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. Author manuscript; available in PMC: 2009 Nov 19.
Published in final edited form as: Brain Res. 2008 Sep 13;1241:136–147. doi: 10.1016/j.brainres.2008.09.006

Pregnant rats show enhanced spatial memory, decreased anxiety, and altered levels of monoaminergic neurotransmitters

AH Macbeth a,b,*,1, C Gautreaux a, VN Luine a,b,*
PMCID: PMC2652572  NIHMSID: NIHMS82922  PMID: 18823955

Abstract

Spatial memory, anxiety and central monoaminergic activities were measured in non-pregnant (NP) and pregnant females during two time periods of pregnancy: gestational day 7–9 (GD7, GD9) & gestation day 16–18 (GD16, GD18). Pregnant females discriminated between object locations on both test days on an object placement task, whereas NP females were unable to discriminate between locations. Pregnant females displayed decreased anxiety on the elevated plus maze on GD9 compared to NP females, followed by increased anxiety-like behavior on the elevated plus maze on GD18. Monoamine levels and activity (as indexed by turnover ratio) were measured in prefrontal cortex (PFC), CA1 and CA3 regions of the hippocampus (areas important for memory), and medial preoptic area (mPOA, an area important in display of maternal behaviors). In the PFC, NP females generally had higher monoamine levels and turnover ratios; however, norepinephrine (NE) turnover was higher in pregnant females at GD18. In the CA1 and CA3 regions of the hippocampus, monoamine levels and turnover ratios were generally higher during pregnancy, particularly on GD9. In the mPOA, pregnancy was associated with increases in NE activity, a previously unreported finding. The present study expands upon existing research indicating that pregnancy is beneficial to spatial memory and may decrease anxiety. Changes in monoamine levels and activity in specific brain regions indicate that the dopamine, norepinephrine and serotonin systems may contribute to the observed behavioral differences.

Keywords: pregnancy, spatial memory, anxiety, monoamines

I. Introduction

Existing research into behavioral changes in new dams during pregnancy and lactation has focused primarily on the development and display of maternal behaviors (i.e. licking, grooming, and pup retrieval), which seem to arise from the hormonal changes occurring during pregnancy and postpartum periods (Rosenblatt et al., 1988), as well as from experience with pups (Fleming and Sarker, 1990). However, pregnancy and reproductive experience are also associated with “non-maternal” behavioral alterations that may also be essential for offspring care (Kinsley et al., 1999). Specifically, reproductive experience has been shown to 1) decrease the behavioral and neural responses typically activated by stressful or anxiety-provoking stimuli; 2) alter hippocampal morphology and spatial memory; and 3) affect monoaminergic neurotransmitter concentrations throughout the brain (see details and references below).

In studies reported to date, anxiety-like behavior is decreased in pregnant and parous (given birth and lactated) females in the open field (Wartella et al., 2003) and on the elevated plus maze (Neumann, 2001; Lonstein, 2005; Byrnes & Bridges, 2006; de Brito Faturi et al., 2006). Additionally, late pregnant, lactating, and parous females express significantly less c-fos mRNA in the hypothalamus, medial amygdala and lateral septum after restraint stress in comparison to nulliparous females (da Costa et al., 1996; Wartella et al., 2003), providing neurochemical support to the behavioral results suggesting dampened stress/anxiety responses due to pregnancy. Evolutionarily speaking, resistance to stressful and anxiety-provoking stimuli may be necessary to accept and care for formerly fear-inducing pups (Fleming & Luebke, 1981).

In the hippocampus, a brain region particularly important for spatial memory (Potvin et al., 2006), morphological changes occur during pregnancy, the postpartum period, and after weaning. Specifically, late pregnant and lactating rats have elevated dendritic spine density on CA1 pyramidal cells as compared to nulliparous females, and are likely due to an altered hormonal milieu (Kinsley et al., 2006). Furthermore, elevated dendritic spine density on CA1 pyramidal cells is maintained after pups have been weaned and persists through the second litter (Pawluski & Galea, 2006). However, prior to weaning primiparous females (first litter) show decreased cell proliferation in the dentate gyrus (Pawluski & Galea, 2007), indicating that hippocampal morphological changes may not be specific to the CA1 region, and that pregnancy does not always result in elevated dendritic spine density (Pawluski & Galea, 2007).

In accord with the morphological data, pregnancy enhances spatial memory. Rats between days 7–10 of pregnancy have shorter path lengths and latencies to escape on the Morris water maze than non-pregnant females (Galea et al., 2000). Pregnant females are also better able to learn the new location of the hidden platform of the Morris water maze when moved compared to virgin females (Bodensteiner et al., 2006). First pregnancy and mothering in particular seem to enhance spatial memory, as pregnant-only and primiparous females make significantly fewer working memory or reference memory errors on the radial arm maze than nulliparous or multiparous females (Pawluski et al., 2006a; Pawluski et al., 2006b).

