- Email: [email protected]
Visual behavior of monocularly deprived kittens treated with 6-hydroxydopamine
Developmental Brain Research, 24 (1986) 21-29 Elsevier
21
BRD 50305
Visual Behavior of Monocularly Deprived Kittens Treated with 6-Hydroxydopamine BARBARA GORDON, JEFFREY MORAN*, PAUL TROMBLEY and JENNIFER SOYKE
Department of Psychology and Institute of Neuroscience, University of Oregon, Eugene, OR 97403 (U. S. A.) (Accepted June 4th, 1985)
Key words: visual development - - visual behavior - - monocular deprivation - - 6-hydroxydopamine
Several investigators have reported that treating the visual cortex with 6-hydroxydopamine (6-OHDA) preserves the ability of a monocularly deprived eye to drive cells in the visual cortex. If 6-OHDA provides useful protection from the effects of monocular deprivation, it should also prevent the behavioral blindness that normally accompanies monocular deprivation. To test this prediction we compared the visual behavior of monocularly deprived kittens pretreated with 6-OHDA with that of kittens similarlydeprived, but not drug-treated. Kittens were trained on a visual discrimination task before drug treatment or suture. Starting at about 5 weeks of age the kittens were given 6-OHDA via ventricular cannula, given vehicle solution, or given no treatment at all. At about 6 weeks of age all kittens were monocularly deprived for one week. When the deprived eye was opened at 7 weeks of age, most kittens not receiving 6OHDA were blind when tested with the deprived eye. In contrast, none of the kittens receiving 6-OHDA intraventricularly were blind when tested with the deprived eye. 6-OHDA had no effect on performance with the non-deprived eye. We conclude that 6-OHDA protects vision through the monocularly deprived eye without impairing vision through the non-deprived eye.
INTRODUCTION M o d e r n work on plasticity in the m a m m a l i a n visual system began with Wiesel and H u b e l ' s p a p e r in 1963 on monocular deprivation31: if one eye of a kitten is deprived of vision for m o r e than a few days during the first weeks of life, subsequent stimulation of that eye fails to activate cells in the primary visual cortex. A l t h o u g h the environmental conditions controlling this ocular d o m i n a n c e shift have subsequently been described in some detail (for reviews see refs. 24 and 28), little research has been directed towards the underlying cellular mechanisms until recently. In 1979, Kasamatsu and colleagues~7,18 found that monocular deprivation did not cause an ocular dominance shift if the visual cortex was treated with 6-hydroxydopamine ( 6 - O H D A ) , a neurotoxin that selectively destroys catecholaminergic cells a n d terminals5.6. They also found that exogenous norepinephrine (NE) could prevent the effects of 6O H D A t6-18. They suggested that N E acts as a neuro-
modulator controlling plasticity and that destroying N E terminals prevents the plastic changes that normally result from m o n o c u l a r deprivation. Kasamatsu and colleagues' results and interpretations have been subject to several criticisms. Two of these criticisms provided the motivation for the current study. First, although some of these findings have been replicated2.3.s, failures to replicate have also been r e p o r t e d 1. Second, 6 - O H D A itself m a y damage geniculocortical or intracortical connections. In this case it might preserve responses from the deprived eye not by eliminating plasticity but by eliminating the binocular competition that normally accompanies monocular deprivation. U n d e r some conditions eliminating binocular competition by enucleating the experienced eye greatly attenuates the effects of monocular deprivation 4.20. In o r d e r to address these criticisms we m e a s u r e d the visual acuity of monocularly deprived kittens treated with 6 - O H D A . W e chose to measure acuity because several groups of investigators have found that the acuity of a deprived eye is roughly related to
* Present address: Laboratory of Neuropsychology, NIMH, Bethesda, MD 20205, U.S.A. Correspondence: B. Gordon, Department of Psychology, University of Oregon, Eugene, OR 97403, U.S.A. 0165-3806/86/$03.50 O 1986 Elsevier Science Publishers B.V. (Biomedical Division)
its ability to drive cells in area 1715-21,3°. Finding that a monocularly deprived animal t r e a t e d with 6 - O H D A had good acuity in its deprived eve would provide corroboration for the results of Kasamatsu and colleagues using a m e t h o d very different from theirs, A negative result would not, of course, prove Kasamatsu and colleagues wrong because some cells in the primary visual cortex may retain input from the deprived eye and yet be unable to influence m o t o r centers used in performing the visual task. If 6 - O H D A damages geniculocortical or intracortical connections serving both eyes. it should reduce the quality of vision through both eyes These deficits might be detectable by measures of visual acuity, even though a b n o r m a l cell responses have not been r e p o r t e d in area 17 of monocularly deprived kittens treated with 6 - O H D A 2,~,~,~- ~. On the other h and. if 6 - O H D A provides genuine protection from the effects of monocular deprivation, it should prevent the blindness that normally accompanies monocular deprivation t2,23,:4,2<27 without decreasing the acuity of the non-deprived eye. MATERIALS AND METHODS
Behavioral apparatus and procedure Eighteen kittens were tested in an apparatus similar to that described by Mitchell et al. 22. T h e apparatus consisted of a small box and a platform T h e box stood a short distance (13.5-20 cm~ above the platform. The platform was divided into two sides by a low partition. The floor of each side of the platform was actually a t r a p d o o r . Both t r a p d o o r s were normally kept closed and locked. A p h o t o g r a p h of a square-wave grating (light and dark phases = 40 cd/m 2 and 6.8 cd/m 2. respectively) was placed on one t r a p d o o r . The other t r a p d o o r contained a gray p h o t o g r a p h of luminance equivalent to the m e a n of the grating (23 cd/m2}. T h e positions of the two p h o t o g r a p h s could be interchanged. The kitten's task was to j u m p out of the box and onto the side of the platform that held the grating. Correct responses were r e w a r d e d with a taste of baby food; incorrect responses (jumping to the grayl were punished with a loud " N o ! " from the experimenter. The kittens ate their rewards avidly, even during and after 6 - O H D A t r e a t m e n t . Each kitten first learned this discrimination with the t r a p d o o r
open on the side containing the uniform gray stimulus. The other side of the platform contained a very low spatial frequency grating (0. tl cycles/degree~. A f t e r learning this simple discriminauon, training was continued with both doors closed and locked. All kittens rapidly learned the discrimination with both trapdoors closed: we could then esthnate the kittens' acuity by increasing the spatial frequency of the grating until the kittens could no longer discriminate between stripes and the uniform gray. After learning the discriminatinn, kittens were trained 7 days a week. 0 . 5 - 2 h/da~, They were deprived of food by taking them awax from their mothers or removing food from their cages for a few hours before training or tesnng. Every session began with an easily discriminated grating. If the kitten scored 5 out of the first 7 trials correct, the frequency of the grating was increased by 1/3 octave and 7 m o r e trials were run. If the kitten gave fewer than 5 correct responses, the spatial frequency was decreased by l/3 octave and 7 more trials were run. On the days when an acmty m e a s u r e m e n t was required I see Schedule of experiments, belowl this p r o c e d u r e was r e p e a t e d until 2i trials were collected at two grating frequencies t/3 octave apart such that the kitten gave 15 or more correct responses at the lower frequency and fewer than 15 correct responses at the higher frequency. The mean of these two frequencies was defined as the kitten's acuity for that session, The probability of 15 correct responses occurring by chance is less than 0.05 {binomial test}. On days when no acuitx measure was required, each kitten was trained for 0 . 5 - 2 h in o r d e r to maintain its familiarity with the task, When testing an eye that had been monocularly deprived, testing began with the t r a p d o o r which he ld the gray photograph p r o p p e d open about 45 ° and with the lowest spatial frequency {0.11 cvcles/degreel on the other (closed} t r a p d o o r . If the kitten scored 5 out of 7 trials correct, the t r a p d o o r holding the gray stimulus was closed and the sequence of trials continued as described above. If the kitten failed to score 5 trials correct, the e x p e r i m e n t continued with the t r a p d o o r open and the (I.11 cycles/degree grating. If a kitten failed to achieve 15 out o f 2t trials correct with the t r a p d o o r open. we considered it blind Distinguishing the open t r a p d o o r was an easier discrimination than distinguishing the lowest fre-
23 quency from a uniform gray. This 'trapdoor open' test allowed the kitten to demonstrate some visual ability when its acuity could not be measured. In order to decrease the likelihood that the kittens would develop a preference for jumping to one particular side, the positions of the grating and the gray photographs were interchanged pseudorandomly; i.e., the stimulus positions were random except that the photographs were left in one position for no more than two trials in a row. In the event that a kitten did develop a side preference, as evidenced by 3 consecutive errors to one side, several trials were given in which the grating always appeared on the side ignored by the kitten. These trials did not contribute to the acuity measure.
