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Normal mice were studied at postnatal days 35-40 (P35-P40) or at P60. Monocular deprivations lasted for 20 or 40 d beginning at P17-P19 (Table 1). Animals were prepared for electrophysiological recordings following the procedure described by Gordon and Stryker (1996)
The Journal of Neuroscience, June 1, 1999, 19(11):4388-4406
Anatomical Correlates of Functional Plasticity in Mouse Visual Cortex
Antonella Antonini1, Michela Fagiolini1, 2, and Michael P. Stryker1 1 W. M. Keck Foundation Center for Integrative Neuroscience, Department of Physiology, University of California, San Francisco, California 94143-0444, and 2 Laboratory for Neuronal Circuit Development, Brain Science Institute, RIKEN, Saitama 351-01, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
Much of what is known about activity-dependent plasticity comes from studies of the primary visual cortex and its inputs in higher mammals, but the molecular bases remain largely unknown. Similar functional plasticity takes place during a critical period in the visual cortex of the mouse, an animal in which genetic experiments can readily be performed to investigate the underlying molecular and cellular events. The experiments of this paper were directed toward understanding whether anatomical changes accompany functional plasticity in the developing visual cortex of the mouse, as they do in higher mammals. In normal mice, transneuronal label after an eye injection clearly delineated the monocular and binocular zones of area 17. Intrinsic signal optical imaging also showed monocular and binocular zones of area 17 but revealed no finer organization of ocular dominance or orientation selectivity. In normal animals, single geniculocortical afferents serving the contralateral eye showed great heterogeneity and no clustering consistent with the presence of ocular dominance patches. Growth and elaboration of terminal arbor continues beyond postnatal day 40 (P40), after the peak of the critical period. After prolonged monocular deprivation (MD) from P20 to P60, transneuronal labeling showed that the projection serving the ipsilateral eye was severely affected, whereas the effect on the contralateral eye's pathway was inconsistent. Optical imaging also showed profound effects of deprivation, particularly in the ipsilateral pathway, and microelectrode studies confirmed continued functional plasticity past P40. Reconstruction of single afferents showed that MD from P20 to P40 promoted the growth of the open eye's geniculocortical connections without causing the closed eye's contralateral projection to shrink, whereas MD from P20 to P60 caused an arrest of growth of deprived arbors. Our findings reveal numerous similarities between mouse and higher mammals in development and plasticity, along with some differences. We discuss the factors that may be responsible for these differences.
Key words: area V1; optical imaging; monocular deprivation; critical period; area 17; development; visual cortex; transneuronal labeling; axonal reconstruction
INTRODUCTION
The organization, development, and plasticity of the primary visual cortex are of interest for its role in understanding visual perception, but the cortex is of even more widespread interest as a model system for studying how patterns of neural activity operate to organize connections in the developing brain. Much of our understanding of how neural connections are modified by sensory experience stems from the remarkable rearrangements of physiological responses and anatomical connections produced by blurring or occluding vision in one eye during a sensitive period in early life (Hubel et al., 1977
; LeVay et al., 1980
In animals with binocular vision, there is a region of visual cortex within which single neurons respond to stimulation of both eyes and a more peripheral representation that responds to visual stimulation only through the contralateral eye. In many species, inputs representing the two eyes that arise from the lateral geniculate nucleus (LGN) and project to the binocular zone of visual cortex are arranged in alternating patches in layer IV, from which they preferentially activate the radial columns of cortical cells above and below, an arrangement referred to as ocular dominance columns (Hubel and Wiesel, 1965
Understanding the mechanisms responsible for activity-dependent plasticity is a major goal of neuroscience because these same mechanisms are thought to guide the development of precise connections in many regions of the normal brain (Goodman and Shatz, 1993
Recently, we extended the earlier findings of Dräger (1975
In this paper we examine the arrangement of geniculocortical inputs labeled transneuronally and the shapes of individual afferent arbors serving the contralateral eye, both during normal development and after plasticity induced by monocular deprivation. We relate the anatomical findings to visual cortical plasticity assayed with two different physiological measures: intrinsic signal optical imaging and extracellular microelectrode recording. Our findings reveal numerous similarities between the mouse and higher mammals in development and plasticity, as well as some differences. We discuss the factors that may be responsible for these differences.
