Behavioural Brahl Research, 51 (1992) 203-209 9 1992 Elsevier Science Publishers B.V. All rights reserved. 0166-4328/92/$05.00
203
BBR 01370
Visual performance of pigeons following hippocampal lesions V e r n e r P. B i n g m a n ~ a n d W i l l i a m H o d o s b "Department of Psychology, Bowling Green State University, Bowling Green, OH 43403 (USA) and bDepartment of Psychology, University of Maryland, College Park, )liD 20742 (USA) (Received 28 April 1992) (Revised version received 24 July 1992) (Accepted 4 August 1992)
Key words: Visual acuity; Size-difference threshold; Animal psychophysics; Hippocampus; Lesion; Pigeon
The effect of hippocampal lesions on performance in two psychophysical measures of spatial vision (acuity and size-difference threshold) was examined in 7 pigeons. No difference between the preoperative and postoperative thresholds of the experimental birds was found. The visual performance of pigeons in the psychophysical tasks failed to reveal a role of the hippocampal formation in vision. The results argue strongly that the behavioral deficits found in pigeons with hippocampal lesions when tested in a variety of memory-related spatial tasks is not based on a defect in spatial vision but impaired spatial cognition.
INTRODUCTION
The avian dorsomedial forebrain, or hippocampal formation (HP), is a paired structure consisting of a medial hippocampus and dorsomedial parahippoeampus 16. Studies examining HP function under natural t-4' semi-natural z3 and laboratory conditions 9'21'22 have identified the hippocampal formation as a structure critical for memory, in particular but not exclusively spatial memory. Further, the larger HP found in avian species in which complex, memory-based spatial behavior is part of their natural history has been explained by assuming that more complex spatial behavior requires larger cognitive processing capacity, which in turn requires a larger hippocampal formation 18'z~ In summary, interpretation of HP function has focussed primarily on a role in spatial cognition. An alternative hypothesis that has yet to be systematically explored, however, is that HP lesion actually may lead to one or more perceptual impairments, which might explain the observed behavioral deficits. In a number of the studies that have examined the relationship between hippocampal function and spatial cognition in birds, visual stimuli were assumed to be used bY the experimental animals to coordinate their behavior. Because HP-ablated birds showed no apparent impairment in visually guided behavior such as
pecking for seeds, experimenters tended to conclude that such birds did not suffer any visual perceptual impairmentz3. Untested, however, is the possibility that more subtle visual impairments, not easily detected by such casual observation, may in part explain the observed behavioral deficits. In support of this possibility are reported anatomical connections between HP and known or suspected visual processing areas of the avian telencephalons'6. In the present study we examined the effect of HP ablation on visual performance in two psychophysical measures of spatial vision: visual acuity and sizedifference threshold. Visual acuity reflects the subjects' ability to detect high spatial frequencies. Size-difference thresholds are determined, to a great extent, by the subjects' ability to detect low spatial frequencies. HP ablation was found not to affect threshold performance in either of the tasks. These data demonstrate that HP lesions, which result in behavioral deficits in tasks of memory and spatial cognition, do not affect performance on two sensitive measures of spatial vision. Thus, the data support hypotheses emphasizing cognitive rather than sensory deficits following hippocampal injury.
MATERIALS AND METHODS
S,bjects: acuity Correspondence: V.P. Bingman, Department of Psychology, Bowling Green State University, Bowling Green, OH 43403, USA.
The subjects were 4 White Carneaux pigeons
(Cohmlba livia) that were at least 2 years old at the start
204 of the study and had been maintained at 75-80~o of their free-feeding weights during the study.
photometer or a Simpson Model 408 photometer. Both meters were calibrated against a certified luminance standard traceable to the U.S. Bureau of Standards.
Apparatus: acuity The apparatus has previously been described in detail 13. Briefly, the experimental chamber was a 3-key Lehigh Valley Electronics pigeon chamber, the rear wall of which had been modified to allow an external light source to be focused on a diffusing screen behind the center key. The subjects viewed the stimulus through the center key which was constructed from a 1.0-mmthick glass microscope slide. The glass key was made opaque, except for a 1.5-cm-diameter circular area at its center. The light source that illuminated the center key was a Sylvania CBA lamp mounted in a slide projector. A 5.0 x 5.0 cm white, rear-view projection screen was placed directly in front of the projector lens and a 10 x 15 cm sheet of white translucent Plexiglas was positioned 15 cm behind the center key. Both served to diffuse and attenuate the intensity of the light from the projector. The luminance of the center key was maintained at 70 cd/m 2. The side keys were conventional Plexiglas pecking keys 2.5 cm in diameter. The stimuli were a series of 6, diagonally ruled, square-wave gratings, the nominal spatial frequencies of which were 1.0, 2.0, 4.0, 8.0, 12.0, and 20.0 lines/ mm. Each grating was paired with a blank stimulus constructed from Bausch and Lomb glass neutraldensity filters (nominal density = 0.3) or glass squares cut from 1.0-mm-thick microscope slides. Kodak Wratten No. 96 gelatin neutral-density filters (optical density 0.1-0.6) and, where necessary, microscope coverglass (optical density 0.04-0.07) was added to the gratings and/or the blank stimuli to match the luminances within each pair. The luminance difference within a stimulus pair did not exceed 0.02 log unit. This difference is well below the luminance difference threshold for pigeons tested in a comparable apparatus 1~ TWO sets of stimulus gratings and luminancematched neutral density filters were contained in a motor-driven wheel that was mounted between the diffusing screen and the center-viewing key. A solenoidoperated shutter mounted just behind the center key was closed between trials to prevent subjects from detecting the motion of the wheel when it changed positions. The interior of the chamber was painted fiat white and illuminated.by a ceiling-mounted day-light fluorescent lamp which was diffused by a sheet of white translucent Plexiglas. At the pigeon's eye level, the chamber illumination varied from 43.04 lux in the darkest corner to 225.96 lux directly below the lamp. All luminance measures were made using a Photovolt Model 520-M
