Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Apr 15.
Published in final edited form as: Behav Brain Res. 2013 Jan 15;243:239–246. doi: 10.1016/j.bbr.2013.01.009

Males, but not females, lose tyrosine hydroxylase fibers in the medial prefrontal cortex and are impaired on a delayed alternation task during aging

Nioka C Chisholm a,c,2, Taehyeon Kim a, Janice M Juraska a,b,1
PMCID: PMC3594341  NIHMSID: NIHMS444422  PMID: 23327742

Abstract

The structure of the prefrontal cortex (PFC) is particularly vulnerable to the effects of aging, and behaviors mediated by the PFC are impaired during aging in both humans and animals. In male rats, behavioral deficits have been correlated with a decrease in dopaminergic functioning. However, studies have found that anatomical changes associated with aging are sexually dimorphic, with males experiencing greater age-related loss than females. The present study investigated the effects of sex and aging on performance of a delayed alternation t-maze, a task mediated by the medial prefrontal cortex (mPFC), and on tyrosine hydroxylase (TH) immunoreactivity in this brain region using adult (7 months) and aged (21 months) male and female F344 rats. There was a sex by age interaction in performance of the delayed alternation task such that adult males performed better than aged males, but aged females were not different than adult females. Adult males performed better than adult females across all delays; however, this sex difference was reversed during aging and aged males performed worse than aged females. In addition, TH immunoreactivity decreased during aging in layers 2/3 in the male, but not female mPFC. Thus females were less sensitive to the effects of aging on the prefrontal dopaminergic system and on performance of a delayed alternation task. These effects may be due to decreases in testosterone in aging males, as well as the protective effects of ovarian hormones, which continue to be secreted after cessation of the estrous cycle in aging females.

Keywords: working memory, prefrontal cortex, aging, dopamine, sex differences, t-maze

1. Introduction

The prefrontal cortex (PFC) has been identified as a brain region that is particularly vulnerable to the effects of aging. The largest decline in human brain metabolism during aging occurs in the PFC [1], and this region has a greater loss of gray matter volume than other brain areas [2, 3]. Decreases in synaptic density, spine density, and dendritic arborization have also been found in the aged human frontal cortex [4–7] as well as the aged medial PFC (mPFC) of rodents [8–10]. In addition to the anatomical changes, several studies have shown that the functions of the PFC, including behavioral flexibility, attention, and memory, are impaired during aging in humans (reviewed in [11] and rats [10, 12–14]).

While age-related changes occur in several neurotransmitter systems, the prefrontal dopaminergic system is of particular importance because of its role in maintaining cognitive function (reviewed in [15]). Numerous studies have found that aging results in a compromised dopaminergic system in humans, non-human primates and rodents (reviewed in [16]). Importantly, several of these age-related changes are greatest in the PFC. For example, human PET and autopsy studies have found decreases in D1 [17, 18] and D2 receptors in both the frontal cortex and hippocampus [19], with the fastest rate of decline found in the frontal cortex [20, 21]. In addition, there is a decrease in dopamine synthesis during aging of human males and this was the greatest in the dorsal lateral PFC [22], a region homologous to the rodent mPFC [23]. Similar to the human research, endogenous levels of dopamine decrease in the cerebral cortex of male rhesus monkeys and this decrease is the greatest in the PFC [24]. A recent study in rodents examined the relationship between changes in the prefrontal dopaminergic system and age-related cognitive decline in male rats. They found that males were impaired on delayed alternation, a task mediated in part by the mPFC, and that tyrosine hydroxylase (TH) fibers were decreased in the mPFC during aging [12], highlighting the importance of understanding how aging affects the prefrontal dopaminergic system.

It is notable that most of the literature on aging only examines males, especially since there is some evidence that aging impacts males to a greater degree than females. For example, performance on a delayed recognition test is impaired during aging in male rhesus monkeys but not females [25, 26] and on a repeated acquisition water maze task, aged male rats, but not aged females were impaired as compared to adults [27]. Likewise, age-related changes in cortical neuroanatomy tend to be more profound in males. Male rats undergo a greater loss of dendritic spines and branches than females, and male, but not female rats, lose neurons during aging in the mPFC [9, 28]. Alterations in neocortical choline acetyltransferase and glutamic acid occurred in male mice as early as 17 months, whereas changes in females did not occur until 25 months of age [29]. In addition, age-related changes in the basal forebrain cholinergic system and the striatal dopaminergic system of rodents are sexually dimorphic and region specific [30–33]. Although there is evidence for losses in the dopaminergic system in the PFC of both aged males and females, comparisons of the sexes have not been done [12, 31, 34].