Reproductive experience also alters monoamine neurotransmitter concentrations. Dopamine (DA), norepinephrine (NE) and serotonin (5HT) are altered in the mPOA during the lactation period, which may aid in subsequent display of maternal behaviors (Lonstein et al., 2003). Levels and activity of NE and its metabolite MHPG are elevated in the hippocampus during late pregnancy (Desan et al., 1988; Glaser et al., 1992); as NE modulates vigilance, attention and memory (Lapiz & Morilak, 2006), elevated levels during late pregnancy and parturition may enhance the female’s ability to pay attention to her offspring. NE and MHPG may also act to reduce the responsiveness of the hypothalamic-pituitary-adrenal axis to stress during late pregnancy and parturition in both rats (Douglas et al., 2005) and humans (Altemus et al., 2004; Teixeira et al., 2005). Thus, monoaminergic neurotransmitters, particularly NE and its metabolite, seem important for attention, memory, and reduced stress response.

We report here on cognitive functioning and monoamine levels and activity during pregnancy. Non-pregnant and pregnant females were assessed for 1) spatial memory in the object placement (OP) task; 2) anxiety as measured on the elevated plus maze (EPM); and 3) levels of monoaminergic neurotransmitters in three brain regions implicated in memory (CA1 and CA3 regions of the hippocampus, and pre-frontal cortex: PFC)) and the mPOA, which is implicated in maternal behavior.

2. Results

2.1. Effect of pregnancy on object placement

OP performance was assessed twice during pregnancy in virgin, non-pregnant (NP) and pregnant females: on gestation day 7 (GD7) and gestation day 16 (GD16), with a 2-h inter-trial delay on both days. Figure 1A shows data from the sample trial (T1). There was no significant effect of group, day, or group x day interaction (p > 0.05 for all) indicating equal overall exploration between NP and pregnant females on both test days. Figure 1B shows data from the retention trial (T2). There was a significant main effect of group: F1,24 = 5.28, p < 0.05, but no effect of day or group x day interaction. On both test days, pregnant females had significantly higher exploration ratios (GD7: 0.69, GD16: 0.64) than NP females (GD7: 0.50, GD16: 0.55), whose performance did not differ from chance (0.50: equal exploration of both familiar and novel object locations).

Figure 1. Effect of pregnancy on object placement.

Figure 1

(A) Mean exploration times (± SEM) during the sample trial (T1) are shown for NP (□ n = 6) and pregnant (■ n = 8) females. Data were analyzed by two-way ANOVA (group x day). Groups did not differ in overall object exploration. (B) Mean exploration ratios (± SEM) during the retention trial (T2). The exploration ratio is calculated as [time spent in the novel location/(time in familiar location + time in novel location)] and analyzed by two-way ANOVA (group x delay). There was a significant group effect (p < 0.05), with significantly higher exploration ratios in pregnant females compared to NP females on both test days. Dashed line at 0.50 indicates chance performance.

2.2. Effect of pregnancy on elevated plus maze

Data from the EPM is presented in Table 1. A three-way repeated-measures MANOVA (group x day x arms) revealed a significant main effect of arms [F2,46 = 390.1, p < 0.001]. Over both groups and test days, all animals had a significantly higher percentage of entries and time spent into the closed arms of the EPM, compared with the open arms. There was also a significant day x arms interaction [F2,46 = 15.8, p < 0.001], and a significant group x day x arms interaction [F2,46 = 6.05, p < 0.01]. The MANOVA revealed that NP and pregnant females significantly differed only on GD9, with NP females spending a significantly lower percentage of time in the open arms than pregnant females (p < 0.05). Across test days, NP females did not significantly differ in percent of entries into or time in the open or closed arms across test days (p > 0.10 via t-test). In contrast, pregnant females spent a significantly lower portion of time in, and had fewer entries into, the open arms of the EPM from GD9 to GD18 (p < 0.001 for both via t-test). Accordingly, the percentage of time in, and entries into, the closed arms of the EPM significantly increased from GD9 to GD18 (p < 0.001 for both via t-test).

Table 1.

Performance of NP and pregnant females on elevated plus maze (EPM).

Percent of total entries into open arms Percent of time spent in open arms Percent of total entries into closed arms Percent of time spent in closed arms
NP females GD9
GD18
22 ± 3 %
13 ± 6 %
2 ± 1 %
8 ± 4 %
78 ± 3 %
87 ± 6 %
88 ± 1 %
92 ± 4 %
Pregnant females GD9
GD18
30 ± 5 %
14 ± 5 %***
25 ± 6 %
7 ± 6 %***
70 ± 5 %
86 ± 5 %***
75 ± 6 %
93 ± 6 %***

Data are given as mean ± SEM for NP (n = 6) and pregnant (n = 8) females. Data were analyzed by three-way (group x day x arms) MANOVA. Pregnant females spent a significantly greater percentage of time in the open arms compared with NP females only on GD9 (rows 1 & 3; column 2). Across the two test days, NP females did not differ in number or duration of entries. In contrast, pregnant females significantly differed on all four measurements from GD9 and GD18, with a lower percentage of entries and time in the open arms on GD9 compared to GD 18 (Columns 1 and 2), and a higher percentage of entries and time in the closed arms on GD9 compared to GD18 (Columns 3 & 4),

***

p < 0.001 between GD9 and GD18.

p < 0.05 between NP and pregnant females.

