Ectopic growth of hippocampal mossy fibers in a mutated GAP-43 transgenic mouse with impaired spatial memory retention

Abstract

In a previous study, it was shown that transgenic mice, designated G-NonP, forget the location of a water maze hidden platform when tested 7 days after the last training day (Holahan and Routtenberg (2008) Hippocampus 18:1099-1102). The memory loss in G-NonP mice might be related to altered hippocampal architecture suggested by the fact that in the rat, 7 days after water maze training, there is discernible mossy fiber (MF) growth (Holahan et al. (2006) Hippocampus 16:560-570; Rekart et al. (2007) Learn Mem 14:416-421). In the present report, we studied the distribution of the MF system within the hippocampus of naïve, untrained, G-NonP mouse. In WT mice, the MF projection was restricted to the stratum lucidum of CA3 with no detectable MF innervation in distal stratum oriens (dSO). In G-NonP mice, in contrast, there was an ectopic projection terminating in the CA3 dSO. Unexpectedly, there was nearly a complete loss of immunostaining for the axonal marker Tau1 in the G-NonP transgenic mice in the MF terminal fields indicating that transgenesis itself leads to off-target consequences (Routtenberg (1996) Trends Neurosci 19:471-472). Because transgenic mice overexpressing nonmutated, wild type GAP-43 do not show this ectopic growth (Rekart et al., in press) and the G-NonP mice overexpress a mutated form of GAP-43 precluding its phosphorylation by protein kinase C (PKC), the possibility exists that permanently dephosphorylated GAP-43 disrupts normal axonal fasciculation which gives rise to the ectopic growth into dSO.

Figure 1

Transgenic GAP-43 mRNA localization in A) the WT mice and B) G-NonP transgenic mice. Transgene GAP-43 mRNA signals in the WT mice were, as expected, at background levels. Of particular relevance to the mossy fiber axonal distribution found in the G-NonP mice, GAP-43 transgene mRNA occurred at high levels in the granule cells of the hippocampus (1B1).

Figure 2

Timm’s heavy metal staining from the dorsal hippocampus shows that G-NonP mice (B) have a larger mossy fiber-stained area in the stratum lucidum (SL) and distal stratum oriens (dSO) of CA3 than WT (A). This is observed throughout the entire rostral-caudal extent of the dorsal hippocampus. Cresyl violet counter stain shows pyramidal cell layer (SP). C. Quantification of the Timm’s staining in dorsal hippocampus at the mid-caudal level (asterisk) confirms that G-NonP mice have a larger Timm’s-stained area in SL and dSO compared to the WT group. Data are presented as the mean area of staining. Scale bar = 200 μm. ** p < 0.01, *** p < 0.001. D. Immunohistofluorescent labeling for the zinc transporter protein ZnT3 (red). Scale bar = 200 μm. E. Timm’s heavy metal staining from the dorsal hippocampus shows that G-Perm mice have no ectopic MF in dSO and thus a similar distribution of mossy fiber terminal fields as WT.

Figure 3

Immunohistochemical localization of the endogenous (mouse) and transgenic (chicken) GAP-43 protein, the ZnT3 protein and their merged images. A) WT mice. Staining in the WT mouse revealed no traces of GAP-43 staining in the mossy fiber pathways as labeled with the ZnT3 antibody. B) G-NonP mice. There was a striking pattern of elevated GAP-43 staining in the CA3 SL and dSO region of the G-NonP mice which appeared to overlap with the ZnT3 staining. This points to an ectopic distribution of GAP-43 in the G-NonP mice located in the mossy fiber axonal terminals. SLM: stratum lacunosum moleculare, IML: inner molecular layer of the granule cells (GC), SR: stratum radiatum, SP-MF, supra-pyramidal mossy fiber pathway, IIP-MF: infra/intra-pyramidal mossy fiber pathway.

Figure 4

Immunohistofluorescent labeling for the axon-specific microtubule associated protein Tau1 (A) and the presynaptic terminal marker, synaptophysin (B) using DAPI (blue) counterstain to indicate nuclei. Images are from the dorsal hippocampus of a G-NonP and WT mouse. Note the complete absence of Tau1 staining in the SL of the G-NonP mouse as compared to the high level of staining in the same region of the WT mouse. Note that the synaptophysin staining in the G-NonP mouse was comparable to that seen in WT. Scale bar = 200 μm.