**4. Estrogens and spines**

The hippocampal region not only contains a high density of spines, but these spines are plastic, i.e. their numbers fluctuate depending upon the state of the host. A dramatic 30%

An Integrative Review of Estradiol Effects on Dendritic Spines and Memory over the Lifespan 187

2008; Frick, 2008); however, the changes are often small in magnitude. Nonetheless, estradiol administration to Ovx rats or mice has been shown to enhance performance of radial arm maze, T-maze, Morris water maze, and object recognition/placement tasks (see Dohanich, 2002 for review). A critical consideration is that hormones do not usually enhance all aspects of cognition. For example, learning to play a card game like Bridge (*acquisition* of the extensive rules of play) is different from playing the game and remembering which cards have been played (*short-term/working memory*). Thus, acquisition/learning is different than memory, and it appears that estradiol may not enhance both (Luine, 2008). In understanding memory in animal models, it should be noted that the rats face the same issues when they learn a task and then use memory to solve or complete the task. Thus, it is important to consider both learning and memory when examining the influence of gonadal hormones on cognitive function. In this review, we focus on the role of hormone-dependent changes in spines in mediating memory function. Thus, we have applied tasks which utilize memory to female rats, but there are also different types of memory. Spatial memory is the most widely assessed form of memory in rodents, and a variety of tasks have been developed for its measurement, for example, radial arm maze, Morris water maze and object placement. These strongly hippocampal-dependent tasks rely on the innate ability of rodents to know and defend a territory by utilization of salient environmental landmarks to establish a cognitive map which resides within hippocampal neurons or networks. Thus, if the hippocampus is ablated, the rats can no longer perform spatial tasks (Broadbent et al, 2004). We have applied the object placement task because this task, unlike other tasks, requires minimal learning and it also does not entail use of positive (food) or negative (drowning) rewards which might inadvertently influence performance through stress or other influences. Object placement relies on the observation that rats seek novelty and readily explore their environment. When a delay is interspersed between presentation of objects in a new as compared to an old location, then memory can be assessed by determining whether the subjects spend more time exploring the object in the new location than in the old location (Ennaceur et al, 1997). The task is conducted as depicted in Figure 4. Rats spend three minutes exploring two identical objects on an open field in the sample trial (T1). After inter-trial delays of 1 to 4 h, the subject is returned to the field where one object has been moved to a different location (retention trial or T2). The time spent exploring the object in the old and in the new location is noted. Spending significantly more time exploring at the new vs. the old location indicates that the rat remembers the old location and hence explores at the novel location, i.e. significantly discriminates between locations. Object placement memory can also be reported using the exploration ratio (time exploring new place/time exploring old + time exploring new place) where ratios of 0.5 indicate chance performance (poor memory) and ratios higher than 0.5 indicate that subjects remember the old location and significantly discriminate between the locations. This task can also be configured as a visual memory task which relies on visual associations by the prefrontal cortex as well as integration of the PFC with hippocampal memory circuits (Broadbent et al, 2004; Ennaceur et al, 1997). See Figure 5. The sample trial is the same as in object placement, but in the retention trial, a new object is switched for one of the two identical objects. Scoring in the retention trial is as in object placement. Estradiol, given to Ovx rats or mice, has been shown to enhance memory in these tasks by a number of laboratories (Luine et al, 2003; Li et al,

2004; Walf et al, 2006, 2007; Frye et al, 2007; Scharfman et al, 2007; Jacome et al, 2010).

The axon terminal contains vesicles which release their contents , then bind to the post synaptic receptor and then initiate a series of events that induce structural changes in the dendritic spine. Drawn by Dr. A. Bornstein

Fig. 3. Schematic representation of the events that occur at the axo-spinous synapse.

change in spine density has been shown in the hippocampus during the 4-5 day estrus cycle of the female rat in concert with the changes in estradiol and progesterone (Woolley, et al, 1990). These changes occur in the CA1, but not the CA3 or dentate gyrus region of the hippocampus and allow for changes in neural traffic through frontal cortex and the hippocampus itself. Ovariectomy (Ovx) is associated with decreased spine density in CA1 (Gould et al. 1990, Wallace et al, 2006), and it has been recently demonstrated that the PFC also undergoes spine (Wallace et al, 2006) and spine synapse (Leranth et al, 2003) loss following gonadectomy. Since memory function undergoes similar changes with alterations in gonadal hormones (see Estrogens and memory below), dendritic spines in CA1 and the medial PFC may be important in mediating gonadal hormone influences on cognitive function in females.

