Plasticity in Cat Visual Cortex Restored by Electrical Stimulation of the Locus Coeruleus
Takuji Kasamatsu 1,*, Kazushige Watabe 1,**, Paul Heggelund 2 and Erling SchOller 2
1Division of Biology 216-76, California Institute of Technology, Pasadena, CA 91125 (U.S.A.) and 2Neurobiology Laboratory, University of Trondheim, N-7055 DragvoU- Trondheim (Norway)
(Received August 13th, 1984; Revised version received and accepted January 21st, 1985)
Key words: Neuronal plasticity - - eat visual cortex - - activation of locus coeruleus
SUMMARY
It has been proposed that the presence of noradrenaline (NA)-containing terminals and NA-related receptors within the visual cortex is necessary to maintain the high level of neuronal plasticity in the immature visual cortex of kittens. In the present study we wanted to show whether electrical stimulation of the locus coeruleus (LC), which contains the somata of these cortical NA fibers, can restore neuronal plasticity to the normally aplastic visual cortex of juvenile and adult cats. We consistently found a significant loss of binocular cells in the visual cortex of mature animals which had monocular vision for only 12 h dispersed over 6 days (2 h a day, otherwise kept in the dark) in combination with concurrent LC stimulation. This result was interpreted as indicating that endogenous NA released from NA terminals restored suscep- tibility to monocular vision in the mature visual cortex. We next examined how long the restored plasticity lasts in the same animals after the LC stimulation was ended. The animals revived from the first recording session were either returned to the same daily schedule of brief monocular exposure (light/dark = 2/22 h) as before, or subjected to the usual monocular lid suture and kept in a cat colony environment (light/dark = 16/8 h). The LC electrodes had been removed and no more electrical stimulation was delivered at this stage. In the animals subjected to reiteration of brief monocular exposure, the state of reduced binocularity gradually returned to normal over a period of 2-3 weeks after stopping LC stimulation. We calculated that the revived plasticity disappeared at an average rate of a 22 % loss every 7 days. This result sharply contrasted with the result obtained in the animals subjected to usual monocular lid suture. In this test the state of reduced binocularity continued for at least the next 3 weeks, suggesting that the restored plasticity was sustained throughout a period of 3 weeks (longest term tested). The different results obtained in the two paradigms may be explained by the different strength of binocular imbalance in the two tests imposed on the visual cortex in which neuronal plasticity was restored partially.
* Correspondence: T. Kasamatsu. Present address: Smith-Kettlewell Institute of Visual Sciences, 2200 Webster Street, San Francisco, CA 94115, U.S.A.
** Present address: Department of Physiology, Aichi-Gakuln University Dental School, Nagoya 464, Japan.
We have studied roles of the central noradrenaline (NA) system in the regulation of neuronal plasticity in cat visual cortex. We have used changes in ocular dominance, which usually occur following brief monocular lid suture, as a simple yet reliable assay of visuocortical plasticity 54. Chemical lesions of the catecholamine (CA)-containing terminals within kitten visual cortex, caused by a CA-related neurotoxin, 6-hydroxydo- pamine (6-OHDA) s,16,5° abolished visuocortical plasticity 14"z5. Neuronal plasticity was then restored into such visual cortex by direct and continuous perfusion of the cortex with exogenous NA 27'44. The lowest yet effective concentration of NA for restoring plasticity was estimated as approximately 0.3/~M at the site of recording 26. Exogenous NA also restored plasticity, at least in part, in adult visual cortex 27 which is generally thought to have lost this type of neuronal plasticity due to maturation beyond the usual duration of the postnatal susceptible period ~5. Since fl-adrenergic receptors appear to be involved in visuocortical plasticity 18'19"46, we have further examined postnatal onto- geny of endogenous monoamines as well as their receptors in cat visual cortex. We found that a total number of specific fl-adrenergic receptor binding sites attained a conspicuous peak above the adult value at 5-13 weeks of age, independent of a continual increase in endogenous NA toward the adult value 17.
Recently, other groups of researchers partially confirrned our early results in kitten visual cortex which had been locally perfused with 6-OHDA; they observed a partial blockade of the expected shift in ocular dominance after brief monocular depriva- tion 1L42. Furthermore, in the visual cortex of kittens which had been exposed to unidirectionally moving vertical gratings, the cortical 6-OHDA perfusion significantly decreased experience-dependent modifiability of directional selectivity of individual cortical neurons 1~. The latter results argue for generality of the involvement of NA ter- minals in the regulation of visuocortical plasticity. Recently, however, "negative" results have been reported with 6-OHDA used in paradigms other than those of direct cortical perfusion; the usual shift in ocular dominance took place following brief monocular deprivation, despite a substantial reduction in endogenous NA in kitten visual cor- tex 1,3,4'12 (see refs. 21-23 for further discussion).
In view of the apparent discrepancy surrounding the use of 6-OHDA, we have taken the following two ways to provide further support to the NA hypothesis. One is to duplicate the original fmding of the disappearance of neuronal plasticity in kitten visual cortex by means other than using 6-OHDA. The other, more importantly, is to show that endogenous NA within the visual cortex is directly related to the high level of plasticity in preparations which are totally free from the influence of any exogenous chemicals. We recently showed that a functional blockade of fl-adrenergic receptors in kitten visual cortex with fl-adrenergic antagonists blocked, in a dose-dependent manner, the expected shift in ocular dominance following brief monocular lid suture 46 (also manuscripts in preparation). Thus the first test yielded a satisfactory answer.
