Max-Planck-Institut für Hirnforschung, Neurophysiologische Abteilung, Frankfurt am Main and , 1 Forschergruppe Visuelle Entwicklung und Plastizität, Leibniz-Institut für Neurobiologie, Magdeburg, Germany
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Abstract |
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Introduction |
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What are the mechanisms governing the development of ocular dominance columns? It is generally assumed that the deprivation-induced reorganization of ocular dominance columns is driven by activity-dependent competition between the geniculocortical afferents of the two eyes (Stryker, 1986, 1991
; Goodman and Shatz, 1993
), whereby the temporal patterning of neuronal activity conveys the essential information (Stryker and Strickland, 1984
). Whether competition can also change the initial columnar layout, i.e. the spacing of adjacent columns, or whether this is determined and maintained by activity-independent factors intrinsic to the cortex is still a matter of debate (Jones et al., 1991
). Larger than normal spacing in strabismic cats (Löwel, 1994
) and in cats raised with alternating monocular occlusion (Tieman and Tumosa, 1997
) suggested that the degree of correlation between activity patterns conveyed by the two eyes may influence periodicity as predicted by theoretical work (Goodhill, 1993
; Goodhill and Löwel, 1995
). Although this correlation could be reduced in monocularly deprived as compared to normal cats, it is less clear from a theoretical point of view whether this factor plays a role for their columnar layout (Goodhill and Willshaw, 1994
; Goodhill and Löwel, 1995
; Wolf et al., 2000
). We therefore complemented our qualitative description of the ocular dominance patterns with a quantitative analysis of pattern layout to decide this question empirically. In particular, using a two-dimensional, nearest-neighbor analysis for quantitative measurements, we assessed the spacing of deprived eye columns statistically and, for comparison, reference columns of normal and strabismic cats which have been published previously (Löwel and Singer, 1987
, 1992
, 1993a
,b
; Löwel, 1994
; Schmidt et al., 1997
; Löwel et al., 1998
).
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Materials and Methods |
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For all surgical procedures, a short-term anesthesia was induced with an i.m. injection of ketamine hydrochloride (15 mg/kg; Ketavet, Upjohn GmbH, Heppenheim) and xylazine hydrochloride (2.5 mg/kg; Rompun, Bayer, Leverkusen). All monocularly deprived (MD) cats had one eye sutured at the age of 810 days (the left eye in cats MD1 and 2; the right eye in cats MD3 and 4). To this end, the distal lid margins of this eye were excised and then sutured. A small opening at the medial canthus was left for drainage of wound secretion and application of antibiotic ointment (Gentamicin, Hoechst). In cats S1S8, exotropic squint was induced at the age of 18 days, as previously described (Löwel and Singer, 1992; Schmidt et al., 1997
). For transneuronal labeling of ocular dominance columns cats received eye injections at ages between 2 and 4 months. Skin and sclera were incised beneath the upper bone margin of the orbit and some vitreous humor was aspirated with a syringe. [³H]proline (2.5 mCi, sp. act. 9395 Ci/mmol; Amersham, Braunschweig), dissolved in NaCl (50 µl), was injected with a Hamilton pipette into the non-deprived (right) eye of cat MD1 and into the deprived eye of cats MD2 (left), MD3 (right) and MD4 (right). Cats S3, S4, S8, N3 and N4 had their non-squinting or normal (right) eyes, cat S7 its squinting (left) eye injected. The cut was carefully closed with metal clips. After 1214 days, the time the tracer needs for transneuronal transport from the retina to the visual cortex, the cats were anesthetized as described above and then given a lethal dose of i.p. pentobarbital (180 mg/kg; Nembutal, WDT, Hannover).