Behavioral procedure: acuity The procedure used was a two-alternative forced choice described in previous near-field acuity studies 13'14. A brief description follows. After a period of adaptation to the vivarium, the birds were reduced gradually to 7 5 - 8 0 ~ of their freefeeding weights. When the birds were pecking a diffuse white center key reliably for a grain reinforcer, successive discrimination training began using the grating of lowest spatial frequency (1.0 line/mm) and a luminancematched blank disc. The birds were required to choose between the two side keys. The correct choice, which resulted in a reward for the bird, was a function of whether a grating was present. After the bird pecked the center key 10 times, the internal shutter of the slide projector closed, darkening the center key, and both side keys were then illuminated. A single peck on the correct side key resuited in access to the mixed grain. A 5-s time-out followed incorrect side key responses: the response keys were dark and inoperative, while the chamber illumination remained constant in order to maintain the subject's level of light adaptation. In addition to the timeout, correction of an incorrect response also was required before the next trial began. As soon as the bird reached a performance level of 90% correct on the 1.0 line/mm grating vs. blank the computer program was switched from the training program to the psychophysical program. A variant of the psychophysical method of constant stimuli was used to obtain each subject's spatial acuity. For acuity, the blank stimulus was compared against each of the six gratings that varied in spatial frequency. Each psychophysical session consisted of 14 blocks of 24 trials each for a total of 336 trials; sessions were run 5 days per week. Within a block one grating and the comparison blank were presented 12 times each in a pseudo-random fashion s. The subject viewed only one stimulus at a time as it pecked the center key 10 times; thus, the bird was required to detect if a grating was present. Left-key pecks were required if the center key had displayed the blank while the presence of a grating required a right-key response. A random 5 0 ~ of the bird's correct responses were followed by the availability of the unconditioned reinforcer; however, all correct responses were followed by illumination of the feeder light for a length of time equal to the magazine time. Incorrect responses were followed by the 5-s time-out
205 and a correction trial, in which the same grating or blank was displayed on the center key until the bird pecked the appropriate side key. Illumination of the feeder light accompanied the end of the correction trial. The data from correction trials were discarded and, hence, were not used to calculate the percentage of correct responses for any stimulus in a given block. The first block of each session was similar to the discrimination training procedure in that the lowest spatial frequency grating (1.0) line/mm and blank were presented to the subject. This block functioned as a 'warm-up' period for the animal, and the data were not used in the determination of acuity. The second block, which was considered the 'assessment' block, contained the same stimulus pair as the first block. The bird's performance in this block determined whether or not the session was going to be a psychophysical one: if the bird made 90~o or more correct responses, then the remaining gratings were presented to the subject during the psychophysical session. Otherwise, the subject was presented with the same stimuli (1.0 line/mm grating vs. blank) for the remainder of the session to give the bird more experience with the basic discrimination task, and no acuity was calculated. During the psychophysical session, assuming that the bird 'passed assessment', the other five gratings were paired with the standard in the next five blocks (blocks 3-7) in order of increasing spatial frequency (2.0, 4.0, 8.0, 12.0, 20.0 lines/mm). The second sequence of blocks (blocks 8-14) began immediately upon completion of the first. Again, the animal was given a 'warm-up' period (block 8), which allowed any strong response biases to diminish that may have developed during the more difficult discriminations in the first set of stimulus presentations~2; data from this second warm-up block also were discarded. In blocks 9-14, the second series of gratings were presented in the same order as the first series. The data obtained from the two block sequences were combined to arrive at the threshold value for the psychophysical session. The percentage of correct responses was plotted as a function of spatial frequency for each session. A computer-derived psychometric function was obtained via a linear interpolation between two data points, one above and one below the 75~o line. From this psychometric function acuity was determined. For acuity, the visual angle subtended by a single bar at threshold was estimated using the average viewing distance of pigeons (66.5 mm) from the surface of the gratings to the anterior nodal point of the eye 19. Psychophysical testing was continued until the bird's performance on the tasks met a preoperative criterion
of stability: this corresponded to a range of acuity for at least five consecutive sessions that did not vary by more than + 25~o from mean acuity for those sessions and, in addition, the threshold values were not progressively decreasing. Birds were then subjected to surgical ablation of the hippocampal formation. Psychophysical testing resumed after a recovery period of 5-7 days. The psychophysical procedure used to test the birds postoperatively was the same as the one used prior to surgery. Again, the birds were tested 5 days per week until their performance was stable.
Subjects: size difference thleshold The subjects were 3 White Carneaux pigeons that were at least 2 years of age at the start of the study and had been maintained at 7 5 - 8 0 ~ their free-feeding weights during the study.
Apparatus: size difference threshold A full description of the apparatus may be found in Hodos, Weiss and Bessette ~5. In brief, the apparatus consisted of a three-key Lehigh Valley Electronics pigeon chamber, with the wall of the experimental chamber modified to allow light from an externally located slide projector to be focused on the center key. The center key was a Plexiglas rear-projection screen. The stimuli were placed in the carousel tray of the slide projector and were projected onto the back of the center key. The luminance on the center key with no stimulus in place was 72.6 cd/m 2. Each of the three response keys was 25 mm in diameter. The three keys were arranged in a horizontal row. The stimuli in the present experiment were a series of black annuli on white backgrounds. The diameter of the standard annulus was 3 mm; the diameter values of the comparison stimuli were 3.5, 4, 5, 7, 10, and 15 mm. The difference between the inner and outer circumference of the annulus was 0.5 mm. The contrast between the white image of the annulus and the dark surround was 9 1 ~ . Each comparison annulus was paired with a standard annulus that had been matched for luminance. Two sets of stimuli were mounted in the carousel tray, one set for each half of the experimental session. An industrial-control computer (Action Instruments Basic Controller 2.2) controlled the slide projector, the sequence of stimulus presentations, and all other aspects of the apparatus and data collection.
Behavioral procedure: size difference threshoM The behavioral procedure used to measure size difference thresholds was similar to that used for acuity with the following exceptions.