2.3. Effect of pregnancy on monoamine levels and activity

Monoamine and metabolite concentrations (DA and metabolites DOPAC and HVA, NE and metabolite MHPG, 5HT and metabolite 5-HIAA) were measured in four brain regions: PFC, CA1, and CA3 hippocampus, and mPOA. In each brain region, data were analyzed by a two-way ANOVA (group x neurochemical). Turnover ratios (metabolite/monoamine) were calculated as a measure of monoaminergic activity and analyzed by a two-way ANOVA (group x turnover ratio). The ANOVA results for individual significant interactions (group x neurochemical or group x turnover ratios) in all four regions are listed in Table 2. Post-hoc analysis was carried out using Tukey’s LSD test.

Table 2.

ANOVA results (group x neurochemical) for monoamine, metabolite measurements and turnover ratios in four brain regions.

Frontal Cortex CA1 CA3 mPOA

Monoamine F2,13 p value Monoamine F2,12 p value Monoamine F2,13 p value Monoamine F2,13 p value
DA 5.64 < .05 DA 0.83 > .05# DA 5.64 < .05 DA 0.19 > .05#
DOPAC 29.91 < .01 DOPAC 69.87 < .001 DOPAC 29.91 < .01 DOPAC 2.86 > .05#
HVA 41.26 < .01 HVA 22.76 < .001 HVA 41.26 < .01 HVA 0.33 > .05#
NE 8.34 < .01 NE 135.87 < .001 NE 8.34 < .01 NE 7.46 < .01
MHPG 28.32 < .01 MHPG 24.06 < .001 MHPG 28.32 < .01 MHPG 15.20 < .001
5HT 69.09 < .01 5HT 17.48 < .001 5HT 69.09 < .01 5HT 0.10 > .05#
5-HIAA 93.28 < .01 5-HIAA 0.46 > .05# 5-HIAA 93.28 < .01 5-HIAA 0.35 > .05#

Turnover ratio F2,13 p value Turnover ratio F2,12 p value Turnover ratio F2,13 p value Turnover ratio F2,13 p value

HVA/DA 1.59 > .05# HVA/DA 16.11 < .001 HVA/DA 1.59 > .05# HVA/DA 0.35 > .05#
DOPAC/DA 5.20 < .05 DOPAC/DA 12.41 < .001 DOPAC/DA 5.20 < .05 DOPAC/DA 2.02 > .05#
MHPG/NE 5.74 < .05 MHPG/NE 3.82 < .05 MHPG/NE 5.74 < .05 MHPG/NE 10.75 < .001
5-HIAA/5HT 5.74 < .05 5-HIAA/5HT 6.64 < .05 5-HIAA/5HT 5.74 < .05 5-HIAA/5HT 0.19 >.05#

Comparisons were made between NP and pregnant females for measured monoamines and metabolites, and calculated turnover ratios (metabolite/monoamine). A p value of < .05 was accepted for significance;

#

indicates comparisons that were not significant.

2.3.1. Monoamine levels and activity in the PFC

In the PFC, there was a significant main effect of group [F2,91 = 186.1, p < 0.001], neurochemical [F6,91 = 546.1, p < 0.001] and a significant group x neurochemical interaction [F12,91 = 21.8, p < 0.001]. In general, NP females had significantly higher levels of monoamines and metabolites as compared to pregnant females (p < 0.001 compared to both pregnant groups). Post hoc analysis revealed that, compared to GD9 and GD18 females, NP females had significantly higher levels of DA (p < 0.05), DOPAC (p < 0.001), and HVA (p < 0.001; Figure 2A). Both NP and GD9 females had significantly higher levels of NE than did GD18 females (p < 0.01; Figure 2C), but only NP females had significantly higher levels of the NE metabolite MHPG (p < 0.001; Figure 2C) compared to GD18 females. Finally, NP females had significantly higher levels of 5HT and metabolite 5-HIAA (p < 0.001; Figure 2E).

Figure 2. Effect of pregnancy state on monoamines, metabolites and turnover ratios in prefrontal cortex.

Figure 2

Mean concentration (± SEM) for NP (□ n = 5), GD9 ( Inline graphic n = 7) and GD18 (■ n = 4) females. Concentrations are pg/μg total protein. DA, dopamine; DOPAC, 3,4-dihydroxy-phenylacetic acid; HVA, homovanillic acid; NE, norepinephrine; MHPG, 3-methoxy-4-hydroxyphenylglycol; 5HT, serotonin; 5-HIAA, 5-hydroxy indole acetic acid. Data were analyzed by one-way ANOVA [(group x neurochemical) or (group x turnover ratio)] *p < .05, **p < .01 from both other groups for each neurochemical or turnover ratio.

For turnover ratios, there was a significant main effect of group [F2,52 = 4.33, p < 0.05], ratios [F3,52 = 326.7, p < 0.001], and a significant group x turnover ratio interaction [F6,52 = 19.9, p < 0.001]. Generally, GD18 females had significantly different turnover ratios than GD9 females (p < 0.01). Post hoc analyses revealed that NP females had significantly higher turnover ratios of DOPAC/DA compared to both pregnant groups (p < 0.05; Figure 2B); groups did not differ in turnover ratios of HVA/DA (Figure 2B). GD18 females had significantly higher turnover ratios of MHPG/NE compared to GD9 and NP females (p < 0.01; Figure 2D). Finally, GD9 females had significantly higher turnover ratios of 5-HIAA/5HT than GD18 females (p < 0.01; Figure 2F).