Some of these results have been presented in abstract (Fagiolini et al., 1997
MATERIALS AND METHODS
All mice studied were C57Bl/6 (BNT) and were housed in the University of California (San Francisco, CA). Experimental procedures were approved by the institutional animal care and use committee.
Tracer injections into the LGN, anterograde labeling, and reconstruction of geniculocortical arbors
Tracer injections. The aim of the experiment was to reconstruct single geniculocortical axons serving the contralateral eye and innervating the binocular portion of the visual cortex in normal and monocularly deprived mice. To this end, anterograde tracers were injected at LGN sites identified electrophysiologically.
A small flap of bone (3 × 3 mm) was removed over the rostral half of the hemipheres, and the dura was left intact. To protect the visual cortex from damage, care was taken not to expose fully the caudal portion of the brain. A glass pipette filled with the tracer was positioned under visual guidance onto the surface of the brain at the stereotaxic coordinates anteroposterior 2.0-2.3 posterior to bregma and mediolateral 3-3.3 from the midline (Franklin and Paxinos, 1997
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Table 1. Arbors reconstructed in normal and MD animals Transneuronal labeling of geniculocortical projections
Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP, 1.5%, 2-3 µl; Sigma) in saline was injected into one eye. After a survival time of 3-5 d, the animals were perfused with 0.1 M phosphate buffer followed by 2% glutaraldehyde in 0.1 M phosphate buffer. The cerebral hemispheres were flattened, sandwiched between two glass slides, and cryoprotected overnight in 30% sucrose in 0.1 M phosphate buffer containing 2% glutaraldehyde. The next day the flattened hemispheres were cut tangentially at 40 µm and reacted by the tetramethylbenzydine method (Mesulam, 1978Monocular deprivation
Animals were anesthetized with 2.0-2.5% halothane in a 1:1 mixture of N2O/O2. The area around the eye was wiped with 70% ethanol, and the superior and inferior eyelids were trimmed along the margins and then sutured with 7-0 sterile surgical silk (Ethicon, Somerville, NJ). The animals were checked every day for the first 4-5 d to make sure that the eyes remained closed. Subsequently, when the scar tissue had permanently sealed the eyelids, animals were checked every other day. The eye was reopened for several hours for the tracer injection into the LGN and the intraocular injection of rhodamine dextran and subsequently resutured. Table 1 lists for each animal the duration of the MD and the age at perfusion.Single-unit recordings
Five normal mice between P45 and P60, three mice monocularly deprived from P20 to P40, three mice deprived from P20 to P60, and three mice deprived from P40 to P60 were used for single-unit electrophysiological recordings. The animals were prepared for electrophysiological recordings in a manner similar to that described by Gordon and Stryker (1996)n7) + 2/3(n2
Intrinsic signal optical imaging
Four normal mice between P35 and P40 and four P40 mice monocularly deprived for 20 d were used in optical imaging experiments. The animals were prepared as for single-unit recordings, with the addition of computer-controlled shutters in front of each eye. The dura was left intact, and the exposed brain was covered with 3% agarose and sealed with a clear glass coverslip to ensure a flat surface. Details of the optical imaging apparatus and protocols have been published previously (Crair et al., 1997
RESULTS
The visual cortex in the mouse: localization and normal histology
Electrophysiological recordings have revealed a single continuous representation of the monocular and binocular segments of the visual field within the primary visual cortex of the mouse (Dräger; 1975
Figure 1 shows two hemispheres from a P38 mouse in which serial sections have been processed with Nissl, acetylcholinesterase, and myelin stains and placed in register. In Nissl-stained coronal sections, the primary visual area can be distinguished by the presence of densely packed granule cells in layer IV (Valverde, 1968
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Figure 1. A, C, E, Consecutive 50 µm sections through the posterior pole of the cerebral hemisphere. B, D, F, Consecutive sections from the opposite hemisphere of the same mouse in which an electrolytic lesion (red arrow) was made after electrophysiological recordings to mark the border between areas 17 and 18a. Area 17 is recognizable in Nissl staining by the presence of small granule cells in layer IV (A, black arrow). High acetylcholinesterase activity in layer IV is also a marker for the primary visual cortex (C, D, light staining), as is the pattern of myelin staining (E, F). The medial border of the visual cortex is clearly identified in all three stains (arrowhead). Furthermore, there is a good match between the 17-18a border recognized in the three stains and that identified electrophysiologically (B, D, F). The pair of arrows on the left point to the binocular portion of the visual cortex clearly recognizable in all three preparations.