206 The procedure was two-alternative forced choice as described earlier ~5. Successive discrimination training began using the 3-mm and 15-mm annuli. The side key that yielded a reward for the bird was a function of the annulus size projected on the screen. As soon as the bird reached a performance level of 90~o correct on the 15-mm vs. 3-mm discrimination problem, the computer program was switched from the training program to the psychophysical program. Within each block of a psychophysical session, one comparison and the standard stimulus were presented. The bird was required to compare each stimulus with its memory of the standard. Left key pecks were required in the presence of the standard annulus while the presence of one of the larger stimuli required a right key response. Incorrect responses were followed by correction trials in which the same annulus was displayed. For the first block of each psychophysical session, the subject was presented the 3-mm and 15-mm annuli. The second or 'assessment' block contained the same stimulus pair. If the bird made 90~o or more correct responses in this block, then the remaining comparison stimuli were presented. Otherwise, the subject was presented with the same 15-mm vs. 3-mm annuli for the remainder of the session. Assuming the bird 'passed assessment' the other five annuli were paired with the standard in the next five blocks in order of decreasing size (10, 7, 5, 4, and 3.5 mm). A similar second sequence of blocks followed. The percentage of correct responses was plotted as a function of the diameter value of the comparison stimulus (in mm) for each session.
Surgery Within 48 h of satisfying the preoperative stability criterion, the subjects underwent stereotaxic surgery. TheY were anesthetized with sodium pentobarbital (10 mg/kg) i.m. followed by ketamine (5 mg/kg) i.m. The ketamine doses were repeated as necessary to maintain a surgical depth of anesthesia. The intended incision site was infiltrated with xylocaine and, following incision and retraction of the scalp, a high-speed, gas-turbine drill was used to penetrate the skull. Two small bone flaps were removed in order to allow the electrode access to the brain. The electrodes were stainless-steel insect pins that had been insulated with epoxy paint. The exposed tips were 5.0 mm in length. The electrode was positioned in the brain according to the stereotaxic coordinates obtained from the atlas of Karten and H o d o s 16. The intended target regions were the hippocampus and parahippocampus. Bilateral, anodal electrolytic lesions were made in the target region.
TABLE I Hippocampal lesion parameters Lesion 1
Lesion 2
Lesion 3
Anterior coordinate Lateral coordinate Vertical coordinate
3.8 0.3 12.2
3.8 0.5 13.2
Duration (s) Intensity (mA)
15 3
20 3
3.3 1.0 Just ventral to brain surface 20 3
For each side of the brain, three lesions were made with the electrode oriented parallel to the anterior-posterior axis of the brain. The lesion parameters are summarized in Table I.
Histology Upon completion of postoperative testing the subjects were deeply anesthetized with sodium pentobarbital intravenously and were perfused via the left ventricle with normal saline followed by Heidenhain's solution (without mercuric chloride). The bird's head was severed from the body, the calvarium was removed, and the brain was fixed overnight in Heidenhain's solution. After fixation the brain was placed in the stereotaxic instrument and blocked in situ as described by Karten and Hodos 16. It then was removed from the skull and placed in 10~o formal saline for additional fixation and storage. Several days later the brain was washed in tap water, dehydrated via a progressive ethanol series, cleared in xylene, and embedded in paraffin. The brains were frontally cut at 10 tim. Every fifth section through the telencephalon was mounted using a diluted albumin-glycerol solution and subsequently stained with a modified version of the Kl~ver-Barrera ~7 stain for the visualization of cell bodies and myelinated axons. Each brain was examined microscopically for evidence of necrosis, gliosis, cell loss, and retrograde degeneration. Lesion reconstructions The lesions were reconstructed, without knowledge of the behavioral data, on standard drawings of the pigeon brain derived from the Karten and Hodos atlas 16. The volume of each lesion and the percentage of damage to the hippocampus, parahippocampus and neighboring regions were measured with a Summagraphics Bit-Pad digitizing tablet interfaced to a Kaypro 10 microcomputer. Hippocampal boundaries described in Erichsen, Bingman and Krebs 7 were used to compute hippocampus and parahippocampus volumes.
207 4.0
RESULTS
3.5
Anatomical Table II presents a summary of the quantitative analysis of the lesion reconstructions. Data are given for the percent of damage to the hippocampus and parahippocampus. Three percentages are given. These are the percentage of damage to the right hemisphere (R~o), the percentage of damage to the left hemisphere (L~o) and a weighted index of the bilateral damage (W~). The latter is the product of R~o and L ~ divided by 1001 ~. Also given in Table II is the extent of HP damage (hippocampus and parahippocampus) as a percentage of total lesion volume. As may be seen in Table II, 6 of 7 birds sustained approximately 5 0 ~ or greater damage to all components of liP (L~o and R~o for hippocampus and parahippocampus, Table II), with "4 birds (D896, D888, D903 and D908) sustaining almost complete damage to the hippocampus. HP damage accounted for most of the total lesion damage sustained by the birds. It was also found that W ~ scores for hippocampus and parahippocampus damage was larger for the birds in the size-difference task than the birds in the acuity task (hippocampus t5=2.71, P 0.10; immediate post-op vs. stable post-op t 3 = 1.46, P>0.10). Pearson product-moment correlations between the mean postoperative threshold changes of the birds and the W~o values of (a)hippocampus (Table II), (b)parahippocampus (Table II), (c)hippocampus and parahippocampus combined, and (d)total lesion volume were not significant (P> 0.10).
TABLE II
Sutmnary of the hippocampal lesion vohtme analysis Subject
Acuity study D831 D833 D847 D896 Size difference threshold study D888 D903 D908
tlippocampus
Parahippocampus IV%
L%
R%
IV%
Hippocampal damage as percent total lesion vohtme
53 57 15 88
36 31 4 77
54 50 36 62
45 48 30 61
24 24 11 38
65 57 64 74
100 97 87
100 97 71
94 79 77
55 79 55
52 62 43
77 87 90
L%
R%
67 54 25 87
100 I00 82
208 1.0
0.8
_= P
Lu --
~ 0.6
~ ~.