2.3.2. Monoamine levels and activity in the CA1 hippocampus

In the CA1 hippocampus there was a significant effect of group [F2,84 = 21.90, p < 0.001], neurochemical [F6,84 = 88.79, p < 0.001], and a significant group x neurochemical interaction [F12,84 = 24.39, p < 0.001]. In general, GD9 females had significantly different levels of monoamines and metabolites than NP or GD18 females. Post hoc analysis revealed differences in the DA metabolites: DOPAC levels were higher in NP females than GD9 and GD18 females (p < 0.001) and HVA levels were higher in GD9 females than NP or GD18 females (p < 0.001; Figure 3A). All three groups significantly differed in levels of NE, with lowest levels in NP females, median levels in GD9 females, and highest levels in GD18 females (p < 0.001 for all comparisons; Figure 3C). Additionally, GD9 females had significantly higher levels of the NE metabolite MHPG than both other groups (p < 0.001; Figure 3C), but lower lower levels of 5HT than NP and GD18 females (p < .01; Figure 3E). No differences were seen between group in levels of DA or 5-HIAA (p > 0.05 for both; Figure 3A, 3E).

Figure 3. Effect of pregnancy state on monoamines, metabolites and turnover ratios in CA1 hippocampus.

Figure 3

Mean concentration (± SEM) for NP (□ n = 4), GD9 ( Inline graphic n = 7) and GD18 (■ n = 4). Concentrations are pg/μg total protein. Data were analyzed by one-way ANOVA [(group x neurochemical) or (group x turnover ratio)] *p < .05, **p < .01, ***p < .001 from both other groups (unless otherwise indicated) for each neurochemical or turnover ratio (see Figure 2 for abbreviations)

For turnover ratios, there was a significant main effect of group [F2,48 = 10.58, p < 0.001], turnover ratio [F3,48 = 30.02, p < 0.001], and a group x turnover ratio interaction [F6,48 = 5.29, p < 0.001]. Generally, GD18 females had significantly lower turnover ratios compared to NP and GD9 females (p < 0.01). Specifically, NP females had significantly higher turnover ratios of DOPAC/DA than GD9 or GD18 females (p < 0.01) and GD9 females had significantly higher turnover ratios of HVA/DA than NP or GD18 females (p < 0.001; Figure 3B). and significantly higher turnover ratios of MHPG/NE than GD18 females (p < 0.05; Figure 3D). but significantly higher turnover ratios of 5-HIAA/5HT than GD18 females (p < 0.05; Figure 3F).

2.3.3. Monoamine levels and activity in the CA3 hippocampus

In the CA3 hippocampus there was a significant effect of group [F2,91 = 35.72, p < 0.001], neurochemical [F6,91 = 367.18, p < 0.001], and a group x neurochemical interaction [F12,91 = 18.58, p < 0.001]. Post hoc analysis revealed no differences in levels of DA or metabolite DOPAC (p > 0.05), but GD9 females had significantly higher levels of the DA metabolite HVA compared to NP and GD18 females (p < 0.001; Figure 4A). There were also no differences in levels of NE (p > 0.05), but both NP and GD9 females had significantly higher levels of the NE metabolite MHPG (p < 0.01; Figure 4C) and 5HT (p < 0.01; Figure 4E) compared to GD18 females. GD9 females had significantly higher levels of the 5HT metabolite 5-HIAA compared to NP and GD18 females (p < 0.01; Figure 4E).

Figure 4. Effect of pregnancy state on monoamines, metabolites and turnover ratios in CA3 hippocampus.

Figure 4

Mean concentration (± SEM) for NP (□ n = 7), GD9 ( Inline graphic n = 7) and GD18 (■ n = 5) females. Concentrations are pg/μg total protein. Data were analyzed by one-way ANOVA [(group x neurochemical) or (group x turnover ratio)] *p < .05, **p < .01 from both other groups for each neurochemical or turnover ratio (see Figure 2 for abbreviations)

For turnover ratios, there was a significant main effect of group [F2,52 = 17.03, p < 0.001], turnover ratios [F3,52 = 22.80, p < 0.001] and a group x turnover ratio interaction [F6,52 = 5.92, p < 0.001]. GD9 females had significantly higher turnover ratios of HVA/DA than both NP and GD18 females (p < 0.001; Figure 4B), but GD9 MHPG/NE ratios were significantly higher only from GD18 females (p < 0.001; Figure 4D). Both GD9 and GD18 females had significantly higher 5-HIAA/5HT turnover ratios than NP females (p < 0.01; Figure 4F). DOPAC/DA ratios did not significantly differ across groups (p > 0.05; Figure 4B).

2.3.4. Monoamine levels and activity in the mPOA

In the mPOA there was a significant effect of group [F2,98 = 6.69, p < 0.01], neurochemical [F6,98 = 605.94, p < 0.001] and a significant group x neurochemical interaction [F12,98 = 13.73, p < 0.001]. There were no significant differences between NP, GD9 and GD18 females in levels of DA or metabolites DOPAC and HVA (p > 0.05; Figure 5A), or levels of 5HT and metabolite 5-HIAA (p > 0.05; Figure 5E). However, post-hoc analysis revealed that NP females had significantly higher levels of NE compared to GD9 (p < 0.05) and GD18 (p < 0.01) females (Figure 5C), but lower levels of the NE metabolite MHPG as compared to GD9 and GD18 females (p < 0.001; Figure 5C).