Microelectrode recordings confirmed the histological identification of area 17. Electrode penetrations revealed a topographic map as reported previously (Dräger, 1975
Transneuronal labeling of thalamocortical input in normal animals
Area 17 is also defined by its receipt of a direct projection from the LGN. We studied the pattern of termination of the geniculocortical pathway using the transneuronal transport of WGA-HRP injected into one eye (Itaya and van Hoesen, 1982
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Figure 2. Extent of the mouse visual cortex in the coronal plane, as demonstrated by the transneuronal transport of WGA-HRP injected into one eye. The series of coronal sections through the hemispheres contralateral and ipsilateral to the eye injected with WGA-HRP are presented from posterior to anterior; the distance from the posterior edge of the hemispheres is indicated on the left. The images have been obtained by scanning the photographic negatives of the histological sections. The light intensity and contrast of the images of the ipsilateral hemisphere (right column) have been enhanced to better visualize the ipsilateral projections.
In the caudal pole of the forebrain, area 17 curved around the ventral surface of the hemisphere, whereas in successive posteroanterior sections, it covered progressively more lateral portions of the hemisphere. Furthermore, no projection from the ipsilateral eye was found in the rostral-most sections where the lower, monocular portion of the visual field is represented.
It has been reported that in rodents the LGN projections terminate exclusively in area 17 and do not invade the surrounding areas 18b and 18a (Ribak and Peters, 1975
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Figure 3. Aligned, consecutive 40 µm sections from a P43 animal in which one eye was injected with WGA-HRP. Top, Nissl stain. Area 17 is recognizable cytoarchitectonically by the presence of small and closely packed cells in layer IV, more evident in the monocular (m) than in the binocular region, enclosed by the two gray arrows (b). The transneuronal WGA-HRP labeling (bottom) is found mainly in area 17. Note the densest geniculocortical projection in the monocular region. Very pale transneuronal labeling is also present laterally to area 17 (up to the white arrow, area 18a).
In a few coronal sections from some experiments (Fig. 3, bottom), the binocular zone in the hemisphere contralateral to the injected eye appeared paler than the monocular zone. This result is in agreement with the report by Dräger (1974)
The whole extent of the primary visual area, labeled transneuronally after an intraocular injection of WGA-HRP, can be appreciated in tangential sections through the flattened hemisphere. Figure 4 shows the hemispheres contralateral and ipsilateral to the injected eye in a P40 mouse. On the contralateral side, the labeled area had a triangular shape with a sharp border on the medial side. In the hemisphere ipsilateral to the injected eye, transneuronal label was always present and restricted to a patch in the lateral portion of the hemisphere.
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Figure 4. Flattened surfaces of the posterior portion of the two hemispheres demonstrating the extent of the primary visual area as shown by the transneuronal labeling of geniculocortical terminals (dark areas) after an intraocular injection of WGA-HRP. On the side contralateral to the injected eye, the labeling is very intense, covering both the monocular and binocular regions of the visual cortex. On the side ipsilateral to the injected eye, the labeling is more restricted; its intensity and boundaries varied from animal to animal. This area of labeling defines the binocular zone. The figures are negatives of dark-field photomicrographs of a single section each.