Groups The experimental group received 6-OHDA. The control group did not. The right eye of every kitten in both groups was sutured shut at about 43 days of age. The left eye was untouched. The number of animals in each group at each point of the experiment is given in Table 1. Ventricular injection group (n = 7). Animals in this group received injections of 6 - O H D A via ventricular cannulas. Cannulas were constructed of 19 mm lengths of 20-gauge hypodermic stock fitted with a removable cap and implanted in the left lateral ventricle under ketamine (30 mg/kg) and acepromazine (4 mg/kg) anesthesia. The cannula was placed stereotaxically at AP +11.0, L 3.0 and its tip was lowered TABLE I
Number of animals in each group tested for acuity at each stage of the experiment 6-OHDA yentricular cannula Initial binocular testing Non-deprived eye prior to opening deprived eye Non-deprived eye after deprived eye measures Deprived eye, first day of testing Deprived eye, second day of testing Both eyes occluded
Controls
7
5
7
11
7 7
11 11
7 7
10' 10"
* One animal was in a 'dark' cage with a light leak. We used the deprived eye data collected on the day of eye opening, but discarded the remainder of the data from this animal.
11-11.5 mm below the surface of the brain. The cannula was cemented to the skull with dental acrylic. Prior to applying the acrylic, the skull was coated with cyanoacrylate glue to increase adhesion of the acrylic. Controls (n = 11). All kittens in this group received right eye suture. Three animals received no other surgery or treatment. Two animals were implanted with 2 ml osmotic minipumps containing only vehicle Solution (0.4% ascorbic acid in physiological saline). Six animals were implanted with ventricular cannulas. These animals received injections on the same schedule as the 6-OHDA ventricular injection group, but received vehicle solution rather than 6O H D A . The results from all control animals were similar and have been combined.
Statistical analyses All statistical tests comparing the groups on their performance with the deprived eye were non-parametric because we could not assign a number to an animal's performance if it was blind or if it could perform only the trapdoor-open discrimination. We made 3 types of comparisons. First, we used the Mann-Whitney U-test (MWU) to find which groups were different 29. This test uses rank order. We assigned the lowest ranks to those animals which were blind and assigned the next higher rank to those animals which could only perform the discrimination with the trapdoor open. Second, we used Fisher's exact probability test (FEP) 29 to find out whether the groups differed in the proportion of animals that were blind. Third, we used the sign test 29 to determine whether the animals performed better with their deprived or with their non-deprived eyes. Schedule of experiments A typical experimental schedule for one kitten is shown in Table II. For all kittens, training was begun at 28 days of age. By 33-37 days of age all kittens had learned the discrimination. At this time we determined the binocular acuity of 12 animals (7 experimental and 5 control animals); 6 animals were not tested at this time. All animals were then assigned to either the 6-OHDA or the control group, the appropriate surgery performed, and drug administration begun. At 42-44 days of age, the right eye of each animal was sutured under ketamine anesthesia (35
24 TABLE 11
Typical experimental schedule. animal V. C. 3 Age ~daysl
Event
10 28 33
Eyes open Begin training on task Animal can perform task; implant cannula; begin 6-OHDA administration: continue training Suture right eye; continue training Discontinue 6-OHDA administration: continue training Acuity measure of left, non-deprived eye (start with easily discriminated grating) Reopen sutured, right eye; acuity measure of right, deprived eye (start with trapdoor open): place in dark room Acuity measure of right, deprived eye (start with trapdoor open); place in dark room Acuity measure of left, non-deprived eye (start with easily discriminated grating); place in dark room 'Acuity measure' with both eyes occluded
42 44 48 49 50 51 52
mg/kg). E x p e r i m e n t a l animals received at least 83% of their total dose of 6 - O H D A prior to suture. The acuity of the left. unsutured eye was determined 6 days after suture, the day before the right, sutured eye was r e o p e n e d . The sutured eye of 9 animals (2 ventricular injection, 7 controls) was o p e n e d under k e t a m i n e anesthesia. Vision through this eye was tested the following day. In 9 animals (5 ventricular injection, 4 controls) the right, sutured eye was o p e n e d under halothane anesthesia and vision through this eye was tested half an hour later. T h e r e were no differences between the results o b t a i n e d with these two p r o c e d u r e s and the results have been combined. To minimize recovery after the d e p r i v e d eye was o p e n e d , all animals lived in lightproof cages, coming out only for
tion schedule described by K a s a m a t s u and Pettigrewl7 Because this dose m a d e the animals eat poorly and lose weight, the r e m a m i n g 5 animals received a total of 4.8 mg 6 - O H D A (16 mg/ml, in 0. lq~ ascorbic acid in Ringer's) in 6 equal doses over a 6-day period. These latter animals were considerably healthier. The behavioral results were similar regardless of total 6 - O H D A dose.
Assays o f drug effectiveness For the animals receiving 6 - O H D A . the presence of side effects (e.g. urination, defecation, circling, hissing) indicated that the drug had reached the ventricles. In o r d e r to get a quantitauve estimate of depletion of N E we used high-performance liquid chrom a t o g r a p h y with electrochemical detection ( H P L ( ' EC) to measure the amount of NE in the cortices of 2 control and 5 ventricular injection animals (4.8 mg total dose J. Samples were taken from frontal, parietal. and occipital cortex on both sides of the midline as well as from the lateral geniculate nuclei. We used the p r o c e d u r e described by Cassone el al, v except that our mobile phase consisted ol 0.1 M sodium acelate. 0,02 M citric acid m o n o h y d r a t e . |00 mg/l octvl sodium sulfate. 50 mg/1 E D T A , 10('4- methanol. RESULI'S
testing. To assess changes in vision over time and variability in p e r f o r m a n c e , the acuity of the deprived eye was retested on the day after its initial test. T h e n , on the next day, the acuity of the n o n - d e p r i v e d eye was tested a second time. Finally, to be certain that occlusion of the untested eye was a d e q u a t e , we tested the kittens with both eyes occluded.
Bilateral distribution o f 6 - O H D A in cortex We have two pieces of evidence that 6 - O H D A injected unilaterally into a lateral ventricle effects cortex in both hemispheres equally, First. at the end ol the experiment. 0.1 cc ot radio o p a q u e dye (sodium diatrizoate, Winthrop) was injected into the cannulas of several kittens and an X-ray taken immediately thereafter. These X-rays showed that the dye diffused throughout the ventricles on both sides of the brain. Second. injecnon of 6 - O H D A into the left lateral ventricle depletes m o n o a m i n e s to similar levels in the cortex of both hemispheres ( T r o m b l e y , Allen, Sovke, Blaha. Lane and G o r d o n . J. Neurosci.. in press).
Drug doses and schedules Two animals received a total of l0 mg of 6 - O H D A (16 mg/ml, in 0.1% ascorbic acid in Ringer's solution) over a 7-day period following the administra-
Initial acuity measure Prior to any drugs or surgeries, a subset of the ant~ reals (7 experimental and 5 control ~was tested binocularly and found able to perform the acuity task. The
25
1.0
initial acuities of kittens subsequently placed into the experimental and control groups were not statistically different (P > 0.50, MWU; see Fig. 1).
l
Performance with deprived eye First testing day (Figs. 2 and 3). After 7 days of
0.5
/////A
o
VIllA
0
D
D
Pre-drug D = C =
Post-drug, Pre-eye opening
drug control
C
19
C
Post-eye opening
Fig. 1, Average performance with the non-deprived eye of the ventricular cannula and control animals at 3 stages of the experiment. The left histogram gives performance of 7 animals treated with 6-OHDA and 5 control animals tested with both eyes open, after the animals had learned the task, but prior to any drug treatment. The middle histogram gives the performance with the non-deprived eye the day before reopening the deprived eye. The right histogram gives the performance with the non-deprived eye the day after testing the deprived eye. Open bars are means from drug-treated animals. Hatched bars are means for controls. Because all animals had measurable acuity, standard errors were plotted.
Drug (N = 7)
Control (N = 11)
1.0
0.8
C¢¢C~
E
monocular deprivation the experimental animals saw much better than the animals in the control group. All 7 of the animals with ventrieular injections of 6O H D A could at least make the discrimination with the trapdoor open; all but one animal had measurable acuity. Only 3 of the 11 control animals had measurable acuity through the deprived eye. The remaining 8 were blind. The Mann-Whitney U-test confirms that the animals with ventricular injections of 6-OHDA did better than the control animals (P < 0.02). Even if the analysis is based only on whether or not the animals could perform trapdoor open test for blindness, we find that more control than ventricular cannula animals were blind (P < 0.005, FEP). Second test day (Fig. 4). By the second test day the two groups did not differ significantly in their acuities or in their abilities to perform the trapdoor open task (P > 0.05, MWU; P > 0.05, FEP, for all comparisons). Animals in the control group performed substantially better on the second day than they had on the previous day (P < 0.02, MWU). Six of the 10 were at least able to perform the trapdoor open test.