Figure 5. Effect of pregnancy state on monoamines, metabolites and turnover ratios in mPOA.

Figure 5

Mean concentration (± SEM) for NP (□ n = 4), GD9 ( Inline graphic n = 7) and GD18 (■ n = 6) females. Concentrations are pg/μg total protein. Data were analyzed by one-way ANOVA [(group x neurochemical) or (group x turnover ratio)] *p < .05, **p < .01, ***p < 0.001, #p = .06 from both other groups (unless otherwise indicated) for each neurochemical or turnover ratio (see Figure 2 for abbreviations).

For turnover ratios, there was a significant main effect of group [F2,56 = 11.48, p < 0.001], turnover ratios [F3,56 = 104.06, p < 0.001] and a significant group x turnover ratio interaction [F6,56 = 10.22, p < 0.001]. Groups did not differ in turnover ratios of DOPAC/DA or HVA/DA (p > 0.05: Figure 5B), or turnover ratios of 5-HIAA/5HT (p > 0.05; Figure 5F). In contrast, NP females had significantly lower turnover ratios of MHPG/NE as compared to GD9 (p < 0.01) and GD18 (p < 0.001) females. There was also a non-significant trend towards a higher turnover ratio of MHPG/NE in GD18 as compared to GD9 females (p = 0.06; Figure 5D).

3. Discussion

The current study compared behaviors and neurochemicals in NP and pregnant females at two time periods during pregnancy: GD 7–9 and GD16–18. Compared to NP females, pregnant females demonstrated significantly better spatial memory at both GD7 and GD16, and significantly less anxiety in the EPM on GD9, but not on GD18. Differences in monoaminergic levels and activity in various brain regions may underlie the observed behavioral differences. Generally, lower monoamine levels and turnover ratios in the PFC, but higher monoamine levels and turnover ratios in the hippocampus, are seen in pregnant females and could contribute to the observed spatial memory enhancements.

3.1. Pregnancy significantly enhanced spatial memory

The current study presents findings that pregnant females outperform NP females on the OP task, an assessment of spatial memory. At both time points during pregnancy, GD7 and GD16, NP females demonstrated chance performance (exploration ratios similar to 0.50), indicating lack of location discrimination. Failure to discriminate object location is likely not due to differences in exploration, as NP and pregnant females explored objects equally during the sample trial (Figure 1A). In contrast, pregnant females were able to perform the OP task equally well with a 2-h inter-trial delay on both test dates (GD7 and GD16), spending approximately 66% of their time in the new object location, significantly better than chance (Figure 1B). Interestingly, the difference in exploration ratios between NP and pregnant females was less on GD16 (only 13% different), than on GD7 (27% different). One possibility for this difference is the hormonal milieu of the animal. Previous work in our lab (Bisagno et al., 2003; Luine et al., 2003) has also shown that the ability to distinguish object location with delays 2 hours and greater are generally not seen unless estradiol levels are elevated, such as occurs during pregnancy. However, the rise in estrogen levels does not generally occur until Day 19 of pregnancy (Rosenblatt et al., 1988). As our testing took place on GD16, the full benefit of estrogen on memory may not yet have been seen, accounting for the slight differences in performance by the pregnant group from GD7 to GD16.

These results confirm previous findings that females just ending the first week of pregnancy have significantly better spatial memory on the Morris water maze as compared to nulliparous females (Galea et al, 2000). However, this study is the first to indicate that females in the third week of pregnancy also out-perform non-pregnant females, in direct contrast to previous studies in which females in the third week did not demonstrate enhanced spatial memory in the Morris water maze (Galea et al., 2000; Bodensteiner et al., 2006). We have also tested pregnant females on the OP task with a 4-h inter-trial delay (data not shown); the results indicate that pregnant females are only able to discriminate between familiar and novel object locations at longer inter-trial delays in a later stage of pregnancy. Overall, our data indicate that pregnancy enhances spatial memory as tested on the OP task; further study is needed to fully draw out the relationship between pregnancy and spatial memory.

Day of testing, as well as task used, may underlie differences from previous studies (Galea et al., 2000; Bodensteiner et al., 2006). In both, the Morris water maze was used, a task that has been shown to increase stress in males as measured by increased corticosterone production and release throughout the amygdala, hippocampus and hypothalamus (Aguilar-Valles et al., 2005). While stress in males generally leads to decrements in performance on spatial tasks (Luine et al., 1994), restraint stress does not decrease spatial task performance in females (Bowman et al., 2001; Luine, 2002). Furthermore, females with reproductive experience given one exposure to restraint stress demonstrated reduced activity in brain areas normally activated by stress (Wartella et al., 2003). However, pregnant females given restraint stress from day 15 through gestation demonstrated significantly impaired performance on the Morris water maze as compared to mothers not given stress, both two weeks after weaning and at 22 months of age (Lemaire et al., 2006), indicating that increased stress during pregnancy can impair spatial memory ability. Thus, repeated stress in females in late pregnancy, such as that given with repeated testing in the water maze, may decrease performance on a spatial task. More data needs to be obtained on a variety of spatial tasks to fully understand the relationship between pregnancy and spatial memory.