Transneuronal labeling of the geniculocortical projection in monocularly deprived animals
In many other species, prolonged monocular deprivation alters the distribution and intensity of transneuronally labeled geniculocortical projections in layer IV of the visual cortex (Shatz and Stryker, 1978
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Figure 5. Tangential sections through the flattened surface of the hemispheres demonstrating transneuronal labeling of geniculocortical terminals after an injection of WGA-HRP into nondeprived eye (ltd3, ltd2, ltd6, ltd7) or deprived eye (ltd4, ltd8, ltd5, ltd1). All animals were deprived for 40 d, ending at P60. Note in the hemisphere ipsilateral to the injected eye the strong transneuronal labeling when the nondeprived pathway was labeled and the reduced labeling when the deprived eye was injected. In contrast, a clear effect of MD on the projections serving the contralateral eye was observed only in ltd1, in which the deprived eye was injected with WGA-HRP. In this case, the lateral third of the visual cortex, presumably corresponding to the binocular zone, was less strongly labeled.
In a group of eight P40 animals monocularly deprived for 20 d, the contralateral projections from either the deprived or nondeprived eyes appeared within the normal range. The labeling of the ipsilateral projection from the deprived eye was not consistently less intense than normal, except in one of the four cases, in which it was very pale.
The transneuronal labeling technique is useful for studying the anatomical characteristics and the area covered by the projection of the entire population of geniculocortical afferents. However, finer features of the projection, including changes in the size of afferent arbors, cannot be analyzed with this method. Serial reconstruction of individual afferents allows detailed analysis of this projection and the assessment of changes in arbors during development and after deprivation.
Labeling and reconstruction of geniculocortical afferent arbors
The organization of the geniculocortical pathway and its plasticity after deprivation have been studied in the visual cortex of the cat by analyzing the morphology of single geniculocortical arbors (Humphrey et al., 1985
Geniculate afferent arbors to the visual cortex were labeled anterogradely by tracer injections into the LGN and were serially reconstructed in three dimensions. Our aim was to reconstruct a homogeneous population of axon arbors. It was therefore important to consistently label axons serving one particular eye, and the identification of the eye of origin of the labeled arbors was further essential in assessing the effects of monocular deprivation. Because the portion of the LGN receiving projections from the ipsilateral eye is quite small and often contains fibers of passage from the contralateral retina, a tracer injection aimed at neurons receiving input exclusively from the ipsilateral eye is technically difficult. Therefore, we chose to make injections of the tracer into the dorsal portion of the LGN, which contains neurons responding exclusively to the contralateral eye.
Biocytin or Pha-L tracer injections for labeling geniculocortical afferents were made at the dorsal border of the anterior half of the LGN, which contains the representation of the binocular portion of the upper visual field. In all cases, the site of injection had been physiologically characterized by single- or multi-unit extracellular recordings through the pipette containing the tracer. The published data on the topographic organization of the rat LGN (Reese and Cowey, 1983
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Figure 6. A, Series of anteroposterior coronal sections through the LGN contralateral and ipsilateral to a monocular injection of fluorescent dextran. The portion of the LGN receiving ipsilateral retinal fibers is confined to a small patch in the rostral half of the LGN. Dorsal is up and to the right in ipsilateral sections; up and to the left in contralateral sections. B, Example of a biocytin injection and its relation to the ipsilateral retinal projections. Left panel, Scan of fluorescent photomicrograph of an LGN coronal section showing the rhodamine dextran-labeled terminals arising from the ipsilateral eye. Right panel, Scan of a confocal image of the same section after biocytin histochemistry showing the biocytin injection site. The rhodamine-labeled area from the fluorescent photomicrograph has been superimposed to show that the injection site, located next to the pia in the dorsolateral portion of the LGN, did not overlap with the "ipsilateral patch."
Geniculocortical afferents were well labeled by both tracers, as shown for biocytin in Figure 7. The densest labeling was found in layer IV. However, the superficial layers also received a strong LGN projection, and often, axonal branches ran 100-200 µm tangentially below the pial surface (Fig. 7B). Ramifications in layers V and VI were usually sparse. Afferents were distributed homogeneously in the area of labeling, and there was no sign of subdivision into eye-specific domains such as the ocular dominance patches in higher mammals. Arbors were chosen for reconstruction in zones of relatively sparse labeling, as shown in Figure 7, B and C.
