Endogenous FGF-2 is important for cholinergic sprouting in the denervated hippocampus

Abstract

To investigate the molecular mechanisms of cholinergic sprouting in the hippocampus after removal of entorhinal cortical inputs, we evaluated trophic factor gene expression in the denervated hippocampus. Despite the proposed role for nerve growth factor (NGF) in this sprouting, we observed no change in NGF mRNA or protein at several postlesion time points. In contrast, FGF-2 mRNA was increased within 16 hr. FGF-2 immunoreactivity was localized within GFAP-positive hypertrophic astrocytes distributed specifically within the denervated outer molecular layer after the lesion. To address the functional significance of this increase in FGF-2, we assessed the magnitude of cholinergic sprouting in animals receiving chronic intracerebroventricular infusions of neutralizing antibodies specific for FGF-2 and compared it with that observed in lesioned animals receiving infusate controls. Animals given FGF-2 antibodies displayed a marked reduction in cholinergic sprouting as compared with controls. In fact, many of these animals exhibited virtually no sprouting at all despite histological verification of complete lesions. These results suggest that endogenous FGF-2 promotes cholinergic axonal sprouting in the injured adult brain. Furthermore, immunocytochemical localization of receptors for FGF-2 (i.e., FGFR1) on projecting basal forebrain cholinergic neurons suggests that FGF-2 acts directly on these neurons to induce the lesion-induced sprouting response.

Fig. 1.

Autoradiogram of RT-PCR products from control and lesioned (16 hr) rat hippocampus. At the top,CON and LES indicate control and lesioned RNA samples, respectively. The numbers at thetop (1–4) indicate the position of the lane for each of the four samples in both groups.Numbers down the right side indicate the position of 123 base pair markers on the autoradiogram.Names and arrows on theleft denote the PCR products predicted to correspond to the individual bands. Question marks indicate PCR products not expected from the original pool of primers used, presumably resulting from chance nonspecific priming of unrelated mRNAs present in the tissue extracts. Because several of the resulting PCR products are very similar in size and overlap on the gel, more than one individual set of primers may contribute to band intensity (e.g., GAD and FGF-2 products are close enough to cause uncertainty in conclusively predicting the identity of clustered bands). In these cases, follow-up analysis (see Fig. 2 and Results) was necessary to confirm the identity and intensity of the PCR products.

Fig. 2.

Autoradiograms of RT-PCR products from a second round of analysis of control and lesioned (16 hr) hippocampal RNA. Primer sets for species of interest or those giving products of uncertain origin from the first examination of control and lesioned hippocampal RNAs were combined in a pair-wise manner and reexamined in individual gels. CON and LES indicate mRNAs from control and lesioned animals, respectively. Mindicates the 123 base pair marker lane, and 1–4indicate the four different mRNA samples in each group.Numbers on the left side show base pair markers. Names and arrows at theright indicate the name and location of the various PCR products. All reactions were run with the RPL27A (L27) internal control primer set. Top left, BDNF and NT-3 analysis; top right, FGF-2 and FGFR1 analysis; middle left,trkB and GAD analysis; middle right, GFAP analysis; bottom center, NGF and additional FGF-2 analysis.

Fig. 3.

Graph showing quantification ofGFAP and NGF mRNA levels in hippocampal tissue obtained from animals killed at various early time points after unilateral PP transection. RT-PCR was performed on hippocampal tissue from n = 4 animals at each postlesion time point. Values are presented as the mean percentage of unoperated control values (0 hr, after normalizing to the RPL27A internal control bands within each sample). Error bars correspond to ± SEM. Asterisk indicates difference from unoperated control value (0 hr); p < 0.05.

Fig. 4.

Color photomicrographs of double-labeling immunocytochemistry showing distribution of FGF-2 (brown) and GFAP (pink) immunoreactivity (IR) within hippocampal area CA2 (A) and dentate gyrus (B) and immunofluorescence confocal images localizing FGF receptor-1 (FGFR1) and choline acetyltransferase (ChAT) within the medial septal nucleus (C–F) of adult rats. A, Colocalization of FGF-2-IR (brown) and GFAP-IR (pink) within area CA2 demonstrates that both the pyramidal cells in CA2 (P,arrowhead) and adjacent astrocytes (arrow) express FGF-2. Scale bar, 50 μ.B. Seven days after perforant pathway lesion,FGF-2 (brown) is expressed by GFAP-IRastrocytes (pink) both around the granule cell layer (G) of the dentate gyrus and in the dentate molecular layer (ML). Hypertrophic astrocytes expressing FGF-2 are especially prominent in the outer ML, the region in which the terminals of the lesioned perforant pathway (PP) were located. Scale bar, 50 μ. C, The fluorescent nuclear counterstain propidium iodide stains neurons, glia, and endothelial cells in this field of view in the medial septum. Thearrows and arrowhead are in registration and show the same location within the field of view forC–F. Scale bar, 25 μ for C–F.D, FGFR1 (green) immunofluorescence is localized to a number of cells showing a neuronal morphology and fine processes. E, ChATimmunofluorescence (blue) shows a number of positive neurons (arrows) that are a subset of the number of neurons indicated in C and D. A primary dendrite of a ChAT-IR neuron is indicated by thearrowhead. F, A merge ofC–F demonstrates colocalization of FGFR1 on ChAT-IR cell bodies (arrows) and processes (arrowhead), indicating that cholinergic neurons in the medial septum of the adult rat express FGFR1. In addition, many noncholinergic (ChAT-negative) neurons express FGFR1.

Fig. 5.

Photomicrographs of hippocampal tissue sections processed for (A) immunocytochemistry for normal serum and (B–H) histochemistry for AChE 14 d after unilateral PP transection. A, Immunoreactivity is observed within hippocampal parenchyma, demonstrating successful antibody penetration into this region after chronic ICV infusion.B, Contralateral (unlesioned) hippocampus from an animal receiving chronic anti-FGF-2 infusions displays the normal pattern of AChE-positive fibers within the outer molecular layer (OML, arrow) and inner molecular layer (IML) of the dentate gyrus. This pattern on the contralateral side is consistent between animals regardless of infusate (CSF, normal serum, or anti-FGF-2). C, Lesioned hippocampus from an animal receiving control infusate exhibits an increase in density of AChE-positive fibers in the OMLof the dentate gyrus. D, Lesioned hippocampus from an animal receiving neutralizing antibodies to FGF-2 displays no obvious increase in AChE-positive fiber density within the OMLof the dentate gyrus as compared with the contralateral side (B). E–H are high-power views of the OMLs from these same animals. Subsequent quantification of fiber density (see Fig. 6) was performed on tissue viewed at this high-power magnification. Scale bars: 500 μm in A; 50 μm inB–D; 12.5 μm in E–H.

Fig. 6.

Histogram showing percentage of increase in AChE-positive fiber density in the denervated molecular layer (ML) of animals receiving ICV infusions of anti-FGF-2 or control infusates (i.e., CSF or normal serum) for 14 d after unilateral PP transection. Values for individual animals are presented as the percentage of increase in AChE-positive fiber density in the denervated ML as compared with the contralateral side. Student’s ttests demonstrated no statistical difference between mean values for animals receiving CSF or normal serum (t = −1.45,p > 0.05), so these groups were combined to make a single control group and subsequently were compared to values from animals receiving anti-FGF-2. Open triangles identify individual animals receiving control infusate, and open circles identify individual animals receiving neutralizing antibodies to FGF-2. Bold horizontal line identifies mean value for each group (CONTROL andANTI-FGF-2). Asterisk indicates difference from control values, p < 0.05.