0.4
7
0.2
0.0 I'R E - O P
P(],~T-O P I"IR S T $
PO:~ T.OP L,'~~;r S
Fig. 2. Effects of hippocampal lesions on size-difference thresholds (the smallest increase in the diameter o f a standard 3.0 mm annulus that can be detccted reliably). The left ordinate indicates mean sizedifference threshold in millimcters. The bars represent the stable mean preoperative threshold, mean threshold for the first five postoperative test days and the stable mean postoperative threshold as well as standard errors for the three birds tested.
difference thresholds of the three birds studied. No differences were found between the preoperative, immediate postoperative or stable postoperative thresholds of the pigeons (pre-op vs. immediate post-op t2= 1.32, P>0.10; pre-op vs. stable post-op t2=0.68, P > 0.10; immediate post-op vs. stable post-op tz = 0.42, P>0.10). Pearson product-moment correlations between the postoperative threshold changes of the birds and the W?/o values of (a)hippocampus (Table II), (b) parahippoeampus (Table II), (c) hippocampus and parahippocampus combined and (d)total lesion volume were not significant.
DISCUSSION
The spatial-visual performance of pigeons in the psychophysical tasks examined in this study failed to reveal a role for the hippocampal formation in vision. The postoperative performance of the experimental birds was indistinguishable from their preoperative performanee. Indeed, the postoperative acuity and size difference thresholds of the birds with HP lesions matched nicely normative data for acuity and size difference threshold obtained in tmlesioned pigeons t~ The results argue strongly that the behavioral deficits found in pigeons with HP lesions when tested in a variety of memory-relatcd tasks is not based on a defect in spatial vision but on the cognitive dctnands of the tasks. The homing pigeons with HP lesions that took more time to retnrn home' or navigated poorly near their l o f t 4
seemed to have done so not because they had difficulty seeing landmarks but because they had difficulty using landmarks to navigate a course home. One important consideration is whether the HP damage obtained in tile present study was similar to that obtained in studies where cognitive deficits have been reported. The extent of HP damage described in the present study was similar to or exceeded that reported in studies where navigational deficits in homing pigeons have been observed t-a, and is similar to damage sustained by pigeons in other behavioral tasks where HP lesion was found to cause deficits 9"z~-3z. For example, Bingman and Mench 4 have found that HP lesions primarily restricted to either the hippocampus or parahippocampus are similarly effective in impairing the navigational behavior of homing pigeons. Whether sustaining primarily hippocampal or parahippocampal damage, the homing impaired birds in the study of Bingman and Mench 4 experienced less damage to HP than the birds in the present study. A possible criticism of the study reported here might be that it measured visual performance based on nearfield stimuli, and that at least for the homing behavior of pigeons, distant stimuli would be the most relevant. Several points argue against this hypothesis: ( a ) H P lesions have resulted in performance deficits 2z on tasks in which the discriminative stimuli were as close as they were in the present study, or on the order of 25-50 cm away 9'zt'2a. In other words, deficits following HP lesions are not necessarily limited to situations in which visual stimuli are viewed from a distance. (b) The data demonstrate that the retina and primary visualprocessing areas of the brain are functioning normally with regard to spatial vision despite damage to the hippocampal formation. Although an examination of the visual performance of homing pigeons using distant stimuli would be desirable, the near-field data reported here provide the best support to date that demonstrate that HP is not involved in sensory aspects of vision. Although the tasks used in the present study arc designed to measure visual performance, they also have memory components. In the acuity task, the animal must remember to peck one side key in the presence of a grating and the other side key when a grating is not prcscnt. The memory demands in the size-threshold task are even greatcr. For size-threshold, the animal must compare the size of a presented stimulus with the size of a stored representation of a similarly shaped stimulus. If the stimulus is perceived as being the same size as that stored in memory it pecks one side key, if it is different it pecks the other side key. Following HP lesions, pigeons displayed no impairment in their ability to perform either of these tasks. Indeed, their ira-
209 mediate postoperative performance did not differ significantly from their performance immediately prior to surgery. This result further reinforces observations in previous studies that indicate that the hippocampal formation plays no role in memory tasks based on simple stimulus-response associations 2"9"23.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the excellent technical assistance of Ms. Brenda Bessette and Mr. Eddie Penland. This work was supported by an NIMH postdoctoral research fellowship to VPB and Grant EY00735 from the National Eye Institute to W.H.
REFERENCES 1 Bingman, V.P., Ioa16, P., Casini, G. and Bagnoli, P., Hippocampal ablated homing pigeons show a persistent impairment in the time taken to return home, J. Comp. Physiol. A., 163 (1988) 559563. 2 Bingman, V.P., Ioal6, P., Casini, G. and Bagnoli, P., Unimpaired acquisition of spatial reference memory, but impaired homing performance in hippocampal ablated pigeons, Behav. Brabz Res., 27 (1988) 179-188. 3 Bingman, V., Ioa16, P., Casini, G. and Bagnoli, P., The avian hippocampus: evidence for a role in development of homing pigeon navigational map, Behav. Neurosci., 104 (1990) 906-911. 4 Bingman, V.P. and Mench, J.A., Homing behavior ofhippocampus and parahippocampus lesioned pigeons following shortdistance releases, Behav. Brabl Res., 40 (1990) 227-238. 5 Bradley, P., Davies, D. and Horn, G., Connections of the hyperstriatum ventrale of the domestic chick (Gallus domesticus), J. Anat., 140 (1985) 577-589. 6 Casini, G., Bingman, V.P. and Bagnoli, P., Connections of the pigeon dorsomedial forebrain studied with WGA-HRP and H 3 proline, J. Comp. NeuroL, 245 (1986) 454-470. 7 Erichsen, J., Bingman, V. and Krebs, J., The distribution ofneuropeptides in the hippocampal region of the pigeon (Cohonba livia): a basis of regional subdivisions,,/. Comp. NeuroL, 245 (1991) 478-492.