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Figure 7. A, Scan of a photomicrograph of a coronal section through the visual cortex showing dense cortical labeling of biocytin-filled geniculocortical afferents. Note the abundant innervation not only of layer IV but also of the supragranular layers. B, C, Branches of biocytin-labeled geniculocortical arbors presented as a collage of photomicrographs combining serial focal planes. Note in B the ramifications running beneath the pial surface. Note in C branches running in layer IV. Scale bars: A, 200 µm; B, 50 µm; C, 20 µm.
A few labeled geniculocortical afferents were consistently found to arise from the white matter and arborize 300-700 µm lateral to the lateral edge of the dense area of labeling. This region is most probably area 18a, although we could not verify it on the basis of cytoarchitectonic features, because the reaction products of the biocytin prevented a good Nissl counterstaining. The presence of a few geniculocortical afferents in area 18a is in agreement with the light labeling observed in the transneuronal experiments (Fig. 3). Branches from axons arborizing within area 17 were not seen to extend into this lateral region, except in a single case (Fig. 8, mo2h) in which a separate trunk arose from the white matter lateral to area 17.
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Figure 8. Computer reconstructions of geniculocortical arbors in area 17 comparing normal P40 (A) and P60 (B) mice. The arrowheads indicate the boundary of layer IV. All arbors are presented in coronal view, and the most complex arbors are also presented in surface view after a 90° rotation along an axis passing through layer IV (mo2a, mo2g, mo4b, mo4c, insets). The line above coronal views indicates the pial surface. The scale is for all arbors.
Geniculocortical afferent arbors in normal animals
Single geniculocortical afferents were serially reconstructed in mice at two different ages, at P40 (which is at the end of the critical period of susceptibility to the effects of a brief period of monocular deprivation; Gordon and Stryker, 1996
Fourteen geniculocortical arbors were reconstructed in P40 animals. These arbors showed great variability in size and shape, as illustrated in Figure 8A. Some afferents arborized extensively and formed a dense patch of terminal branches (mo2g), whereas other afferents gave rise to only a few branches (mo1b). Arbors differed also in the laminar pattern of arborization. In most arbors the majority of collaterals resided in layer IV, with a few branches extending into the superficial layers and layers V and VI (mo2a, mo2g). In two arbors, the predominant arborization was in layer I. One example, mo3b, is shown in Figure 8A. Extensive ramifications in the infragranular layers were also found in six arbors, examples of which are shown in Figure 8A (mo2h, mo2e). On average, the extent of layer IV covered by a single geniculocortical afferent was 493 µm (range, 200-1700 µm) in the mediolateral axis and 395 µm (range, 50-640 µm) in the anteroposterior axis.
The five geniculocortical arbors reconstructed at P60 showed the same variability that was present at P40 (Fig. 8B). Three afferents arborized densely in layers IV and III (Fig. 8B, mo4b, mo4c, mo4d); the remaining two were rather sparsely ramified with most branches located in the superficial layers (Fig. 8B, mo4a, mo4e). The mean extension of the terminal arborization was 581 µm along the mediolateral axis (range, 370-960 µm) and 408 µm along anteroposterior axis (range, 280-480 µm). At both ages, the terminal fields of geniculocortical arbors were not organized in distinct patches.
Quantification of the total length and number of branch points of the portion of the afferents ramifying both in layer IV and supragranular layers indicates that arbors at P60 were larger and more complex than arbors at P40 (total length, p < 0.02; branch points, p < 0.03), suggesting continued growth between these two ages. As illustrated in Figure 11, A and B, the total length of 8 of 14 arbors at P40 was <6000 µm, whereas the smallest arbor at P60 had a total length of 6100 µm. Moreover, 12 of 14 arbors at P40 had <40 branch points, whereas only 1 of the 5 arbors reconstructed at P60 had so few. The two groups did not differ in their mediolateral or anteroposterior extents.
In summary, geniculocortical arbors in normal mice are very heterogeneous, and their growth continues after the peak of the critical period.
