**8. Conclusions**

192 Sex Steroids

spine density in CA1 have been determined in several studies with variable results. In a recent study, we found that dendritic spine density was decreased on the apical branch of CA1 neurons on the day of birth in dams when compared with the virgin females (Frankfurt et al, 2011) whereas Kinsley et al. (2006) demonstrated that dendritic spine density on the apical branch of CA1 neurons was greatest in late pregnancy and during lactation (day 5) when compared with virgin rats in different stages of estrous. Brusco et al. (2008) demonstrated that, starting at day four postpartum, there were no differences in either spine density or spine type in CA1 between postpartum and virgin Wistar rats. The differences between these studies may be attributed to the fact that the animals were examined at different postpartum times and

As indicated earlier, Estradiol Benzoate (EB) treatment for two days increases CA1 apical spine density (Gould et al, 1990,), and estrogens and other gonadal hormones regulate the density of synapses on these CA1 spines (Parducz et al, 2006; MacLusky et al, 2005); however, neither learning nor memory was assessed in these studies. Conrad and colleagues (McLaughlin et al, 2008) examined the of effects two doses of EB, 5 and 10 ug, given twice to Ovx rats on object placement and other cognitive tasks as well as spine densities. The higher doses resulted in significant discriminations in object placement and a doubling of spine density in the apical dendrites of CA1 (but not in the basal dendrites). Interestingly, if rats were Ovx for ten weeks without any hormonal replacement, then estradiol did not alter

We have reported that Ovx females chronically fed regular rat chow (Purina LabDiet), which contains high levels of a variety of phytoestrogens, have better memory function and greater dendritic spine density in some brain areas than Ovx rats fed chow low in phytoestrogens (Teklad 2016) (Luine et al, 2006). Phytoestrogens are plant derived estrogens which have a much lower affinity for the estrogen receptor than estradiol but nonetheless exert some estrogenic effects. Following 7 weeks on the diets, the high phytoestrogen diet group significantly discriminated between objects at old and new locations while the Ovx rats fed the low phytoestrogen diet could not. Interestingly, Ovx rats fed either diet could not significantly discriminate between old and new objects after 6, 8 or 9 weeks on the diets. Thus, phytoestrogens were insufficient to enhance object recognition memory which again suggests that behaviors mediated by the medial prefrontal cortex maybe very sensitive to losses in circulating estrogens. Apical spine density was assessed in pyramidal cells in CA1 and the PFC, areas where we previously saw differences between Ovx and gonadally intact rats. Spine density of the low phytoestrogen diet group was 32% lower in CA1 and 21% lower in the PFC than the high phytoestrogen group. Comparison of the two experiments utilizing Ovx rats (Ovx vs. intact rats; low vs. high phytoestrogen diet in Ovx rats) suggests that a reduction of 20-30% in CA1 apical spines is sufficient to impact spatial memory function but that larger declines are necessary in PFC in order to affect recognition memory. However, the relationship between spine density and memory may not be direct since spines were

decreased in the low phytoestrogen diet group but object recognition memory was not.

Working with groups at Rockefeller University (Li et al, 2003), we found a somewhat different pattern of estrogen treatment in mice vs. rats. Ovx mice received 1 ug of EB daily for 5 days and then received object placement testing. EB treatment enhanced object placement, but the density of spines in apical CA1 was not increased. Interestingly, the density of mushroom

therefore the gonadal hormone levels also differ.

**7.2 Replacement of Estradiol to Ovx rats** 

spine density (memory was not assessed).

The data presented here show that estrogen effects on memory are associated with dendritic remodeling in the hippocampus and PFC. It is impossible, at present, to state conclusively which dendritic spines are involved in mediating a given function because most of the studies done to date include confounding variables. For example, some evidence suggests that learning (Beltran-Campos, 2011) or forming associative memories (Leuner and Shors 2003) increases spine density in CA1, but Beltran-Campos, in the same study, found that spines were not increased by training in Ovx rats, only in gonadally intact rats. Moreover, Frick et al (2004) reported that behavioral training in the water maze interfered with the ability of estradiol to increase CA1 spine synapse density in Ovx rats. While an influence of the stress of swimming cannot be discounted, the above studies indicate a complex interaction between hormones, memory and spines. Since there is some evidence of different hormonal or learning effects on different spines, counting of mushroom, thin and filapodial spines might be informative for future studies. Also critical is the comparison of behaviorally tested vs. non-behaviorally tested subjects and how long the subjects have been without circulating estrogens as well as how long after behavioral testing spine density is analyzed. Nonetheless, estrogenic facilitations of memory functions regulated by the hippocampus and prefrontal cortex provide a rich context in which to examine the mechanisms underlying memory consolidation and retrieval. Using estradiol as a physiological probe, it should be possible to identify the intracellular signals whereby spines are generated, strengthened or shed, and how newly generated spines promote memory consolidation and may be ultimately integrated into memory networks.