At age 23 months, cats S1S4, N1, N2 and N5 had a venous catheter implanted into the humeral vein under mask anesthesia with a mixture of N2O/O2 (70%/30%) and halothane (12%) and one eye occluded with a black contact lens provided with additional black tape coverage (Löwel and Singer, 1993b). After full recovery from anesthesia (
5 h), [14C]2-deoxyglucose (100120 µCi/kg, sp. act., 300310 mCi/mmol; Amersham) was injected i.v. and the cats were allowed to freely move around in the laboratory for effective monocular stimulation. Strabismic cats S5S8 were also prepared for a 2-DG experiment under anesthetized and paralyzed conditions (2-DG data of cats S7 and S8 were not used in the following analyses; details of anesthesia and stimulation are provided in the original papers: S5/6 (Löwel et al., 1987
; Löwel and Singer, 1993b
) and S7/8 (Löwel et al., 1998
). Cats S5 and S6 were stimulated monocularly through the right eye, while the left eye was covered with a black contact lens and an additional black patch. Visual stimulation consisted of moving square wave gratings covering the central 20° of the visual field. A 1.5° wide strip along the vertical meridian was stimulated with horizontal contours only, whereas the orientation of the grating in the remaining visual field changed every 5 s in 45° steps (spatial frequency, 1, 0.5 and 0.15 cycles/degree; velocity 2 degree/s) (Löwel and Singer, 1993b
).
Histological Procedures
The occipital poles of the brains of both proline and 2-DG injected cats containing visual cortices and lateral geniculate nuclei (LGN) were removed. The LGN were frozen in methylbutane cooled to 35°C. The non-fixated cortices were flat-mounted (Freeman et al., 1987; Löwel et al., 1987
) before freezing them on dry ice. To provide landmarks for later reconstruction, three or more holes were melted into the tissue with hot needles before cutting 26 µm thick serial cryostat sections at 16°C. Blocks containing the visual cortex were cut parallel to the cortical surface; those containing the LGN were cut in the frontal plane. Sections were mounted on glass slides, dried on a hot plate and exposed to X-ray films for either 3 weeks to visualize 2-DG labeling (Structurix D7W, Agfa Gevaert) or for 816 weeks to visualize proline labeling (Hypofilm-³H, Amersham, Braunschweig). In the case of double-labeling, sections were first exposed to reveal 14C-labeling and then postfixed with 4% paraformaldehyde, washed to remove all 2-DG and then re-exposed to ³H-sensitive film (Löwel et al., 1988
).
Even after preparing flat-mounts, single sections never contained the complete pattern of [³H]proline-labeled layer IV afferents. To obtain the overall two-dimensional distribution of ocular dominance columns, a photomontage of all label-containing regions was made (Löwel and Singer, 1987).
Spillover Estimation
Since we used X-ray films instead of photo emulsions, we could not evaluate spillover on the original sections by counting silver grains in LGN neurons (LeVay et al., 1978). To get a rough estimate of the contribution of spillover of radioactive label in the LGN laminae to cortical labeling, we made optical density measurements on proline-autoradiographs of the LGN sections at three different horizontal eccentricities. Spillover was then computed as described previously (LeVay et al., 1978
) as follows. We determined the relative density of labeling in laminae A and A1 after subtracting the density of background label (depicted from unlabeled tissue parts). Subsequently, we computed the amount of label in the lamina (A or A1) which is not supposed to be innervated by the injected eye in relation to labeling in the other laminae (A1 or A, respectively) innervated by the injected eye (Table 1
). Since we did not differentiate between label in ganglion cell bodies and fibers of passage, our measurements may have overestimated actual spillover.
Quantitative Analysis
In addition to the qualitative description, we quantitatively analyzed the patterns of ocular dominance in monocularly deprived cats using a two-dimensional, nearest-neighbor analysis (Shapiro et al., 1985; Murphy et al., 1998
). For comparative reasons, we reanalyzed previously published ocular dominance patterns of normally raised and strabismic cats with the same algorithm.
Autoradiographs were digitized in 18.75-fold magnification with an image processing system (Imago II, Compulog) and displayed in grey values between 0 and 255. Subsequently, the centers of ocular dominance columns were determined as the local minima of grey values (the pixel with the darkest labeling) in the images. To obtain plausible minima, images were converted to floating point arrays and low-pass filtered using a Butterworth filter of third order at a cutoff of 25 pixels (550 µm). This particular cutoff was located above 95% of the area-under-the-curve of the one-dimensional power spectra in all analyzed images in order to assure that filtering operated outside the signals range of spatial frequencies. One-dimensional power spectra resulted from averaging the power over iso-frequencies in two-dimensional power spectra obtained by fast Fourier transformation of the images. After filtering, local minima were computed by comparing the value of each pixel with the immediately surrounding pixels.
