Proc. Natd. Acad. Sci. USA Vol. 89, pp. 1832-1836, March 1992 Neurobiology
Cortical local circuit axons do not mature after early deafferentation (barrels/mouse/plasticity/somatosensory/cerebral cortex)
JAMES S. MCCASLAND*, KERRY L. BERNARDO, KARL L. PROBST, AND THOMAS A. WOOLSEY Division of Experimental Neurology and Neurological Surgery and McDonnell Center for Studies of Higher Brain Function, Washington University School of Medicine, St. Louis, MO 63110
Communicated by Oliver H. Lowry, November 13, 1991
ABSTRACT The processes underlying development, refinement, and retention of the intracortical connections critical for the function of the mammalian brain are unknown. Horseradish peroxidase-labeled fibers in mouse somatosensory barrel cortex, which is patterned like the whiskers on the contralateral face from which it receives inputs, were evaluated by automated image analysis. The sensory nerve to the whiskers was sectioned on p ntal day 7, after the whisker map is set. The deprived barrel cortices, examined in adults, showed drastically diminished intracortical projections relative to normal contrdos, although the map of the whiskers in the cortex was unchanged. This demonstrates anatomically that the normal pattern of intracortical connections, like the normal sensory map, is dependent upon the sensory periphery four synapses away.
thetized, perfused transcardially with oxygen-saturated artificial cerebrospinal fluid (ACSF) at 320C, and decapitated. The brains were removed quickly and washed with iced ACSF. The hemispheres were separated in the midline, and the cortices were removed, gently flattened between microscope slides, and transferred to a slice chamber. Injection and histological procedures were standardized. A picospritzer was used to inject calibrated volumes of HRP in dimethyl sulfoxide focally into supragranular layers of the whiskerdefined barrel map, in vitro, by means of a template (23). After 4-6 hr of incubation, the slices were fixed with 1.25% glutaraldehyde/1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), frozen sectioned at 75 gm parallel to the pia, processed with 3,3'-diaminobenzidine as the chromagen, counterstained with 0.125% thionin, dehydrated in alcohols, cleared with xylene, and fixed on coverslips with Permount. The 4 control and 5 experimental cortices quantitated were selected from nearly 100 to meet the following criteria: (i) complete section of the 10 verified by examining the facial skin, (ii) position of injection localized in barrels of the C, D, and E rows (see Fig. 2 for a drawing of the map), and (iii) depth of injection confined to the supragranular layers. Sections were evaluated on a motorized microscope system (24). For each section 8192(x) x 8192 (y) x 6(z) picture elements (pixels), representing a volume of 2 x 2 x 0.075 mm, were analyzed. Image-processing software detected neuronal processes (16) and other programs produced montages from raw or processed images to make pictures of the injection sites and HRP-filled fibers. The section containing the largest diameter of the injection site was identified (15, 16) and the area of the densest reaction product in this section was used to create a "mask" over the injection site by thresholding the computer-generated montage (see Fig. 1 A' and B'). The distribution of fibers outside the mask was mapped. Maps from 10-12 sections in a series were serially aligned by an automated procedure (25).
The functional role of intracortical connections from local circuit neurons has been investigated intensively (1-8). In visual cortex, horizontal connections mainly from pyramidal cells subserve convergence of visual inputs for integration (9). In visual and other cortices, intrinsic activity functions to extract features from stimuli, to sharpen receptive fields, and to improve signal-to-noise ratios (10-12). Yet little is known about the factors on which local intracortical connections depend for normal development. The anatomy of layer IV of the mouse somatosensory cortex mimics the whiskers on the contralateral face (refs. 13 and 14; see Fig. 2). The whiskers are innervated by the infraorbital branch of the trigeminal nerve (10) and are arranged in five rows that project through several relays to groups of cells, called barrels, that are also arranged in five rows. The pattern of local intracortical connections in barrel cortex of normal animals is asymmetrical, with major projections originating in and confined to a barrel row and minor projections to the adjacent anterior barrel row (refs. 15 and 16; see Figs. 1 and 2). Nerve section on postnatal day (PND) 1 dramatically alters the anatomical representations of the whiskers in three brainstem nuclei, in the ventrobasal thalamus, and in barrel cortex, whereas section on PND7 does not alter these representations (17-21). In this experimental context, the axonal tracer horseradish peroxidase (HRP) was injected into supragranular layers of the barrel cortex. If peripheral inputs are necessary for the development of normal intracortical connections after PND7, then cutting the nerve should alter these patterns without changing the barrel map.