0.6 ,~O/CXX
g 2
0.4
Drug (N = 7) ~Ox'XXX x~CxQ,C'X K~XX
0.2
2.01.8 1.6
0 %" "~ o
7
Fig. 2. Performance using the deprived eye on the first test day after eye opening. The left, non-deprived eye of each animal was occluded and the animal forced to use its right, deprived eye. The ordinate of each graph gives the proportion of animals failing into each category. Along the abscissa of each graph are 3 categories. Animals in the "blind" category could perform neither the acuity task nor the simpler discrimination with the trapdoor open under the gray photograph. Animals in the "trapdoor open" category could not perform the acuity task but could make the discrimination with the trapdoor open. Animals in the "acuity measure" column could perform the acuity task and thus had an acuity of 0.11 cycles/degree or greater.
Control (N = ~1)
2.8_E_
~" "5
A o
o
D
1,4 1.2 1.0
o~
o
C~
o o
o
0.8 o.s 0.4
~
o
o ~x
0.2 0 TOO
8lind Animol
I
I
I
i
i
i
i
~1
I
2
3
4
5
6
7
1
2
i
a
I
3
4
5
6
7
8.
9
* no
10
111
•
Fig. 3. Performances of individual animals. Each animal's results are shown in a single column. The filled triangle gives the performance with the deprived eye on the first day of testing and the filled circle the performance on the second day of testing. The open triangle gives the performance with the non-deprived eye the day before suture opening, and the open circle gives the performance with the non-deprived eye after deprived eye testing.
26 Drug (N = 7 )
Control
(N =
10)
the day it was opened than they had with the non-deprived eye on the previous day (P < 0.01, sign test).
1.0
o E
~
"d g
~:
P
~-
0.8
Performance with both eyes covered With both eyes occluded, none of the animals could even detect the open trapdoor.
0.6 (XX~X~
o.4
Assays of drug effectiveness
0.2
0 "o
~-o
Fig. 4. Performancethrough the deprived eye on second testing day. Conventions as in Fig. 2. We presume that this improvement resulted from the visual experience they obtained in the first testing day. All animals with ventricular injection of 6-OHDA continued to make the trapdoor open discrimination. Five of the 7 animals in this group had measurable acuity (Figs. 3 and 4). Although the 6 - O H D A animals appeared to perform slightly worse on the second day than the first, their performance was not statistically different on the two days (P > 0.4, MWU). Furthermore, even the poorer performance of the 6O H D A animals on the second day of testing was better than the performance of the control animals on the first day (P < 0.02. MWU).
All the animals receiving 6 - O H D A via ventricular injection showed side effects (loud meowing, circling, hiding in corners, etc.). The average concentration of NE in two undepleted control brains was 81 _+ 14 ng/g per tissue. In 5 animals receiving 6O H D A via ventricular injection, the average concentration was 8.3 + 2.7 ng of NE/g per tissue. When each of the animals with ventricular injection was compared to the mean normal concentration, depletion varied from 81 to 98%. Fig. 5 shows sample HPLC records. DISCUSSION Our results indicate that intraventricular 6 - O H D A largely prevents the behaviorally measured blindness that usually results from monocular deprivation. Immediately after reopening the deprived eye, animals
©
Performance with non-deprived eye When using the non-deprived eye, all animals were able to perform the acuity task and their acuittes did not deteriorate from their pretreatment levels (Figs. I and 3). If there is any trend at all, the animals' acuity with the non-deprived eye seems to improve with age. There was no difference, on any day of testing, between the acuities of the two groups when using this eye (P > 0.05. MWU).
Comparison between deprived and non-deprived eye There was no difference between the performance of the drug-treated animals with the deprived eye and their performance with the non-deprived eye (P > 0.05, sign test). In contrast, animals in the control group showed evidence of visual deprivation: i.e.. they performed more poorly with the deprived eye
05 n&
n~
!
A mlr~
Je 4 rrtln
l ~j
B
Fig. 5. Sample HPLC records. Arrowspoint to the NE peak. A: record from a kitten depleted by intraventricular 6-OHDA. The peak represents 4.7 ng/g of NE. B: record from a controt kitten. Note that the sensitivity of themaehine is only one-fifth that in record A. The peak represents 62 ng/g of NE. The amount of NE in the sample cannot be calculated directly from the height of the peaks. The final calculations must take account of the peaks produced by standards and the weight of the tissue in the sampte.
27 treated with 6-OHDA perform as well with this eye as they do with the non-deprived eye. In contrast, control animals perform much more poorly with the deprived than with the non-deprived eye.
Alternative explanations Because all animals were blind when tested with both eyes occluded, it is unlikely that the occlusion of the untested eye was inadequate or that the kittens had developed an effective strategy to win at our pseudorandom task. Because all animals underwent similar surgeries for reopening the deprived eye, it is unlikely that control animals performed poorly simply because they did not feel well after surgery. We do not think that 6-OHDA causes non-specific brain damage that results in a decrease in plasticity. Using the deprived eye, the animals receiving 6OHDA actually performed better than the controls; it is difficult to explain how a toxic reaction would actually improve performance. Furthermore, when using the non-deprived eye, performance was similar for 6-OHDA animals and controls. These results are not consistent with the view that the effects are due to non-specific brain damage but are consistent with the view that 6-OHDA prevents plasticity 17,18. Similarly, we do not think that 6-OHDA prevents the effects of monocular deprivation by creating a pseudo-binocular deprivation that prevents the development of competition between synapses serving the deprived eye and synapses serving the non-deiprived eye. Although we could find no behavioral studies of short-term binocular suture or dark-rearing, animals dark-reared for several months are blind when their eyes are first opened 30. Dark-rearing for only 3-6 days causes a marked decrease in the responsiveness of the visual cortex 14. These results predict that if 6-OHDA causes an effect akin to binocular deprivation, it should decrease the acuity of both eyes. Instead, we found that both the deprived and non-deprived eye acuities of kittens treated with 6OHDA were similar before and after treatment. In contrast, the performance of the control animals was extremely poor on the first day the deprived eye was tested. Because our animals never performed as well as those studied by Mitchell 22 (see below) and were somewhat variable from day to day, we cannot rule out the possibility that the 6-OHDA animals had small decreases in acuity that we did not detect.
Comparison with other measures of acuity Our measured acuities are lower than those of Mitchell.et al. 22. At 5 weeks of age his animals could discriminate 1.2 cycles/degree while our controls could only discriminate 0.62 cycles/degree. By 7 weeks his animals could discriminate 2.5 cycles/degree while our controls could still only discriminate 0.78. On the other hand, Elberger 13 has also measured the acuity of young kittens and obtained results similar to ours. At about 7 weeks of age her kittens' acuity averaged slightly more than 0.5 cycles/degree. We do not know how to account for these differences among laboratories.
Comparison with physiology Several groups of investigators have provided support for the assumption that the acuity of an eye roughly reflects the number of cortical cells driven by that eye 15,2~,30.Therefore, it is fair to say that our behavioral results are qualitatively in agreement with the physiological results of Kasamatsu and colleagues 16117and in disagreement with those of Adrien et al.l. Like Kasamatsu and colleagues, we find that the visual system is less responsive to monocular deprivation if the brain is treated with 6-OHDA. They reported that the ocular dominance histograms from monocularly deprived animals treated with 6-OHDA were close to normal. We found that the performance of the drug-treated animals was better than the performance of the controls on the first day of deprived eye testing. In addition, the deprived eye performance on the first day of testing was not statistically different from non-deprived eye performance on the previous day. On the other hand, it is probably not possible to make detailed quantitative comparisons between acuity and ocular dominance histograms. The acuity of the deprived eye may be controlled, at least partially, by the spatial frequency sensitivity of the cells driven by that eye. Our results do not provide information about the anatomic site where 6-OHDA protects visual acuity. Because intraventricular injection allows the drug to work diffusely throughout the brain, we are unable to pinpoint the critical site or sites of action. Our results suggest that 6-OHDA does, indeed, decrease plasticity. Our finding that 6-OHDA also depletes NE does not, however, prove that NE is
necessary for plasticity. T h e best e v i d e n c e for this in-
tion of the n o r m a l signal-to-noise ratio in the c o r t e x
t e r p r e t a t i o n c o m e s f r o m K a s a m a t s u and colleagues"
can d e c r e a s e plasticity. All of these results suggest
demonstration
re-
that 6 - O H D A p r o b a b l y d o e s not e x e r t its effect via
t r e a t e d c o r t e x Is,l~.