Geniculocortical afferent arbors in monocularly deprived animals
The aim of this series of experiments was to determine whether the physiological effects of MD (Dräger, 1978
P40 animals monocularly deprived for 20 d around the peak of the critical period
Six arbors serving the deprived eye (Fig. 9A) and six arbors serving the nondeprived eye (Fig. 9B) were reconstructed in animals deprived for 20 d and perfused at P40. Both groups of arbors had patterns of cortical innervation similar to arbors reconstructed in normal age-matched controls. For example, axon collaterals were found in the superficial layers, including layer I, as well as in layer IV.
Two of the deprived arbors had a rather dense arborization in layer IV (Fig. 9A, d5da, d8db). One arbor (Fig. 9A, d3db) ramified in both the supragranular and infragranular layers but not in layer IV. The remaining two arbors had very narrow and poorly ramified terminal fields (Fig. 9A, d5db, d8da). On average, the extension of the arbors in layer IV and the supragranular layers was 491 µm (range, 200-1060 µm) in the mediolateral axis and 547 µm (range, 240-880 µm) in the anteroposterior axis. As a group, arbors serving the nondeprived eye (Fig. 9B) were more homogeneous than both normal and deprived arbors. With one exception (Fig. 9B, d7ndc), these arbors qualitatively appeared to be as richly ramified as the densest arbors reconstructed in normal animals. The average extension of the terminal arborization was 388 µm along the mediolateral axis (range, 300-520 µm) and 400 µm along anteroposterior axis (range, 240-600 µm). The scattergrams in Figure 11 provide quantification of the total length (see Fig. 11A) and number of branch points (see Fig. 11B) for arbors reconstructed in normal animals and for both deprived and nondeprived arbors reconstructed in MD animals. The mean value of each group is also plotted in the same graph. In P40 animals, no statistical differences were found in either total length or number of branch points between deprived arbors and arbors reconstructed in normal, age-matched controls. Moreover, although on average the nondeprived arbors were longer (1.8 and 1.4 times, respectively) and more ramified (1.8 and 1.7 times, respectively) than both deprived and normal arbors, because of arbor variability only the differences between nondeprived and normal arbors was significant (p < 0.05 for both parameters). Deprived and nondeprived arbors did not differ in either mediolateral or anteroposterior extensions, and both groups were not different from normal arbors.
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Figure 9. Single, serially reconstructed geniculocortical arbors in area 17 in P40 animals monocularly deprived for 20 d starting at P20. A, Arbors serving the deprived eye. B, Arbors serving the nondeprived eye. The arrowheads indicate the boundary of layer IV. The scale is for both groups of arbors.
P60 animals monocularly deprived for 40 d
Seven geniculocortical arbors serving the deprived eye (Fig. 10A) and six arbors serving the nondeprived eye (Fig. 10B) were reconstructed in P60 animals monocularly deprived for 40 d. Both groups of arbors exhibited similar variability in size and complexity as normal arbors reconstructed at P60. All arbors, deprived and nondeprived, had their main arborization in layer IV. Three deprived arbors (Fig. 10A, d4da, d4db, d4dd) extended collateral branches toward layer I, as did, with one exception (Fig. 10B, d1ndb), all nondeprived arbors.
For the deprived arbors, the mean extension of the terminal arborization was 401 µm along the mediolateral axis (range, 350-655 µm) and 405 µm along anteroposterior axis (range, 200-640 µm). For the nondeprived arbors, the mean extension of the terminal arborization was 588 µm along the mediolateral axis (range, 260-800 µm) and 693 µm along anteroposterior axis (range, 520-1000 µm). The nondeprived arbors differed significantly from the deprived arbors only in the anteroposterior extension (p < 0.02). The quantification of the total length and number of branch points for deprived and nondeprived arbors at P60, along with results from P60 normal arbors, is shown in the scattergram of Figure 11, A and B. The mean total length of nondeprived and normal arbors was 1.6 times larger than that of deprived arbors. Deprived arbors were statistically different from nondeprived arbors in total length (p < 0.03), and the difference from normal arbors was close to significant (p = 0.06). Moreover, deprived arbors at P60 were similar in length and complexity to deprived arbors at P40 (p > 0.7), indicating a lack of growth after P40. Nondeprived arbors did not differ statistically from normal arbors at P60.
