Environmental lead exposure during early life alters granule cell neurogenesis and morphology in the hippocampus of young adult rats

Abstract

Exposure to environmentally relevant levels of lead (Pb(2+)) during early life produces deficits in hippocampal synaptic plasticity in the form of long-term potentiation (LTP) and spatial learning in young adult rats [Nihei MK, Desmond NL, McGlothan JL, Kuhlmann AC, Guilarte TR (2000) N-methyl-D-aspartate receptor subunit changes are associated with lead-induced deficits of long-term potentiation and spatial learning. Neuroscience 99:233-242; Guilarte TR, Toscano CD, McGlothan JL, Weaver SA (2003) Environmental enrichment reverses cognitive and molecular deficits induced by developmental lead exposure. Ann Neurol 53:50-56]. Other evidence suggests that the performance of rats in the Morris water maze spatial learning tasks is associated with the level of granule cell neurogenesis in the dentate gyrus (DG) [Drapeau E, Mayo W, Aurousseau C, Le Moal M, Piazza P-V, Abrous DN (2003) Spatial memory performance of aged rats in the water maze predicts level of hippocampal neurogenesis. Proc Natl Acad Sci U S A 100:14385-14390]. In this study, we examined whether continuous exposure to environmentally relevant levels of Pb(2+) during early life altered granule cell neurogenesis and morphology in the rat hippocampus. Control and Pb(2+)-exposed rats received bromodeoxyuridine (BrdU) injections (100 mg/kg; i.p.) for five consecutive days starting at postnatal day 45 and were killed either 1 day or 4 weeks after the last injection. The total number of newborn cells in the DG of Pb(2+)-exposed rats was significantly decreased (13%; P<0.001) 1 day after BrdU injections relative to controls. Further, the survival of newborn cells in Pb(2+)-exposed rats was significantly decreased by 22.7% (P<0.001) relative to control animals. Co-localization of BrdU with neuronal or astrocytic markers did not reveal a significant effect of Pb(2+) exposure on cellular fate. In Pb(2+)-exposed rats, immature granule cells immunolabeled with doublecortin (DCX) displayed aberrant dendritic morphology. That is, the overall length-density of the DCX-positive apical dendrites in the outer portion of the DG molecular layer was significantly reduced up to 36% in the suprapyramidal blade only. We also found that the area of Timm's-positive staining representative of the mossy fibers terminal fields in the CA3 stratum oriens (SO) was reduced by 26% in Pb(2+)-exposed rats. These findings demonstrate that exposure to environmentally relevant levels of Pb(2+) during early life alters granule cell neurogenesis and morphology in the rat hippocampus. They provide a cellular and morphological basis for the deficits in synaptic plasticity and spatial learning documented in Pb(2+)-exposed animals.

Figure 1

Experimental design and timeline of Pb²⁺ exposure and BrdU administration.

Figure 2

Effect of Pb²⁺ exposure on cell proliferation in the hippocampus DG. The figure depicts BrdU-positive cells one day after the last BrdU administration in the DG of a control (a) and Pb²⁺-exposed (b) rat. BrdU labeled cells were often arranged in clusters (Figure 2a, insert). A lower number of BrdU labeled cells was detected throughout the entire extent of the dorsal dentate gyrus (c). Each value is the mean ± sem of n=7 different control animals and n=9 Pb²⁺-exposed animals. SPB= suprapyramydal blade; IPB= infrapyramydal blade.

Figure 3

(a) Co-localization of BrdU (red) with DCX (green) and GFAP (blue). (b) No significant differences in percent co-localization were found between the treatment groups in the percentages of new cells labeled with either a neuronal or astrocytic phenotype. Approximately 20% of cells in control or Pb²⁺-exposed rats did not co-localize with either marker. Mean values were obtained from a total of 4 different animals from both control and Pb²⁺-exposed groups.

Figure 4

Effect of Pb²⁺ exposure on granule cell survival in the dorsal DG. The figure depicts BrdU-postive cells four weeks after the last BrdU administration in the DG of a control (a) and Pb²⁺-exposed (b) rat. A reduction in the number of BrdU labeled cells was observed throughout the extent of the dorsal DG (c). Each value is the mean ± sem of n=6 different control animals and n=5 Pb²⁺-exposed animals. SPB= suprapyramydal blade; IPB= infrapyramydal blade.

Figure 5

DCX-positive neurons in the dorsal DG (a,b) and their fiber length densities in the outer molecular layer of the dentate gyrus (c,d) in control and Pb²⁺-exposed rats. Boxed areas in (a) and (b) are represented in (c) and (d) at higher magnification. In control animals (a) DCX-positive neurons displayed vertically oriented primary dendrites that branch within the IML, extending their fine dendrites into the OML generally reaching the hippocampal commisure. In Pb²⁺-treated rats (b), primary dendrites of DCX-positive neurons appeared shorter and formed their arbors within the GCL proper. Their fine terminals generally reached the IML or MML but rarely the OML. In Pb²⁺-exposed rats, the density of the DCX-labeled fiber length was diminished in the OML of the suprapyramidal blade throughout the dorsal DG (f). This effect was not observed in the infrapyramidal blade (e). Each value is the mean ± sem of n=6 different control animals and n=6 Pb²⁺-exposed animals. OML= outer molecular layer; MML= medial molecular layer; IML= inner molecular layer.

Figure 6

Representative images of DXC-positive newly born cell morphology in the dorsal DG of control (a) and Pb²⁺-exposed rats (b-d). In DCX-positive cells of the DG from control animals, vertically oriented primary apical dendrites extended trough the GCL and branched in the ML (a). On the other hand in Pb²⁺-exposed animals, DCX-positive cells frequently had an irregular orientation of apical dendrites (see arrow heads in b and c), a “bushy” appearance or significant branching within the borders of GCL (see star in b). In addition, numerous apical dendrites often appeared dystrophic with a “spiral-like appearance (see arrows in c and d).

Figure 7

Timm’s and Nissl staining (a,b) in the hippocampus of control (a) and Pb²⁺-exposed rats (b). In control animals, Timm’s stained mossy fibers were prominent in both the stratum lucidum (SL) and stratum oriens (SO) of the CA3 region (a). In Pb²⁺-exposed rats, reduced Timm’s staining was detected in the CA3-SO. The stratum pyramidale (SP) is easily identified by Nissl staining. (b). The area of Timm’s staining in the SO in the CA3 region was significantly diminished in Pb²⁺ -exposed animals throughout the dorsal hippocampus (c). Each value is the mean ± sem of n=6 different control animals and n=6 Pb²⁺-exposed animals.