The present study is concerned with the second strategy. We wanted to restore
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neuronal plasticity in the mature and normally aplastic visual cortex by endogenous NA released in response to electrical stimulation of NA-containing cells in the locus coeruleus (LC), thus leaving the visual cortex totally intact. This is an extension of our previous study in adult visual cortex; neuronal plasticity was partly restored to the aplastic adult cortex by directly perfusing the cortex with exogenous NA :7. Furthermore, we asked an important question which has not been asked before: how long does neuronal plasticity last in the adult cortex once revived by activation of the central NA system? For the In'st time in the present paradigm we were able to look straight- forwardly for an answer to the above question by recording from the same animals twice or more at various time intervals after stopping LC stimulation. A preliminary report has appeared elsewhere :9.
MATERIALS A N D M E T H O D S
A total of 35 recordings were made in the visual cortex of 19 animals, 14 juvenile (17-37 weeks of age) and 5 adult cats (60 weeks of age or older). Fifteen of them were derived from our quarantined colony of a partially inbred line of tabbies in T.K.'s laboratory. The remaining four were from the laboratory of P.H.
Implantation of stimulation electrodes in the LC To obtain the appropriate stereotaxic coordinates of the cat LC, we first studied
response properties of isolated single neurons extracellularly recorded from the fight dorsal tegmentum in half the animals. They were anesthetized with 1.0-1.5 ~ halothane mixed into N20 and 02 (2 : 1) for necessary surgical operations, and maintained with a mixture of N20, 02 and CO2 (75 : 22.5 : 2.5) throughout the recording 51. Cut wounds and pressure points were infiltrated with 2~/o carboeaine-HCl. Paralysis was attained with Flaxedil (loading dose, 40 mg; maintaining dose, 7.5 mg/kg/h). Antidromic spike activity evoked in response to electrical stimulation of the ascending NA bundle (AP 0 mm; L 2 mm; H -1.5 ram) 9"35, as well as orthodromic responses evoked by various natural stimuli, were examined in the right LC. The latter included strong flashing light, sound by hand clapping and noxious stimuli with moderate strength given to the pad of the forelegs. Body temperature was maintained at 37.5 °C and heart beat was monitored continuously. The details of this physiological identification of NA cells in the eat LC, followed by anatomical confm-nation, were already reported 52. Using the LC coordinates (P 1.5-4.0 mm; L 2.0-3.0 mm; H -1.0 to -3.0 mm) thus obtained as a guide, we next implanted a pair of gross electrodes obliquely through the cerebellum. They were made of two insulated wires glued together side-by-side with a tip separation of ~ 1.0 mm. Their exposed tip (~ 0.5 mm) was placed in or very close to a cluster of NA cells in the left LC (Fig. If). In addition to the implantation of the chronic bipolar electrodes, the right eyelid was sutured shut in order to examine whether the shift in ocular dominance takes place in the visual cortex.
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Brief monocular exposure and LC stimulation Immediately after the animal's recovery from gas anesthesia and paralysis, the
animal was placed in total darkness. Starting from the next morning the monocularly lid-sutured, awake animal was, once a day for 6 days, placed in an observation box and exposed to various visual objects in a usual laboratory environment. Concurrently, electrical stimulation consisting of a train of 4 square pulses (0.05 ms width, 50 Hz, ~ 1.5 mA) was given to the LC electrodes every 3.3 s. The intensity of stimulation current was set and stayed below the level with which slight twitches of masseter muscles, whiskers, or pinnae were observed, usually at one side of the animal's face, due presumably to the current spread to the ipsilateral mesencephalic trigeminal nucleus and its fibers. No gross changes were observed in the animal's behavior during LC stimulation, which lasted for 2 h. The polarity of the current was changed after the first hour. In 3 animals in which one visual cortex had been pretreated with the local perfusion of 4 mM 6-OHDA for a week, stimulation electrodes were implanted bilater- ally into both LCs. These LC electrodes were simultaneously stimulated with the same parameters as mentioned above. A 21-week-old cat in which the implanted LC elec- trodes were accidentally removed the next morning served as a sham-operated control. Including this animal, a total of 3 animals were subjected to the monocular exposure paradigm alone to serve as control for the effect of LC stimulation.
Plotting of receptive fields On the 7th day, single-unit recordings were carried out, following the standard
procedures in our laboratory 24'25'27'28. Briefly, visual receptive fields were plotted by a custom-made rear projection visual stimulator fitted with a joystick. Tungsten-in-glass microelectrodes 34 were introduced in the postlateral gyrus ipsilateral to the side of LC stimulation (see Table I). They were angled by 5-10 ° anteriorly and 5 ° medially, so that sampling of single units was made across laminar and columnar boundaries more than a few times. We always recorded 30 visually active cells at about every 100 #m along a long electrode track (> 3 mm). Each of these 30 cells was classified into one of the 7 ocular dominance groups following Hubel and Wiesel's scheme TM. In addition, we studied other standard receptive field properties of visually active cells as before 24,25,27,28. Throughout recordings the animals were anesthetized with a gas mixture of N20/O2/CO2 (75 : 22.5 : 2.5) and paralyzed by continual venous infusion of Flaxedil (7.5 mg/kg/h) (see above for detail during exploratory recordings).