N E d e p l e t i o n . This c o n c l u s i o n d o e s not. h o w e v e r ,
that
exogenously supplied
stored plasticity to a 6 - O H D A Nevertheless,
3 laboratories
NE
have challenged
the
view that N E is n e c e s s a r y and sufficient for plasticity:
imply that the effect of 6 - O H D A is ~'non-specific". It does suggest, t h o u g h , that a t t e m p t s to u n d e r s t a n d
( l ) D a w et al. d e p l e t e d cortical N E by cutting the
the cellular basis of 6 - O H D A ' s
dorsal n o r a d r e n e r g i c b u n d l e or by t r e a t i n g animals
search for s o m e a d d i t i o n a l m e c h a n i s m
action will h a v e to
with N - ( 2 - c h t o r o e t h y l ) - N - e t h y l - 2 - b r o m o b e n z y l a m i n e (DSP-4). N e i t h e r of t h e s e m a n i p u l a t i o n s d e c r e a s e d plasticityg-ll;
(2)
Bear
and
D a n i e l s 2 and
Bear
ACKNOWLEDGEMENTS
et al.3 t r e a t e d n e o n a t a l kittens with i n t r a p e r i t o n e a l We
thank
Rose
Lane,
Vincent
Cassone
and
6 - O H D A . This p r o c e d u r e p r o d u c e d lasting N E d e p l e t i o n but failed to p r o t e c t the p r i m a r y visual cor-
Charles B l a h a for t e a c h i n g us to d o H P L C . W e t h a n k
tex against the effects of m o n o c u l a r d e p r i v a t i o n ; (3)
Alvse
Shaw and C y n a d e r ~5 h a v e p r o v i d e d e v i d e n c e that the
Mark Mettler. M a l c o l m M a n n e s s and S t e v e B y l s m a
Rail,
Matthew
Harrison
Leah
Tufanelli,
p r e s e n c e of N E is not sufficient for plasticity. T h e y
for testing s o m e of the animals. I'his r e s e a r c h was
o b t a i n e d a d e c r e a s e in plasticity by i n j e c t i n g gluta-
s u p p o r t e d by N I H G r a m 5 R O 1 EY04050. J . M was
m a t e , not 6 - O H D A .
s u p p o r t e d by N I H T r a i n i n g G r a n t G M 07257.
T h e y suggest that any disrup-
REFERENCES 1 Adrien, J., Buisseret, P.. Fregnac, Y., Gary-Bobo, E., hnbert, M., Tassin, J.-P. and Trotter, Y., Noradrenaline et plasticit6 du cortex visuel du chaton: un reexamen, C.R. Acad. Sci., Ser. D., 295 (1982) 745-750, 2 Bear. M.F. and Daniels, J.D., The plastic response to monocular deprivation persists in kitten visual cortex after chronic depletion of norepinephrine, J. Neurosci., 3 (1983) 407-416. 3 Bear, M.F.. Paradiso, M.A,. Schwartz, M., Nelson, S.B.. Carnes, K,M. and Daniels, J.D., Two methods of catecholamine depletion in kitten visual cortex yield different effects on plasticity, Nature (Londont, 302 (1983) 245-247. 4 Blakemore, C. and Hawken. M.J., Rapid restoration ol functional input to the visual cortex of the cat after brief monocular deprivation. J. Physiol. (London), 327 (1982) 463-487. 5 Breese, G.R. and Cooper, B.R., Chemical lesioning: catecholamine pathways. In R.D. Myers lEd.), Methods in Psychobiology, Academic Press. New York, 1977, pp. 27-48. 6 Breese, G.R. and Traylor, l . D , , Depletion of brain noradrenaline and dopamine by 6-hydroxydopamine, Br. ,1, Pharmacol., 42 (1971) 88-99. 7 Cassonc, V.M., Lane, R.F. and Menaker, M., Daily rhythms of serotonin metabolism in the medial hypothal amus of the chicken: effects of pinealectomy and exogenous melatonin, Brain Res., 289 (1983) 129-134. 8 Daw, N.W., Rader, R.K., Robertson. T.W. and Ariel, M., The effects of 6-hydroxydopamine on visual deprivation in the kitten striate cortex, J. Neurosci., 3 (1983)907-914. 9 Daw, N,W.. Robertson, T.W., Rader, R.W. and Videen, Y.O., Effect of lesions of the dorsal adrenergic bundle on plasticity in the visual cortex, lnvest. Ophthalmol. (Vis. Sci., Suppl., 24 (1983) 140. 10 Daw, N.W., Robertson, T.W., Rader, R.W., Videen,
I.O and ('oscia. C.J. Substantial reduction oI cortical noradrenaline by lesions of the dorsal noradrenergic pathway does not prevent effects of monocular deprivation, J Neurosci. 4 (1984) 1354-1360. 17 Daw. N.W.. Videen. T.O., Robertson. T.W. and Rader K.. An evaluation of the hypothesis that noradrenaline affects plasticity in the developing visual cortex. In A. Fine led.), The Visual System. Symposium to Honor E.F. MacNichol. A.R. Liss, Philadelphia, 1985, pp. 133-144. 12 Dews. P.B, and Wiesel T.N.. Consequence of monocular de privation on visual behaviour in kittens. J Physiol. ,'London j, 206 (1970) 437-455. 13 Elberger. A.J., The corpus callosmn is a cmical factor fo~ developing maximum visual acmtv Dev. Brain Res. 5 11982~ 350-352. 14 Freeman. R.D.. Mallach. R and ttartley. S,. Responsivlty of normal kitten striate cortex deteriorates after brief binocular deprivation, J. Neuroph.vsiol.. 45 (1981~ 1074-1084 15 Giffin, F. and Mitchell, D.E.. The rate of recovery of vision after early monocular deprivation in kittens, J. Physiol. cLondom 274 (1978] 511-537. 16 Kasamatsu. T.. Neuronal plasticity maintained by the central norepinephrine system in the cat visual cortex. In J.M Sprague and A.N. Epstein (Eds. ~, Progress in Psychobiology and Physiological Psychology, Vol. 10. Academic Press, New York. [983, pp. 1-111. 17 Kasamatsu T. and Pettigrew. J.D.. Preservation of binocularity after monocular deprivation in the striate cortex ol kittens treated with 6-hydroxydopamine, J. Comp. Neurol. 5119791139-162 18 Kasamatsu. T.. Pettigrew. J.D. anti Ary. M., Restorauon of visual cortical plasticity by local microperfusion of norepmephrine. J. Comp. Neurol. 185 (1979) 163-182,. 19 Kasamatsu. T., Pettigrew, J.D. and Ary. M.. Cortical rccoverv from effects of monocular deprivation: acceleranon with norepinephrine and suppressmn with 6-hydroxydopamine J. Neurophysiol., 45 119811 254-266
29 20 Kratz, K.E. and Spear, P.D., Post-critical period reversal of effects of monocular deprivation in the striate cortex in cats, Neurophysiology, 39 (1976) 501-511. 21 Mitchell, D.E., Cynader, M. and Movshon, J.A., Recovery from the effects of monocular deprivation, J. Comp. Neurol., 176 (1977) 53-63. 22 Mitchell, D.E., Giffin, F., Wilkinson, F., Anderson, P. and Smith, M.L., Visual resolution in young kittens, Vision Res., 16 (1976) 363-366. 23 Movshon, J.A., Reversal of the behavioural effects of monocular deprivation in the kitten, J. Physiol. (London), 261 (1976) 175-187. 24 Movshon, J.A. and Van Sluyters, R.C., Visual neural development, Ann. Rev. Psychol., 32 (1981) 477-522. 25 Shaw, C. and Cynader, M., Disruption of cortical activity prevents ocular dominance changes in monocularly deprived kittens, Nature (London), 308 (1984) 731-734.
26 Sherman, S.M., Visual field defects in monocularly and binocularly deprived cats, Brain Res., 49 (1973) 25-45. 27 Sherman, S.M., Permanence of visual perimetry deficits in monocularly and binocularly deprived cats, Brain Res., 73 (1974) 491-501. 28 Sherman, S.M. and Spear, P.D., Organization of visual pathways in normal and visually deprived cats, Physiol. Rev., 62 (1982) 738-855. 29 Siegel, S., Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, 1956. 30 Timney, B., Mitchell, D.E. and Cynader, M., Behavioral evidence of prolonged sensitivity to effects of monocular deprivation in dark-reared cats, J. Neurophysiol., 43 (1980) 1041-1054. 31 Wiesel, T.N. and Hubel, D.H., Single cell responses in striate cortex of kittens deprived of vision in one eye, J. Neurophysiol., 26 (1963) 1003-1007.
