequencies, waveswere evident as they followedthe stimulion a RESULTS 1:1 basis(Fig. 3a). There was a fusion of this re- Dark Adaptation.-- The ERGs from dark adap- sponse as intensities and flicker frequencies in- tation tests for both subjectsshowed very early creased,with a subsequentwaveform taking over at low-amplitude responseswhich peaked at around high intensitiesand high flicker frequencies(Fig. 1-2 min into dark adaptation (Figs. la, 2a). Also, 3b). The Great Horned Owl also displayed the late appearing (between20 and 30 rain into dark abovepattern, but with lessclarity (Fig. 4).

hme I0'00

15 O0 O3O

20'00 I O0

25 O0 2 O0

red 300

500 blue

white IOo •uvT._. • 50 msec

Figure2. ERGsduring dark adaptationin the Great Horned Owl. Time indicatesminutes into dark adaptation. Stimulus: 1.076 x 104mL attenuatedwith #2 neutral densityfilter and #26 red filter; 20 msecduration. a) Slightregeneration of photopicb-wave after 1-2 minutesinto dark adaptation.b) Slightregeneration of scotopicb-wave after 20-30 minutesinto dark adaptation. 64 STZWNJ. AULT VOL. 18, NO. 2

neutroi dentlt¾ filter

4

• 2 •

b

12 HZ 30 Hz IOOpV'• I00 m,,ec 100'uVL 2Hz 100,uV-L• 5Hz 500 msec 200 m*•ec

Figure3. FlickerERGs of theEastern Screech Owl. a) Arrowsindicate one-to-one response of waveformsto individual flickersat lowintensity and low flickerfrequency. b) Arrowsindicate one-to-one response of waveformsto individualflickers at high intensityand high flickerfrequency. 1.076 x 104mL light sourceattenuated with indicatedneutral density filters. Histology. -- The retinal layers of the Eastern A few cones were also seen in owl retinas. These ScreechOwl could be clearlydiscerned histologi- were identified by their nuclei, which are typically cally (Fig. 5). Retinaswere composedprimarily of rounder than rod nuclei and lie a•jacent to the rods; indicated by the elongated and cylindrical external limiting membrane. In all sections,cones morphologyof their outer segmentsin the receptor were alwaysfew in number and were greatly out- layer. The nuclei in the outer nuclear layer were numbered by the high densityof rods.

alsoidentified as rod nucleibecause they were typi- DISCUSSION cally more elongatedand were fairly evenlydistri- buted throughout the outer nuclear layer (Walls The typicalERG waveformis composedof the a, 1942; Duke-Elder 1958). The rod nuclei of the b and c waves.The initial negative deflection (a- Great Horned Owl were extremelyelongated and wave) is followed by a positivedeflection (b-wave) closelypacked. normallyof greater amplitude.The late-occuring

neutral density f,lter a

12 Hz

IOO.•VL IOO msec

30 Hz

Figure4. FlickerERGs of theGreat Horned Owl. a) Arrowsindicate one-to-one response of waveformsto individual flickersat lowintensity and low flickerfrequency. b) Arrowsindicate one-to-one response of waveformsto individualflickers at highintensity and high flicker frequency. 1.076 x 104mL lightsource attenuated with indicatedneutral density filters. SUMMER 1984 OWL ELECTRORETINOGRAMS 65

Figure5. a). Layersof theEastern Screech Owl retina. A) cartilaginouscup; B) pigmentepithelium; C) receptorlayer; D) externallimiting membrane; E) outernuclear layer; F) outerplexiform layer; G) inner nuclearlayer; H) innerplexiform layer; I) ganglioncell layer; J) nervefiber layer; and K) internallimiting membrane (200x); b) EasternScreech Owl and c) Great HornedOwl retinas.cn = conenuclei; onl = outer nuclearlayer (primarilyrod nuclei).(787.5x and 500xrespectively).

positive deflection (c-wave) is not commonly suggestinggeneration by a cone component. As evaluated in comparative studies. Brown (1968) dark adaptationprogressed, these early responses explained that the a-wave is produced in the re- diminished and were replaced by the scotopicre- ceptor cell layer, the b-wave through bipolar cell sponses.After-25-30 rain, there was still no com- activity,and the c-waveby metabolicactivity of the plete regeneration of the scotopicresponses in pigment epithelium. The a- and b-wavescan be either owl, denotingthat manyof the rodswere not further subdividedinto photopic(cone generated) yet adapted to the dark. The reduced effect of the and scotopic(rod generated)components. In gen- blue light on the scotopicsystem also verified this eral, the photopic componentshave shorter !aten- sinceblue light is primarily a rod stimulator.These cies and steeper slopesthan scotopiccomponents observationssuggested a retina predominated by (Armington 1974). These patternswere evident in rods, but with a small conecomponent. However, it the owls studied (Figs. 1, 2) could meanthat the initial light adaptingintensity The dark adaptation results revealed typical wastoo high. This seemsunlikely sincean absence patterns associatedwith retinas composedpredo- of completeregeneration of the scotopicresponse minantlyof rods.The late regeneratingwave forms was also observed in the Great Horned Owl which were most likely scotopicb-waves suggesting that wasexposed to a lower light-adaptingintensity. rods were regenerating after having been bleached The shift from scotopicto photopic systems during light adaptation.Latency of theseb-waves during the flicker procedureswas indicated by a suggestedthe responsewas from the scotopicsys- decreasein latencyand an increasein amplitude of tem. Longer latency or implicit time (approxi- the initial b-wave as flicker frequenciesand inten- ,mately 100 msec)is indicativeof a scotopicrather sities were increased. The initial a-wave also became than photopicb-wave (approximately 50-70 msec). more prominent at higher flicker-frequenciesand Earlier low-amplitude responseswere probably intensities,providing further indication of the shift cone responsesbecause of their early appearance to the photopicsystem (Armington 1974).The rods during dark adaptation.Latency of theseresponses and cones were also able to follow the individual wasalso shorter than the scotopicresponses, again flickering stimuli. At low intensitiesand low flicker 66 STEVENJ. AULT VOL 18, NO. 2