Tests for persistence of changes in ocular dominance A total of 11 animals were revived at least once from the initial recording (see
Table I). Monocular lid suture was resumed on the same fight eye. Seven of them were returned to the darkroom and the daily light exposure was reiterated following the same schedule as before (light/dark = 2/22 h) but this time without accompanying LC stimu- lation. In a 23-week-old cat (Fig. 3) in this group, the second week of monocular exposure was still accompanied by LC stimulation so as to see if longer-term stimulation
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of the LC induced a stronger effect. This animal was eventually treated the same as the others. The remaining 4 animals were subjected, after being revived from the In'st recording, to a usual form of monocular lid suture. They were kept caged in our cat colony (light/dark -- 16/8 h) with the absence of LC stimulation. All revived animals were then recorded similarly after various time intervals in 1-3 weeks.
Histology At the deepest end of each electrode penetration, a microlesion was made by passing
a DC current (~ - 5 #A for 10 s). Later, microelectrode tracks in the visual cortex as well as the sites of electrical stimulation in the brainstem were examined in Nissl-stained histology as described before 24'25'27'28.
lndices measured and statistics To quantify changes seen in the distribution of ocular dominance we calculated the
following two indices for each original ocular dominance histogram: (1)binocularity (B) was defined as the proportion of the number of binocular cells (groups 2-6) to the total number of visually active cells recorded to construct an ocular dominance histo- gram; and (2) contra vs ipsi ratio (C/I), which concerned the relative strength of input from the two eyes, was a measure of the number of cells predominated by input from the contralateral eye (group 1-3 cells) divided by that from the ipsilateral eye (group 5-7 cells), excluding group 4 cells which by definition received balanced excitatory input from the two eyes. In the case ofmonocularly exposed or deprived animals, the D/ND was used instead of the C/I. The former was calculated in the same manner as the latter: the number of cells receiving predominant input by that from the contralateral, deprived eye (D, group 1-3 cells) was divided by that from the ipsilateral, non-deprived eye (ND, group 5-7 cells). Whenever necessary, means and standard deviations of these indices were calculated, by averaging individual values which were based on single ocular dominance distributions, and used to represent the respective composite histograms.
The means and standard deviations for the two indices in the visual cortex (area 17) of normal cats were calculated by averaging the respective values from original ocular dominance histograms (n--15) published by previous investigators in the last 20 years 5-7'10'13"14"32'33'36'41'43'53. They are:
B = 0.75 + 0.10 and C/I -- 1.52 + 0.73.
Accordingly, the confidence limits of these values were obtained at 95 % :
0.81 > B > 0.69 and 1.94 > C/I > 1.10.
The rejection limits at 5% (df~ = 1, df 2 -- 14) were as follows:
0.98 > B > 0.53 and 3.19 > C/I > 0.
For each of the indices derived from single ocular dominance histograms in Figs. 1 and 3, the deviation of its values from normal was evaluated in terms of the confidence and rejection limits mentioned above.
÷ q- -t- -F -t- -I- -t- q- q- Jr -F q- q- -I- q- q- -I- F
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372
F-tests 49 were used to compare the means of the corresponding indices (B and C/I) between normal and each of composite histograms in Figs. 2,4,5 and 6, and among the latter themselves. We assumed that the distribution of values of each index calculated for a given experimental condition can be transformed into the normal distribution and that the two standard deviations for any pair of means which are to be statistically compared by the F-test are comparable. The correlation coefficient (r) was calculated between age and binocularity for data shown in Fig. 2 and its significance was analyzed by F-tests. Mann-Whitney U-tests 47 were also included in analyzing data shown in Fig. 4.
RESULTS
Changes in ocular dominance after brief monocular vision A 17-week-old juvenile cat received monocular vision 2 h a day for 6 days together
with electrical stimulation of the LC (no. 1 in Table I). The recording was made in the visual cortex, ipsilateral to the side of LC stimulation as well as the monocularly exposed eye. Despite the age of this animal which had outgrown the usual duration of the susceptible term as well as the brevity of monocular exposure, we noted a large decrease in binocularity (B = 0.33) and a sign of the shift in ocular dominance toward the exposed eye at the expense of input from the deprived eye (D/ND = 0.22). These indices showed significantly smaller values than those obtained in the normal visual cortex (outside the 95 Yo confidence limits). This result is shown in Fig. la. The electrode track in the visual cortex was reconstructed histologically (Fig. ld). The site of electrical stimulation was found near the principal part of the LC (Fig. le).
