Perinatal gonadectomy exerts regionally selective, lateralized effects on the density of axons immunoreactive for tyrosine hydroxylase in the cerebral cortex of adult male rats

Abstract

The catecholamine innervation of the cerebral cortex is essential for its normal operations and is implicated in cortical dysfunction in mental illness. Previous studies in rats have shown that the maturational tempo of these afferents is highly responsive to changes in gonadal hormones. The present findings show that perinatal hormone manipulation also has striking, region- and hemisphere-specific consequences for cortical catecholamines in adulthood. The effect of perinatal gonadectomy on catecholamines was examined in representative sensory, motor, and association cortices of adult male rats by combining hormone manipulation with immunocytochemistry for tyrosine hydroxylase, a rate-limiting enzyme in catecholamine biosynthesis. Qualitative and quantitative comparison of immunoreactivity in rats perinatally gonadectomized or sham-operated revealed complex changes in gonadectomized subjects; in cingulate cortex, TH immunoreactivity was strongly and bilaterally diminished, in sensory and motor cortices, axon density was decreased in left hemispheres, but was minimally affected on the right, and in a premotor cortex, gonadectomy was without significant effect in either hemisphere. Corresponding analyses in gonadectomized rats supplemented with testosterone revealed a protective influence, albeit one in which TH immunoreactivity so showed regional and hemispheric variability in responsiveness to hormone replacement. These complex patterns of TH sensitivity suggest highly asymmetric hormone stimulation of cortical catecholamines. Such discriminative action may contribute to sex differences in the functional maturation and lateralization of the cortex and may also have bearing on disorders such as dyslexia, which show sexual dimorphisms, and in which functional laterality of the cortex may be particularly at issue.

Fig. 1.

Schematic diagram showing a lateral view of the rat cerebral cortex (top) and a representative cross section (bottom) taken at the level of approximately the anteroposterior midpoint of the septal nucleus. In the coronal section, the locations of area Cg1 and Par 1 of Zilles (1990) and of areas AgM and AgL (Donoghue and Wise, 1982) are shown. Qualitative analyses were performed at representative rostrocaudal levels within each of these four areas. Quantitative measures were derived from sections matching the cross section depicted. In each section used for quantitative study, camera lucida drawings of tyrosine hydroxylase-immunoreactive fibers were obtained from layer II/III and from layer V of all four areas. In total, these drawings subtended virtually the entire span of areas Cg1, AgM, and AgL present within the section; the stippled area offset with dashed lines within area Par1 illustrates the approximate location of the subportion of this region from which quantitative measures were obtained. olf, Olfactory bulb; cc, corpus callosum; cd, caudate nucleus.

Fig. 2.

Representative camera lucida drawings of tyrosine hydroxylase-immunoreactive fibers within the dorsal anterior cingulate cortex (area Cg1). The left (L) and right (R) hemispheres of a control (CTRL) animal, an animal gonadectomized on the day of birth (GDX-SO), and an animal gonadectomized at birth and supplemented with testosterone proprionate (GDX-TP) are shown. The approximate borders of cortical layers are marked by the roman numeralsappearing on the left. The immunostaining represented was obtained using a commercially available antibody purchased from Chemicon. Comparison across the animal groups represented illustrates the stark loss of axon density in both hemifields of the cingulate cortex of gonadectomized animals and the appearance of more normal levels of innervation in gonadectomized animals that received injections of testosterone proprionate. In all animal groups, seemingly normal, layer-specific patterns of tyrosine hydroxylase-immunoreactive fiber orientation are preserved.wm, White matter.

Fig. 3.

Scatterplots of mean pixel density measures derived from camera lucida drawings of tyrosine hydroxylase-immunoreactive fibers (Chemicon) in layers II/III and V of the left and right anterior dorsal cingulate hemifields of control (CTRL), gonadectomized (GDX-SO), and gonadectomized, testosterone proprionate-supplemented (GDX-TP) animals. The points plotted (triangles) correspond to raw data points that are inclusive of all measures obtained from each of the six individual animals comprising each of the three experimental groups.Horizontal bars mark the group means of the pixel density measures. The decreases in axon density in GDX-SO animals are proportionately similar across layers and hemispheres, and values in both are significantly less then controls. Treatment of gonadectomized animals with testosterone proprionate yields statistically normal innervation density in layer II, but produces axon density levels that fall between and are significantly different from GDX-SO and control values in layer V.