Changes in gonadal hormones may influence the process of aging in both males and females, given that gonadal hormones are known to affect the prefrontal dopaminergic system in adults. Gonadectomy results in an increase in TH fibers in the mPFC of adult male rats and this is restored to normal levels with testosterone treatment [35]. In contrast, a reduction in TH fibers was observed after ovariectomy of adult female non-human primates, and this is returned to levels observed in control animals after treatment with estradiol [36, 37]. Results from adult males might suggest that aged males would have increased TH fibers in the mPFC because testosterone is decreased in aged males [38]; however, the Mizoguchi et al. [12] study observed a decrease in TH fibers indicating that the aging male PFC responds differently than the adult PFC to alterations in gonadal hormones. To date, no studies have examined how the dopaminergic system in the male PFC responds to testosterone treatment during aging. We have recently shown that long-term estrogen treatment results in a greater density and volume of TH fibers in the mPFC of aging ovariectomized females [39], indicating that estradiol administered soon after ovariectomy has similar effects in both adult and aged animals. Because aging intact female rats continue to secrete low levels of ovarian hormones [9, 40], it is possible that the decreases in TH observed in the mPFC of male rats [12] may not occur to the same degree in female rats. Both sex specific changes during aging in the prefrontal dopaminergic system and behavior mediated by the PFC were investigated in the present study. Adult and aging rats of both sexes were tested on a delayed alternation task and TH fibers were quantified in the mPFC.

2. Methods

2.1 Subjects

Subjects were Fischer 344 rats, obtained from the NIA aging farm, at 6 months of age (males, n =7; females, n =7) and 17 months of age (males, n=7; females, n=7). Animals from the same group were pair- or triple-housed in clear cages in a temperature controlled environment on a 12:12-hr light–dark cycle. Food and water were available ad libitum to all animals, except during behavioral procedures. All rats were handled once a week and checked for health problems (tumors). Animal care and experimental procedures were in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee.

2.2 Behavioral Procedures

The first day of testing occurred when the adult animals were 7 months old and aged animals were 21 months. Food restriction began five days prior to introduction to the maze. Initially, animals were fed two pellets of rodent chow daily but the amount of food was altered to keep each animal within 85–90% of their starting body weight.

2.2.1 Apparatus

The T-maze consisted of an approach alley (15 cm wide × 45 cm long) and two goal arms (15 cm wide × 45 cm long). The start box (15cm wide × 26cm long) opened into the approach alley. The walls of the maze were 15 cm high and were constructed of black plexiglass. Manually operated sliding doors were positioned at the entrance to each goal arm. The orientation and position of the maze were not changed during the experiment.

2.2.2 Habituation

Animals were exposed to the maze during four 10 minute sessions over 4 days. During this time, each arm was baited with sunflower seeds and animals were allowed to roam the maze freely for 10 minutes.

2.2.3 Training

Animals were placed in the start box and were given a forced run. During the forced fun, one arm of the maze was blocked so that the animal could not choose that arm and the other arm was baited. The arm that was baited during the forced run was randomly assigned. After the animal had eaten the sunflower seed in the baited arm, they were immediately placed back in the start box for a choice run. During the choice run, both arms were open but the animal was only rewarded when choosing the arm opposite to the one that it had visited during the forced run. For example, on the forced run if the right arm was blocked and the left arm was baited, during the choice run, both arms would be open, but the animal would only be rewarded for choosing the right arm. A forced trial followed by a choice trial is referred to as a pair of trials. Animals were run in squads of 5–6 animals which resulted in a 7–12 minute delay between pairs of trials.

Because animals were initially slow at completing each forced and choice run, animals completed 3 pairs of trials a day until they were able to finish each forced run within one minute. Two aged males and one aged female failed to meet this requirement even after 21 days of training, so that each group had the following number of subjects: adult males (n =7) and adult females (n =7); aged males (n=5) and aged females (n=6). After animals were successfully completing the 3 pairs of trials a day, the number of trials was increased to 6 pairs of trials a day. Training continued until animal reached a criterion of 5/6 correct across two days.

2.2.4 Delayed Alternation

Once animals reached criterion, they began the delayed alternation portion of the task. The procedures were the same as training, except that after each forced run, animals were placed in a holding cage where they would experience one of the delays. Delays of 10, 30, and 60 seconds, and 5 minutes were presented. After each pair of trials animals were returned to their home cage and the next pair of trials started after a 7–12 minute delay. Each animal completed 5 pairs of trials per day in which the same delay was used for all 5 pairs. The delays were presented to all animals in the same sequence (10sec, 30sec, 60sec, 5min, 10sec, 30sec, 60sec, 5min). Testing continued for 16 days and this resulted in 20 pairs of trials at each delay.