8 Fellows, B.J., Chance stimulus sequences for discrimination task, PsychoL B,dl., 67 (1967) 87-92. 9 Good, M., The effects of hippocampal-area parahippocampalis lesions on discrimination learning in the pigeon, Behav. Brahz Res., 26 (1987) 171-184. 10 Hodos, W., Bessette, B., Macko, K. and Weiss, S., Normative data for pigeon vision, Vision Res., 25 (1985) 1525-1527. 11 Hodos, W. and Bobko, P., A weighted index of bilateral brain lesions, .L Neurosci. Meth., 12 (1984) 43-47. 12 Hodos, W. and Bonbright, J., The detection of visual intensity differences by pigeons, .L Exp. Anal. Behav., 18 (1972) 471479. 13 Hodos, W. and Leibowitz, R.W., Near-field visual acuity of pigeons: effects of scoptopic adaptation and wavelength, Vision Res., 17 (1977) 463-467. 14 Hodos, W., Leibowitz, R.W. and Bonbright, J.C., Near-field visual acuity of pigeons: effects of head location and stimulus luminance, J. Exp. Anal. Behav., 25 (1976) 129-141. 15 Hodos, W., Weiss, S. and Bessette, B., Size-threshold changes after lesions of the visual telencephalon in pigeons, Behav. Brain Res., 21 (1986) 203-214. 16 Karten, H. and Hodos, W., A Stereotaxic Atlas of the Bra#l of the Pigeon, Cohtmba livia, Johns Hopkins Press, Baltimore, MD, 1967. 17 Kltlver, H. and Barrera, E., A method for combined staining of cells and fibers in the nervous system,.L Neuropathol Exp. Ne,troL, 12 (1953) 400-403. 18 Krebs, J., Sherry, D., Healy, S., Perry, V. and Vaccarino, A., Hippocampal speizialization of food storing birds, Proc. NatL Acad. Sci. USA, 86 (1989) 1388-1392. 19 Macko, K. and Hodos, W., Near-field acuity after visual system lesions in pigeons, I. Thalamus, Behav. Brahs Res., 13 (1984) 1-14. 20 Rehk~mper, G., Haase, E. and Frahm, H., Allometric comparison of brain weight and brain structure volumes in different breeds of the domestic pigeon, Cohmzba livia f.d. (fantails, homing pigeons, strassers), Brahl, Behav. EvoL, 31 (1988) 141-149. 21 Reilly, S. and Good, M., Enhanced DRL and impaired forcedchoice alternation performance following hippocampal lesion in the pigeon, Behav. Brain Res., 26 (1987) 185-197. 22 Sahgal, A., Hippocampal lesions disrupt recognition memory in pigeons, Behav. Brahl Res., 11 (1984) 47-58. 23 Sherry, D. and Vaccarino, A., The hippocampus and memory for food caches in black-capped chickadees, Behav. Neurosci., 103 (1989) 308-318.
203
BBR 01370
Visual performance of pigeons following hippocampal lesions V e r n e r P. B i n g m a n ~ a n d W i l l i a m H o d o s b "Department of Psychology, Bowling Green State University, Bowling Green, OH 43403 (USA) and bDepartment of Psychology, University of Maryland, College Park, )liD 20742 (USA) (Received 28 April 1992) (Revised version received 24 July 1992) (Accepted 4 August 1992)
Key words: Visual acuity; Size-difference threshold; Animal psychophysics; Hippocampus; Lesion; Pigeon
The effect of hippocampal lesions on performance in two psychophysical measures of spatial vision (acuity and size-difference threshold) was examined in 7 pigeons. No difference between the preoperative and postoperative thresholds of the experimental birds was found. The visual performance of pigeons in the psychophysical tasks failed to reveal a role of the hippocampal formation in vision. The results argue strongly that the behavioral deficits found in pigeons with hippocampal lesions when tested in a variety of memory-related spatial tasks is not based on a defect in spatial vision but impaired spatial cognition.
INTRODUCTION
The avian dorsomedial forebrain, or hippocampal formation (HP), is a paired structure consisting of a medial hippocampus and dorsomedial parahippoeampus 16. Studies examining HP function under natural t-4' semi-natural z3 and laboratory conditions 9'21'22 have identified the hippocampal formation as a structure critical for memory, in particular but not exclusively spatial memory. Further, the larger HP found in avian species in which complex, memory-based spatial behavior is part of their natural history has been explained by assuming that more complex spatial behavior requires larger cognitive processing capacity, which in turn requires a larger hippocampal formation 18'z~ In summary, interpretation of HP function has focussed primarily on a role in spatial cognition. An alternative hypothesis that has yet to be systematically explored, however, is that HP lesion actually may lead to one or more perceptual impairments, which might explain the observed behavioral deficits. In a number of the studies that have examined the relationship between hippocampal function and spatial cognition in birds, visual stimuli were assumed to be used bY the experimental animals to coordinate their behavior. Because HP-ablated birds showed no apparent impairment in visually guided behavior such as
pecking for seeds, experimenters tended to conclude that such birds did not suffer any visual perceptual impairmentz3. Untested, however, is the possibility that more subtle visual impairments, not easily detected by such casual observation, may in part explain the observed behavioral deficits. In support of this possibility are reported anatomical connections between HP and known or suspected visual processing areas of the avian telencephalons'6. In the present study we examined the effect of HP ablation on visual performance in two psychophysical measures of spatial vision: visual acuity and sizedifference threshold. Visual acuity reflects the subjects' ability to detect high spatial frequencies. Size-difference thresholds are determined, to a great extent, by the subjects' ability to detect low spatial frequencies. HP ablation was found not to affect threshold performance in either of the tasks. These data demonstrate that HP lesions, which result in behavioral deficits in tasks of memory and spatial cognition, do not affect performance on two sensitive measures of spatial vision. Thus, the data support hypotheses emphasizing cognitive rather than sensory deficits following hippocampal injury.
MATERIALS AND METHODS
S,bjects: acuity Correspondence: V.P. Bingman, Department of Psychology, Bowling Green State University, Bowling Green, OH 43403, USA.
The subjects were 4 White Carneaux pigeons
(Cohmlba livia) that were at least 2 years old at the start
204 of the study and had been maintained at 75-80~o of their free-feeding weights during the study.
photometer or a Simpson Model 408 photometer. Both meters were calibrated against a certified luminance standard traceable to the U.S. Bureau of Standards.