#### **5. Estrogens and memory**

A substantial literature has demonstrated that gonadal hormones, mainly estradiol, influence cognition function during development, at adulthood and during aging (Luine,

The axon terminal contains vesicles which release their contents , then bind to the post synaptic receptor and then initiate a series of events that induce structural changes in the dendritic spine. Drawn by Dr.

change in spine density has been shown in the hippocampus during the 4-5 day estrus cycle of the female rat in concert with the changes in estradiol and progesterone (Woolley, et al, 1990). These changes occur in the CA1, but not the CA3 or dentate gyrus region of the hippocampus and allow for changes in neural traffic through frontal cortex and the hippocampus itself. Ovariectomy (Ovx) is associated with decreased spine density in CA1 (Gould et al. 1990, Wallace et al, 2006), and it has been recently demonstrated that the PFC also undergoes spine (Wallace et al, 2006) and spine synapse (Leranth et al, 2003) loss following gonadectomy. Since memory function undergoes similar changes with alterations in gonadal hormones (see Estrogens and memory below), dendritic spines in CA1 and the medial PFC may be important in mediating gonadal hormone influences on cognitive

A substantial literature has demonstrated that gonadal hormones, mainly estradiol, influence cognition function during development, at adulthood and during aging (Luine,

Fig. 3. Schematic representation of the events that occur at the axo-spinous synapse.

A. Bornstein

function in females.

**5. Estrogens and memory** 

administration to Ovx rats or mice has been shown to enhance performance of radial arm maze, T-maze, Morris water maze, and object recognition/placement tasks (see Dohanich, 2002 for review). A critical consideration is that hormones do not usually enhance all aspects of cognition. For example, learning to play a card game like Bridge (*acquisition* of the extensive rules of play) is different from playing the game and remembering which cards have been played (*short-term/working memory*). Thus, acquisition/learning is different than memory, and it appears that estradiol may not enhance both (Luine, 2008). In understanding memory in animal models, it should be noted that the rats face the same issues when they learn a task and then use memory to solve or complete the task. Thus, it is important to consider both learning and memory when examining the influence of gonadal hormones on cognitive function. In this review, we focus on the role of hormone-dependent changes in spines in mediating memory function. Thus, we have applied tasks which utilize memory to female rats, but there are also different types of memory. Spatial memory is the most widely assessed form of memory in rodents, and a variety of tasks have been developed for its measurement, for example, radial arm maze, Morris water maze and object placement. These strongly hippocampal-dependent tasks rely on the innate ability of rodents to know and defend a territory by utilization of salient environmental landmarks to establish a cognitive map which resides within hippocampal neurons or networks. Thus, if the hippocampus is ablated, the rats can no longer perform spatial tasks (Broadbent et al, 2004). We have applied the object placement task because this task, unlike other tasks, requires minimal learning and it also does not entail use of positive (food) or negative (drowning) rewards which might inadvertently influence performance through stress or other influences. Object placement relies on the observation that rats seek novelty and readily explore their environment. When a delay is interspersed between presentation of objects in a new as compared to an old location, then memory can be assessed by determining whether the subjects spend more time exploring the object in the new location than in the old location (Ennaceur et al, 1997). The task is conducted as depicted in Figure 4. Rats spend three minutes exploring two identical objects on an open field in the sample trial (T1). After inter-trial delays of 1 to 4 h, the subject is returned to the field where one object has been moved to a different location (retention trial or T2). The time spent exploring the object in the old and in the new location is noted. Spending significantly more time exploring at the new vs. the old location indicates that the rat remembers the old location and hence explores at the novel location, i.e. significantly discriminates between locations. Object placement memory can also be reported using the exploration ratio (time exploring new place/time exploring old + time exploring new place) where ratios of 0.5 indicate chance performance (poor memory) and ratios higher than 0.5 indicate that subjects remember the old location and significantly discriminate between the locations. This task can also be configured as a visual memory task which relies on visual associations by the prefrontal cortex as well as integration of the PFC with hippocampal memory circuits (Broadbent et al, 2004; Ennaceur et al, 1997). See Figure 5. The sample trial is the same as in object placement, but in the retention trial, a new object is switched for one of the two identical objects. Scoring in the retention trial is as in object placement. Estradiol, given to Ovx rats or mice, has been shown to enhance memory in these tasks by a number of laboratories (Luine et al, 2003; Li et al, 2004; Walf et al, 2006, 2007; Frye et al, 2007; Scharfman et al, 2007; Jacome et al, 2010).