Next, Delaunay triangulations were applied to determine the nearest-neighboring columns (Shapiro et al., 1985). This algorithm tries to find the largest point (local minimum)-free circle with a columnar center inside its convex hull (Guibas and Stolfi, 1985
). Voronoi polygons connecting all centers with the nearest-neighboring centers immediately outside the circle were fitted to the image and all distances were counted. To get as many counts as possible, we analyzed the labeling pattern in the entire area 17. Very long distances occurring as border artefacts at the outer envelopes and distances accidentally crossing more than two labeled column diameters were interactively removed from the data set as non-sense distances. All other distances of one hemisphere were counted and entered the statistical analysis (2001200 per hemisphere). Since distance distributions in single hemispheres did not always reveal a single maximum, we discarded the maximum as a descriptive value (see Fig. 1
). We chose the median rather than the mean of the distance distributions for statistical comparison in order to avoid possible influences of asymmetric extreme values: some distance distributions had remained with a small positive right tail, although we had corrected for non-sense distances.
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Results |
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Qualitative Observations
Layout of ocular dominance columns of the deprived eye
In three monocularly deprived cats (MD2, 3 and 4), [³H]proline was injected into the deprived eye. On the bright-field reproductions of the proline-autoradiographs, labeled regions appeared dark grey to black (Figs 1 and 2). The dark domains innervated by the deprived eye were shrunken and the unlabeled light grey regions representing the non-deprived eye were enlarged compared to the pattern observed in normally raised cats
compare Figures 1A,B and 2A,B
with 4B
and previously published images (Löwel and Singer, 1987
).
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Deprived eye afferents occupy more cortical territory in the contralateral compared to the ipsilateral hemisphere (compare Figs 1A and 1B, and 2A and 2B): in contralateral hemispheres, there are at least some continuous (beaded) bands, whereas ipsilaterally, only isolated islands of deprived eye domains are visible.
Single columns in the reconstructed images have variable sizes (see, for example, Fig. 1A), regardless of their eccentricity in the visual field representation. Consistent with previous investigations (Shatz and Stryker, 1978
) we observed that deprived eye domains are wider at the base of layer IV compared to more superficial regions, leading to a pyramidal shape of ocular dominance columns in the vertical plane. We therefore ascribe irregular patch sizes on the flat-mount sections to differences in section depth and angle, rather than to true differences in column diameter.
Layout of Ocular Dominance Columns of the Normal (Non-deprived) Eye in Monocularly Deprived Cats
Cat MD1 had received a [³H]proline injection into the non-deprived eye. Reconstruction of the pattern of afferences revealed an almost homogeneous dark labeling of both areas 17 and 18 in the hemisphere ipsilateral to the deprived eye (contralateral to the non-deprived/normal eye; Fig. 3A). Only some faint patches corresponding to unlabeled deprived eye afferents are visible in the center of the anterior third of the labeled area (see Fig. 3C
). Even after shorter exposure times of the autoradiographs, patterns of interdigitating ocular dominance columns could not be visualized in other regions.
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Optic Disc and Monocular Segment (MS)
Both optic disc and MS representations show marked alterations in monocularly deprived compared to normally raised animals (Fig. 5). The optic disc representations identifiable as demarcated oval regions are homogeneously labeled on the ipsilateral and pale, nearly label-free on the contralateral side of a proline injection (Löwel and Singer, 1987
). In monocularly deprived cats, the optic disc representations appear as a rather narrow, darkly labeled slab (Figs 1A, 2A and 5A,B
) and measure 0.40.6 x 2.02.9 mm, a value much smaller than 0.80.9 x 2.62.9 mm usually observed in normally raised cats (Figs 4B and 5C) (
Löwel and Singer, 1987
). Assuming an ellipsoid shape, optic disc representations of deprived eyes thus occupy up to 50% less cortical territory than those of normal eyes in normally raised cats.