21
BRD 50305
Visual Behavior of Monocularly Deprived Kittens Treated with 6-Hydroxydopamine BARBARA GORDON, JEFFREY MORAN*, PAUL TROMBLEY and JENNIFER SOYKE
Department of Psychology and Institute of Neuroscience, University of Oregon, Eugene, OR 97403 (U. S. A.) (Accepted June 4th, 1985)
Key words: visual development - - visual behavior - - monocular deprivation - - 6-hydroxydopamine
Several investigators have reported that treating the visual cortex with 6-hydroxydopamine (6-OHDA) preserves the ability of a monocularly deprived eye to drive cells in the visual cortex. If 6-OHDA provides useful protection from the effects of monocular deprivation, it should also prevent the behavioral blindness that normally accompanies monocular deprivation. To test this prediction we compared the visual behavior of monocularly deprived kittens pretreated with 6-OHDA with that of kittens similarlydeprived, but not drug-treated. Kittens were trained on a visual discrimination task before drug treatment or suture. Starting at about 5 weeks of age the kittens were given 6-OHDA via ventricular cannula, given vehicle solution, or given no treatment at all. At about 6 weeks of age all kittens were monocularly deprived for one week. When the deprived eye was opened at 7 weeks of age, most kittens not receiving 6OHDA were blind when tested with the deprived eye. In contrast, none of the kittens receiving 6-OHDA intraventricularly were blind when tested with the deprived eye. 6-OHDA had no effect on performance with the non-deprived eye. We conclude that 6-OHDA protects vision through the monocularly deprived eye without impairing vision through the non-deprived eye.
INTRODUCTION M o d e r n work on plasticity in the m a m m a l i a n visual system began with Wiesel and H u b e l ' s p a p e r in 1963 on monocular deprivation31: if one eye of a kitten is deprived of vision for m o r e than a few days during the first weeks of life, subsequent stimulation of that eye fails to activate cells in the primary visual cortex. A l t h o u g h the environmental conditions controlling this ocular d o m i n a n c e shift have subsequently been described in some detail (for reviews see refs. 24 and 28), little research has been directed towards the underlying cellular mechanisms until recently. In 1979, Kasamatsu and colleagues~7,18 found that monocular deprivation did not cause an ocular dominance shift if the visual cortex was treated with 6-hydroxydopamine ( 6 - O H D A ) , a neurotoxin that selectively destroys catecholaminergic cells a n d terminals5.6. They also found that exogenous norepinephrine (NE) could prevent the effects of 6O H D A t6-18. They suggested that N E acts as a neuro-
modulator controlling plasticity and that destroying N E terminals prevents the plastic changes that normally result from m o n o c u l a r deprivation. Kasamatsu and colleagues' results and interpretations have been subject to several criticisms. Two of these criticisms provided the motivation for the current study. First, although some of these findings have been replicated2.3.s, failures to replicate have also been r e p o r t e d 1. Second, 6 - O H D A itself m a y damage geniculocortical or intracortical connections. In this case it might preserve responses from the deprived eye not by eliminating plasticity but by eliminating the binocular competition that normally accompanies monocular deprivation. U n d e r some conditions eliminating binocular competition by enucleating the experienced eye greatly attenuates the effects of monocular deprivation 4.20. In o r d e r to address these criticisms we m e a s u r e d the visual acuity of monocularly deprived kittens treated with 6 - O H D A . W e chose to measure acuity because several groups of investigators have found that the acuity of a deprived eye is roughly related to
* Present address: Laboratory of Neuropsychology, NIMH, Bethesda, MD 20205, U.S.A. Correspondence: B. Gordon, Department of Psychology, University of Oregon, Eugene, OR 97403, U.S.A. 0165-3806/86/$03.50 O 1986 Elsevier Science Publishers B.V. (Biomedical Division)
its ability to drive cells in area 1715-21,3°. Finding that a monocularly deprived animal t r e a t e d with 6 - O H D A had good acuity in its deprived eve would provide corroboration for the results of Kasamatsu and colleagues using a m e t h o d very different from theirs, A negative result would not, of course, prove Kasamatsu and colleagues wrong because some cells in the primary visual cortex may retain input from the deprived eye and yet be unable to influence m o t o r centers used in performing the visual task. If 6 - O H D A damages geniculocortical or intracortical connections serving both eyes. it should reduce the quality of vision through both eyes These deficits might be detectable by measures of visual acuity, even though a b n o r m a l cell responses have not been r e p o r t e d in area 17 of monocularly deprived kittens treated with 6 - O H D A 2,~,~,~- ~. On the other h and. if 6 - O H D A provides genuine protection from the effects of monocular deprivation, it should prevent the blindness that normally accompanies monocular deprivation t2,23,:4,2<27 without decreasing the acuity of the non-deprived eye. MATERIALS AND METHODS
Behavioral apparatus and procedure Eighteen kittens were tested in an apparatus similar to that described by Mitchell et al. 22. T h e apparatus consisted of a small box and a platform T h e box stood a short distance (13.5-20 cm~ above the platform. The platform was divided into two sides by a low partition. The floor of each side of the platform was actually a t r a p d o o r . Both t r a p d o o r s were normally kept closed and locked. A p h o t o g r a p h of a square-wave grating (light and dark phases = 40 cd/m 2 and 6.8 cd/m 2. respectively) was placed on one t r a p d o o r . The other t r a p d o o r contained a gray p h o t o g r a p h of luminance equivalent to the m e a n of the grating (23 cd/m2}. T h e positions of the two p h o t o g r a p h s could be interchanged. The kitten's task was to j u m p out of the box and onto the side of the platform that held the grating. Correct responses were r e w a r d e d with a taste of baby food; incorrect responses (jumping to the grayl were punished with a loud " N o ! " from the experimenter. The kittens ate their rewards avidly, even during and after 6 - O H D A t r e a t m e n t . Each kitten first learned this discrimination with the t r a p d o o r
open on the side containing the uniform gray stimulus. The other side of the platform contained a very low spatial frequency grating (0. tl cycles/degree~. A f t e r learning this simple discriminauon, training was continued with both doors closed and locked. All kittens rapidly learned the discrimination with both trapdoors closed: we could then esthnate the kittens' acuity by increasing the spatial frequency of the grating until the kittens could no longer discriminate between stripes and the uniform gray. After learning the discriminatinn, kittens were trained 7 days a week. 0 . 5 - 2 h/da~, They were deprived of food by taking them awax from their mothers or removing food from their cages for a few hours before training or tesnng. Every session began with an easily discriminated grating. If the kitten scored 5 out of the first 7 trials correct, the frequency of the grating was increased by 1/3 octave and 7 m o r e trials were run. If the kitten gave fewer than 5 correct responses, the spatial frequency was decreased by l/3 octave and 7 more trials were run. On the days when an acmty m e a s u r e m e n t was required I see Schedule of experiments, belowl this p r o c e d u r e was r e p e a t e d until 2i trials were collected at two grating frequencies t/3 octave apart such that the kitten gave 15 or more correct responses at the lower frequency and fewer than 15 correct responses at the higher frequency. The mean of these two frequencies was defined as the kitten's acuity for that session, The probability of 15 correct responses occurring by chance is less than 0.05 {binomial test}. On days when no acuitx measure was required, each kitten was trained for 0 . 5 - 2 h in o r d e r to maintain its familiarity with the task, When testing an eye that had been monocularly deprived, testing began with the t r a p d o o r which he ld the gray photograph p r o p p e d open about 45 ° and with the lowest spatial frequency {0.11 cvcles/degreel on the other (closed} t r a p d o o r . If the kitten scored 5 out of 7 trials correct, the t r a p d o o r holding the gray stimulus was closed and the sequence of trials continued as described above. If the kitten failed to score 5 trials correct, the e x p e r i m e n t continued with the t r a p d o o r open and the (I.11 cycles/degree grating. If a kitten failed to achieve 15 out o f 2t trials correct with the t r a p d o o r open. we considered it blind Distinguishing the open t r a p d o o r was an easier discrimination than distinguishing the lowest fre-
23 quency from a uniform gray. This 'trapdoor open' test allowed the kitten to demonstrate some visual ability when its acuity could not be measured. In order to decrease the likelihood that the kittens would develop a preference for jumping to one particular side, the positions of the grating and the gray photographs were interchanged pseudorandomly; i.e., the stimulus positions were random except that the photographs were left in one position for no more than two trials in a row. In the event that a kitten did develop a side preference, as evidenced by 3 consecutive errors to one side, several trials were given in which the grating always appeared on the side ignored by the kitten. These trials did not contribute to the acuity measure.
