Altered Calmodulin Response to Light in the Suprachiasmatic Nucleus of PAC 1 Receptor Knockout Mice Revealed by Proteomic Analysis Jan Fahrenkrug,* ,1 Jens Hannibal, 1 Bent Honoré, 2 and Henrik Vorum 2 1 Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, DK-2400 Copenhagen NV, Denmark; and 2 Institute of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark Received July 2, 2004; Accepted July 29, 2004 Abstract In mammals circadian rhythms are generated by a light-entrainable oscillator located in the hypothalamic suprachiasmatic nucleus (SCN). Light signals reach the SCN via a monosynaptic neuronal pathway, the retino- hypothalamic tract, originating in a subset of retinal ganglion cells. The nerve terminals of these cells contain the classical neurotransmitter glutamate and the neuropeptide pituitary adenylate cyclase-activating polypep- tide (PACAP), and there is evidence that these two transmitters interact to mediate photoentrainment of the oscillator in the SCN. To elucidate light-provoked PACAPreceptor signaling we used proteomic analysis. Wild- type mice and mice lacking the PAC 1 receptor (PAC 1 –/– ) were light stimulated at early night, and the SCN was examined for proteins that were differentially expressed using two-dimensional gel electrophoresis and iden- tification by tandem mass spectrometry. The most striking finding, which was subsequently confirmed by West- ern blotting, was a significant reduction of calmodulin (CaM) in wild-type mice as compared with PAC 1 –/– mice. Analysis at the mRNA level by quantitative in situ hybridization histochemistry was inconclusive, indicating that a translational mechanism might be involved. The findings indicate that PAC 1 receptor signaling in the SCN in response to light stimulation induces a down-regulation of CaM expression and that CaM is involved in the photic-entrainment mechanism. Index Entries: Calmodulin; two-dimensional gel electrophoresis; knockout; PAC 1 receptor; photic entrain- ment; pituitary adenylate cyclase-activating polypeptide; proteomics; suprachiasmatic nucleus. Introduction In mammals daily physiological and behavioral rhythms are governed by a biological clock located in the suprachiasmatic nucleus (SCN) of the hypo- thalamus (Klein et al., 1991). The molecular machin- ery driving the brain’s biological clock consists of a number of clock genes, which, in double autoregu- latory feedback loops, interact and regulate their own transcription within the individual SCN neurons (Reppert and Weaver, 2002). To maintain synchrony with the solar day/night cycle, the clock is adjusted daily by light, a process called photoentrainment (Roenneberg and Foster, 1997); but the mechanisms by which external light cues modify the activity of the pacemaker in the SCN are not completely under- stood. The major input pathway mediating infor- mation on environmental light and darkness originates in a subpopulation of retinal ganglion cells that project to the SCN via the retinohypothalamic Journal of Molecular Neuroscience Copyright © 2005 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/02/25:251–258/$30.00 Journal of Molecular Neuroscience 251 Volume 25, 2005 *Author to whom all correspondence and reprint request should be addressed. E-mail: bbhjanf@inet.uni2.dk ORIGINAL ARTICLE