PHOSPHORYLATION OF Ca 2 /CALMODULIN-DEPENDENT PROTEIN KINASE TYPE II AND THE -AMINO-3-HYDROXY-5-METHYL-4- ISOXAZOLE PROPIONATE (AMPA) RECEPTOR IN RESPONSE TO A THREONINE-DEVOID DIET J. W. SHARP, a * C. M. ROSS, a T. J. KOEHNLE b AND D. W. GIETZEN a a Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, 1 Shields Avenue, Davis, CA 95616, USA b Department of Neuroscience, University of Pittsburgh, 446 Crawford Hall, Pittsburgh, PA 15260, USA Abstract—The anterior piriform cortex (APC) functions as a chemosensor for indispensable amino acid deficiency and re- sponds to this deficiency with increased activity, as indicated by observations including averaged evoked-potentials and c- fos expression in the APC. Little is known of the intracellular signaling mechanisms that mediate this deficiency-related in- crease in neuronal excitability, but previous studies have shown effects on intracellular Ca 2 in deficient APC slices in vitro. In the present study we hypothesized that indispens- able amino acid deficiency increases intraneuronal Ca 2 , re- sulting in autophosphorylation of calcium/calmodulin- dependent protein kinase type II (CaMKII) in vivo. Results demonstrated that phosphorylation levels of CaMKII (pCaMKII) in APC neurons increased at 20 and 40 min after a single meal of threonine-devoid diet. Phosphorylation of the -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit (GluR1) at the serine 831 (S831) site was modestly increased in the APC in response to a threonine- devoid meal. The GluR1 subunit also showed increased phosphorylation at the 845 (S845) site, suggesting additional signaling mechanisms. Although phosphorylation of CaMKII was sustained, phosphorylation of the GluR1 subunit re- turned to control levels by 40 min. These effects of amino acid deficiency did not occur throughout the brain as neither CaMKII nor GluR1 showed increased phosphorylation in the neocortex. These findings support the notion that calcium and gluta- mate signaling in the APC, but not throughout the brain, are triggered during early responses to amino acid deficiency. They also suggest that longer-term changes in APC neurons in response to such a deficiency may be mediated at least in part by CaMKII. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: indispensable amino acids, signal transduction, AMPA, anterior piriform cortex, CaMKII, nutrient deficiency. Rats detect dietary deficiencies of indispensable amino acids (IAAs) within 20 min and change their feeding be- havior by reducing food intake (Gietzen et al., 1986; Koehnle et al., 2003). Subsequently, they develop a learned aversion to the deficient diet (reviewed in Gietzen, 1993). Evidence suggests that the primary chemosensory area for detection of IAA deficiency lies within the CNS, specifically within the anterior piriform cortex (APC; Leung and Rogers, 1971; Beverly et al., 1990; Gietzen, 1993; Russell et al., 2003). The sensing of IAA deficiency is post- absorptive and does not depend on olfaction or taste (Harper et al., 1970; Gietzen, 1993; Koehnle et al., 2003). Yet, little is known of the intracellular signaling mecha- nisms that are evoked in response to IAA deficiency or of the neuronal mechanisms that result in altered feeding behavior. Previously, we have demonstrated increased phosphorylation of mitogen-activated protein kinase (MAP kinase) in APC neurons and increased neuronal Ca 2+ influx in the APC in response to changes of IAA concen- trations (Magrum et al., 1999; Sharp et al., 2002). In- creased duration and frequency of calcium influx pulses have been shown to initiate calcium/calmodulin-dependent protein kinase type II (CaMKII) phosphorylation (De Kon- inck and Schulman, 1998). Following calcium influx and binding to calcium-calmodulin, CaMKII is activated via au- tophosphorylation (De Koninck and Schulman, 1998; Han- son and Schulman, 1992; Meyer et al., 1992). CaMKII is a protein kinase that is highly concentrated in the brain and consists of  (52 kDa) and  (60 kDa) subunits coded for by  and  genes, with the  subunit being most abundant (Brocke et al., 1999); the  subunit phosphorylated at Threonine286 has been shown to be crucial for learning (Giese et al., 1998). CaMKII is present in neuronal cyto- plasm and is associated with post-synaptic neuronal den- sities. It comprises as much as 1% of total forebrain protein (Kelly, 1991; Murray et al., 1995). Many studies show that MAP kinase and CaMKII play key roles in the genesis and maintenance of the long-term neuronal potentiation (LTP) learning model (Barria et al., 1997; Caputi et al., 1999; Derkach et al., 1999; Fukunaga *Corresponding author. Tel: +1-530-752-8073. E-mail address: jwsharp@ucdavis.edu (J. W. Sharp). Abbreviations: ABC, avidin– biotin-complex; AMPA, -amino-3- hydroxy-5-methyl-4-isoxazole propionate; APC, anterior piriform cortex; APC lot , area of APC medial to lateral olfactory tract; APC vr , ventral–rostral area of the APC; BTD, basal threonine-devoid; CaMKII, calcium/calmodulin-dependent protein kinase II; CaMKII,  subunit of CaMKII; DAB, diaminobenzidine; GluR1, subunit of the AMPA receptor; GS-PBS, goat serum dissolved in PBS; HRP, horseradish peroxidase; IAA, indispensable amino acid; IgG, immunoglobulin G; LTP, long-term potentiation; MAP kinase, mitogen-activated protein kinase; pAb, primary antibody; PBS, phosphate buffered saline; pCaMKII, phosphorylated calcium/calmodulin-dependent protein ki- nase II; p-CaMKII, phosphorylated CaMKII; PFA-PBS, paraformal- dehyde dissolved in PBS; PKA, cAMP-dependent protein kinase A; PVDF, polyvinylidene difluoride; S831, serine 831; S845, serine 845; TBS-T, Tris-buffered saline with 0.1% Tween-20. Neuroscience 126 (2004) 1053–1062 0306-4522/04$30.00+0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.03.066 1053