Bcl-2 protein as a marker of neuronal immaturity in postnatal primate brain

Abstract

The distribution of neurons expressing immunoreactivity for the protein Bcl-2 was studied in the brain of squirrel monkeys (Saimiri sciureus) of various ages. Several subsets of small and intensely immunoreactive neurons displaying an immature appearance were disclosed in the amygdala and piriform cortex. The piriform cortex exhibited clusters of various forms in which Bcl-2+ neurons appeared linked to one another by their own neurites. The subventricular zone, which is known to harbor the largest population of rapidly and constitutively proliferating cells in the adult rat brain, was intensely stained, particularly at the basis of the lateral ventricle. A long and dorsoventrally oriented Bcl-2+ fiber fascicle was seen to emerge from the subventricular zone, together with numerous Bcl-2+ cells that formed a densely packed column directed at the olfactory tubercle. In adult and aged monkeys, the small and intensely labeled neurons were progressively replaced by larger and more weakly stained neurons in the amygdala and piriform cortex. In contrast, Bcl-2 immunostaining did not change with age in the subventricular zone and olfactory tubercle, the islands of Calleja of which were markedly enriched with Bcl-2. The dentate gyrus contained only a few layers of intensely labeled granule cells in juvenile monkeys, but the number of these layers increased markedly in adult and aged monkeys. These findings suggest that Bcl-2 can serve as a marker of both proliferating and differentiating neurons and indicate that such immature neurons may be much more widespread than previously thought in postnatal primate brain.

Fig. 1.

Western blot analysis of monkey brain extracts for Bcl-2 protein. Thirty and 35 μg of amygdala (A-30 andA-35) and hippocampus (H-30 andH-35) protein extracts were loaded on SDS-PAGE gels. Anti-Bcl-2 antibody (clone 124) recognizes a single major band of ∼26 kDa in both samples.

Fig. 2.

Confirmation of the specificity of the anti-human Bcl-2 antibody (clone 124) for Bcl-2 protein by transfection studies in monkey (vero) cells. A specific Bcl-2 immunofluorescence was detected only in vero cells transfected by a pRC/CMV expression vector containing the human Bcl-2 cDNA sequence (middle). No immunostaining was observed in untransfected cells (top) or in cells transfected by a pRC/CMV expression vector containing the human Bcl-XL cDNA sequence (bottom). Scale bar, 50 μm.

Fig. 3.

Variations in the pattern of Bcl-2 immunostaining of the amygdala among juvenile, adult, and aged squirrel monkeys. Frontal sections are from midamygdaloid levels in the three cases, and the material is shown at both low (A, C, E) and higher (B, D, F) magnifications. A, B, In juvenile monkeys, the immunostaining is particularly prominent in the basomedial sector of the amygdala, and many labeled fiber fascicles emerge from the positive cells and ascend dorsally within the amygdala (A). This immunostaining is the result of the accumulation of numerous intensely stained small and round neurons (B). C, D, In adult animals, Bcl-2 immunostaining is decreased by comparison with juveniles (C), as a result of a loss in the number of small, intensely immunoreactive neurons. However, note the presence of some larger and more weakly immunostained neurons located more deeply in the amygdala (D). E, F, In the aged monkey, Bcl-2 immunostaining is relatively intense (E). This is largely caused by a marked increase in the number of large and moderately stained neurons, with only a few small and intensely immunoreactive neurons remaining along the lateral border of the amygdala (F). Scale bars:A, C, E, 500 μm; B, D, F, 200 μm.

Fig. 4.