3.2. Pregnancy significantly decreased anxiety

A significant increase in number and duration of entries into the open arm of the EPM is a reliable indicator of decreased anxiety (Pellow et al., 1985; Lonstein, 2005). In the current study, pregnant females displayed decreased anxiety-like behavior compared with NP females on GD9 as evidenced by an increase in time spent in the open arms of the EPM. The two groups did not differ in any measures of anxiety-like behavior on GD18. These findings uphold previous reports of reduced anxiety-like behaviors in pregnant females on the open field (Wartella et al., 2003), as well as decreased anxiety in the EPM during the first week of lactation (Lonstein, 2005), after a single (Byrnes & Bridges, 2006) or multiple litters (Walf & Frye, 2008), and throughout the lifespan (Love et al., 2005; but see Byrnes & Bridges, 2006; Macbeth et al., 2008).

The data indicates that anxiety-like behaviors are increased in pregnant females at a later stage of pregnancy, a previously unreported finding. From GD9 to GD18, pregnant females had a 53% decrease in percent entries into open arms and spent 72% less time in the open arms; they had a corresponding 19% increase in percent entries and time in the closed arms (Table 2). While NP females displayed similar behaviors, the difference from GD9 to GD18 was not significant in this group (possibly due to a smaller group size). This study is the first to find differences in anxiety-like behavior on the EPM across two time points of gestation. The EPM is often administered one time only, due to possible anxiolytic effects of repeated exposure to the EPM (Lonstein, 2005). As anxiety-like behavior instead increased in the pregnant group from GD9 to GD18, the finding is likely not an artifact of the testing paradigm. Indeed, the hormonal milieu of the female may underlie the increased anxiety in pregnant females. Progesterone levels are significantly higher on GD18 compared to GD9 (Rosenblatt et al., 1988); progesterone has been shown to be anxiogenic on the EPM (Galeeva & Tuohimaa, 2001; Galeeva et al., 2003; but see Mora et al., 1996). Furthermore, estradiol levels are beginning to rise on GD18, and are slightly higher than on GD9 (Rosenblatt et al., 1988). Unfortunately, it remains unclear whether estradiol is anxiolytic (Walf & Frye, 2007), anxiogenic (Patisaul & Bateman, 2008) or has no effect on anxiety (Luine et al., 2006); the differences may be due to whether estradiol acts on the alpha or beta estrogen receptor (Lund et al., 2005), or dose and timing of administration (see Walf & Frye, 2006 for review). Therefore, we cannot say with certainty what effect rising estradiol levels might have on EPM performance.

3.3. Pregnancy altered monoamine levels and activity

In the current study, significant changes in monoamine and metabolite levels, as well as turnover ratios, were found in the PFC, CA1 and CA3 regions of the hippocampus, and mPOA, of pregnant females compared to NP females. In the PFC, NP females had between 30–88% higher levels of all monoamines and metabolites measured, compared to GD9 and GD18 females. Therefore, decreased levels and activity of DA, NE, and 5HT in the PFC may contribute to better performance on a spatial working memory task, as pregnant females displayed significantly better spatial memory. The PFC in general seems to work with the hippocampus to aid in performance on spatial working memory tasks (Jones & Wilson, 2005; Sloan et al., 2006). Elevated DA and NE levels in the PFC of male rats are associated with poorer working memory (Kobori et al., 2006). Similarly, DA can increase inhibition in the PFC of primates, which can impair working memory (Kroner et al., 2006). Thus, lower levels of DA and NE may be most beneficial for working memory performance; the current study lends support to this idea.

In contrast to the PFC, in the CA1 and CA3 regions of the hippocampus, monoamine and metabolite levels, as well as turnover, were generally higher during pregnancy, with the greatest number of changes measured on GD9. The CA1 and CA3 hippocampal subregions are required for various types of memory, including episodic memory (Hunsaker et al., 2008), temporal memory (Hoge & Kesner, 2007), and spatial memory (Lee et al., 2005). Monoamines in these hippocampal subregions play a role in spatial memory performance. Recently, DA has been shown to be necessary for certain forms of hippocampal synaptic plasticity. Specifically, activation of D1 and D5 dopamine receptors lowered the threshold at which long-term potentiation and depression occurred in CA1 synapses (Lemon & Manahan-Vaughan, 2006). While in the current study DA levels did not differ in the CA1 or CA3 of NP and pregnant females, increased metabolism of DA (as measured by increased HVA levels) in both hippocampal regions of GD9 females could contribute to enhanced spatial memory. NE also plays a role in memory formation. Injections of NE into the hippocampus immediately post-training on an inhibitory-avoidance task facilitated memory for the task (Bevilaqua et al., 1997). Similarly, injections of NE into the CA1 enhanced long-term memory for an inhibitory avoidance task (Izquierdo et al., 1998). In the present study, both GD9 and GD18 females had respectively 43% and 74% higher levels of NE in the CA1 hippocampus, which could have aided in spatial memory. These findings contrast with a previous study in which NE was significantly decreased in the hippocampus throughout pregnancy (Smolen et al., 1987). The regions chosen for analysis and day of pregnancy at which samples were obtained could account for these differences.