An Integrative Review of Estradiol Effects on Dendritic Spines and Memory over the Lifespan 189

As indicated earlier, some cognitive functions appear optimal in the presence of circulating gonadal hormones, but whether maintenance of dendritic spines at critical levels contributes to gonadal hormone influences on cognition has not received extensive investigation; however, we assessed the effects of gonadectomy on recognition memory (object recognition) and spatial memory (object placement) and spine density in the medial prefrontal cortex (PFC) and in hippocampal sub-regions (Wallace et al, 2006). Prior to Ovx, rats could significantly discriminate between old and new objects and between objects in new and old locations when tested 4 hours after first viewing the objects. One week following surgery, Ovx females showed impaired object recognition memory because they could not discriminate between old and new objects (ratio less than 50%) while gonadally intact females still discriminated in the task (Wallace et al, 2006). At this time interval post Ovx, place memory showed different results because both gonadally intact and Ovx rats could significantly discriminate between objects in new and old locations (ratios of approximately 0.65), however by four weeks post-Ovx, rats could not significantly discriminate locations (See Figure 6). These results suggest that gonadal hormones contribute to the performance of the memory tasks. Moreover,

The exploration ratio + SEM is shown for gonadally intact, sham (solid circles) and Ovx (open circles) rats before Ovx (0 week) and weeks 1-7 post Ovx. Dashed line at 0.5 indicates chance performance of task (same amount of time spent exploring the object at the old and new locations). Two way ANOVA (group x week) of ratios showed no significant main effects but a significant group x week interaction (F(1,16)=2.22, p<0.047). Post hoc testing by t-tests showed significant differences between groups at

weeks 4, 5, 6 and 7, by at least p < 0.02. Adapted from Wallace et al. (2006) Fig. 6. The effect of ovariectomy on object placement memory task.

**6. Estrogens, memory and spines 6.1 Declines in estrogen following Ovx** 

The rat is shown on an open field with objects to be explored.

Fig. 4. Depiction of the object placement memory task.

The rat is shown on an open field with objects to be explored.

Fig. 5. Depiction of the object recognition memory task.

#### **6. Estrogens, memory and spines**

188 Sex Steroids

The rat is shown on an open field with objects to be explored. Fig. 4. Depiction of the object placement memory task.

The rat is shown on an open field with objects to be explored. Fig. 5. Depiction of the object recognition memory task.

#### **6.1 Declines in estrogen following Ovx**

As indicated earlier, some cognitive functions appear optimal in the presence of circulating gonadal hormones, but whether maintenance of dendritic spines at critical levels contributes to gonadal hormone influences on cognition has not received extensive investigation; however, we assessed the effects of gonadectomy on recognition memory (object recognition) and spatial memory (object placement) and spine density in the medial prefrontal cortex (PFC) and in hippocampal sub-regions (Wallace et al, 2006). Prior to Ovx, rats could significantly discriminate between old and new objects and between objects in new and old locations when tested 4 hours after first viewing the objects. One week following surgery, Ovx females showed impaired object recognition memory because they could not discriminate between old and new objects (ratio less than 50%) while gonadally intact females still discriminated in the task (Wallace et al, 2006). At this time interval post Ovx, place memory showed different results because both gonadally intact and Ovx rats could significantly discriminate between objects in new and old locations (ratios of approximately 0.65), however by four weeks post-Ovx, rats could not significantly discriminate locations (See Figure 6). These results suggest that gonadal hormones contribute to the performance of the memory tasks. Moreover,

The exploration ratio + SEM is shown for gonadally intact, sham (solid circles) and Ovx (open circles) rats before Ovx (0 week) and weeks 1-7 post Ovx. Dashed line at 0.5 indicates chance performance of task (same amount of time spent exploring the object at the old and new locations). Two way ANOVA (group x week) of ratios showed no significant main effects but a significant group x week interaction (F(1,16)=2.22, p<0.047). Post hoc testing by t-tests showed significant differences between groups at weeks 4, 5, 6 and 7, by at least p < 0.02. Adapted from Wallace et al. (2006)

Fig. 6. The effect of ovariectomy on object placement memory task.