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Spillover in the LGN Laminae
Spillover between laminae A and A1 ranged from 16 to maximal 40% in ipsilateral nuclei and from 30 to 50% in nuclei contralateral to the injected eye (mean values; see Table 1). There was no difference between cats with deprived eye or non-deprived eye injections. Although we may have overestimated spillover (see Materials and Methods), average spillover ratios of four cats (ipsi, 24%; contra, 45%) were in the range of ratios observed previously (LeVay et al., 1978
) for that age group (6595 days; ipsi, 1423%; contra, 3748%).
Quantitative Analysis of Intercolumnar Spacing
Since distance distributions measured by nearest-neighbor analysis from different hemispheres were rather symmetric and differed only slightly in shape (see Figs 1C,D, 3D and 4C,D for individual examples and Fig. 4E
for summary histograms), we compared average values rather than distributions. For inter-individual comparisons, the medians (see Materials and Methods) of the counted distances of each hemisphere (seven monocularly deprived, 10 normal and 14 strabismic hemispheres) are plotted in Figure 6A
. The pattern of afferents in the left hemisphere of cat MD1 revealed too few discernible columns and was discarded from quantitative analysis.
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Median spacing ranged from 843 to 890 µm in monocularly deprived cats, from 718 to 988 µm in normal cats and from 870 to 1015 µm in strabismic cats. The largest values were observed in strabismic cats, the smallest values in normally raised cats. Spacing measurements overlapped between all three groups (Fig. 6A). To test the hypothesis that columnar spacing is different in animals with different visual experience, distance distributions of rearing groups were compared pairwise using the MannWhitney U-test. Intercolumnar spacing in the group of strabismic cats (n = 14 hemispheres of seven cats; average, 942 µm) was significantly higher than in normally raised controls (n = 10 hemispheres of five cats; average, 847 µm; Mann Whitney U, P = 0.019) and monocularly deprived animals (n = 7 hemispheres of four cats; average, 868 µm; MannWhitney U, P = 0.012). However, spacing in monocularly deprived animals did not differ significantly from that in normally raised animals (MannWhitney U, P > 0.999; Fig. 6B
).
Spacing and Litter Membership
Column spacing turned out to be strongly influenced by membership of a specific litter (ANOVA, F = 19.26; P = 0.0001, Fig. 6C). Testing the interdependency of the two parameters rearing condition and litter membership was, however, not possible since litter members were always subjected to equal rearing conditions. To nevertheless get an estimate of how litter membership might have influenced our previous analyses, we computed the relative variance of median column spacing within litters and compared it to the variance within the three rearing groups. The respective coefficients of variance (CoV) were determined by dividing the standard deviation by the mean of the median distances for each group separately (Fig. 6D
). The CoV among median distances is largest within the normal control group (0.114) as compared to a relatively small coefficient within all different litters (0.034). The group of squinters (0.052) shows less than half of the intra-group variance of normal animals. Among monocularly deprived cats, relative variance is orders of magnitude smaller than in the normally raised cats (0.019) and below the average variance within litters (0.034). These data suggest that genetic relationship has a pronounced influence on the intercolumnar distance, at least in the sense that littermates reacted very similarly to a specific type of altered visual experience.
Interhemispheric differences in column spacing vary between 4 and 77 µm (mean, 28 µm) in individual cats and the direction of the difference is not eye-specific in squinting or monocularly deprived cats. Differences are largest among squinters, in agreement with the rather broad distribution of distances in these cats. Still, interhemispheric differences in individuals tend to be smaller than differences between group averages of squinting and normal cats (95 µm) or monocularly deprived cats (74 µm).