Examples of the immature features displayed by Bcl-2-positive neurons in amygdala (A–C) and piriform cortex (D–F) of juvenile monkeys. A, Low-power view of the intensely immunostained neurons lying at the basis of the amygdala (AM), along the fiber layer (FL) that separates the amygdala from the adjoining entorhinal cortex (CT). B, C, High-power view of two clusters of immunoreactive neurons, the exact location of which in the amygdala is indicated by insets in A.D, A small collection of Bcl-2-positive neurons linked together by their long and linear processes in the piriform cortex.E, A small round group of closely packed immunoreactive neurons displaying a peculiar arrangement in the piriform cortex.F, Elongated immunoreactive structure in the piriform cortex composed of closely packed Bcl-2-positive cells embedded in a tubular sheath that appears to be formed by the cells own processes. Scale bars: A, 200 μm; B, C, 50 μm;D, 25 μm; E, F, 15 μm.

Fig. 5.

Variations in patterns of Bcl-2 immunostaining in the hippocampus of juvenile (A, B, E) and adult squirrel monkeys (C, D, F). At this level, Bcl-2 immunoreactivity is largely confined to perikarya of granule cell layer and neuropil of the molecular layer, leaving the polymorph layer relatively free of immunostaining. A, B, In juvenile monkeys, the granule cell layer is thin, and the neuropil of the molecular layer is moderately stained. C, D, In adult monkeys, the granular layer is thicker, and the immunostaining in certain sectors of the molecular layer is more intense than in juveniles. E, F, High-power views of similarly intense immunoreactive neurons in the granular layer in juveniles (E) and in adult (F) monkeys. CA2,CA3, Fields 2 and 3, respectively, of Ammon’s horn (cornu ammonis); DG, dentate gyrus with its molecular (M), granular (G), and polymorph (P) layers. Scale bars: A, C, 500 μm; B, D, 200 μm; E, F, 100 μm.

Fig. 6.

A, Intense Bcl-2 immunoreactivity encountered in the islands of Calleja (arrows), which are scattered within the olfactory tubercle (OT), and in the subventricular zone (arrowhead) at the basis of the lateral ventricle (LV). B, High-power view of the multitude of small and intensely stained neurons encountered in one of the island of Calleja (inset inA). C, A fascicle formed by numerous Bcl-2-immunostained fibers that stem from the ventral tip of the lateral ventricle and course along the medial border of nucleus accumbens (NA). The location of the caudate nucleus (CD) and internal capsule (IC) is also indicated. D, High-power view of Bcl-2-positive cells in the subventricular zone located at the ventral tip of the lateral ventricle (arrowhead in C).E, The Bcl-2-positive glial septum located along the midline in the lower brainstem. Photomicrographs were taken from sections of different juvenile monkey brains. Scale bars: A, C, 500 μm; B, 50 μm; D, 100 μm; E, 125 μm.

Fig. 7.

Cells in the SVZ of an adult monkey expressing both Bcl-2 and nestin, as visualized on the same section with a double-immunofluorescence procedure. The doubly labeled cells display a green FITC fluorescence indicative of Bcl-2 (A), as well as a Texas Red fluorescence indicative of nestin (B). The photomicrograph inC is a double exposure of the same group of cells, theyellow color of which confirms the colocalization of Bcl-2 and nestin. Scale bar, 30 μm.

Fig. 8.

Variations of the pattern of Bcl-2 immunostaining in the cortex of juvenile, adult, and aged squirrel monkeys. A, B, In the temporal cortex of juveniles (A), Bcl-2-positive neurons are confined to supragranular layers, particularly layer II, and in infragranular layers, particularly layer V and, to a lesser extent, layer VI, whereas in the adults and aged animals (B) the immunoreactive neurons are concentrated in infragranular layers. C, Layer II in juvenile monkeys contains both type A and type B Bcl-2-immunopositive neurons. D, Type A neurons are intensely stained with clearly visible processes. E, Layers V–VI in adult and aged monkeys are populated by small pyramidal neurons with well delineated apical dendrites. F–H, The moderate Bcl-2 immunostaining observed in the supragranular layers of the insular (IN) and temporal (TE) cortices of juveniles monkeys (F) becomes very weak in adult monkeys (G) and virtually absent in the aged monkey (H). Scale bars: A, B, F, G, H, 250 μm; C, E, 100 μm; D, 20 μm.