2.3 Histology

All groups were placed on free feed for three weeks following behavioral testing. Then all rats were deeply anesthetized with sodium pentobarbital (2 mg/kg of a 65 mg/ml solution), and intracardially perfused with Ringer’s wash (2 minutes) followed by a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for three minutes. Brains were removed and stored in a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 20 hours, followed by cryoprotection in 30% sucrose for three days. Because time in fixation can affect the amount of staining observed, this was closely monitored and remained the same for all animals. Brains were coronally sectioned at 30 μm using a freezing microtome and stored at 4°C in 30% glycerol, 30% ethylene glycol, 30% distilled water, and 10% PBS.

2.3.1 Immunohistochemistry

Sections from all animals (including those excluded from behavioral testing so that n =7 per group) were immunoreacted according to procedures in Kritzer et al. [35] with a few modifications. Free floating sections containing the mPFC were rinsed in 0.1 M PBS, pH 7.4 (3 × 15 minutes), incubated in 1% H2O2 in PBS for 30 min. Sections were then rinsed in Tris-buffered saline (TBS 3 × 15 minutes), pH 7.4, placed in blocking solution (TBS containing 10% normal swine serum; NSS) for 2 hours, and incubated in primary antiserum (diluted in TBS containing 1% NSS, two days, 4°C). Anti-TH antibodies (Chemicon International, Temecula, CA) were used at a working dilution of 1:1000. Following incubation in primary antibody, sections were rinsed in TBS, placed in biotinylated secondary antibodies (Vector, Burlingame, CA; 2 h room temperature; 1:200), rinsed in TBS, and then incubated in avidin–biotin-complexed horseradish peroxidase for two hours at room temperature (ABC; Vector, Burlingame, CA). Sections were then rinsed in TBS, pH 7.4 and reacted by using diaminobenzidine fast Tabs (Sigma, Saint Louis, MO). Control sections, in which the primary antibody was omitted, were processed using the same procedures. Sections were immediately mounted and allowed to dry overnight. The following day slides were dehydrated and coverslipped.

2.3.2 Fiber Density

Examination of TH immunoreactive fibers was carried out in the right hemisphere of two PFC-containing sections. For each subject, the first section containing the PFC (the most anterior part of the PFC) was used for section 1 and the second section was 280 microns posterior to the first section. Images were acquired every .125 microns throughout the thickness of the tissue using a Zeiss Axiovert 200M fluorescence microscope (Carl Zeiss, Thornwood, NY) and compressed using Axiovision software (Figure 1A). Individual images were observed prior to compression to insure antibody penetration throughout the tissue. Within each layer of the PFC (1, 2/3, and 5/6), three pictures were taken (two in the PL one in the IL). A total of 18 pictures were taken per animal (9 per section). Layers of the mPFC can be identified by fiber orientation (Figure 2). Layer 1 consists of a thick band of fibers that runs in the dorsal to ventral direction and marks the boundary of Layer 1 and Layer 2. Care was taken to make sure that images used for Layers 2/3 did not contain any of this type of fiber. Likewise, fibers in layers 2/3 tended to run laterally where as fibers in layers 5/6 had a more diverse orientation that was easily identifiable in the images. Using these differences between the layers, all images were checked to insure they only contained fibers of the respective layers. The images acquired were 240 microns by 160 microns. In some images, layer 1 was less than 240 microns wide and contained some of layer 2; therefore these images were cropped to prevent layer 2 from being included in the analysis of layer 1. For layers 2/3 and layers 5/6, pictures were taken within the middle of the layers.

Figure 1.

Figure 1

Open in a new tab

A. Tyrosine hydroxylase immunostained fibers in a Z-stacked image that has been compressed using Axiovision software. Image pixel density (the percent of the image in black) was measured in two ways: as a binary image (B), taking thickness into account, and as a skeletonized image (C), reducing the thickness to 1 pixel wide.

Figure 2.

Figure 2

Open in a new tab

TH immunostained fibers in layer 1(A) layers 2/3 (B) and layers 5/6 (C) that illustrate the differences between the layers.