Apparatus: acuity The apparatus has previously been described in detail 13. Briefly, the experimental chamber was a 3-key Lehigh Valley Electronics pigeon chamber, the rear wall of which had been modified to allow an external light source to be focused on a diffusing screen behind the center key. The subjects viewed the stimulus through the center key which was constructed from a 1.0-mmthick glass microscope slide. The glass key was made opaque, except for a 1.5-cm-diameter circular area at its center. The light source that illuminated the center key was a Sylvania CBA lamp mounted in a slide projector. A 5.0 x 5.0 cm white, rear-view projection screen was placed directly in front of the projector lens and a 10 x 15 cm sheet of white translucent Plexiglas was positioned 15 cm behind the center key. Both served to diffuse and attenuate the intensity of the light from the projector. The luminance of the center key was maintained at 70 cd/m 2. The side keys were conventional Plexiglas pecking keys 2.5 cm in diameter. The stimuli were a series of 6, diagonally ruled, square-wave gratings, the nominal spatial frequencies of which were 1.0, 2.0, 4.0, 8.0, 12.0, and 20.0 lines/ mm. Each grating was paired with a blank stimulus constructed from Bausch and Lomb glass neutraldensity filters (nominal density = 0.3) or glass squares cut from 1.0-mm-thick microscope slides. Kodak Wratten No. 96 gelatin neutral-density filters (optical density 0.1-0.6) and, where necessary, microscope coverglass (optical density 0.04-0.07) was added to the gratings and/or the blank stimuli to match the luminances within each pair. The luminance difference within a stimulus pair did not exceed 0.02 log unit. This difference is well below the luminance difference threshold for pigeons tested in a comparable apparatus 1~ TWO sets of stimulus gratings and luminancematched neutral density filters were contained in a motor-driven wheel that was mounted between the diffusing screen and the center-viewing key. A solenoidoperated shutter mounted just behind the center key was closed between trials to prevent subjects from detecting the motion of the wheel when it changed positions. The interior of the chamber was painted fiat white and illuminated.by a ceiling-mounted day-light fluorescent lamp which was diffused by a sheet of white translucent Plexiglas. At the pigeon's eye level, the chamber illumination varied from 43.04 lux in the darkest corner to 225.96 lux directly below the lamp. All luminance measures were made using a Photovolt Model 520-M
Behavioral procedure: acuity The procedure used was a two-alternative forced choice described in previous near-field acuity studies 13'14. A brief description follows. After a period of adaptation to the vivarium, the birds were reduced gradually to 7 5 - 8 0 ~ of their freefeeding weights. When the birds were pecking a diffuse white center key reliably for a grain reinforcer, successive discrimination training began using the grating of lowest spatial frequency (1.0 line/mm) and a luminancematched blank disc. The birds were required to choose between the two side keys. The correct choice, which resulted in a reward for the bird, was a function of whether a grating was present. After the bird pecked the center key 10 times, the internal shutter of the slide projector closed, darkening the center key, and both side keys were then illuminated. A single peck on the correct side key resuited in access to the mixed grain. A 5-s time-out followed incorrect side key responses: the response keys were dark and inoperative, while the chamber illumination remained constant in order to maintain the subject's level of light adaptation. In addition to the timeout, correction of an incorrect response also was required before the next trial began. As soon as the bird reached a performance level of 90% correct on the 1.0 line/mm grating vs. blank the computer program was switched from the training program to the psychophysical program. A variant of the psychophysical method of constant stimuli was used to obtain each subject's spatial acuity. For acuity, the blank stimulus was compared against each of the six gratings that varied in spatial frequency. Each psychophysical session consisted of 14 blocks of 24 trials each for a total of 336 trials; sessions were run 5 days per week. Within a block one grating and the comparison blank were presented 12 times each in a pseudo-random fashion s. The subject viewed only one stimulus at a time as it pecked the center key 10 times; thus, the bird was required to detect if a grating was present. Left-key pecks were required if the center key had displayed the blank while the presence of a grating required a right-key response. A random 5 0 ~ of the bird's correct responses were followed by the availability of the unconditioned reinforcer; however, all correct responses were followed by illumination of the feeder light for a length of time equal to the magazine time. Incorrect responses were followed by the 5-s time-out
205 and a correction trial, in which the same grating or blank was displayed on the center key until the bird pecked the appropriate side key. Illumination of the feeder light accompanied the end of the correction trial. The data from correction trials were discarded and, hence, were not used to calculate the percentage of correct responses for any stimulus in a given block. The first block of each session was similar to the discrimination training procedure in that the lowest spatial frequency grating (1.0) line/mm and blank were presented to the subject. This block functioned as a 'warm-up' period for the animal, and the data were not used in the determination of acuity. The second block, which was considered the 'assessment' block, contained the same stimulus pair as the first block. The bird's performance in this block determined whether or not the session was going to be a psychophysical one: if the bird made 90~o or more correct responses, then the remaining gratings were presented to the subject during the psychophysical session. Otherwise, the subject was presented with the same stimuli (1.0 line/mm grating vs. blank) for the remainder of the session to give the bird more experience with the basic discrimination task, and no acuity was calculated. During the psychophysical session, assuming that the bird 'passed assessment', the other five gratings were paired with the standard in the next five blocks (blocks 3-7) in order of increasing spatial frequency (2.0, 4.0, 8.0, 12.0, 20.0 lines/mm). The second sequence of blocks (blocks 8-14) began immediately upon completion of the first. Again, the animal was given a 'warm-up' period (block 8), which allowed any strong response biases to diminish that may have developed during the more difficult discriminations in the first set of stimulus presentations~2; data from this second warm-up block also were discarded. In blocks 9-14, the second series of gratings were presented in the same order as the first series. The data obtained from the two block sequences were combined to arrive at the threshold value for the psychophysical session. The percentage of correct responses was plotted as a function of spatial frequency for each session. A computer-derived psychometric function was obtained via a linear interpolation between two data points, one above and one below the 75~o line. From this psychometric function acuity was determined. For acuity, the visual angle subtended by a single bar at threshold was estimated using the average viewing distance of pigeons (66.5 mm) from the surface of the gratings to the anterior nodal point of the eye 19. Psychophysical testing was continued until the bird's performance on the tasks met a preoperative criterion
of stability: this corresponded to a range of acuity for at least five consecutive sessions that did not vary by more than + 25~o from mean acuity for those sessions and, in addition, the threshold values were not progressively decreasing. Birds were then subjected to surgical ablation of the hippocampal formation. Psychophysical testing resumed after a recovery period of 5-7 days. The psychophysical procedure used to test the birds postoperatively was the same as the one used prior to surgery. Again, the birds were tested 5 days per week until their performance was stable.