Spacing and Weight/Age and Length of Area 17
To exclude the possibility that our observation of the influence of rearing and litter membership on spacing resulted from a sampling artefact with regard to age, body weight or area length in our cat population, we computed correlation coefficients between these parameters and intercolumnar spacing. None of the interactions disturbed the relation with the factors litter membership or rearing (data not shown) and only significant observations are described. Column spacing seemed to be smaller in older and heavier cats as compared to younger, less heavy cats (ANOVA: age, F = 7.41, R = 0.45, P = 0.01; weight, F = 4.9, R = 0.38, P = 0.003). The length of area 17 was positively correlated with intercolumnar spacing in normal cats (ANOVA: n = 10, F = 158, R = 0.98, P = 0.0001), thus indicating larger column spacing in larger areas in adulthood. Interestingly, this strong relationship was not observed in cats with abnormal visual experience (ANOVA: n = 20, F = 1.5, R = 0.28, P = 0.23).
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Discussion |
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In agreement with former studies in monkeys and cats, domains serving the deprived eye occupied much less territory than those serving the normal eye (Hubel et al., 1977; Shatz and Stryker, 1978
; LeVay et al., 1980
; Hata and Stryker, 1994
; Horton and Hocking, 1997
; Crawford, 1998
; Hata et al., 2000
). This is compatible with electrophysiological recordings showing that only 21% of the neurons in layer IV
7% in other layers
are dominated by the deprived eye (Hubel and Wiesel, 1970
; Blakemore and Van Sluyters, 1974
; Shatz and Stryker, 1978
; Movshon and Van Sluyters, 1981
).
Interestingly, shrinkage of deprived eye territory extended also into exclusively monocularly driven domains, such as the representations of the optical disc and of the MS. The same observation was previously made in primary visual cortex of monocularly deprived macaque monkeys (von Noorden et al., 1976; Horton and Hocking, 1997
). Part of this shrinkage may be explained by competition of deprived and non-deprived eye afferents at the borders of these monocular representations, whereby the open eye gains more cortical territory than normally (Antonini and Stryker, 1998
). In addition, sensory disuse could have interfered with the stabilization and elaboration of geniculocortical axons. This observation is interesting given that electrophysiological studies so far showed that the deprived pathway is impaired only within the representation of the binocular part of the visual field (Sherman, 1973
; Sherman et al., 1974
; Sherman and Guillery, 1976
; Wilson and Sherman, 1977
). Support for the shrinkage by disuse hypothesis comes from investigations in binocularly deprived cats which revealed that sensory disuse, in the absence of competition, is sufficient to impair cortical functions (Wiesel and Hubel, 1965
). The present results complement these observations and suggest an influence of sensory disuse on geniculocortical connectivity.
In agreement with a previous suggestion (Shatz and Stryker, 1978), our data indicate that overlap between afferents representing the two eyes in layer IV is greater in monocularly deprived cats compared to normally raised animals. This observation is supported by electrophysiological studies showing that in deprived eye domains, monocular neurons with different eye dominance are intermingled and that even some binocularly driven cells occur (Shatz and Stryker, 1978
). This overlap further correlates with electrophysiological recordings describing a substantial amount of functionally binocular neurons also outside layer IV in monocularly deprived cats (Shatz and Stryker, 1978
; Freeman and Ohzawa, 1988
).
Eccentricity Dependent Variations of Intercolumnar Competition?
In all monocularly deprived cats, density of labeling was higher contralateral than ipsilateral to the injected eyes. This is in accordance with previous anatomical (Hubel et al., 1977; Shatz and Stryker, 1978
; LeVay et al., 1980
; Horton and Hocking, 1997
), electrophysiological (Hubel and Wiesel, 1962
; Blakemore and Pettigrew, 1970
; Albus, 1975
; Shatz and Stryker, 1978
) and 2-deoxyglucose (Löwel et al., 1993a, b) studies, which all report a prevalence of the contralateral eye representation. This bias may also explain the observation that unlabeled deprived eye patches are hardly discernible in the hemisphere contralateral to the labeled normal eye (Shatz and Stryker, 1978
). In this study we noted, in addition, that segregation of afferents from the two eyes was less pronounced in the representation of the peripheral visual field. We are confident that the lack of distinct columns in the peripheral visual field representation contralateral to the injected normal eye of cat MD1 does reflect a decreased segregation of non-deprived eye afferents and is not the result of methodological artefacts. Labeling in cat MD1 is comparable to that in other cats, as indicated by a similar quality of staining in the LGN autoradiographs and by the good expression of columns in the cortex ipsilateral to the injected eye. With proline labeling, the density of cortical labeling is often weaker in regions representing the central than the peripheral visual field (LeVay et al., 1978
; Löwel and Singer, 1987
). Therefore, one could argue that fading of contrast in the periphery might be due to saturation when exposure times are chosen that render optimal contrast in the center. However, on unsaturated films an increase in label density should not eliminate contrast. Moreover, peripheral label in cat MD1 appeared homogeneous even after short exposure times. We therefore exclude saturation as a cause of the eccentricity effect.