Image J was used to measure TH fibers in two ways: first as a binary image, quantifying the number of pixels in black (Figure 1B), and then as a skeletonized image, in which the thickness of each fiber was reduced to 1 pixel wide which measures fiber length (Figure 1C). Both of these methods quantify the length of fibers within the tissue, not the optical density of the precipitate within the fiber. Skeletonized images were analyzed because previous studies that have quantified TH fibers used this type of image [36]; binary images were quantified to assess whether fiber thickness might differ between groups. The same number of images were analyzed for each animal.

2.4 Statistical analysis

Performance on delayed alternation was analyzed using a three-way (2 Sex × 2 Age × 4 Delay) ANOVA with repeated measures on delay. The total pixel density of TH immunoreactive fibers in both binary and skeletonized images was analyzed using a two-way ANOVA for layer 1, layers 2/3, and layers 5/6 separately. The three animals that were not included in behavioral analysis were included in the analysis of TH, and the values for these animals were within the range of their respective groups. Two-tailed t-tests were used for all post-hoc comparisons. The binary image density of TH fibers in each layer and the total number correct across all delays were analyzed using Pearson correlations for each age group separately.

3. Results

3.1 Delayed Alternation Performance

Because some animals were slow to complete pairs of trials during initial training they were given more days consisting of 3 pairs of trials a day. This was most often observed in the aged animals. Therefore, the days to criterion were not statistically compared.

The ANOVA revealed a significant effect of age (F(1,21) =13.992 p =.001), and a significant interaction between age and sex (F(1,21) = 15.269 p =.001). There was also a significant effect of delay (F(3,63) = 10.150 p <.01) such that increasing the delay significantly reduced performance. There was not a significant main effect of sex alone. Post-hoc tests found that adult males made more correct responses than adult females across all delays (p <.01). However, aged females made more correct responses than aged males across all delays, resulting in a reversal of this sex difference (p <.04) (Figure 3). Adult males performed significantly better than aged males at all delays except the five minute delay for which there was a trend for fewer correct responses in the aged animals (p <.01; p <.09). Adult females did differ from aged females at any of the delays (Figure 4).

Figure 3.

Figure 3

Open in a new tab

Average number of correct alternations across all delays (mean+ SEM). There was an effect of age (p =.001), and an interaction between age and sex (p = .001). Adult males made more correct choices than adult females across all delays. Aged females made more correct choices than aged males across all delays. ** p <.02, * p <.05

Figure 4.

Figure 4

Open in a new tab

Total number of correct alternations at each delay (mean+ SEM). Adult males performed better than aged males at all delays except the five minute delay. Performance in aged females did not differ from adult females at any of the delays. There was also an effect of delay (p <.01) such that increasing the inter-trial delay reduced performance. * p<.05

3.2 Tyrosine Hydroxylase

3.2.1 Layer 1

Analysis of the skeletonized images revealed a significant effect of sex within layer 1 (F (1, 24) = 4.960, p < .04) and an age × sex interaction (F (1, 24) = 5.985, p <.03) (Figure 5A). Post-hoc tests demonstrated that aged females had significantly higher TH pixel densities than aged males (p <.02) and adult females (p <.05). Analysis of layer 1 binary images found a trend for an age × sex interaction (F (1, 24) = 3.787, p <.07) (Figure 5B).

Figure 5.

Figure 5

Open in a new tab

The density of TH fibers in Layer 1 (mean+ SEM). In skeletonized images (A), there was an effect of sex (p < .04) and an age × sex interaction (p <.03). Aged females had higher TH pixel densities than aged males and adult females. In binary images (B) there was a trend for an age × sex interaction (p <.07). ** p <.02, * p <.05

3.2.2 Layers 2/3

Analysis of skeletonized images revealed a significant age × sex interaction F (1, 24) = 6.371, p <.02) (Figure 6A). Post-hoc analysis found that aged males had lower TH pixel densities than adult males (p <.04) and aged females (p <.01). Aged females were not different than adult females. Similar to analysis of skeletonized images, analysis of the binary images found a significant age × sex interaction (F (1, 24) = 5.635, p <.03). Post-hoc tests found that aged males had significantly lower TH fiber densities than adult males (p<.04) and aged females (p <.02) (Figure 6B).

Figure 6.

Figure 6

Open in a new tab

The density of TH fibers in layers 2/3 (mean+ SEM). In skeletonized images (A), there was a significant age × sex interaction (p < 0.02). Aged males had lower TH pixel densities than adult males and aged females. Aged females were not different than adult females. In binary images (B), there was a significant age × sex interaction (p < 0.03). Aged males had lower TH fiber densities than adult males and aged females. ** p <.01, * p<.05

3.2.3 Layers 5/6

Analysis of skeletonized images revealed a significant effect of sex (F (1, 24) = 4.306, p <.05) (females > males) (Figure 8A). Similar to analysis of skeletonized images, analysis of the binary images found a trend for an effect of sex (F (1, 24) = 3.738, p <.07) (Figure 8B).

