Circulating insulin-like growth factor I mediates exercise-induced increases in the number of new neurons in the adult hippocampus

Abstract

Although the physiological significance of continued formation of new neurons in the adult mammalian brain is still uncertain, therapeutic strategies aimed to potentiate this process show great promise. Several external factors, including physical exercise, increase the number of new neurons in the adult hippocampus, but underlying mechanisms are not yet known. We recently found that exercise stimulates uptake of the neurotrophic factor insulin-like growth factor I (IGF-I) from the bloodstream into specific brain areas, including the hippocampus. In addition, IGF-I participates in the effects of exercise on hippocampal c-fos expression and mimics several other effects of exercise on brain function. Because subcutaneous administration of IGF-I to sedentary adult rats markedly increases the number of new neurons in the hippocampus, we hypothesized that exercise-induced brain uptake of blood-borne IGF-I could mediate the stimulatory effects of exercise on the adult hippocampus. Thus, we blocked the entrance of circulating IGF-I into the brain by subcutaneous infusion of a blocking IGF-I antiserum to rats undergoing exercise training. The resulting inhibition of brain uptake of IGF-I was paralleled by complete inhibition of exercise-induced increases in the number of new neurons in the hippocampus. Exercising rats receiving an infusion of nonblocking serum showed normal increases in the number of new hippocampal neurons after exercise. Thus, increased uptake of blood-borne IGF-I is necessary for the stimulatory effects of exercise on the number of new granule cells in the adult hippocampus. Taken together with previous results, we conclude that circulating IGF-I is an important determinant of exercise-induced changes in the adult brain.

Fig. 1.

Infusion of an anti-IGF-I antiserum inhibits exercise-induced brain uptake of circulating IGF-I. A,Control sedentary animals show very low IGF-I labeling in the brain. Rats running in a treadmill for 15 d and receiving a simultaneous infusion of a nonblocking normal rabbit serum (NRS) show marked increases in IGF-I labeling throughout the brain. However, animals undergoing the same exercise schedule but receiving an infusion of an anti-IGF-I antiserum do not show increased brain IGF-I labeling. The cerebellar cortex and the hippocampus are shown as representative areas accumulating IGF-I after exercise (see references for further details). Arrows indicate typically labeled neurons.Mol, Cerebellar molecular layer; PC,Purkinje cell layer; Gr, cerebellar granule cell layer;Py, hippocampal pyramidal cell layer;StR, hippocampal stratum radiatum. B,Levels of IGF-I in the CSF of exercising animals receiving an infusion of NRS are 68% higher than in control, sedentary animals. However, exercising rats receiving an anti-IGF-I infusion do not show increased CSF IGF-I levels; n = 3 per group.C, The anti-IGF-I antibody used for blocking uptake of IGF-I blocks interaction of IGF-I with its receptor. The presence of the anti-IGF-I antiserum (20% in culture medium) completely blocks IGF-I-induced receptor tyr-phosphorylation in cerebellar neuronal cultures (right). When 20% NRS was used instead, IGF-I receptor was tyr-phosphorylated in response to IGF-I (left), indicating normal biological activity of IGF-I. Cerebellar cultures were lysed 3 min after addition of 100 nm IGF-I, immunoprecipitated (IP) with a polyclonal anti-IGF-I receptor antibody, and blotted (WB) with a monoclonal anti-pTyr antibody.

Fig. 2.

Characterization of BrdU-labeled cells in the granule cell layer of the adult hippocampus. A, Typical BrdU-positive cells in the dentate gyrus of an adult rat hippocampus, as revealed with DAB visualized with Nomarski optics. Immunopositive nuclei are easily distinguishable and clearly separated, making them well suited for stereology counts. B, A higher magnification of the area marked in A to show BrdU⁺ nuclei in greater detail. Orientation inA is dorsal (D), ventral (V), medial (M), and lateral (L). C, A BrdU⁺cell (red nucleus) within the granule cell layer also shows IGF-I receptor labeling (green signal surrounding the nucleus). D, Representative BrdU⁺ nuclei in the hippocampus of an animal after 15 d of treadmill exercise and simultaneous infusion of nonblocking rabbit serum. E, BrdU⁺nuclei in the hippocampus of an exercised animal receiving a simultaneous infusion of anti-IGF-I blocking antibody. Note that the number of BrdU nuclei is considerably lower after anti-IGF-I treatment. In both D and E, image contrast is sharp to delineate easily the granule cell layer. F,Identification of BrdU-positive nuclei (green) as neurons using double fluorescence immunohistochemistry for β-tubulin III (red cytoplasm). Cells shown are located in the inner part of the granule layer. G, Whereas several GFAP-positive astrocytes (green) appeared in the dentate hilus with BrdU-positive nuclei (red), no GFAP-positive astrocytes appeared inside the granule layer.G, Granule cell layer; H, dentate hilus.Arrows indicate positive cells. H–L,Serial confocal microscopy images along the z-axis of the same neuron showing colocalization of BrdU (yellow–green patches) in the nucleus and β-tubulin (red) in the cytoplasm to demonstrate the specificity of both signals.

Fig. 3.

Peripheral IGF-I increases the number of hippocampal BrdU⁺ cells and is necessary for exercise-stimulated increases in hippocampal BrdU⁺cells in adult rats. A, Treatment for 7 d with a subcutaneous infusion of IGF-I (50 mg · kg⁻¹ · d⁻¹) results in significantly increased number of BrdU⁺hippocampal granule cells. BrdU⁺ cells in the granule cell layer of the hippocampus are significantly increased in animals killed either 24 hr (p < 0.05) or 3 weeks (p < 0.005) after the last BrdU injection to determine short-term and long-term effects of IGF-I on BrdU⁺ cell numbers, respectively. *p < 0.05; ***p < 0.005 versus controls. B, C, Animals undergoing exercise training for 15 d and simultaneously receiving an infusion of nonblocking rabbit serum (NRS + exercise) show significant increases in BrdU⁺ cells both 24 hr (B) as well as 2 weeks (C) after the last BrdU injection as compared with nonexercising rats (controls) receiving an infusion of saline. However, both 24 hr survival as well as 2 week survival of BrdU⁺ cells are significantly reduced when exercising animals receive an infusion of blocking anti-IGF-I antiserum (anti-IGF-I + exercise). Furthermore, infusion of anti-IGF-I to normal sedentary rats (control + anti-IGF-I) results in a significant decrease in long-term survival of BrdU⁺ cells. *p < 0.01 versus control; **p < 0.01 versus NRS + exercise; ***p < 0.01 versus control. Data are the mean ± SEM of the number of BrdU⁺ cells per cubic millimeter.