Altogether we studied 15 animals, including the animal mentioned above, with various ages from 17 weeks old to over 3 years old. Although 4 of the 15 animals showed the ocular dominance histogram with a moderate sign of the shift as exemplified in Fig. la, the ocular dominance in 4 other animals was predominated by group 1 cells, which corresponded to the deprived eye. In the remaining 7 animals, monocular cells increased in number but the proportion of group 1 and group 7 cells stayed about the same. Thus, a composite ocular dominance histogram, obtained by averaging the number of cells in each ocular dominance group, has a W-shaped pattern as a whole (Fig. 2, right inset). Binocularity for these 15 experimental animals varied from 0.30 to 0.57, giving an average value of 0.41 (S.D. + 0.082). This was significantly smaller than the average binocularity of 0.75 (S.D. + 0.10) in the normal visual cortex (F = 102.1, df I = 1, dfz = 28, P < 0.005). On the other hand, the average D/ND ratio was 1.38 (range, 0.22-5.3; S.D. + 1.26), being not different from 1.52 (S.D. + 0.73) in normal
(F = 0.13, df~ = 1, df 2 = 28, P > 0.10). In 3 control animals which were monocularly exposed for a total of 12 h following
the same paradigm as others but without LC stimulation throughout, the distribution of ocular dominance in fact stayed normal, having an average binocularity
10- ~a
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373
d
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Fig. 1. Brief electrical stimulation delivered to an area around the locus coeruleus (LC) induced changes in ocular dominance in the visual cortex of a juvenile cat (no. 1 in Fig. 4 and Table I) which was monocularly exposed (leR eye) to a usual laboratory environment only during the LC stimulation, (2 h a day for 6 days and otherwise kept in total darkness), a: the recording (Rec. 1), which lasted for 11 h, showed a significant decrease in the proportion of binocular cells (B = 0.33) as well as a sign of the shift in ocular dominance toward the monocularly exposed left eye (D/ND = 0.22). These values were smaller than those in the normal visual cortex (95% confidence limits; 0.81 > B > 0.69, 1.94 _> C/I > 1.10). B, binocularity is defined as the ratio of group 2-6 cells to total visually responsive cells. D/ND, a shift index is defined as the ratio of the number of cells dominated by the deprived eye (groups 1-3) to that by the non-deprived, i.e. monocularly exposed eye (groups 5-7), excluding group 4 cells. Each ocular dominance histogram was compiled from 30 visually active cells (n = 30). Ocular dominance groupings were based on Hubel and Weisers scheme 14. GL and U refer to activity of lateral geniculate axons recorded in the cortex and visually unresponsive cells, respectively• b: the animal was revived from anesthesia and paralysis. Monocular exposure was then resumed for the next 7 days without accompanying LC stimulation to examine how long the changes in ocular dominance lasted in this animal. Results in the second recording (Rec. 2) still showed reduced binocularity (B = 0.47) with a suggestion of shifted ocular dominance (D/ND = 0.69). Although the B value and the D/ND ratio were still smaller than normal, the difference in the latter may not be significant (rejection limits at 5 To ; 0.98 _> B -> 0•53, 3.19 _> C/I -> 0). c: timing of LC stimulation, monocular exposure and two recordings are shown in terms of the postnatal age in weeks, d and e: drawing of histology sections which contained a small lesion at the deepest end of each microelectrode track• Arrows indicate the entry points of microelectrodes on the surface of the postlateral gyrus (area 17). Scale bar, 1 nun. f: drawing of a histology section which contained a microlesion at the deepest end of bipolar electrodes (arrow) used to stimulate the LC neurons• Scale bar, 1 ram. 5 ME, mesencephalic trigeminal nucleus; BC, brachium conjunctivum; CB, cerebellum; V4, 4th ventricle; DRM, dorsal nucleus of the raphe, median division; TDC, dorsal tegmental nucleus, central division.
Pos tna ta l Age in W e e k s ( a t f irst record ing)
Fig. 2. Binocularity (ordinate) obtained at the first recording, right after stopping brief monocular exposure combined with unilateral LC stimulation, is shown for each of 15 experimental animals (n = 15) whose postnatal age varied from 18 weeks old to over 3 years old at the time of recording (abscissa). There was a moderate correlation between the reduction in binocularity and age for animals younger than 38 weeks of age. Average binocularity (with standard deviation) was 0.41 (+0.082) for this group of animals, significantly lower than that (B = 0.78 + 0.042) obtained in 3 control cats (n = 3) which had been subjected to the same monocular vision but without accompanying LC stimulation. The D/ND ratio, however, was not significantly different between the two groups (1.38 _+ 1.26 vs 1.26 + 0.37). Seven of these animals appear in Fig. 4. The average distribution of ocular dominance for these 15 experimental and 3 control animals is shown by the respective composite histogram (experimental, right; control, lett). A vertical bar refers to a standard deviation of the average number of cells in a given group of ocular dominance. n indicates a total number of visually active cells included in each histogram. Other abbreviations are the same as appeared in Fig. 1.
(0.78 + 0.042) and a D/ND ratio (1.26 + 0.37) which were not statistically different from normal ( F = 0.14, F = 0.24, dfl = 1, df 2 = 16, P > 0.10).
In the present study, there seemed to be a moderate effect of the animal's age on the extent of decrease in binocularity in response to brief monocular exposure combined with the LC stimulation. Excluding 5 adult animals, there was a weak positive correla- tion between the age of animals and binocularity in the remaining 10 animals younger than 38 weeks of age (r = 0.64, F = 5.45, dfl -- 1, df2 = 8, P < 0.05). These results are shown in Fig. 2.
375
Persistence of the state of reduced binocularity Next, recording from the same animals more than once, we wanted to answer the
question of how long the change in ocular dominance might last in the mature cortex. The animals revived from the In'st recording were subjected to either of the following two conditions: (1)reiteration of the brief monocular exposure and otherwise kept in the dark as done before (fight/dark = 2/22 h); or (2) usual monocular lid suture and kept in our cat colony (light/dark -- 16/8 h). In both cases the implanted bipolar electrodes had been removed upon the revival of animals and no LC stimulation was accompanied at this second stage of study.