Groups The experimental group received 6-OHDA. The control group did not. The right eye of every kitten in both groups was sutured shut at about 43 days of age. The left eye was untouched. The number of animals in each group at each point of the experiment is given in Table 1. Ventricular injection group (n = 7). Animals in this group received injections of 6 - O H D A via ventricular cannulas. Cannulas were constructed of 19 mm lengths of 20-gauge hypodermic stock fitted with a removable cap and implanted in the left lateral ventricle under ketamine (30 mg/kg) and acepromazine (4 mg/kg) anesthesia. The cannula was placed stereotaxically at AP +11.0, L 3.0 and its tip was lowered TABLE I
Number of animals in each group tested for acuity at each stage of the experiment 6-OHDA yentricular cannula Initial binocular testing Non-deprived eye prior to opening deprived eye Non-deprived eye after deprived eye measures Deprived eye, first day of testing Deprived eye, second day of testing Both eyes occluded
Controls
7
5
7
11
7 7
11 11
7 7
10' 10"
* One animal was in a 'dark' cage with a light leak. We used the deprived eye data collected on the day of eye opening, but discarded the remainder of the data from this animal.
11-11.5 mm below the surface of the brain. The cannula was cemented to the skull with dental acrylic. Prior to applying the acrylic, the skull was coated with cyanoacrylate glue to increase adhesion of the acrylic. Controls (n = 11). All kittens in this group received right eye suture. Three animals received no other surgery or treatment. Two animals were implanted with 2 ml osmotic minipumps containing only vehicle Solution (0.4% ascorbic acid in physiological saline). Six animals were implanted with ventricular cannulas. These animals received injections on the same schedule as the 6-OHDA ventricular injection group, but received vehicle solution rather than 6O H D A . The results from all control animals were similar and have been combined.
Statistical analyses All statistical tests comparing the groups on their performance with the deprived eye were non-parametric because we could not assign a number to an animal's performance if it was blind or if it could perform only the trapdoor-open discrimination. We made 3 types of comparisons. First, we used the Mann-Whitney U-test (MWU) to find which groups were different 29. This test uses rank order. We assigned the lowest ranks to those animals which were blind and assigned the next higher rank to those animals which could only perform the discrimination with the trapdoor open. Second, we used Fisher's exact probability test (FEP) 29 to find out whether the groups differed in the proportion of animals that were blind. Third, we used the sign test 29 to determine whether the animals performed better with their deprived or with their non-deprived eyes. Schedule of experiments A typical experimental schedule for one kitten is shown in Table II. For all kittens, training was begun at 28 days of age. By 33-37 days of age all kittens had learned the discrimination. At this time we determined the binocular acuity of 12 animals (7 experimental and 5 control animals); 6 animals were not tested at this time. All animals were then assigned to either the 6-OHDA or the control group, the appropriate surgery performed, and drug administration begun. At 42-44 days of age, the right eye of each animal was sutured under ketamine anesthesia (35
24 TABLE 11
Typical experimental schedule. animal V. C. 3 Age ~daysl
Event
10 28 33
Eyes open Begin training on task Animal can perform task; implant cannula; begin 6-OHDA administration: continue training Suture right eye; continue training Discontinue 6-OHDA administration: continue training Acuity measure of left, non-deprived eye (start with easily discriminated grating) Reopen sutured, right eye; acuity measure of right, deprived eye (start with trapdoor open): place in dark room Acuity measure of right, deprived eye (start with trapdoor open); place in dark room Acuity measure of left, non-deprived eye (start with easily discriminated grating); place in dark room 'Acuity measure' with both eyes occluded
42 44 48 49 50 51 52
mg/kg). E x p e r i m e n t a l animals received at least 83% of their total dose of 6 - O H D A prior to suture. The acuity of the left. unsutured eye was determined 6 days after suture, the day before the right, sutured eye was r e o p e n e d . The sutured eye of 9 animals (2 ventricular injection, 7 controls) was o p e n e d under k e t a m i n e anesthesia. Vision through this eye was tested the following day. In 9 animals (5 ventricular injection, 4 controls) the right, sutured eye was o p e n e d under halothane anesthesia and vision through this eye was tested half an hour later. T h e r e were no differences between the results o b t a i n e d with these two p r o c e d u r e s and the results have been combined. To minimize recovery after the d e p r i v e d eye was o p e n e d , all animals lived in lightproof cages, coming out only for
tion schedule described by K a s a m a t s u and Pettigrewl7 Because this dose m a d e the animals eat poorly and lose weight, the r e m a m i n g 5 animals received a total of 4.8 mg 6 - O H D A (16 mg/ml, in 0. lq~ ascorbic acid in Ringer's) in 6 equal doses over a 6-day period. These latter animals were considerably healthier. The behavioral results were similar regardless of total 6 - O H D A dose.
Assays o f drug effectiveness For the animals receiving 6 - O H D A . the presence of side effects (e.g. urination, defecation, circling, hissing) indicated that the drug had reached the ventricles. In o r d e r to get a quantitauve estimate of depletion of N E we used high-performance liquid chrom a t o g r a p h y with electrochemical detection ( H P L ( ' EC) to measure the amount of NE in the cortices of 2 control and 5 ventricular injection animals (4.8 mg total dose J. Samples were taken from frontal, parietal. and occipital cortex on both sides of the midline as well as from the lateral geniculate nuclei. We used the p r o c e d u r e described by Cassone el al, v except that our mobile phase consisted ol 0.1 M sodium acelate. 0,02 M citric acid m o n o h y d r a t e . |00 mg/l octvl sodium sulfate. 50 mg/1 E D T A , 10('4- methanol. RESULI'S
testing. To assess changes in vision over time and variability in p e r f o r m a n c e , the acuity of the deprived eye was retested on the day after its initial test. T h e n , on the next day, the acuity of the n o n - d e p r i v e d eye was tested a second time. Finally, to be certain that occlusion of the untested eye was a d e q u a t e , we tested the kittens with both eyes occluded.
Bilateral distribution o f 6 - O H D A in cortex We have two pieces of evidence that 6 - O H D A injected unilaterally into a lateral ventricle effects cortex in both hemispheres equally, First. at the end ol the experiment. 0.1 cc ot radio o p a q u e dye (sodium diatrizoate, Winthrop) was injected into the cannulas of several kittens and an X-ray taken immediately thereafter. These X-rays showed that the dye diffused throughout the ventricles on both sides of the brain. Second. injecnon of 6 - O H D A into the left lateral ventricle depletes m o n o a m i n e s to similar levels in the cortex of both hemispheres ( T r o m b l e y , Allen, Sovke, Blaha. Lane and G o r d o n . J. Neurosci.. in press).
Drug doses and schedules Two animals received a total of l0 mg of 6 - O H D A (16 mg/ml, in 0.1% ascorbic acid in Ringer's solution) over a 7-day period following the administra-
Initial acuity measure Prior to any drugs or surgeries, a subset of the ant~ reals (7 experimental and 5 control ~was tested binocularly and found able to perform the acuity task. The
25
1.0
initial acuities of kittens subsequently placed into the experimental and control groups were not statistically different (P > 0.50, MWU; see Fig. 1).
l
Performance with deprived eye First testing day (Figs. 2 and 3). After 7 days of
0.5
/////A
o
VIllA
0
D
D
Pre-drug D = C =
Post-drug, Pre-eye opening
drug control
C
19
C
Post-eye opening
Fig. 1, Average performance with the non-deprived eye of the ventricular cannula and control animals at 3 stages of the experiment. The left histogram gives performance of 7 animals treated with 6-OHDA and 5 control animals tested with both eyes open, after the animals had learned the task, but prior to any drug treatment. The middle histogram gives the performance with the non-deprived eye the day before reopening the deprived eye. The right histogram gives the performance with the non-deprived eye the day after testing the deprived eye. Open bars are means from drug-treated animals. Hatched bars are means for controls. Because all animals had measurable acuity, standard errors were plotted.
Drug (N = 7)
Control (N = 11)
1.0
0.8
C¢¢C~
E
monocular deprivation the experimental animals saw much better than the animals in the control group. All 7 of the animals with ventrieular injections of 6O H D A could at least make the discrimination with the trapdoor open; all but one animal had measurable acuity. Only 3 of the 11 control animals had measurable acuity through the deprived eye. The remaining 8 were blind. The Mann-Whitney U-test confirms that the animals with ventricular injections of 6-OHDA did better than the control animals (P < 0.02). Even if the analysis is based only on whether or not the animals could perform trapdoor open test for blindness, we find that more control than ventricular cannula animals were blind (P < 0.005, FEP). Second test day (Fig. 4). By the second test day the two groups did not differ significantly in their acuities or in their abilities to perform the trapdoor open task (P > 0.05, MWU; P > 0.05, FEP, for all comparisons). Animals in the control group performed substantially better on the second day than they had on the previous day (P < 0.02, MWU). Six of the 10 were at least able to perform the trapdoor open test.