Another process mimicking reduced segregation is spillover of radioactive tracer across LGN laminae from labeled fibers of the contralateral eye that pass through lamina A1 and terminate in lamina A (LeVay et al., 1978; Shatz and Stryker, 1978
). Our measurements indicated that such spillover had occurred, but did not differ between deprived or non-deprived eye injections (LGN autoradiographs, not shown). Since we measured spillover on films and not directly on sections, we may have overestimated its magnitude (see Materials and Methods) by including labeled fibers of passage and not only cell bodies (LeVay et al., 1978
). Thus spillover ratios most probably did not exceed 50% and this ratio should not have prevented the visualization of segregated afferents.
Taken together, the arguments suggest that the lack of modulation in labeling intensity at large eccentricities and contralateral to non-deprived eye injections reflects low or absent segregation of afferents. This eccentricity dependent difference in labeling pattern was not observed after deprived-eye injections. Thus, in contrast to normally raised cats (Hata and Stryker, 1994) and monocularly deprived monkeys (LeVay et al., 1980
; Horton and Hocking, 1997
), (i) geniculocortical afferents of the two eyes in monocularly deprived cats are not complementary and (ii) their overlap seems to be expressed to varying degrees on contra- and ipsilateral sides. When studying plasticity of geniculocortical afferents and its pharmacology, it might thus be important to consider that layout differences between ipsi- and contralateral eye afferents and between central and peripheral visual field representation can be observed in monocularly deprived cats, especially ipsilateral to the open eye, even without pharmacological intervention.
Complementary support for an eccentricity dependent variation in the segregation of OD columns comes from one macaque monkey who was monocularly deprived by lid suture very early on (at 1 week of age). After a non-deprived eye injection, segregated ocular dominance columns appeared also predominantly in the representation of the fovea and faded completely towards the periphery monkey 3 (Horton and Hocking, 1997
). In animals deprived at successively later ages, this trend became less and less evident [see also (LeVay et al., 1980
)]. This observation had been attributed mainly to a difference in column spacing between foveal and more peripheral visual field representations: in the macaque fovea, columns are widely spaced, whereas in the periphery columns are much more narrowly spaced. Thus, spreading of non-deprived eye afferents into deprived eye domains was suggested to prevent the visualization of domain segregation assuming an equal radius of the spread of non-deprived afferents in central and peripheral regions. In principle, this might also be an explanation for the lack of segregation we observed in the peripheral visual field representation of monocularly deprived cats.
However, we did not find a clear difference in column spacing between central and peripheral visual field representations in cat area 17 (data not shown), as has been reported for monkey primary visual cortex. Thus, differences in column spacing most likely do not account for the observed retinotopic gradient in segregation in cat MD1. Alternatively, an eccentricity dependent gradient in cortical maturation might explain our observations. In cats, geniculocortical afferents start to segregate around eye opening and thus 12 weeks after birth (LeVay et al., 1978
; Crair et al., 2001
), so that the developmental scenario is different from that in the monkey, who is already born with columns (Rakic, 1977
, Horton and Hocking, 1996a
; Crowley and Katz, 2000
). Once ocular dominance columns are laid out, nondeprived eye afferents may no longer be able to invade the center of a deprived eye domain, but if they have not yet left the deprived eyes territory in the course of the normal segregation process, non-deprived eye projections may stabilize. This would imply that the retinotopic gradient we observed in the segregation of non-deprived eye afferents reflects delayed maturation of domains devoted to the peripheral visual field.