Fig. 4.

Representative bright-field photomicrographs illustrating the morphology and orientation of axons immunoreactive for tyrosine hydroxylase in layer II of the anterior dorsal cingulate cortex (area Cg1, A–C) and layer III of the primary motor cortex (area AgL,D–F) in controls (CTRL), gonadectomized rats (GDX-SO), and rats gonadectomized and supplemented with testosterone proprionate (GDX-TP). All sections were immunoreacted for tyrosine hydroxylase (antibody from Chemicon) and are counterstained for Nissl substance with 1% cresyl violet. None of the cellular staining depicted in any of the panels corresponds to tyrosine-immunopositive somata. In both regions represented, the same features of axon morphology and orientation that characterize control animals are also found in the immunoreactivity of gonadectomized and gonadectomized, testosterone-supplemented animals; distinctive features such as the short, tortuous axons segments in layer II of the cingulate cortex (A–C), and the long radial fibers that are prominent in layer III of the primary motor cortex (D–F) are preserved across experimental groups. Clear decreases in the number of immunoreactive axon segments, however, readily distinguish the GDX-SO animal from control and GDX-TP cases. Scale bars:A–C, 100 μm; E,F, 500 μm.

Fig. 5.

Scatterplots of the percentages of axon arbor measured at a single focal plane that are <15 μm in two-dimensional length in the supragranular and infragranular layers of cingulate (Cg1), premotor (AgM), primary motor (AgL), and primary somatosensory (Par1) cortex. For each plot, points represent raw data inclusive of all measures obtained from each of the six control animals (large black squares), the six gonadectomized animals (small open squares), and the six gonadectomized animals supplemented with testosterone proprionate (small open circles) analyzed in this study. The relative high percentage of short fiber segments in layer II of the cingulate cortex is consistent with its signature pattern of innervation by short, highly branched, randomly oriented axon segments. In all other regions and layers, shortest axon segments, which could include segments oriented steeply with respect to the plane of the tissue section and, thus, most significantly foreshortened in two-dimensional drawings, comprised ∼10% or less of the total axon arbor sampled. Data collected for all three animal groups is largely overlapping. Area AgM represents an exception in which a disproportionate number of GDX-SO animals had more short axon segments than GDX-TP or control animals.

Fig. 6.

Representative camera lucida drawings of tyrosine hydroxylase-immunoreactive fibers within the primary motor cortex (areaAgL). The left (L) and right (R) hemispheres of a control (CTRL) animal, an animal gonadectomized on the day of birth (GDX-SO), and an animal gonadectomized at birth and supplemented with testosterone proprionate (GDX-TP) are shown. The approximate borders of cortical layers are marked by the roman numerals appearing on the left. The immunostaining represented was obtained using a commercially available antibody purchased from Chemicon. Comparison across the animal groups represented illustrates the pronounced reduction in axon density in the left primary motor hemifield in gonadectomized animals, and the appearance of more normal levels of innervation in the right hemifield of gonadectomized animals, and in both hemispheres of gonadectomized animals that received injections of testosterone proprionate. In all animal groups, seemingly normal, layer-specific patterns of tyrosine hydroxylase-immunoreactive fiber orientation are preserved in both hemispheres. wm, White matter.

Fig. 7.