An Integrative Review of Estradiol Effects on Dendritic Spines and Memory over the Lifespan 191

**Group PFC Apical PFC Basal CA1 Apical CA1 Basal CA3 Apical CA3 Basal**  Young 8.13 + 0.40 7.80 + 0.41 7.87 + 0.58 5.37 + 0.21 5.42 + 0.36 5.52 + 0.29 Aged 6.86 + 0.52\* 7.19 + 0.31 6.59 + 0.39\* 5.96 + 0.15 6.30 + 0.39 4.79 + 0.36 Entries are mean number of spines/10µm + S.E.M. for 6-8 rats/group. Young is 4 months and aged is 21

Von Bohlen et al (2006) also reported decreased CA1 spine density in a group of aged rats of both sexes that had impaired Morris water maze performance. However, our data is different from the only other published study in females. Markham et al. (2005), who applied Golgi techniques, reported no changes in CA1 spine density in rats of a similar age as ours (19-22 mo.) but of a different strain, Long Evans. Nonetheless, it is notable that Markham and Juraska (2002) reported decreased spine density in the PFC of their aged females. In neither of these studies was memory assessed. An important variable which may have contributed to our demonstration of decreased hippocampal spine density with aging is the reproductive history of the females utilized. The Fischer 344 female rats supplied to us by N.I.A. were virgins. In the other studies (Markham and Juraska, 2002; Juraska et al, 2005) retired Long-Evans breeding dams were used. As discussed in detail below, rats that have been pregnant and reared pups (multiparous) generally have better memory abilities than female rats that have never experienced motherhood (virgin or nulliparous; see Macbeth and Luine, 2010 for review). For example, middle-aged rats (12 months old) that have had 4- 5 pregnancies and births demonstrated better object placement performance and other memory tasks than age-matched, virgin rats (Macbeth et al., 2008). Reproductive experience also apparently imparts long lasting effects on memory processes because multiparous females show better spatial memory than age-matched virgins at 24 months of age (Kinsley, 2008; Macbeth and Luine, 2010). Moreover, pregnant or lactating females have greater spine density in CA1 than females at all stages of the estrus cycle (Kinsley, et al, 2006; Kinsley 2008). Whether pregnancy-related spine changes are as enduring as the memory changes is, however, unknown, and needs to be investigated. Thus, the reproductive history of female rats may be an important variable when investigating neural and behavioral function. Overall, our studies indicate that aging in females is accompanied by losses in memory abilities, neural spines and lower estradiol levels. Whether there is a causal relationship

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

months old. \* p < .05 by Student's T-test. Data from Wallace et al, 2007 and Luine et al, 2011.

Table 1. Spine density in young vs aged Fischer 344 rats

among the variables needs further investigation.

**7. Increases in estrogens** 

**7.1 Pregnancy** 

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

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 Morris Water Maze spatial memory task.

#### **6.2 Declines in estrogens with aging**

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 females.


Entries are mean number of spines/10µm + S.E.M. for 6-8 rats/group. Young is 4 months and aged is 21 months old. \* p < .05 by Student's T-test. Data from Wallace et al, 2007 and Luine et al, 2011.

Table 1. Spine density in young vs aged Fischer 344 rats

Von Bohlen et al (2006) also reported decreased CA1 spine density in a group of aged rats of both sexes that had impaired Morris water maze performance. However, our data is different from the only other published study in females. Markham et al. (2005), who applied Golgi techniques, reported no changes in CA1 spine density in rats of a similar age as ours (19-22 mo.) but of a different strain, Long Evans. Nonetheless, it is notable that Markham and Juraska (2002) reported decreased spine density in the PFC of their aged females. In neither of these studies was memory assessed. An important variable which may have contributed to our demonstration of decreased hippocampal spine density with aging is the reproductive history of the females utilized. The Fischer 344 female rats supplied to us by N.I.A. were virgins. In the other studies (Markham and Juraska, 2002; Juraska et al, 2005) retired Long-Evans breeding dams were used. As discussed in detail below, rats that have been pregnant and reared pups (multiparous) generally have better memory abilities than female rats that have never experienced motherhood (virgin or nulliparous; see Macbeth and Luine, 2010 for review). For example, middle-aged rats (12 months old) that have had 4- 5 pregnancies and births demonstrated better object placement performance and other memory tasks than age-matched, virgin rats (Macbeth et al., 2008). Reproductive experience also apparently imparts long lasting effects on memory processes because multiparous females show better spatial memory than age-matched virgins at 24 months of age (Kinsley, 2008; Macbeth and Luine, 2010). Moreover, pregnant or lactating females have greater spine density in CA1 than females at all stages of the estrus cycle (Kinsley, et al, 2006; Kinsley 2008). Whether pregnancy-related spine changes are as enduring as the memory changes is, however, unknown, and needs to be investigated. Thus, the reproductive history of female rats may be an important variable when investigating neural and behavioral function.

Overall, our studies indicate that aging in females is accompanied by losses in memory abilities, neural spines and lower estradiol levels. Whether there is a causal relationship among the variables needs further investigation.