(1) Reiteration of brief monocular exposure In a 23-week-old juvenile cat, which had been subjected to brief monocular exposure
combined with LC stimulation, we found essentially the same change in the ocular dominance distribution as obtained in the younger cat exemplified in Fig. la. However, a moderate reduction of binocularity (B = 0.50) was found in this animal and no sign of the shift was visible (D/ND = 1.1). This result is shown in Fig. 3a. The animal was revived from the f'n'st recording session and monocular exposure combined with LC stimulation was resumed for the following 6 days in order to study whether doubling the duration of the pulsed monocular exposure combined with LC stimulation led to a further change in ocular dominance. We found only a small additional change; the binocularity became 0.47 and there was a slight shift in ocular dominance toward the monocularly exposed eye (D/ND -- 0.60; Fig. 3b). Assuming that wc obtained nearly the maximal effects of monocular exposure in this animal, which was then about 25 weeks old (see Fig. 3e), the daily monocular exposure was resumed for the next 12 days, this time without LC stimulation. At the time of the third recording session, the ocular dominance distribution was closer to normal than before, but binocularity (B = 0.63) was still slightly smaller than the lower end of the 95 % confidence limits (B >_ 0.69) but larger than the lower end of the rejection limits (B > 0.53) at 5% (Fig. 3c). In the following 8 days binocularity increased further up to 0.77 and group 4 ceils predominated in the ocular dominance distribution (Fig. 3d). Thus, the ocular dominance distribution of this animal seemed to have returned to normal some time between 2 and 3 weeks after the end of the LC stimulation.
We similarly studied five more animals (21 weeks to 3 years old) in which the second recording was made about one or three weeks after stopping LC stimulation. The results were essentially the same as described above. The results from these animals, a total of 7 animals including the ones shown in Figs. I and 3, are summarized by right-bound arrows in Fig. 4. We concluded that the state of reduced binocularity caused by LC stimulation in juvenile and adult cats may last for at least I week, with an increasing trend, after stopping such stimulation. More precisely, for 3 animals observed for 12 days or less, the average binocularity (B = 0.53) was still significantly lower than
normal (F = 12.65, df~ = I, df 2 = 16, P < 0.005), although the difference became insignificant for 5 animals (B = 0.73) after 20 days or later (F = 0.15, df I = I, df 2 = 18,
376
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Fig. 3. Four ocular dominance histograms (a -d) were obtained from 4 recordings in a juvenile cat (No. 3 in Fig. 4 and Table I). a: decreased binocularity (B = 0.50) was observed at the first recording (Rec. 1) which was carried out after monocular exposure for a total of 12 h (over 6 days) in conjunction with LC stimulation. There was no shift in ocular dominance (D/ND = 1.1). b: the animal was revived from the first recording, and the daily monocular exposure combined with LC stimulation was resumed and continued for the next 6 days until the second recording (Rec. 2) took place. Only a small additional decrease in binocularity, if any, was obtained by extending the combined treatment (B = 0.47, D/ND = 0.60). c and d: the animal was again revived from the second recording. Persistence of changes obtained at the second recording was examined 12 days (Rec. 3) and 21 days (Rec. 4) after stopping LC stimulation. Binocularity increased gradually toward the normal value over a period of 2-3weeks ; B=0 .47 (Ree. 2 ) ~ B = 0 . 6 3 (Rec. 3) ~ B = 0.77 (Rec. 4). The state of reduced binocularity seemed to continue for a period of at least 2-3 weeks after the end of LC stimulation, e: timing of various treatments is shown in the same fashion
as seen in Fig. lc.
INTERVALS
ANIMALS IN DAYS
no. 1 , 8
2 ~ 7
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7 ~ 24
3 ~ 12
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377
Fig. 4. Persistence of changes in ocular dominance was examined, using change in binocularity as an index, in 11 animals 1-3 weeks after stopping LC stimulation. A juvenile cat (no. 3) was recorded twice aRer stopping LC stimulation. Right-bound arrows show the amount of increase in binocularity attained by 7 animals returned to the briefmonocular exposure routine (light/dark = 2/22 h) after their revival from the first recording. The time interval between two recordings was given in days at the right end. Detailed results for two (no. 1 and no. 3) of the 7 animals in the first group appeared in Figs. 1 and 3, respectively. Left-bound arrows show the amount of decrease in binocularity obtained in 3 animals which had been subjected to a usual form of monocular lid suture and kept in a cat colony environment (light/dark = 16/8 h) aRer being revived from the first recording session. Binocularity happened to be the same for an animal (no. 14) at two recordings (star). The results from these 4 animals appear in Fig. 5.
P > 0.10). The difference in binocularity between the 3 and 5 animals, however, did not reach the significant level, when compared directly, due to the small sample size (F = 5.23, (Ill -- 1, df 2 -- 6, 0.05 < P < 0.10). A nonparametric test, however, showed the significant difference (U-test, U = 1, P = 0.036, one-tailed).
Knowing that there was no difference in the daily rate of increase in binocularity among animals in the two groups (F = 0.67, df~ = 1, df 2 = 6, P > 0.10), we combined all 8 observations to calculate the average rate of increase. It turned out that on the average 7 days were needed to increase binocularity by 0.11 (S.D. + 0.03) from the lowest value of 0.33 toward the highest of 0.83 in the present paradigm. This corresponds to an average rate of a 22% change every 7 days.
