Insulin-Like Growth Factor 1 Is Essential for Normal Dendritic Growth Clara M. Cheng, 1 Ronald F. Mervis, 2 Shui-Lin Niu, 3 Norman Salem Jr., 3 Lee A. Witters, 4 Victor Tseng, 1 Rickey Reinhardt, 1 and Carolyn A. Bondy 1 * 1 Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 2 NeuroStructure Research Laboratories, Columbus, Ohio 3 Laboratory of Membrane Biophysics and Biochemistry, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 4 Department of Medicine and Biochemistry, Dartmouth Medical School, Hanover, New Hampshire This study evaluated somatic and dendritic growth of neurons in the frontoparietal cortex of Igf1–/– brains. Pyramidal neuron density was increased by 25% (P = .005) and soma size reduced by 10% (P  .001). Golgi staining revealed that cortical layer II–III neurons exhib- ited a significant reduction in dendritic length and com- plexity in Igf1 null mice. Dendritic spine density and presumably synaptic contacts were reduced by 16% (P = .002). Similar findings were obtained for cortical layer V and piriform cortex pyramids. Supporting a re- duction in synapses, synaptotagmin levels were reduced by 30% (P  .02) in the Igf1 null brain. Investigation of factors critically involved in dendritic growth and synap- togenesis showed an 50% reduction in cortical CDC42 protein expression (P  .001) and an 10% reduction in brain cholesterol levels (P  .01) in Igf1 null mice. Evi- dence is presented that Igf1 deletion causes disruptions in lipid and microtubule metabolism, leading to impaired neuronal somatic and dendritic growth. Published 2003 Wiley-Liss, Inc. † Key words: cholesterol; synaptogenesis; phospholipid; cdc42; synaptotagmin; cholesterol; mental retardation Insulin-like growth factor 1 (IGF1) has pleiotropic effects on neural tissue in vitro that have been reviewed elsewhere (Feldman et al., 1997). Studies of the murine Igf1-targeted-deletion brain have elucidated IGF1’s neu- rotrophic effects in vivo. We have demonstrated that glucose utilization is significantly reduced in the Igf1–/– brain during early postnatal development, particularly in those structures where IGF1 is normally highly expressed (Cheng et al., 2000). The reduction in glucose use is associated with reduced phosphorylation of Akt and GSK3, decreased GLUT4 expression and hexokinase activity, and reduced glycogen accumulation in growing neurons that normally express IGF1 (Cheng et al., 2000). These findings suggest that IGF1 serves an anabolic, insulin-like role promoting neuronal glucose utilization and growth during brain development (Cheng et al., 2000). IGF1 expression is most abundant in growing pro- jection neurons, which become the largest neurons equipped with the most extensive dendritic trees in the brain (Bondy, 1991). Also, IGF1 is highly expressed dur- ing the time in early postnatal development when dentritic process growth is most exuberant (Bartlett et al., 1991; Bondy, 1991). Because neuronal processes or neuropil occupy a large percentage of brain volume, it seemed possible that impaired dendritic development in the ab- sence of IGF1 may contribute to the reduced brain size and cognitive impairment noted in the human with Igf1 deletion (Woods et al., 1996, 1997). To evaluate this hypothesis, we have used Golgi histological staining and biochemical analyses to evaluate dendritic growth and complexity in the Igf1–/– brain and wild-type (WT) lit- termates. MATERIALS AND METHODS Mice The Igf1–/– mouse line used in this study, which was approved by the NICHD Animal Use and Care Committee, was generated at Genentech, Inc. (San Francisco, CA; Powell- Braxton et al., 1993). This mutation has been bred into a CD1 outbred line for more than 40 generations. Igf1–/– and WT littermate mice were genotyped as described previously (Powell- Braxton et al., 1993). For biochemical studies, mice were sac- rificed by decapitation after CO 2 anesthesia; then, brains were dissected, snap frozen immediately, and stored at –70°C. For morphological study, mice were anesthetized with sodium pen- tobarbital and perfused through the ascending aorta with 4% paraformaldehyde in phosphate-buffered saline (PBS) buffer (pH 7.4) for 45 min. These brains were then postfixed in 10% formalin in PBS buffer overnight. Brains were bisected sagitally, and a half brain from each animal was used for morphological *Correspondence to: Carolyn A. Bondy, MD, Bldg. 10/10N262, 10 Center Dr., NIH, Bethesda, MD 20892. E-mail: bondyc@exchange.nih.gov Received 13 February 2003; Revised 27 February 2003; Accepted 7 March 2003 Journal of Neuroscience Research 73:1–9 (2003) Published 2003 Wiley-Liss, Inc. † This article is a US Government work and, as such, is in the public domain in the United States of America.