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Figure 10. Single, serially reconstructed geniculocortical arbors in area 17 in P60 animals monocularly deprived for 40 d starting at P20. A, Arbors serving the deprived eye. B, Arbors serving the nondeprived eye. The arrowheads indicate the boundary of layer IV. The scale is for both groups of arbors.
Even in normal mice, single geniculocortical arbors serving the contralateral eye are heterogeneous in many aspects, including length, complexity, and pattern of projection. This heterogeneity masks to some extent the effects of deprivation and prevents some of the differences between deprived and nondeprived arbors from reaching statistical significance. An overall picture of the significant changes during development and plasticity of the geniculocortical arbors is illustrated in Figure 11C. For each group of geniculocortical arbors (normal, deprived, and nondeprived at P40 and P60), the mean values for total length and number of branch points have been normalized relative to the mean values of normal arbors at P40. These parameters changed in parallel. The graph shows the significant growth of normal arbors between the age of P40 and P60. It shows also that 20 d of MD tends to allow the nondeprived arbors to become significantly larger and more complex than normal, whereas the deprived arbors are not modified. When the deprivation is maintained for 40 d, these deprived arbors exhibit only slight and insignificant further growth.
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Figure 11. Scattergrams of the total length (A) and number of branch points (B) for arbors reconstructed in normal animals (N) and for both deprived (D) and nondeprived (ND) arbors reconstructed in MD animals. The age at perfusion is also indicated (P40 and P60). The white bar represents the mean value in each group. C, Values of total length (filled bars) and number of branch points (open bars) of arbors in the six experimental groups normalized relative to the mean values of arbors in normal arbors at P40.
Physiological analysis of the visual cortex in normal and monocularly deprived mice
Intrinsic signal optical imaging
Intrinsic signal optical imaging has been used in many species to reveal both the spatial distribution and magnitude of responses to different stimuli in the visual cortex, and it has been used to map orientation columns, ocular dominance columns, and other features of normal cortical organization (Bonhoeffer and Grinvald, 1993
Because optical imaging in normal mice clearly revealed the overall cortical responses to each eye, we studied mice that had been monocularly deprived to determine whether the physiological effects of monocular deprivation would be evident. Figure 12, E, F, I, and J, shows optical responses to stimulation of the two eyes in both hemispheres of a P40 mouse that had been monocularly deprived for 20 d. In the hemisphere ipsilateral to the deprived eye, responses to the deprived (ipsilateral) eye are nearly absent (Fig. 12F), whereas the nondeprived (contralateral) eye activates the cortex strongly (Fig. 12E). The ocular dominance ratio map shows a dark oval region of contralateral dominance encompassing both monocular and binocular zones of area 17 (Fig. 12G). In the hemisphere contralateral to the deprived eye, responses to the nondeprived (ipsilateral) eye are strong (Fig. 12J), much stronger than in normal animals, whereas responses to the deprived (contralateral) eye are weaker than normal, particularly in the binocular zone (Fig. 12I). The ocular dominance ratio map (Fig. 12K) reveals most prominently a nearly white area, indicating dominance by the nondeprived ipsilateral eye over the binocular zone, and shows a darker region, indicating activation by the deprived eye, only in the monocular segment. Similar results were obtained in three other animals (data not shown). In summary, optical imaging revealed consistent changes in the response of the ipsilateral eye's pathways after monocular deprivation and in the relative responses of the two eyes within the binocular zone. Effects of deprivation on the contralateral eye's responses in the monocular zone were seen in some cases, particularly around its edges, but were inconsistent. Intrinsic signal optical imaging provides a reliable method for investigating responses in the visual cortex of the mouse.