(2) Extended monocular exposure
In this test, we subjected four animals (>21 weeks old) to the usual form of monocular lid suture, after reviving them from the first recording. They were kept,
378
without LC stimulation, in our cat colony (light/dark = 16/8 h) for the next 1-3 weeks. In all four animals, binocularity did not increase when we recorded again in the cortex of these animals; in fact, binocularity further decreased in three cats and stayed the same for one (on the average, it decreased from 0.40 to 0.29). The difference did not reach the significant level due to the small sample size (/7 = 2.16, df 1 = 1, df 2 = 6, P > 0.10).
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-'I' rec 1 ~, rec 2
R'eye I [ ;~ ]~L~ I [~ I I ~ / / / / / / / / / / / ~
Fig. 5. Four cats were first treated like others to reduce binocularity by LC stimulation combined with brief monocular exposure for 6 days. After being revived from the first recording, the animal was subjected to left eyelid suture (the right eye was closed for the older 2 cats; see Table I for details) and kept in a usual cat colony environment for the next 1-3 weeks. Instead of the gradual increase in binocularity as seen for other cats exemplified in Figs. 1 and 3, binocularity either stayed the same at the low level or further decreased in these 4 animals. Accordingly, the binocularity (with standard deviation) was significantly smaller than normal at both recordings (Rec. 1, 0.40 _+ 0.086; Rec. 2, 0.29 _+ 0.093). a and b: composite histogram of ocular dominance distribution attained at the first (Ree. 1) and the second recording (Rec. 2), respectively. In the 3 animals which were twice recorded 3 weeks apart, binocularity was significantly smaller than normal at both recordings; that at the second being not significantly different from that at the first. These results suggested that the state of reduced binocularity persisted for at least 3 weeks after stopping the LC stimulation. Both recordings were made from the right hemisphere (R-VC). c: approximate timing of various manipulations in terms of the postnatal age. Note that for the older 2 cats (nos. 15 and 18, Table I) the second recording was made from the hemisphere ipsilateral to the lid-sutured eye. Except for 3 animals (nos. 15, 18 and 11, Table I) which were subjected to bilateral recordings, all animals were recorded from the visual cortex contralateral to the closed eye and ipsilateral to the stimulated LC.
379
It should be stressed, however, that the binocularity of 0.40 obtained at the fn'st recording and 0.29 obtained at the second were both significantly smaller than normal (/7 = 38.2, F = 63.2, dfl = 1, df 2 = 17, P < 0.005). The results from these four juvenile cats are summarized by a pair of composite histograms shown in Fig. 5. The decreasing trend in binocularity is also indicated with left-bound arrows at the bottom of Fig. 4. The binocularity decreased by 0.07 per week (S.D. + 0.05).
In summary, the mature cortex with reduced binocularity produced the opposite answers when further challenged by two ways of monocular deprivation: the state of reduced binocularity gradually returned to normal despite daily reiteration of brief monocular exposure, whereas the binocularity tended to decrease further with the usual form of monocular lid suture.
Where is the site of action ? In order to clarify the immediate consequence of LC stimulation, we carried out the
following study in 3 animals (> 32 weeks old). Their visual cortex had been previously perfused with 4 mM 6-OHDA 26 in only one hemisphere for a week and the other was left untouched. Immediately after the end ofthe cortical 6-OHDA perfusion the animals were exposed to brief monocular vision in conjunction with bilateral stimulation of the LCs. We obtained no indication of changes in ocular dominance (on the average; B = 0.73, D/ND = 1.46) in the hemisphere pretreated with 6-OHDA (/7 = 0.12 for B, F = 0.018 for D/ND, df I -- 1, df2 = 16, P > 0.10), while the other hemisphere of the same animals showed a significant reduction (on the average, B -- 0.42) of binocularity as expected (F = 25.71, df I = 1, df2 = 16, P < 0.005). The D/ND ratio (0.65), however, showed a small reduction (F = 3.78, df I -- 1, df 2 = 16, 0.10 > P > 0.05) due most likely to the fact that the control recording was made from the hemisphere contralateral to the exposed eye. The difference in binocularity between the two hemispheres was also highly significant (F = 21.20, dfl = 1, df 2 = 4, P < 0.01). A pair of composite histo- grams in Fig. 6 summarizes these results. These results suggested that endogenous NA released within the visual cortex in response to LC stimulation was primarily responsible, in conjunction with pulsed monocular exposure, for the observed changes in ocular dominance.
DISCUSSION
In the present study we showed that the significant changes in ocular dominance were induced to the mature visual cortex by brief monocular vision combined with direct activation of NA-containing cells in the LC. Since the visual cortex was left untouched prior to recordings from the cortex in the present paradigm, the interpretation of the present results is more free from the complications which might be present in all previous studies on the role of the NA system in modulating cortical plasticity.