Monoamine concentrations were also analyzed in the mPOA, an area relevant to maternal behavior. Unlike the regions associated with memory, no significant differences in DA or its metabolites, DOPAC and HVA, were found between NP and pregnant females. In contrast, one previous study measured lower DA and DOPAC concentrations from GD10 to GD20 (Lonstein et al., 2003). In both the current and previous studies, no significant differences were seen in turnover rates of DA. Differences in this study and previous could be due to the days at which animals were sacrificed, or small differences in tissue sampling. No significant differences were observed due to pregnancy state in concentrations of serotonin or its metabolite 5-HIAA either, corresponding with previous work (Lonstein et al., 2003). This study is the first to report significant differences in the NE system within the mPOA during pregnancy. NE from brainstem nuclei is a major excitatory input to the paraventricular nucleus of the hypothalamus (PVN), and thus helps to mediate the stress response via the hypothalamic-pituitary-adrenal (HPA) axis (Pacak et al., 1993). During late pregnancy (day 20), decreased NE release is observed in the PVN, which may contribute to reduced HPA activation from stress during late pregnancy (Douglas et al., 2005). Similarly, in women, reduced NE during pregnancy is associated with a relaxed state (Teixeira et al., 2005). Thus, lower levels of NE in mPOA, but increased turnover activity (such as that seen in GD9 and GD18 females compared to NP females) could help to mediate stressors during pregnancy, possibly to prepare the mother to care for formerly fear-inducing pups (Fleming & Luebke, 1981). As shown here, a decreased stress response during pregnancy could enhance spatial memory and reduce anxiety. Furthermore, gonadal hormones such as estradiol and progesterone could also affect monoamine levels throughout the brain (Luine et al., 1998), and thereby indirectly mediate changes in memory and anxiety during pregnancy.

In conclusion, the data in the current study provide additional support for the idea that “non-maternal” behaviors are also altered during pregnancy. The reported changes in cognitive abilities may provide the foundation for parturition and subsequent pup care. Future studies can address whether the changes observed here may also contribute to the enduring alterations seen in the brains of females with reproductive experience (Gatewood et al., 2005; Wartella et al., 2003; Macbeth et al., 2008) and to what extent neural and behavioral modifications aid the mother in offspring care.

4. Experimental procedures

4.1. Animals

Fourteen 60-day-old intact female Sprague-Dawley rats (210–230g) were obtained from Harlan Inc., and double-housed under a 12:12 light:dark cycle (lights on at 5:00 h) with water and food (Purina Rat Chow) available ad libitum. Experiments began after a two-week acclimation period to their home cage, during which all subjects were handled daily by the experimenter. All testing occurred between 1000–1500 hours. All procedures used were approved by the IACUC of Hunter College of the City University of New York.

4.2. Pre-test acclimation and mating

Females were acclimated to the object recognition (OR) and object placement (OP) tasks as described previously (Luine, 2002; Bisagno et al., 2003; Macbeth et al., 2008). These tasks have been used to assess non-spatial recognition memory (OR; Ennaceur & Delacour, 1988) and spatial memory (OP; Ennaceur et al., 1997). Both tasks utilize rodents’ inherent preference for novelty, in that increased investigation of a novel object (OR task) or a novel location (OP), as compared to a familiar object or location, can be used as a measure of memory function (Ennaceur et al., 1997).

Briefly, rats were given four days of exposure to the object recognition (OR) task with increasingly larger inter-trial delays between a sample trial (T1) and a recognition trial (T2). During T1, two identical objects were placed at one end of an open field (70 × 70 × 30cm high); the amount of time the rat explored both objects was recorded for 3 min. During T2, at the end of the inter-trial delay, two objects were again presented, with one familiar object replaced with a novel object. Once all females had demonstrated object recognition (> 65% of time with the novel object), acclimation to the OP task began and consisted of four days of exposure to the OP task with increasingly larger inter-trial delays between T1 and T2. The task was the same as OR, except that during T2 both objects were familiar; one is moved to a novel location. Throughout acclimation and testing, novel objects were presented each day. Exploration of the objects was defined as any time in which the subject sniffed at, whisked at, or looked at the objects from no more than 2 cm away. Objects used were novel for each day of acclimation, and consisted of a variety of plastic containers and soda cans for OR, and glass and ceramic figurines for OP.

Following acclimation, the OP task was administered at two different inter-trial delays: 2-h followed by 4-h on the subsequent day. On both test days, novel objects were used. Based on performance, subjects were divided into two counter-balanced groups. One group was not mated (NP: n = 6; intact females with no sexual or reproductive experience) and the other was mated (n = 8; first sexual and reproductive experience). Pilot data (not shown) suggested that in NP females, day of estrous cycle did not affect performance on OR and OP tasks; therefore vaginal cytology of NP females was not analyzed as a covariate in this study. Mating began for the pregnant group immediately after group assignment (approx. day 91 of age). Three month old male Sprague Dawley rats (~ 350g) were used for mating; each male was placed with two females into a new, clean cage. After confirmation of pregnancy (determined by presence of a copulatory plug on the morning after mating, and subsequent weight gain over the next week), the females were removed from the males’ cages and single-housed into new cages for the duration of the experiment.