frequencies, rods were able to follow individual BOWMAKER,J.K. AND G.R. MARTIN. 1978. Visual pig- flickers, having not yet exceeded their critical ments and colour vision in a nocturnal , Strix aluco flicker fusion frequency. As intensity and/or fre- (Tawny owl). VisionResearch 18:1125-1130. quencywas increased, rods "fused" the stimuli.Fu- BROWN,K.T. 1968. The Electroretinogram:Its Com- sion occurred when the receptorscould no longer ponentsand Their Origins.Vision Research 8:633-677. DUKE-ELDER,S. 1958. Systemof Ophthalmology.Vol. respond to individual flickers on a 1:1 ratio but 1: The Eye in Evolution.St. Louis: C.V. Mosby. insteadresponded to thein asif there wasone con- FITE, K. 1973. Anatomical and Behavioral Correlates of stant stimulus.At high intensitiesand high flicker VisualAcuity in the Great Horned Owl. VisionResearch frequencies,cone responsebecame dominant and 13:219-230. wasable to follow individual flickerssince they pos- HOCKING, g. AND B.L. MITCHELL 1961. Owl Vision. Ibis. sessa higher critical flicker fusion frequency than 103a, 284-288. rods (Armington 1974). Histological results in MARTIN,G.R. 1974. Color Vision in the Tawny Owl combination with the ERG data indicated that the (Strixaluco). Journal of Comparativeand Physiological retinas were predominantly coinposed of rods. Psychology.86, 133-142. MARTIN, G.R. AND 1. GORDON 1974. Increment- This supports the findings of previous workers thresholdSpectral Sensitivity in the Tawny Owl (Strix (Bornshein and Tansley 1961; Hocking and aluco). VisionResearch 14:615-620. Mitchell 1961; Oehme 1961; Fite 1973; Yew et al. 1975. ElectroretinographicallyDeter- 1977; Bowmaker and Martin 1978) who histologi- mined SpectralSensitivity in the Tawny Owl (Strix cally demonstrateda retina coinposedpredomin- aluco).Journal of Comparative and Physiological Psycholo• antly of rods in the Great Horned Owl and other 89:72-78. owl species.However, histologicalresults and ERG OE•ME, H. 1961. Vergleichend-histologiche Unter- data also demonstrated the presence of a cone suchungenan der Retinavon Eulen. Die Zoologischen componentthat wassinall but active. Jahrbucher, Abt. 2, der Anatomieund Ontogenie 79:439-478. My resultsand thoseof Bowmaker and Martin WALLS,G.L. 1942. The Vertebrate Eye. Cranbrook In- (1978) and Martin and Gordon (1975) verify that stitute of Science. Bulletin # 19. the retina of owls,even the mostnocturnal species, YEW, D.T., H.H. Woo AND D.B. MEYER 1977. Further possesscones in numbers large enough to contri- Studieson the Morphologyof the Owl's Retina.Acta bute to the visualprocess. Existence of sucha cone Anatomica 99:166-168. componentcould be the remnants of an ancestral cone-dominated retina. Nocturnal owls such as Departmentof Biology,The University of Akron, Akron, screech and Great Horned Owls are occasionally 44325. Present address: Department of Biology, State University, Pocatello,Idaho 83209. activeduring the day.It isreasonable to assumethat the few cones that are present contribute to the Received8 March 1984; Accepted1 July 1984. owl'svisual processin the brighter illumination of daylight hours.

ACKNOWLEDGMENTS I extendspecial thanks to CharlesJ. Parshall,Animal Specialty Clinic, Richfield,Ohio, for help and expertisewith electroretinog- raphy and to Richard F. Nokes, The Universityof Akron, who assistedwith anesthesiology.F. ScottOrcutt, John H. Olive, Steven P. Schmidt,Edwin W. House,and CarolynWilson made positive critical commentson the manuscript.This studywas supported in part by a Grant-in-Aid of Researchfrom SigmaXi, The Scientific ResearchSociety.

LITERATURE CITED ARMINGTON,J.C. 1974. The Electroretinogram.: Academic Press. BORNSHEIN,H. ANDK. TANSLEY.1961. Elektroretinog- ramm und Netzhautstruktur der Sumpfohreule (Asio fiammeus). Experientia.17: 185-187.