RESULTS Figs. 1 and 2 illustrate injections of similar size, in similar locations in control and experimental brains where the barrels define the somatotopic map (Fig. 1 A, B, and C). In normal mice, injections in the supragranular layers label axons spreading widely to other barrels (Figs. 1A to A" and 2). Projections to other layers vary substantially depending on the layer. For instance, their distribution at the layer II-III boundary is wider and more symmetrical than at the layer V-VI boundary (Fig. 2). For the lesioned animals, the barrel pattern is normal but the pattern of intracortical projections
MATERIALS AND METHODS The experimental, histological, and image-processing methods used have been described (16, 22). Outbred Swiss Webster mice of both sexes had the left 10 sectioned at PND7 under anesthesia. Their care and handling were according to institutional guidelines. At 6 weeks all animals were anes-
Abbreviations: 10, infraorbital branch of the trigeminal nerve; PNDn, postnatal day n; HRP, horseradish peroxidase. *To whom reprint requests should be addressed at: Division of Experimental Neurology and Neurological Surgery, Box 8057, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1832
.,w .~ Neurobiology: McCasland et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
1833
FiG. 1. Photomicrographs of ;qp1111B 75-,.m sections in the plane of and
r5tt ! W 2 i layer IV of mouse barrel through lE
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Cortical local circuit axons do not mature after early deafferentation (barrels/mouse/plasticity/somatosensory/cerebral cortex)
JAMES S. MCCASLAND*, KERRY L. BERNARDO, KARL L. PROBST, AND THOMAS A. WOOLSEY Division of Experimental Neurology and Neurological Surgery and McDonnell Center for Studies of Higher Brain Function, Washington University School of Medicine, St. Louis, MO 63110
Communicated by Oliver H. Lowry, November 13, 1991
ABSTRACT The processes underlying development, refinement, and retention of the intracortical connections critical for the function of the mammalian brain are unknown. Horseradish peroxidase-labeled fibers in mouse somatosensory barrel cortex, which is patterned like the whiskers on the contralateral face from which it receives inputs, were evaluated by automated image analysis. The sensory nerve to the whiskers was sectioned on p ntal day 7, after the whisker map is set. The deprived barrel cortices, examined in adults, showed drastically diminished intracortical projections relative to normal contrdos, although the map of the whiskers in the cortex was unchanged. This demonstrates anatomically that the normal pattern of intracortical connections, like the normal sensory map, is dependent upon the sensory periphery four synapses away.
thetized, perfused transcardially with oxygen-saturated artificial cerebrospinal fluid (ACSF) at 320C, and decapitated. The brains were removed quickly and washed with iced ACSF. The hemispheres were separated in the midline, and the cortices were removed, gently flattened between microscope slides, and transferred to a slice chamber. Injection and histological procedures were standardized. A picospritzer was used to inject calibrated volumes of HRP in dimethyl sulfoxide focally into supragranular layers of the whiskerdefined barrel map, in vitro, by means of a template (23). After 4-6 hr of incubation, the slices were fixed with 1.25% glutaraldehyde/1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), frozen sectioned at 75 gm parallel to the pia, processed with 3,3'-diaminobenzidine as the chromagen, counterstained with 0.125% thionin, dehydrated in alcohols, cleared with xylene, and fixed on coverslips with Permount. The 4 control and 5 experimental cortices quantitated were selected from nearly 100 to meet the following criteria: (i) complete section of the 10 verified by examining the facial skin, (ii) position of injection localized in barrels of the C, D, and E rows (see Fig. 2 for a drawing of the map), and (iii) depth of injection confined to the supragranular layers. Sections were evaluated on a motorized microscope system (24). For each section 8192(x) x 8192 (y) x 6(z) picture elements (pixels), representing a volume of 2 x 2 x 0.075 mm, were analyzed. Image-processing software detected neuronal processes (16) and other programs produced montages from raw or processed images to make pictures of the injection sites and HRP-filled fibers. The section containing the largest diameter of the injection site was identified (15, 16) and the area of the densest reaction product in this section was used to create a "mask" over the injection site by thresholding the computer-generated montage (see Fig. 1 A' and B'). The distribution of fibers outside the mask was mapped. Maps from 10-12 sections in a series were serially aligned by an automated procedure (25).
The functional role of intracortical connections from local circuit neurons has been investigated intensively (1-8). In visual cortex, horizontal connections mainly from pyramidal cells subserve convergence of visual inputs for integration (9). In visual and other cortices, intrinsic activity functions to extract features from stimuli, to sharpen receptive fields, and to improve signal-to-noise ratios (10-12). Yet little is known about the factors on which local intracortical connections depend for normal development. The anatomy of layer IV of the mouse somatosensory cortex mimics the whiskers on the contralateral face (refs. 13 and 14; see Fig. 2). The whiskers are innervated by the infraorbital branch of the trigeminal nerve (10) and are arranged in five rows that project through several relays to groups of cells, called barrels, that are also arranged in five rows. The pattern of local intracortical connections in barrel cortex of normal animals is asymmetrical, with major projections originating in and confined to a barrel row and minor projections to the adjacent anterior barrel row (refs. 15 and 16; see Figs. 1 and 2). Nerve section on postnatal day (PND) 1 dramatically alters the anatomical representations of the whiskers in three brainstem nuclei, in the ventrobasal thalamus, and in barrel cortex, whereas section on PND7 does not alter these representations (17-21). In this experimental context, the axonal tracer horseradish peroxidase (HRP) was injected into supragranular layers of the barrel cortex. If peripheral inputs are necessary for the development of normal intracortical connections after PND7, then cutting the nerve should alter these patterns without changing the barrel map.
RESULTS Figs. 1 and 2 illustrate injections of similar size, in similar locations in control and experimental brains where the barrels define the somatotopic map (Fig. 1 A, B, and C). In normal mice, injections in the supragranular layers label axons spreading widely to other barrels (Figs. 1A to A" and 2). Projections to other layers vary substantially depending on the layer. For instance, their distribution at the layer II-III boundary is wider and more symmetrical than at the layer V-VI boundary (Fig. 2). For the lesioned animals, the barrel pattern is normal but the pattern of intracortical projections
MATERIALS AND METHODS The experimental, histological, and image-processing methods used have been described (16, 22). Outbred Swiss Webster mice of both sexes had the left 10 sectioned at PND7 under anesthesia. Their care and handling were according to institutional guidelines. At 6 weeks all animals were anes-
Abbreviations: 10, infraorbital branch of the trigeminal nerve; PNDn, postnatal day n; HRP, horseradish peroxidase. *To whom reprint requests should be addressed at: Division of Experimental Neurology and Neurological Surgery, Box 8057, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1832
.,w .~ Neurobiology: McCasland et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
1833
FiG. 1. Photomicrographs of ;qp1111B 75-,.m sections in the plane of and
r5tt ! W 2 i layer IV of mouse barrel through lE
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