4.3. Behavioral tests

4.3.1. Object placement

The OP task was administered twice during pregnancy: on GD7 and GD16. At both time periods, the task was administered with a 2-h inter-trial delay. In both the sample trial (T1) and retention trial (T2), the variable of interest was investigation time of the objects (in seconds). For T1, total investigation time of both objects was analyzed using a two-way repeated measures ANOVA (group x day). For T2, investigation time was converted to an exploration ratio, calculated as [time in novel location/(time in familiar location + time in novel location)] and analyzed using a two-way repeated measures ANOVA (group x day). Significance was set at p < 0.05.

4.3.2. Elevated plus maze

The elevated plus maze (EPM) was administered to NP and pregnant females following OP testing at both time periods (e.g. GD9 and GD18) and was conducted as described previously (Macbeth et al., 2007). Briefly, the EPM consisted of two open arms (50 cm x 10 cm) and two closed arms (50 cm x 10 cm x 40cm high). The walls extended from a central neutral area (10 × 10 cm) not counted in exploration or entries. Subjects were placed in the neutral area facing one of the open arms and given 5 minutes to explore the maze. The number and duration of entries into open and closed arms were recorded. A total of three paws inside of an arm were used as criteria for entry. Time spent in the central, neutral area was not recorded. Data from the EPM was analyzed with a three-way repeated measures MANOVA (group x day x arms) on two dependent variables: percentage of entries into open or closed arms and percentage of time in open or closed arms. Percentages were calculated as {[open/(open + closed) X 100] or [closed/(open + closed) X 100]}. Planned comparisons within each group were made with t-tests. Significance was set at p < 0.05 for the MANOVA, and p < 0.025 for the t-tests to control for effects from multiple t-tests.

4.4. Brain tissue

Immediately following the second EPM testing (GD18), all 14 females were sacrificed by decapitation following light anesthesia (a brief exposure (3–5 seconds) to carbon dioxide). Brains were removed immediately (within 30 sec) of removal from the carbon dioxide chamber, blocked just posterior to the prefrontal cortex (PFC; removed and frozen on dry ice) and just anterior to the cerebellum (discarded) then quickly frozen on dry ice and stored at −70°C to prevent degradation of samples until brain neurochemical analysis. As the pregnant females were tested twice during pregnancy it was not possible to analyze neurochemistry from behaviorally tested females in early pregnancy. Thus, an additional eight females were impregnated following same methodology described above, and sacrificed on GD9 without undergoing any behavioral testing. Brains were removed, blocked, and stored in the same manner described above. Frozen brains were sectioned into 300μm sections through the hippocampus and mPOA using a microtome cryostat at −4°C. The frozen PFC, hippocampus, and mPOA sections were sampled with a 500μm-diameter tissue punch (4–6 tissue punches from each animal in the PFC and mPOA, and 10–12 tissue punches from each animal in CA1 and CA3) under a dissecting microscope, on a microscope stage maintained at −4°C (see Palkovits, 1973)

4.5. High performance liquid chromatography

Monoamines and metabolites were measured through high performance liquid chromatography (HPLC) with electrochemical detection (E.C.): dopamine (DA) and metabolites 3,4-dihydroxy-phenylacetic acid (DOPAC) and homovanillic acid (HVA); norepinephrine (NE) and metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG); serotonin (5HT) and metabolite 5-hydroxy indole acetic acid (5-HIAA). The procedure was as described previously (Bisagno et al., 2002; Macbeth et al., 2008). Tissue samples were expelled into a 60μl sodium acetate buffer (pH 5.0) with α-methyl-dopamine as an internal standard. The samples were frozen and thawed, the supernatant drawn off, and the pellet re-suspended in 100μl (PFC and mPOA: 4–6 tissue punches) or 200μl (CA1 and CA3: 10–12 tissue punches) of 2.0N NaOH for protein analysis using Bio-Rad reagent (Bio-Rad Laboratories, Hercules, CA). The supernatant containing the monoamines and metabolites was injected into a Waters Associates chromatographic system (Waters 2690, Milford, Mass), consisting of an automated refrigerated injector, pump, C18 reverse-phase column (Novapak 3 micron; Waters Assoc, Milford Mass), and an ESA Coulochem III detector (+0.48 - +0.50 V potential; Chelmsford, Mass). The mobile phase is described elsewhere (Renner & Luine, 1984; Luine et al., 1990). Peak sharpness was increased by the addition of 100% methanol gradient into the flow (99.5% mobile: 0.5% methanol). Millenium software (Waters Associates) was used to run the chromatography system, in which concentrations of transmitters and metabolites were calculated by reference to standards and internal standard using peak integration. Single sample runs averaged between 12–20 minutes. Monoamine concentrations are expressed as pg/μg total protein. Monoamines, metabolites, and turnover ratios in each brain region (PFC, CA1, CA3, mPOA) were analyzed via a two-way ANOVA (group (NP, GD9, GD18) x neurochemical). Post-hoc analysis was carried out using LSD test; p < 0.05 was set for significance.

Acknowledgments

The authors thank G. Mohan for assistance in behavior testing. This research is supported by S06-GM-60654 and a Research Centers in Minority Institutions award (G12 RR-03037) from the Division of Research Infrastructure of the National Center for Research Resources of NIH, which supports the infrastructure of the Biopsychology program at Hunter College. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR/NIH.

Footnotes

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