0.6 ,~O/CXX
g 2
0.4
Drug (N = 7) ~Ox'XXX x~CxQ,C'X K~XX
0.2
2.01.8 1.6
0 %" "~ o
7
Fig. 2. Performance using the deprived eye on the first test day after eye opening. The left, non-deprived eye of each animal was occluded and the animal forced to use its right, deprived eye. The ordinate of each graph gives the proportion of animals failing into each category. Along the abscissa of each graph are 3 categories. Animals in the "blind" category could perform neither the acuity task nor the simpler discrimination with the trapdoor open under the gray photograph. Animals in the "trapdoor open" category could not perform the acuity task but could make the discrimination with the trapdoor open. Animals in the "acuity measure" column could perform the acuity task and thus had an acuity of 0.11 cycles/degree or greater.
Control (N = ~1)
2.8_E_
~" "5
A o
o
D
1,4 1.2 1.0
o~
o
C~
o o
o
0.8 o.s 0.4
~
o
o ~x
0.2 0 TOO
8lind Animol
I
I
I
i
i
i
i
~1
I
2
3
4
5
6
7
1
2
i
a
I
3
4
5
6
7
8.
9
* no
10
111
•
Fig. 3. Performances of individual animals. Each animal's results are shown in a single column. The filled triangle gives the performance with the deprived eye on the first day of testing and the filled circle the performance on the second day of testing. The open triangle gives the performance with the non-deprived eye the day before suture opening, and the open circle gives the performance with the non-deprived eye after deprived eye testing.
26 Drug (N = 7 )
Control
(N =
10)
the day it was opened than they had with the non-deprived eye on the previous day (P < 0.01, sign test).
1.0
o E
~
"d g
~:
P
~-
0.8
Performance with both eyes covered With both eyes occluded, none of the animals could even detect the open trapdoor.
0.6 (XX~X~
o.4
Assays of drug effectiveness
0.2
0 "o
~-o
Fig. 4. Performancethrough the deprived eye on second testing day. Conventions as in Fig. 2. We presume that this improvement resulted from the visual experience they obtained in the first testing day. All animals with ventricular injection of 6-OHDA continued to make the trapdoor open discrimination. Five of the 7 animals in this group had measurable acuity (Figs. 3 and 4). Although the 6 - O H D A animals appeared to perform slightly worse on the second day than the first, their performance was not statistically different on the two days (P > 0.4, MWU). Furthermore, even the poorer performance of the 6O H D A animals on the second day of testing was better than the performance of the control animals on the first day (P < 0.02. MWU).
All the animals receiving 6 - O H D A via ventricular injection showed side effects (loud meowing, circling, hiding in corners, etc.). The average concentration of NE in two undepleted control brains was 81 _+ 14 ng/g per tissue. In 5 animals receiving 6O H D A via ventricular injection, the average concentration was 8.3 + 2.7 ng of NE/g per tissue. When each of the animals with ventricular injection was compared to the mean normal concentration, depletion varied from 81 to 98%. Fig. 5 shows sample HPLC records. DISCUSSION Our results indicate that intraventricular 6 - O H D A largely prevents the behaviorally measured blindness that usually results from monocular deprivation. Immediately after reopening the deprived eye, animals
©
Performance with non-deprived eye When using the non-deprived eye, all animals were able to perform the acuity task and their acuittes did not deteriorate from their pretreatment levels (Figs. I and 3). If there is any trend at all, the animals' acuity with the non-deprived eye seems to improve with age. There was no difference, on any day of testing, between the acuities of the two groups when using this eye (P > 0.05. MWU).
Comparison between deprived and non-deprived eye There was no difference between the performance of the drug-treated animals with the deprived eye and their performance with the non-deprived eye (P > 0.05, sign test). In contrast, animals in the control group showed evidence of visual deprivation: i.e.. they performed more poorly with the deprived eye
05 n&
n~
!
A mlr~
Je 4 rrtln
l ~j
B
Fig. 5. Sample HPLC records. Arrowspoint to the NE peak. A: record from a kitten depleted by intraventricular 6-OHDA. The peak represents 4.7 ng/g of NE. B: record from a controt kitten. Note that the sensitivity of themaehine is only one-fifth that in record A. The peak represents 62 ng/g of NE. The amount of NE in the sample cannot be calculated directly from the height of the peaks. The final calculations must take account of the peaks produced by standards and the weight of the tissue in the sampte.
27 treated with 6-OHDA perform as well with this eye as they do with the non-deprived eye. In contrast, control animals perform much more poorly with the deprived than with the non-deprived eye.
Alternative explanations Because all animals were blind when tested with both eyes occluded, it is unlikely that the occlusion of the untested eye was inadequate or that the kittens had developed an effective strategy to win at our pseudorandom task. Because all animals underwent similar surgeries for reopening the deprived eye, it is unlikely that control animals performed poorly simply because they did not feel well after surgery. We do not think that 6-OHDA causes non-specific brain damage that results in a decrease in plasticity. Using the deprived eye, the animals receiving 6OHDA actually performed better than the controls; it is difficult to explain how a toxic reaction would actually improve performance. Furthermore, when using the non-deprived eye, performance was similar for 6-OHDA animals and controls. These results are not consistent with the view that the effects are due to non-specific brain damage but are consistent with the view that 6-OHDA prevents plasticity 17,18. Similarly, we do not think that 6-OHDA prevents the effects of monocular deprivation by creating a pseudo-binocular deprivation that prevents the development of competition between synapses serving the deprived eye and synapses serving the non-deiprived eye. Although we could find no behavioral studies of short-term binocular suture or dark-rearing, animals dark-reared for several months are blind when their eyes are first opened 30. Dark-rearing for only 3-6 days causes a marked decrease in the responsiveness of the visual cortex 14. These results predict that if 6-OHDA causes an effect akin to binocular deprivation, it should decrease the acuity of both eyes. Instead, we found that both the deprived and non-deprived eye acuities of kittens treated with 6OHDA were similar before and after treatment. In contrast, the performance of the control animals was extremely poor on the first day the deprived eye was tested. Because our animals never performed as well as those studied by Mitchell 22 (see below) and were somewhat variable from day to day, we cannot rule out the possibility that the 6-OHDA animals had small decreases in acuity that we did not detect.
Comparison with other measures of acuity Our measured acuities are lower than those of Mitchell.et al. 22. At 5 weeks of age his animals could discriminate 1.2 cycles/degree while our controls could only discriminate 0.62 cycles/degree. By 7 weeks his animals could discriminate 2.5 cycles/degree while our controls could still only discriminate 0.78. On the other hand, Elberger 13 has also measured the acuity of young kittens and obtained results similar to ours. At about 7 weeks of age her kittens' acuity averaged slightly more than 0.5 cycles/degree. We do not know how to account for these differences among laboratories.
Comparison with physiology Several groups of investigators have provided support for the assumption that the acuity of an eye roughly reflects the number of cortical cells driven by that eye 15,2~,30.Therefore, it is fair to say that our behavioral results are qualitatively in agreement with the physiological results of Kasamatsu and colleagues 16117and in disagreement with those of Adrien et al.l. Like Kasamatsu and colleagues, we find that the visual system is less responsive to monocular deprivation if the brain is treated with 6-OHDA. They reported that the ocular dominance histograms from monocularly deprived animals treated with 6-OHDA were close to normal. We found that the performance of the drug-treated animals was better than the performance of the controls on the first day of deprived eye testing. In addition, the deprived eye performance on the first day of testing was not statistically different from non-deprived eye performance on the previous day. On the other hand, it is probably not possible to make detailed quantitative comparisons between acuity and ocular dominance histograms. The acuity of the deprived eye may be controlled, at least partially, by the spatial frequency sensitivity of the cells driven by that eye. Our results do not provide information about the anatomic site where 6-OHDA protects visual acuity. Because intraventricular injection allows the drug to work diffusely throughout the brain, we are unable to pinpoint the critical site or sites of action. Our results suggest that 6-OHDA does, indeed, decrease plasticity. Our finding that 6-OHDA also depletes NE does not, however, prove that NE is
necessary for plasticity. T h e best e v i d e n c e for this in-
tion of the n o r m a l signal-to-noise ratio in the c o r t e x
t e r p r e t a t i o n c o m e s f r o m K a s a m a t s u and colleagues"
can d e c r e a s e plasticity. All of these results suggest
demonstration
re-
that 6 - O H D A p r o b a b l y d o e s not e x e r t its effect via
t r e a t e d c o r t e x Is,l~.
