**7. Increases in estrogens**

#### **7.1 Pregnancy**

190 Sex Steroids

recognition memory may be more sensitive to ovarian steroids than spatial memory since performance of object recognition was lost faster after Ovx. However, it is important to consider the possible effects of stress on the results because stressing female rats has been shown to enhance object placement (Bowman et al, 2003). Since corticosterone, released during stress, acts within the hippocampus, it is possible that anesthesia-induced stress in

The Ovx and gonadally intact subjects were sacrificed 7 weeks post Ovx and brain morphology was analyzed following Golgi impregnation. Spines were counted on tertiary apical and secondary basal dendrites of pyramidal neurons in layer II/III of the medial prefrontal cortex (PFC) and in CA1 and CA3 of the hippocampus (See Figure 1). In the PFC and CA1 (but not CA3), Ovx females had lower spine density in both apical and basal dendrites than intact rats, ranging from 17% decreases in apical CA1 to 53% decreases in apical PFC. Thus, poorer memory in the Ovx subjects was associated with lower spine densities in the hippocampus and PFC. Unfortunately, behavior could not be directly correlated with spine density as the subjects were not sacrificed immediately following behavior testing. Similar results have been reported recently by Beltran-Campos et al (2011) who found that CA1 apical dendrite spines were 55% lower in Ovx as compared to intact rats. Moreover, the Ovx rats were impaired in acquisition of the platform location in the

Given that gonadal hormones decrease with age, aged rats also provide interesting subjects for assessing relationships between spines and memory. It is well known that aged rats, as well as aged humans, show declines in both learning and memory as compared to young subjects (Frick, 2008). We examined memory function, brain spine densities and estradiol levels in young (four months old) and aged (21 months old) Fischer 344 rats (Wallace et al, 2007; Luine et al, 2011). Fischer 344 rats are maintained by the National Institute for Aging of the National Institutes of Health as a standard model for studies on the physiological and neural aspects of aging. Consistent with many previous studies on spatial memory using tasks like the radial arm maze (Luine and Hearns, 1990) and its water version (Bimonte et al, 2003), Y and T mazes (Aggleton et al, 1980), Barnes maze (Barrett et al, 2009) and the most widely applied spatial memory task, the Morris water maze (Markowska et al, 1999, Veng et al, 2003) the aged females showed poorer object placement performance; they were unable to discriminate between old and new location with 1.5 h inter-trial delay while young rats could discriminate (Luine et al, 2011). Likewise, aged rats are also impaired in visual memory; they could not discriminate between old and new objects at a one hr inter-trial delay (Wallace et al, 2007). Examination of spine densities in the PFC and hippocampus (Table 1) showed that aged rats had 16% decreases, as compared to young rats, in apical dendrites of the PFC and CA1, but no changes in CA3. This decline in densities with aging was smaller than the decline following Ovx. Moreover, the whole pyramidal neuron was affected following Ovx; both apical and basal dendrites were decreased by Ovx, but only apical dendrites were affected with aging. It is notable that the Fischer 344, aged rats (Luine et al, 2011) still had appreciable circulating estradiol levels, 7.9 + 1 pg/ml serum, a level which is comparable to a young rat at diestrus. With further aging, estradiol levels become negligible, but spine densities have not been examined in this age group so it is not known whether spine densities would further decline with aging to levels seen in young Ovx

this study may have masked any early effects of Ovx on object placement.

Morris Water Maze spatial memory task.

**6.2 Declines in estrogens with aging** 

females.

During pregnancy levels of gonadal hormones are elevated so pregnant dams provide an interesting model for further assessing relationships between memory function and dendritic spines. We found that pregnant females on days 7 and 16 of gestation showed better place memory than virgin females (Macbeth et al, 2008). While spine density was not measured in these subjects, there are several reports of alterations in spine density on pyramidal neurons in CA1 and the PFC with pregnancy. Leuner and Gould (2010) demonstrated that pregnant rats had increased dendritic spine density in both apical and basal branches of neurons in CA1 and the medial PFC as well as enhanced cognitive function 20 days after birth when compared to virgin females. However, the effects of pregnancy on

An Integrative Review of Estradiol Effects on Dendritic Spines and Memory over the Lifespan 193

spines was increased by 38%. Combined with other ICC data, it was suggested that estradiol may be facilitating the spine-maturation process. While this could be a species difference, the longer treatment time compared with McLaughlin et al (2008) is consistent with the notion that

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

estradiol, in general, may facilitate the spine maturation process.

consolidation and may be ultimately integrated into memory networks.

supported by NIH grants GM60654 and RR03037 with supplement from NIA.

We thank many students who have assisted with these studies: Luis Jacome, Claris Gautreaux, Tomoko Inagaki, Govini Mohan, Sara Attala, Ana Costa and Leo Arellanos. Experiments were

Aggleton JP, Blindt HS, Candy JM (1989) Working memory in aged rats. Behav Neurosci

Barrett, GL, Bennie A, Trieu J, Ping S, Tsafoulis C (2009). The chronology of age-related

Beltran-Campos, RA Pado-Alcala, U. Leon-Jacinto, A. Aguilar-Vazquez, GL Quirarte, V

spatial learning impairment in two rat strains, as tested by the Barnes maze. 123:

Ramirez-Amaya, S Diaz-Cintra (2011) Increase of mushroom spine density in CA1 apical dendrites produced by water maze training is prevented by ovariectomy.

**8. Conclusions** 

**9. Acknowledgments** 

103: 975-983.

Brain Research 1369: 119-130.

533-8.

**10. References** 

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 therefore the gonadal hormone levels also differ.

#### **7.2 Replacement of Estradiol to Ovx rats**

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 spine density (memory was not assessed).

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 spines was increased by 38%. Combined with other ICC data, it was suggested that estradiol may be facilitating the spine-maturation process. While this could be a species difference, the longer treatment time compared with McLaughlin et al (2008) is consistent with the notion that estradiol, in general, may facilitate the spine maturation process.