Indirect support for this hypothesis comes from data by Hata et al. who mapped a large part of the ocular dominance pattern of a monocularly deprived cat after proline injection into the non-deprived eye (Hata et al., 2000). The authors began deprivation (lasting only 2 weeks) long after columns have been formed in cats (PND 3538). In this study, the size of the region containing well-segregated columns was much larger than in our case, indicating that segregation of the non-deprived eye extended much further into peripheral visual field representations. This is what one expects if deprivation time has either been too short for non-deprived eye afferents to invade the deprived-eye columns and/or, more likely, if new outgrowth of non-deprived eye afferents into already stabilized peripheral ocular dominance centers of the deprived eye has no longer been possible. Thus, the difference between the data Hata et al. and our study is compatible with a maturational gradient between central and peripheral visual field. To our knowledge, there is no other evidence yet that segregation starts earlier in the central part of area 17. However, there is evidence from other parts of the visual system supporting such a gradient. The orienting response of kittens, which probably involves the superior colliculus, matures later in the more peripheral than in the central visual field (Sireteanu and Maurer, 1982
). Also, peripheral regions of the kittens retina which is immature at birth, mature later than central regions (Donovan, 1966
; Johns et al., 1979
). The central part is fully developed by 45 weeks, but peripheral parts continue to mature until 9 weeks postnatally (Donovan, 1966
).
The fact that in our cats clearly segregated deprived eye columns are especially prominent in the hemisphere contralateral to the deprived eye is consistent with an earlier maturation of the domains of the contralateral eye (Crair et al., 1998; Rathjen and Löwel, 2000
). This is also in agreement with previous studies in cat suggesting an earlier maturation of the crossed versus the uncrossed pathways (Singer and Tretter, 1976
; Anker, 1977
; Singer, 1978
).
Ocular Dominance Column Spacing
In a previous study in our laboratory, intercolumnar distances were investigated by one-dimensional Fourier measurements along vectors perpendicular to the columnar boundaries (Löwel, 1994). Since in normal and especially strabismic cats, a clear alternation of left and right eye columns is mainly observed in the anteriorposterior axis, a one-dimensional analysis is adequate to measure intercolumnar distances in these animals. However, patterns produced by proline injections into monocularly deprived cats revealed discontinuous patches and fewer bands with distinct orientations, making one-dimensional measurements inadequate. We decided to analyze the ocular dominance patterns with a nearest-neighbor analysis using Voronoi triangles, as detailed in Materials and Methods and elsewhere (Murphy et al., 1998
), because this method
in contrast to two-dimensional Fourier transformations
allows the determination of column spacing, even from small layer IV fragments common in proline experiments and the performance of quantitative measurements directly on the original autoradiographs, thus minimizing artefacts.
Rearing
One major finding of the quantitative analyses was that column spacing in monocularly deprived cats was not significantly different from that in normally raised cats. This observation is in agreement with measurements in visually deprived cats (Jones et al., 1996) and monkeys (Crawford, 1998
; Murphy et al., 1998
) revealing no differences between different rearing groups. It is consistent with theoretical predictions by (i) an elastic net model assuming decreased competitive strength of the deprived eye (Goodhill and Willshaw, 1994
) and (ii) models reproducing a squint-induced increase in column spacing, including reduced inter-eye correlations (Goodhill and Löwel, 1995
; Wolf et al., 2000
). However, with only spontaneous activity driving the deprived eye afferents, assumptions about inter-eye correlations are difficult to deduce. In the modelers context, the present results only indicate that reduced inter-eye correlations at least do not prevail over reduced intra-eye correlations in their effect on column spacing.
The re-evaluation of previously published ocular dominance patterns with two-dimensional, nearest-neighbor analysis confirmed the observation of an increased column spacing in the strabismic animals compared to the normally raised controls (Löwel, 1994). It is in agreement with a study in which cats were raised with alternating monocular occlusion (Tieman and Tumosa, 1997
), a preliminary report of strabismic monkeys (Roe et al., 1995
; Murphy et al., 1998
) and developmental models including reduced inter-eye correlations (Goodhill, 1993
; Wolf et al., 2000
).