15. I - - V ~ R - - V ~
~o n
U
"~ lO-
tn
I g s-
O-
380
1 2 3 4 5 6 7 GLU . . . . . . . . . monocularly m o n o c u l a r t y
e x p o s e d e x p o s e d
b i l a t e r a l L C s t i m . . . . . . ; R e c o r d i n g
L-eye ~ C R - e y e
L6-OHDA in L-VC l
, i i , , i i , i i i I , i
32 33 34 36 37 38 58 59 60
Postnatal Age in Weeks
Fig. 6. A control measure which verified the involvement of NA-contalning nerve terminals within the visual cortex in the changes in ocular dominance caused by LC stimulation in conjunction with monocular vision. Three cats were first pretreated with the intracortical perfusion of 4 mM 6-hydroxydopamine (6-OHDA) in the left hemisphere (L-VC) for a week and then for the next 6 days subjected to electrical stimulation of the right and left LC through 2 pairs of chronically implanted bipolar electrodes. Except for bilateral LC stimulation, the animal was treated in the same way as others: the parameters of LC stimulation and the scheme of brief monocular exposure were kept the same. Unit recording was made at a cortical area ~ 2 mm from the site of cannulation in the 6-OHDA-perfused hemisphere and a corresponding area in the opposite, control hemisphere, a: ocular dominance distribution was close to normal in the 6-OHDA-pre- treated, L-VC, (B = 0.73 + 0.027, D/ND = 1.46 + 0.22). b: binocularity was significantly low (0.42 + 0.088), as expected, with a small change in a D/ND ratio (0.65 + 0.16) in the opposite, right hemisphere (R-VC). The former difference was highly significant but not the latter, c: approximate timing of various manipula- tions given to these animals is shown.
Significant loss of binocular cells N o change in ocula r d o m i n a n c e occurs in the n o r m a l visual cor tex o f ma tu re cats
even if cha l lenged by m o n o c u l a r depr iva t ion for a few years. I f any change in ocular
d o m i n a n c e is ob t a ined in the adul t cortex, therefore, it requires an exp lana t ion in its o w n
fight. In all 15 exper imenta l an ima l s s tudied in the p resen t s tudy, the p ropor t ion of
b inocu la r cells was cons i s ten t ly lower t h a n normal . The average b inocular i ty , i rrespec-
tive of the an ima l ' s age w h e n L C s t imula t ion was s tar ted, was as low as 0.41 after very
br ief m o n o c u l a r exposure (12 h) co inc ided with the per iod o f L C s t imulat ion. A com par -
able decrease in b inocu la r i ty was previously repor ted fol lowing m o n o c u l a r lid suture in
381
old kittens (13 weeks old) and adult cats whose visual cortex had been directly perfused with exogenous NA and reared in a cat colony for 1-3 weeks (n = 6, B = 0.36) 27. Despite this significant reduction in binocularity, the D/ND ratio did not change appreciably, suggesting that these two variables are controlled through different mecha- nisms or that the threshold of changes is not the same for the two.
It is conceivable that an increase in the availability of NA in the visual cortex in response to LC stimulation was responsible, in conjunction with monocular vision, for the significant reduction of binocularity. The reduction of binocularity, without a change in the D/ND ratio, obtained in the mature cortex is reminiscent of the similar changes usually seen in young kittens either after very brief periods of monocular deprivation before the shift in ocular dominance eventually takes place 15'4° or at the beginning of recovery of binocularity from the shifted distribution of ocular dominance 28. The changes in binocularity may be used, without referring to underlying mechanisms which are currently unknown, to measure the susceptibility of the mature cortex to monocular vision. Thus, we interpret the significant reduction of binocularity as a sure sign of restoration of neuronal plasticity operating in a given visual cortex.
lnternal control For the 7 animals recorded at least twice in the present study, each served as its own
control: if the changes attained at the first recording were due solely to brief monocular exposure in conjunction with dark rearing, there should have been more changes in the same direction (i.e. smaller binocularity and more shift toward the monocularly exposed eye) at the later recording. The results in the later recording, however, consistently showed an increase in the number of binocular cells (F = 35.57, df~ = 1, df 2 = 7, P < 0.005). Thus, the observed changes in ocular dominance at the first recording must have been caused by brief monocular experience combined with LC stimulation. In support of this interpretation, we obtained no change in ocular dominance whatsoever in 3 control animals which were exposed to brief monocular vision alone for 6 days according to the same 2 h/day schedule (Fig. 2, left).
Primary site of action Because of the widespread projection of the ascending NA fibers in the cat brain,
the effects of LC stimulation obtained in the present study could have been secondary to changes which took place outside the visual cortex rather than due primarily to endogenous NA released within the visual cortex. For example, such a possibility includes the enhanced impulse transmission by LC stimulation in the lateral geniculate nucleus 3°'39'45, which may in turn lead to augmentation of the imposed imbalance between a pair of visual afferents from the exposed eye and the lid-sutured eye. Furthermore, as exemplified in Fig. If, the actual site of electrical stimulation was not always in the middle of the LC. This and other anatomical concerns may leave us with a possibility of stimulating some neuron groups which belong to various neuro- transmitter systems other than NA cells in the LC. Both possibilities are highly unlikely
382
to play major roles in the present study. Nevertheless, we included here a control measure in which we invariably showed that the plasticity-restoring effects of LC stimulation must be mediated through normal NA terminals within the visual cortex, since no such effect was noted in the hemisphere pretreated with 6-OHDA locally.
In the present study we were short of direct evidence for an increase in release of endogenous NA in response to electrical stimulation of the LC. Such evidence, however, is already available in rat cerebral cortex under the similar condition 31,48.