Scatterplots of mean pixel density measures derived from camera lucida drawings of tyrosine hydroxylase-immunoreactive fibers (Chemicon) in layers II/III and V of the left and right primary motor hemifields of control (CTRL), gonadectomized (GDX-SO), and gonadectomized, testosterone proprionate-supplemented (GDX-TP) animals. The points plotted (triangles) correspond to raw data points that are inclusive of all measures obtained from each of six individual animals comprising the three experimental groups. Horizontal bars mark numerical group means of the pixel density measures. The large decreases in axon density in left motor hemifields of GDX-SO animals are proportionately similar across layers and are significantly less than controls in both layers II/III and V. More modest, albeit statistically significant, decreases in density mark the supragranular and infragranular layers of the right motor hemifield of GDX-SO animals. Treatment of gonadectomized animals with testosterone proprionate yields statistically normal innervation density in layers II/III and V of the left primary motor cortex but has seemingly no effect on TH axon density in these layers on the right.

Fig. 8.

Representative camera lucida drawings of tyrosine hydroxylase-immunoreactive fibers within the primary somatosensory cortex (Par1). The left (L) and right (R) hemispheres of a control (CTRL) animal, an animal gonadectomized on the day of birth (GDX-SO), and an animal gonadectomized at birth and supplemented with testosterone proprionate (GDX-TP) are shown. The approximate borders of cortical layers are marked by the roman numerals appearing on the left. The immunostaining represented was obtained using a commercially available antibody purchased from Chemicon. Comparison across the animal groups represented illustrates a striking decrease in tyrosine hydroxylase-immunoreactive axon density in the left somatosensory hemifield in gonadectomized animals and normal appearing innervation in the right hemifield of gonadectomized animals, and in both hemispheres of gonadectomized animals that received testosterone proprionate treatment. In all animal groups, qualitatively normal, layer-specific patterns of tyrosine hydroxylase-immunoreactive fiber orientation are preserved. wm, White matter.

Fig. 9.

Scatterplots of mean pixel density measures obtained from camera lucida drawings of tyrosine hydroxylase-immunoreactive fibers (Chemicon) in layers II/III and V of the left and right primary somatosensory hemifields of control (CTRL), gonadectomized (GDX-SO), and gonadectomized, testosterone proprionate-supplemented (GDX-TP) animals. The points plotted (triangles) correspond to raw data points that are inclusive of all measures obtained from each of six individual animals from each of the three experimental groups. Horizontal bars mark the numerical group means of the pixel density measures. The large decreases in axon density in left somatosensory cortex in GDX-SO animals are proportionately similar in supragranular and infragranular layers, with both values being significantly different from controls. More modest, yet statistically significant, decreases in density also mark the supragranular and infragranular layers of the right somatosensory cortex in GDX-SO animals. Treatment of gonadectomized animals with testosterone proprionate yields statistically normal innervation density in layers II/III and V of the left primary somatosensory cortex but has minimal effect on TH axon density in corresponding layers of the right somatosensory field.

Fig. 10.

Representative camera lucida drawings of tyrosine hydroxylase-immunoreactive fibers within the premotor cortex (areaAgM). The left (L) and right (R) hemispheres of a control (CTRL) animal, an animal gonadectomized on the day of birth (GDX-SO), and an animal gonadectomized at birth and supplemented with testosterone proprionate (GDX-TP) are shown. The approximate borders of cortical layers are marked by the roman numerals appearing on the left. The immunostaining represented was obtained using a commercially available antibody purchased from Chemicon. Comparison across the animal groups represented illustrates the insensitivity of tyrosine hydroxylase-immunoreactive axons to gonadectomy, with and without testosterone proprionate supplementation, in both hemifields. In all animal groups normal, layer-specific patterns of tyrosine hydroxylase-immunoreactive fiber density, distribution, and orientation are preserved. wm, White matter.

Fig. 11.

Scatterplots of mean pixel density measures derived from camera lucida drawings of tyrosine hydroxylase-immunoreactive fibers (Chemicon) in layers II/III and V of the left and right premotor hemifields of control (CTRL), gonadectomized (GDX-SO), and gonadectomized, testosterone proprionate-supplemented (GDX-TP) animals. The points plotted (triangles) correspond to raw data points that are inclusive of all measures obtained from each of six individual animals comprising the three experimental groups. Horizontal bars mark the numerical group means of the pixel density measures. Overall, measures of axon density in control, GDX-SO, and GDX-TP animals are overlapping, and none of the differences in group mean values among the groups are statistically significant.