Subjects: size difference thleshold The subjects were 3 White Carneaux pigeons that were at least 2 years of age at the start of the study and had been maintained at 7 5 - 8 0 ~ their free-feeding weights during the study.
Apparatus: size difference threshold A full description of the apparatus may be found in Hodos, Weiss and Bessette ~5. In brief, the apparatus consisted of a three-key Lehigh Valley Electronics pigeon chamber, with the wall of the experimental chamber modified to allow light from an externally located slide projector to be focused on the center key. The center key was a Plexiglas rear-projection screen. The stimuli were placed in the carousel tray of the slide projector and were projected onto the back of the center key. The luminance on the center key with no stimulus in place was 72.6 cd/m 2. Each of the three response keys was 25 mm in diameter. The three keys were arranged in a horizontal row. The stimuli in the present experiment were a series of black annuli on white backgrounds. The diameter of the standard annulus was 3 mm; the diameter values of the comparison stimuli were 3.5, 4, 5, 7, 10, and 15 mm. The difference between the inner and outer circumference of the annulus was 0.5 mm. The contrast between the white image of the annulus and the dark surround was 9 1 ~ . Each comparison annulus was paired with a standard annulus that had been matched for luminance. Two sets of stimuli were mounted in the carousel tray, one set for each half of the experimental session. An industrial-control computer (Action Instruments Basic Controller 2.2) controlled the slide projector, the sequence of stimulus presentations, and all other aspects of the apparatus and data collection.
Behavioral procedure: size difference threshoM The behavioral procedure used to measure size difference thresholds was similar to that used for acuity with the following exceptions.
206 The procedure was two-alternative forced choice as described earlier ~5. Successive discrimination training began using the 3-mm and 15-mm annuli. The side key that yielded a reward for the bird was a function of the annulus size projected on the screen. As soon as the bird reached a performance level of 90~o correct on the 15-mm vs. 3-mm discrimination problem, the computer program was switched from the training program to the psychophysical program. Within each block of a psychophysical session, one comparison and the standard stimulus were presented. The bird was required to compare each stimulus with its memory of the standard. Left key pecks were required in the presence of the standard annulus while the presence of one of the larger stimuli required a right key response. Incorrect responses were followed by correction trials in which the same annulus was displayed. For the first block of each psychophysical session, the subject was presented the 3-mm and 15-mm annuli. The second or 'assessment' block contained the same stimulus pair. If the bird made 90~o or more correct responses in this block, then the remaining comparison stimuli were presented. Otherwise, the subject was presented with the same 15-mm vs. 3-mm annuli for the remainder of the session. Assuming the bird 'passed assessment' the other five annuli were paired with the standard in the next five blocks in order of decreasing size (10, 7, 5, 4, and 3.5 mm). A similar second sequence of blocks followed. The percentage of correct responses was plotted as a function of the diameter value of the comparison stimulus (in mm) for each session.
Surgery Within 48 h of satisfying the preoperative stability criterion, the subjects underwent stereotaxic surgery. TheY were anesthetized with sodium pentobarbital (10 mg/kg) i.m. followed by ketamine (5 mg/kg) i.m. The ketamine doses were repeated as necessary to maintain a surgical depth of anesthesia. The intended incision site was infiltrated with xylocaine and, following incision and retraction of the scalp, a high-speed, gas-turbine drill was used to penetrate the skull. Two small bone flaps were removed in order to allow the electrode access to the brain. The electrodes were stainless-steel insect pins that had been insulated with epoxy paint. The exposed tips were 5.0 mm in length. The electrode was positioned in the brain according to the stereotaxic coordinates obtained from the atlas of Karten and H o d o s 16. The intended target regions were the hippocampus and parahippocampus. Bilateral, anodal electrolytic lesions were made in the target region.
TABLE I Hippocampal lesion parameters Lesion 1
Lesion 2
Lesion 3
Anterior coordinate Lateral coordinate Vertical coordinate
3.8 0.3 12.2
3.8 0.5 13.2
Duration (s) Intensity (mA)
15 3
20 3
3.3 1.0 Just ventral to brain surface 20 3
For each side of the brain, three lesions were made with the electrode oriented parallel to the anterior-posterior axis of the brain. The lesion parameters are summarized in Table I.
Histology Upon completion of postoperative testing the subjects were deeply anesthetized with sodium pentobarbital intravenously and were perfused via the left ventricle with normal saline followed by Heidenhain's solution (without mercuric chloride). The bird's head was severed from the body, the calvarium was removed, and the brain was fixed overnight in Heidenhain's solution. After fixation the brain was placed in the stereotaxic instrument and blocked in situ as described by Karten and Hodos 16. It then was removed from the skull and placed in 10~o formal saline for additional fixation and storage. Several days later the brain was washed in tap water, dehydrated via a progressive ethanol series, cleared in xylene, and embedded in paraffin. The brains were frontally cut at 10 tim. Every fifth section through the telencephalon was mounted using a diluted albumin-glycerol solution and subsequently stained with a modified version of the Kl~ver-Barrera ~7 stain for the visualization of cell bodies and myelinated axons. Each brain was examined microscopically for evidence of necrosis, gliosis, cell loss, and retrograde degeneration. Lesion reconstructions The lesions were reconstructed, without knowledge of the behavioral data, on standard drawings of the pigeon brain derived from the Karten and Hodos atlas 16. The volume of each lesion and the percentage of damage to the hippocampus, parahippocampus and neighboring regions were measured with a Summagraphics Bit-Pad digitizing tablet interfaced to a Kaypro 10 microcomputer. Hippocampal boundaries described in Erichsen, Bingman and Krebs 7 were used to compute hippocampus and parahippocampus volumes.