Persistence of restored plasticity We expected that the plasticity restored by the LC stimulation would outlast the
stimulation period. Binocularity, in fact, tended to decrease further as expected when the animals with partially restored plasticity were subjected to monocular lid suture and kept under the usual light/dark cycle (16/8 h) in the cat colony, the paradigm which usually causes strong binocular imbalance. However, this decreasing trend of binocularity, as compared with binocularity at the first recording, did not attain the statistical significance due probably to the small sample size (F = 4.0, dfl = 1, df 2 = 2, P > 0.10). At any rate, it should be emphasized that the binocularity was maintained significantly smaller than the normal value (B = 0.75 + 0.10) through the second recording. The current analysis clearly showed that the state of significantly reduced binocularity lasted for at least 3 weeks after stopping the LC stimulation in the 3 of the 4 animals shown in Figs. 4 and 5 (F = 54.68, dfl = 1, df 2 = 16, P < 0.005). Thus, there is no doubt about the peristence of the restored plasticity beyond the period of LC stimulation.
When we tested the same premise by pulsed (2 h daily) monocular exposure, we also found that the state of reduced binocularity indeed continued for some time. However, it gradually returned to normal, despite the maintenance of monocular vision. In this paradigm the mean rate of binocularity increase was calculated as 0.11 per 7 days. Then, in an ideal case in which binocularity was maximally reduced at the In'st recording, for example, cat no. 1 in Figs. 1 and 4 (B = 0.33), the state of reduced binocularity might have lasted for at least the next 3 weeks before it would have reached the lower end of the 95 ~o confidence limits of normal binocularity (0.81 > B >_ 0.69). It might be argued, however, that the reappearance of normal ocular dominance suggests a lack of plasticity rather than its persistence. Alternatively, the result may be interpreted as due to a net outcome of dynamic interactions between the partially restored plasticity, which tends to reduce binocularity further in response to pulsed monocular vision, and the "memory" of binocular afferents, which are secured by well-developed neuropiles and necessarily resistant to any decrease in binocularity. The balance of evidence so far obtained suggests that the latter explanation is more likely than the former as further discussed below. Another common test for the presence of plasticity with a reverse suture method was not made in the present study since our previous study has suggested that the central NA system is unlikely to be involved in a type of neuronal plasticity underlying reverse suture 2.
383
We have shown recently that even such subtle imbalance of binocular input as typically seen in the normally maturing kitten's visual cortex can result in a conspicuous change in the ocular dominance distribution, if the level of plasticity in the cortex is enhanced by exogenous NA 33. This finding suggested to us that the distribution of ocular dominance in a given cortex is primarily governed by the following 3 factors: the extent of binocular imbalance, the strength of plasticity and the amount of time that the effective binocular interaction is maintained (see also ref. 23). This thesis has in fact enjoyed further support rendered by present findings.
Thus, depending on the difference in both the extent of binocular imbalance and its duration, when subjected to the two forms (pulsed or continual) of monocular vision, the visual cortex with partially restored plasticity produced two different pictures in ocular dominance: binocularity tended to return gradually to normal over a period of 2-3 weeks if imposed binocular imbalance was very brief (2 h a day) and necessarily weak, whereas it was well sustained for at least 3 weeks with strong binocular imbalance caused by the usual form of monocular lid suture.
Implication of the present findings The present paradigm made it possible, for the fn'st time, to answer an important
question of how long neuronal plasticity restored in the mature cortex lasts. In our recent study we noted the remarkably fast regeneration of NA fibers which had degenerated at their distal end within the visual cortex foflowing the localized microperfusion with 6-OHDA 37'3s. The regenerative NA fibers were found even in the scar resulting from previous cannulation 2-4 weeks after removal of the cannula. However, since in the present study in which the visual cortex was left totally intact without obvious tissue damage due to cannulation and other factors, which were necessarily involved in previous studies with the localized perfusion method, we were assured that the persistence of plasticity observed over a period of at least 3 weeks was genuine and not complicated by the gliosis and scar formation due to cannulation, subsequent regenera- tive changes of the NA fibers and other factors.
We are further concerned about a practical aspect of the present findings. Provided that one can devise a proper visual training scheme for amblyopic subjects, the persis- tence of neuronal plasticity restored by activation of the central NA system should be beneficial in the treatment of amblyopia (see also Discussion in ref. 20). Furthermore, we have recently described in detail the presence of visual afferents which seem to excite NA-contalning cells in the cat LC and suggested that their likely pathway is through the reticular formation 52. It is thus plausible that properly induced visual signals during visual training sessions may themselves exert a positive influence in enhancing visuo- cortical plasticity, leading to a more effective rehabilitation than otherwise.
384
ACKNOWLEDGEMENTS
We are grateful to Drs. J.D. Pettigrew and E. Keller for helpful discussions. The Lederle Laboratories kindly provided Flaxedil, and Ms. M. Clancy and Ms. L. Dubs helped with histology and drawings. Ms. P. Brown, Ms. C. Hochenedel, Ms. C. Oto, Ms. S. Ovington and Ms. M. King provided the secretarial services. This study was supported by USPHS Grant EY-05549 to T.K. and Grant from the Norwegian Research council for Science and the Humanities to P.H. The completion of this work
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NOTE ADDED IN PROOF
The paper referred to as "manuscript in preparation" (Introduction, 3rd paragraph) was lately submitted for publication and is currently in press.
Kasamatsu, T. and Shirokawa, T., Involvement of/~-adrenoreceptors in the shift of ocular dominance after monocular deprivation, Exp. Brain Res., in press.