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Figure 12. Intrinsic signal optical responses in mouse visual cortex. A-D, Normal mouse. E-H, Hemisphere ipsilateral to deprived eye in MD mouse. I-L, Hemisphere contralateral to deprived eye in MD mouse. Darkness indicates response to visual stimulation as percent change in reflectance as on the scale to the left of each row. A, E, I, Responses elicited from eye contralateral (contra) to the hemisphere imaged. B, F, J, Responses elicited from eye ipsilateral (ipsi) to the hemisphere imaged on scales identical to A, E, and I. C, G, K, Ratio between activation by contralateral and ipsilateral eyes, with darkness indicating greater response to contralateral and lightness indicating greater response to ipsilateral eye. D, H, L, Images of blood vessels on the cortical surface aligned with optical maps in the same row. Monocular and binocular zones of cortex are outlined in a normal case (D), along with electrode penetration sites. Receptive fields of neurons recorded at the leftmost site were in the monocular segment of the visual field; receptive fields at two sites to the right were located successively more central and in the binocular visual field. Note the virtual disappearance of the deprived eye responses in the ipsilateral hemisphere (F) and the increase in the response to the nondeprived eye in its ipsilateral hemisphere (J). Note also the reduction in the deprived eye's response in its contralateral hemisphere (I). Length scale, 1 mm for all images. Rostral is up and caudal is down in all figures. Medial is left in A-D and I-M, whereas medial is right in E-H. Lateral is right in A-D and I-M, whereas lateral is left in E-H.
Single-unit recordings
The critical period of susceptibility to the effects of monocular deprivation in the mouse has been defined using brief periods of eyelid suture. The critical period defined with 4 d eye closure peaks at approximately P26 and declines sharply thereafter, with little or no effect of such brief deprivation evident after P36 (Gordon and Stryker, 1996
Both physiological approaches, optical imaging and single units, reveal strong plasticity during the period before P40.
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Figure 13. CBI (mean and individual values) and ocular dominance in normal and monocularly deprived animals after different deprivation protocols. A, Single neuron responses in normal mice are dominated by the contralateral eye (mean CBI = 0.75). After monocular deprivation, CBIs in the visual cortex ipsilateral to the open eye decrease to 0.45 and 0.39 after 20 and 40 d of MD, respectively, indicating dominance of the ipsilateral eye. Late MD, from P40 to 60, is still able to affect the eye dominance of visual cortical neurons. B-E, Percent of cells assigned to each of the seven ocular dominance classes (Hubel and Wiesel, 1962
DISCUSSION
The experiments described in this paper were directed toward understanding the anatomical changes underlying functional plasticity in the developing visual cortex of the mouse. We begin by describing the location of binocular and monocular segments of the visual cortex in normal histological preparations of the mouse. We then examine geniculocortical input labeled transneuronally and at the level of individual afferent arbors in normal and monocularly deprived animals. Finally, we compare the anatomical plasticity to two different physiological measures of visual responses: intrinsic signal optical imaging and extracellular microelectrode recording.
In normal animals, transneuronal label after an eye injection clearly delineated the monocular and binocular zones of area 17, with no spread across a sharp border shared with area 18b on the medial side but a slight invasion of area 18a laterally. Optical imaging also showed monocular and binocular zones but revealed no finer organization of ocular dominance or orientation selectivity. Single geniculocortical afferents serving the contralateral eye and innervating the binocular segment of area 17 showed great heterogeneity in length, complexity, and pattern of cortical projections. Many individual arbors were large enough nearly to cover the mediolateral extent of the binocular portion of the visual cortex, and they showed no clustering consistent with the presence of ocular dominance patches. Growth and elaboration of terminal arbors continue from P40 to P60, significantly beyond the peak of the critical period (at approximately P26; Gordon and Stryker, 1996
Monocular deprivation ending at P40 appeared to promote the growth of the open eye's contralateral projection without causing the closed eye's contralateral input to shrink. Continued deprivation to P60 prevented the growth of the closed eye's contralateral inputs. A continuation of functional plasticity between P40 and P60 was found in microelectrode studies, and transneuronal labeling showed that the ipsilateral eye's projection was severely affected by prolonged deprivation to P60. Functional imaging showed profound effects of deprivation, particularly in the ipsilateral pathway, even at P40.
The primary visual cortex and its geniculate input in the normal animal
The ability to identify the primary visual cortex and its monocular and binocular zones in any one of three different preparations of normal histological material (Nissl, myelin, or acetylcholinesterase) will prove useful for future studies of cortical plasticity and function in the mouse.
As initially reported by Dräger (1974)
