Variations of Synaptotagmin I, Synaptotagmin IV, and Synaptophysin mRNA Levels in Rat Hippocampus during the Estrous Cycle Marianna Crispino, 1 David J. Stone, 1 Min Wei, Christopher P.Anderson, Georges Tocco, Caleb E. Finch, and Michel Baudry Neuroscience Program, University of Southern California, Los Angeles, California 90089-2520 Received February 1, 1999; accepted July 2, 1999 Periodic changes in ovarian steroid levels during fertility cycles affect learning both in humans and in rats in parallel with electrophysiological and morpho- logical fluctuations in selective neuronal populations. In particular, during the estrous cycle of the female rat, hippocampal CA1 region undergoes cyclic modifi- cations in synaptic density. To investigate the molecu- lar mechanisms involved in synaptic remodeling dur- ing the estrous cycle, we analyzed the expression of three presynaptic markers, synaptotagmin I, synapto- tagmin IV, and synaptophysin, in the female adult rat brain by in situ hybridization. Relative abundance in mRNA for these three markers was quantified at four phases of the estrous cycle: diestrus, proestrus (AM and PM), and estrus. mRNA levels for syt1 exhibited cyclic variations in pyramidal neurons of the CA3 region of hippocampus during the estrous cycle, while mRNA levels for syt4 and SYN were relatively invari- ant in this or other regions of the hippocampus. Be- cause CA3 pyramidal neurons make synaptic contacts in CA1, modulation of syt1 expression in CA3 may participate in the changes in synaptic density ob- served in CA1 during the estrous cycle. Furthermore, both syt1 and SYN mRNA varied cyclically in layer II, but not in layer III of entorhinal cortex, while syt4 remained unchanged throughout the cycle. These data suggest that regular variations in steroid hormone levels during fertility cycles may alter the properties of several networks involved in information process- ing and learning and memory through altered levels of presynaptic proteins.  1999 Academic Press Key Words: synaptotagmin; synaptophysin; estrous cycle; mRNA; in situ hybridization. INTRODUCTION Neuronal circuits undergo constant remodeling in adult brains under normal physiological conditions, as well as in response to challenges from pathological lesions (9, 23). Substantial evidence shows that struc- tural modifications in dendrites and in synaptic connec- tivity of adult neurons are fundamental to ongoing reorganization of neuronal networks (31, 38, 63). Dur- ing the 4- to 5-day estrous cycle of female rats (45), a cyclic pattern of synaptic remodeling occurs in the hypothalamic arcuate nucleus. In this key neuroendo- crine locus, the number of synapses per length of perikaryal membrane transiently decreases; e.g., the number of axosomatic synapses is reduced by 30% within the 24-h period between proestrus and estrus, followed by return of synaptic density to proestrus level within the next 24 h (36). Synaptic changes in the arcuate nucleus are dependent on the preovulatory estradiol surge, as immunoneutralization of circulating estradiol with a specific monoclonal antibody abolished synaptic variations (32). The rat hippocampus also shows physiological cycles of synaptic reorganization and plasticity across the estrus cycle (30, 65): the density of dendritic spines in CA1 increases during diestrus, with a peak at proes- trus when estradiol is highest, and declines during estrus when estradiol decreases and progesterone is elevated (65, 66). Other in vivo experiments with ovariectomized rats showed direct effects of estradiol on dendritic spine density and the importance of progester- one in the precipitant decrease that follows proestrus (16). Moreover, the effect of estradiol on dendritic spines required the activation of NMDA receptors (67). Furthermore, changes in density of dendritic spines on CA1 neurons reflected a parallel cyclic fluctuation of synaptic connectivity between CA1 pyramidal cells and their afferents. Thus, increased spine numbers were accompanied with increased number of spine synapses (66). This finding indicates that estradiol coordinates the regulation of genes coding for proteins participating 1 Both authors contributed equally and share first authorship. 2 Abbreviations used: syt1, synaptotagmin I; syt4, synaptotagmin IV; SYN, synaptophysin; LH, luteinizing hormone; SSC, sodium chloride/sodium citrate; PBS, phosphate-buffered saline. Experimental Neurology 159, 574–583 (1999) Article ID exnr.1999.7186, available online at http://www.idealibrary.com on 574 0014-4886/99 $30.00 Copyright  1999 by Academic Press All rights of reproduction in any form reserved.