207 4.0
RESULTS
3.5
Anatomical Table II presents a summary of the quantitative analysis of the lesion reconstructions. Data are given for the percent of damage to the hippocampus and parahippocampus. Three percentages are given. These are the percentage of damage to the right hemisphere (R~o), the percentage of damage to the left hemisphere (L~o) and a weighted index of the bilateral damage (W~). The latter is the product of R~o and L ~ divided by 1001 ~. Also given in Table II is the extent of HP damage (hippocampus and parahippocampus) as a percentage of total lesion volume. As may be seen in Table II, 6 of 7 birds sustained approximately 5 0 ~ or greater damage to all components of liP (L~o and R~o for hippocampus and parahippocampus, Table II), with "4 birds (D896, D888, D903 and D908) sustaining almost complete damage to the hippocampus. HP damage accounted for most of the total lesion damage sustained by the birds. It was also found that W ~ scores for hippocampus and parahippocampus damage was larger for the birds in the size-difference task than the birds in the acuity task (hippocampus t5=2.71, P 0.10; immediate post-op vs. stable post-op t 3 = 1.46, P>0.10). Pearson product-moment correlations between the mean postoperative threshold changes of the birds and the W~o values of (a)hippocampus (Table II), (b)parahippocampus (Table II), (c)hippocampus and parahippocampus combined, and (d)total lesion volume were not significant (P> 0.10).
TABLE II
Sutmnary of the hippocampal lesion vohtme analysis Subject
Acuity study D831 D833 D847 D896 Size difference threshold study D888 D903 D908
tlippocampus
Parahippocampus IV%
L%
R%
IV%
Hippocampal damage as percent total lesion vohtme
53 57 15 88
36 31 4 77
54 50 36 62
45 48 30 61
24 24 11 38
65 57 64 74
100 97 87
100 97 71
94 79 77
55 79 55
52 62 43
77 87 90
L%
R%
67 54 25 87
100 I00 82
208 1.0
0.8
_= P
Lu --
~ 0.6
~ ~.
0.4
7
0.2
0.0 I'R E - O P
P(],~T-O P I"IR S T $
PO:~ T.OP L,'~~;r S
Fig. 2. Effects of hippocampal lesions on size-difference thresholds (the smallest increase in the diameter o f a standard 3.0 mm annulus that can be detccted reliably). The left ordinate indicates mean sizedifference threshold in millimcters. The bars represent the stable mean preoperative threshold, mean threshold for the first five postoperative test days and the stable mean postoperative threshold as well as standard errors for the three birds tested.
difference thresholds of the three birds studied. No differences were found between the preoperative, immediate postoperative or stable postoperative thresholds of the pigeons (pre-op vs. immediate post-op t2= 1.32, P>0.10; pre-op vs. stable post-op t2=0.68, P > 0.10; immediate post-op vs. stable post-op tz = 0.42, P>0.10). Pearson product-moment correlations between the postoperative threshold changes of the birds and the W?/o values of (a)hippocampus (Table II), (b) parahippoeampus (Table II), (c) hippocampus and parahippocampus combined and (d)total lesion volume were not significant.
DISCUSSION
The spatial-visual performance of pigeons in the psychophysical tasks examined in this study failed to reveal a role for the hippocampal formation in vision. The postoperative performance of the experimental birds was indistinguishable from their preoperative performanee. Indeed, the postoperative acuity and size difference thresholds of the birds with HP lesions matched nicely normative data for acuity and size difference threshold obtained in tmlesioned pigeons t~ The results argue strongly that the behavioral deficits found in pigeons with HP lesions when tested in a variety of memory-relatcd tasks is not based on a defect in spatial vision but on the cognitive dctnands of the tasks. The homing pigeons with HP lesions that took more time to retnrn home' or navigated poorly near their l o f t 4
seemed to have done so not because they had difficulty seeing landmarks but because they had difficulty using landmarks to navigate a course home. One important consideration is whether the HP damage obtained in tile present study was similar to that obtained in studies where cognitive deficits have been reported. The extent of HP damage described in the present study was similar to or exceeded that reported in studies where navigational deficits in homing pigeons have been observed t-a, and is similar to damage sustained by pigeons in other behavioral tasks where HP lesion was found to cause deficits 9"z~-3z. For example, Bingman and Mench 4 have found that HP lesions primarily restricted to either the hippocampus or parahippocampus are similarly effective in impairing the navigational behavior of homing pigeons. Whether sustaining primarily hippocampal or parahippocampal damage, the homing impaired birds in the study of Bingman and Mench 4 experienced less damage to HP than the birds in the present study. A possible criticism of the study reported here might be that it measured visual performance based on nearfield stimuli, and that at least for the homing behavior of pigeons, distant stimuli would be the most relevant. Several points argue against this hypothesis: ( a ) H P lesions have resulted in performance deficits 2z on tasks in which the discriminative stimuli were as close as they were in the present study, or on the order of 25-50 cm away 9'zt'2a. In other words, deficits following HP lesions are not necessarily limited to situations in which visual stimuli are viewed from a distance. (b) The data demonstrate that the retina and primary visualprocessing areas of the brain are functioning normally with regard to spatial vision despite damage to the hippocampal formation. Although an examination of the visual performance of homing pigeons using distant stimuli would be desirable, the near-field data reported here provide the best support to date that demonstrate that HP is not involved in sensory aspects of vision. Although the tasks used in the present study arc designed to measure visual performance, they also have memory components. In the acuity task, the animal must remember to peck one side key in the presence of a grating and the other side key when a grating is not prcscnt. The memory demands in the size-threshold task are even greatcr. For size-threshold, the animal must compare the size of a presented stimulus with the size of a stored representation of a similarly shaped stimulus. If the stimulus is perceived as being the same size as that stored in memory it pecks one side key, if it is different it pecks the other side key. Following HP lesions, pigeons displayed no impairment in their ability to perform either of these tasks. Indeed, their ira-
209 mediate postoperative performance did not differ significantly from their performance immediately prior to surgery. This result further reinforces observations in previous studies that indicate that the hippocampal formation plays no role in memory tasks based on simple stimulus-response associations 2"9"23.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the excellent technical assistance of Ms. Brenda Bessette and Mr. Eddie Penland. This work was supported by an NIMH postdoctoral research fellowship to VPB and Grant EY00735 from the National Eye Institute to W.H.
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