However, the new observation that belonging to the same litter has a very strong influence on column spacing introduces a new perspective on the data set.
Litter Membership
Inter-individual variability was particularly high among normal cats, in agreement with data from normal macaque monkeys (Horton and Hocking, 1996b). In contrast, ocular dominance spacing was very similar (low variance) in the two hemispheres of the same animal and in cats from the same litter. Thus, litter membership and therefore probably genetic factors may play a major role for column spacing. Supporting this idea, a substantial genetic influence on the spacing of orientation columns has recently been observed in cat area 17 (Löwel et al., 2000
).
Since all cats reared with different kinds of visual experience were also littermates, it cannot be excluded that this fact contributed significantly to the consistently wider column spacing in the strabismic group. It is noteworthy in this context that three cats, a strabismic litter, S7/8 (Löwel et al., 1998) and one normal cat, N5 (Schmidt et al., 1997
), which had not been included in the former analysis (Löwel, 1994
), have overlapping spacing values (Table 1
).
Age/Weight
Although our youngest cats were already 67 weeks old and thus at an age at which ocular dominance layout already appears adult-like (LeVay et al., 1977; Rathjen and Löwel, 2000; Crair et al., 2001
), we observed a slight decrease of column spacing with increasing age and body weight. Though indicating that the pattern layout might still change to a limited extent between 8 and 16 weeks of age, this did, however, not affect our above conclusions because different rearing groups behaved similarly.
Length
In normally raised, but not in strabismic and monocularly deprived cats, larger spacings usually occurred in larger areas, which may keep the number of modules analyzing the visual field with the two eyes independent of area size. One explanation could be that a constant relationship between monocular resolution of the visual field and area size needs to be maintained only in cats with binocular receptive fields (normals) in order to ensure a monocular sampling frequency of the visual field, which is invariant of the individual brain size. Further experiments are, however, needed to settle the issue of a possible interaction between area size, column spacing and rearing condition.
Since none of the investigated litters were reared with different paradigms, the present data do not allow one to clearly distinguish between pure effects of rearing and litter membership on column spacing and their interference with age, weight and area size, but the genetic influence seems to be extraordinary high and thus overshadows any other influence. To decide this issue finally, different rearing conditions must be applied to members of the same litter (Rathjen et al., 1999) and a large number of cats must be investigated because effects might be very small.
Mechanisms
Although deprived eye domains in area 17 of monocularly deprived cats are clearly smaller than normal, it is surprising how much territory is still innervated by the deprived eye. Assuming a competitive process, the amount of territory taken by one eye might depend on several factors, such as the overall activity of the afferent fibers, the synchronicity between the activity of the two eyes and the initial density of the afferent fibers of the two eyes. Since afferent activity of the deprived eye is strongly reduced and unstructured, the afferents of the monocularly deprived eye probably cannot greatly benefit from activity- or experience-dependent competition. Thus, one explanation for the even distribution of the small deprived eye columns throughout the visual field representation might be that ocular dominance segregation had started before the onset of visual experience (Crowley and Katz, 2000) and local terminal density was already particularly high, with only few terminals of the other eye around.
In this respect, it is also very interesting that in monocularly deprived cats, functionally intact domains of the deprived eye domains have been found to be co-localized with pinwheel center singularities (Crair et al., 1997b). This co-localization was even stronger than that observed between peaks of monocularity and singularities in normal (Crair et al., 1997a
) or strabismic cats (Löwel et al., 1998
). Specific visual response properties of the neurons in pinwheel center singularities, among other reasons, have been suggested to account for this observation (Crair et al., 1997a
). As the remaining functional domains coincide with the anatomically defined patches of the deprived eye afferents, it is possible that those regions differ from the surrounding cortex with respect to their thalamocortical input pattern and/or to other intrinsic, molecular cues.
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Conclusions |
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Acknowledgments |
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Address correspondence to Kerstin E. Schmidt, Max-Planck-Institut für Hirnforschung, Abteilung Neurophysiologie, Deutschordenstraße 46, D-60528 Frankfurt am Maine, Germany. Email: schmidt{at}mpih-frankfurt.mpg.de.
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References |
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