N E d e p l e t i o n . This c o n c l u s i o n d o e s not. h o w e v e r ,
that
exogenously supplied
stored plasticity to a 6 - O H D A Nevertheless,
3 laboratories
NE
have challenged
the
view that N E is n e c e s s a r y and sufficient for plasticity:
imply that the effect of 6 - O H D A is ~'non-specific". It does suggest, t h o u g h , that a t t e m p t s to u n d e r s t a n d
( l ) D a w et al. d e p l e t e d cortical N E by cutting the
the cellular basis of 6 - O H D A ' s
dorsal n o r a d r e n e r g i c b u n d l e or by t r e a t i n g animals
search for s o m e a d d i t i o n a l m e c h a n i s m
action will h a v e to
with N - ( 2 - c h t o r o e t h y l ) - N - e t h y l - 2 - b r o m o b e n z y l a m i n e (DSP-4). N e i t h e r of t h e s e m a n i p u l a t i o n s d e c r e a s e d plasticityg-ll;
(2)
Bear
and
D a n i e l s 2 and
Bear
ACKNOWLEDGEMENTS
et al.3 t r e a t e d n e o n a t a l kittens with i n t r a p e r i t o n e a l We
thank
Rose
Lane,
Vincent
Cassone
and
6 - O H D A . This p r o c e d u r e p r o d u c e d lasting N E d e p l e t i o n but failed to p r o t e c t the p r i m a r y visual cor-
Charles B l a h a for t e a c h i n g us to d o H P L C . W e t h a n k
tex against the effects of m o n o c u l a r d e p r i v a t i o n ; (3)
Alvse
Shaw and C y n a d e r ~5 h a v e p r o v i d e d e v i d e n c e that the
Mark Mettler. M a l c o l m M a n n e s s and S t e v e B y l s m a
Rail,
Matthew
Harrison
Leah
Tufanelli,
p r e s e n c e of N E is not sufficient for plasticity. T h e y
for testing s o m e of the animals. I'his r e s e a r c h was
o b t a i n e d a d e c r e a s e in plasticity by i n j e c t i n g gluta-
s u p p o r t e d by N I H G r a m 5 R O 1 EY04050. J . M was
m a t e , not 6 - O H D A .
s u p p o r t e d by N I H T r a i n i n g G r a n t G M 07257.
T h e y suggest that any disrup-
REFERENCES 1 Adrien, J., Buisseret, P.. Fregnac, Y., Gary-Bobo, E., hnbert, M., Tassin, J.-P. and Trotter, Y., Noradrenaline et plasticit6 du cortex visuel du chaton: un reexamen, C.R. Acad. Sci., Ser. D., 295 (1982) 745-750, 2 Bear. M.F. and Daniels, J.D., The plastic response to monocular deprivation persists in kitten visual cortex after chronic depletion of norepinephrine, J. Neurosci., 3 (1983) 407-416. 3 Bear, M.F.. Paradiso, M.A,. Schwartz, M., Nelson, S.B.. Carnes, K,M. and Daniels, J.D., Two methods of catecholamine depletion in kitten visual cortex yield different effects on plasticity, Nature (Londont, 302 (1983) 245-247. 4 Blakemore, C. and Hawken. M.J., Rapid restoration ol functional input to the visual cortex of the cat after brief monocular deprivation. J. Physiol. (London), 327 (1982) 463-487. 5 Breese, G.R. and Cooper, B.R., Chemical lesioning: catecholamine pathways. In R.D. Myers lEd.), Methods in Psychobiology, Academic Press. New York, 1977, pp. 27-48. 6 Breese, G.R. and Traylor, l . D , , Depletion of brain noradrenaline and dopamine by 6-hydroxydopamine, Br. ,1, Pharmacol., 42 (1971) 88-99. 7 Cassonc, V.M., Lane, R.F. and Menaker, M., Daily rhythms of serotonin metabolism in the medial hypothal amus of the chicken: effects of pinealectomy and exogenous melatonin, Brain Res., 289 (1983) 129-134. 8 Daw, N.W., Rader, R.K., Robertson. T.W. and Ariel, M., The effects of 6-hydroxydopamine on visual deprivation in the kitten striate cortex, J. Neurosci., 3 (1983)907-914. 9 Daw, N,W.. Robertson, T.W., Rader, R.W. and Videen, Y.O., Effect of lesions of the dorsal adrenergic bundle on plasticity in the visual cortex, lnvest. Ophthalmol. (Vis. Sci., Suppl., 24 (1983) 140. 10 Daw, N.W., Robertson, T.W., Rader, R.W., Videen,
I.O and ('oscia. C.J. Substantial reduction oI cortical noradrenaline by lesions of the dorsal noradrenergic pathway does not prevent effects of monocular deprivation, J Neurosci. 4 (1984) 1354-1360. 17 Daw. N.W.. Videen. T.O., Robertson. T.W. and Rader K.. An evaluation of the hypothesis that noradrenaline affects plasticity in the developing visual cortex. In A. Fine led.), The Visual System. Symposium to Honor E.F. MacNichol. A.R. Liss, Philadelphia, 1985, pp. 133-144. 12 Dews. P.B, and Wiesel T.N.. Consequence of monocular de privation on visual behaviour in kittens. J Physiol. ,'London j, 206 (1970) 437-455. 13 Elberger. A.J., The corpus callosmn is a cmical factor fo~ developing maximum visual acmtv Dev. Brain Res. 5 11982~ 350-352. 14 Freeman. R.D.. Mallach. R and ttartley. S,. Responsivlty of normal kitten striate cortex deteriorates after brief binocular deprivation, J. Neuroph.vsiol.. 45 (1981~ 1074-1084 15 Giffin, F. and Mitchell, D.E.. The rate of recovery of vision after early monocular deprivation in kittens, J. Physiol. cLondom 274 (1978] 511-537. 16 Kasamatsu. T.. Neuronal plasticity maintained by the central norepinephrine system in the cat visual cortex. In J.M Sprague and A.N. Epstein (Eds. ~, Progress in Psychobiology and Physiological Psychology, Vol. 10. Academic Press, New York. [983, pp. 1-111. 17 Kasamatsu T. and Pettigrew. J.D.. Preservation of binocularity after monocular deprivation in the striate cortex ol kittens treated with 6-hydroxydopamine, J. Comp. Neurol. 5119791139-162 18 Kasamatsu. T.. Pettigrew. J.D. anti Ary. M., Restorauon of visual cortical plasticity by local microperfusion of norepmephrine. J. Comp. Neurol. 185 (1979) 163-182,. 19 Kasamatsu. T., Pettigrew, J.D. and Ary. M.. Cortical rccoverv from effects of monocular deprivation: acceleranon with norepinephrine and suppressmn with 6-hydroxydopamine J. Neurophysiol., 45 119811 254-266
29 20 Kratz, K.E. and Spear, P.D., Post-critical period reversal of effects of monocular deprivation in the striate cortex in cats, Neurophysiology, 39 (1976) 501-511. 21 Mitchell, D.E., Cynader, M. and Movshon, J.A., Recovery from the effects of monocular deprivation, J. Comp. Neurol., 176 (1977) 53-63. 22 Mitchell, D.E., Giffin, F., Wilkinson, F., Anderson, P. and Smith, M.L., Visual resolution in young kittens, Vision Res., 16 (1976) 363-366. 23 Movshon, J.A., Reversal of the behavioural effects of monocular deprivation in the kitten, J. Physiol. (London), 261 (1976) 175-187. 24 Movshon, J.A. and Van Sluyters, R.C., Visual neural development, Ann. Rev. Psychol., 32 (1981) 477-522. 25 Shaw, C. and Cynader, M., Disruption of cortical activity prevents ocular dominance changes in monocularly deprived kittens, Nature (London), 308 (1984) 731-734.
26 Sherman, S.M., Visual field defects in monocularly and binocularly deprived cats, Brain Res., 49 (1973) 25-45. 27 Sherman, S.M., Permanence of visual perimetry deficits in monocularly and binocularly deprived cats, Brain Res., 73 (1974) 491-501. 28 Sherman, S.M. and Spear, P.D., Organization of visual pathways in normal and visually deprived cats, Physiol. Rev., 62 (1982) 738-855. 29 Siegel, S., Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, 1956. 30 Timney, B., Mitchell, D.E. and Cynader, M., Behavioral evidence of prolonged sensitivity to effects of monocular deprivation in dark-reared cats, J. Neurophysiol., 43 (1980) 1041-1054. 31 Wiesel, T.N. and Hubel, D.H., Single cell responses in striate cortex of kittens deprived of vision in one eye, J. Neurophysiol., 26 (1963) 1003-1007.