3.2.4 Correlation

In aged animals, the density of TH fibers in layers 2/3 showed a positive correlation with performance on the delayed alternation task (r = .376, p <.05). This was principally driven by the aged female group that had a near significant correlation (r =.6575, p = .0503) between TH fiber density and performance when analyzed alone. The density of TH in other layers of the mPFC was not correlated with performance on delayed alternation in either age group.

4. Discussion

The current study found that the pattern of aging differed between male and female rats both on performance of a delayed alternation task and in the density of TH fibers in the mPFC. Aged male rats performed worse on a delayed alternation task than adult male rats. In contrast to the deficits observed in aged male rats, aged female rats performed at the same level as adult females. In fact, there was a sex difference in the performance of adults rats (male>female) that was reversed in the aged animals. In addition, the density of TH in the mPFC was decreased in males during aging; whereas TH fibers were not decreased in the mPFC of aged females. This is the first study to show that this decrease in TH fibers is sex-specific. Interestingly, there was a significant positive correlation between performance and density of TH fibers in layers 2/3 of the mPFC in the aged animals where the sex differences were most pronounced.

The age-related deficits found in male rats are in agreement with previous literature showing that behaviors mediated by the mPFC are impaired during aging in males [13, 31]. Furthermore similar to the current study, several studies have indicated that cognitive decline during aging is sexually dimorphic. For instance, performance on a delayed recognition test is impaired during aging in male rhesus monkeys but not females [25, 26] and on a repeated acquisition water maze task, aged male rats, but not aged females were impaired as compared to adults [27]. The results from the current study are consistent with the hypothesis that males experience a greater age- related cognitive decline that females. Although animals were food restricted to a similar percentage of their starting body weight, there is the possibility that levels of motivation differed between the groups. Both aged males and females weighed more than their younger counterparts, which can make the motivating effects of food restriction not comparable. Aged animals initially took longer to complete the training trials but this was observed in both males and females, so than the comparison between the sexes appears valid for the aged animals. Once delay testing began, aged animals were completing trials in a similar amount of time as adult animals, so that comparisons between the age groups appear to be justifiable.

Changes in gonadal hormones during aging may play a role in these sex-specific behavioral deficits. For males, aging is associated with a decline in testosterone levels [38] whereas after the cessation of the estrous cycle, intact aging female rats continue to secrete low levels of estrogen and progesterone [9, 40]. Estrogen, progesterone, and androgen receptors are found in several cognitive brain regions, including the PFC [41–43], and studies have found that alterations in gonadal hormones influence performance on tasks mediated in part by the PFC. Removing testosterone in young males impaired acquisition of the delayed t-maze and performance on the radial arm maze [44, 45]. In addition, aged males were impaired on a water radial arm maze, and their performance was improved with testosterone treatment [46].These results suggest that the behavioral deficits observed in the current study in aging male rats may be related to a decrease in testosterone levels and possibly a decline in the estrogen aromatized from testosterone as well. In females, it is known that estrogen treatment also influences cognitive behaviors [47]. For example, estrogen treatment benefited acquisition of a delayed matching to position task in aged female rats [48] and improved performance on a spontaneous alternation task in aged female mice [49]. In a delayed alternation task similar to the one used in the current study, our laboratory has shown that long-term treatment with estradiol and medroxyprogesterone acetate improved acquisition in aged female rats [50]. Therefore the behavioral sex difference observed in aged animals likely results from both decreased testosterone in males and continued secretion of estrogen and progesterone in females.

Several studies have identified an association between performance on the delayed alternation task as well as other working memory tasks, and dopaminergic functioning in the mPFC (reviewed in [51]). In the current study, behavioral performance in the aged rats was correlated with the density of TH in the mPFC where TH fibers were decreased in males, but not in females, during aging. It is important to note that the decrease in TH in layers 2/3 was observed in both skeletonized and binary images. The skeletonized images quantify the length of the fibers while the binary images are a measure that combines both length and thickness. The concordance of effects in these two measures indicates that the differences that were observed did not result from a decrease in fiber thickness but rather a decrease in the total length of fibers. The loss of TH fibers in aged male rats is in agreement with a recent study from Mizoguchi et al. [12] which also found a decrease in skeletonized fibers in the mPFC of aged male rats compared to adult animals and this decrease correlated with performance on a delayed alternation task. The present study, however, is the first study to show that this decrease in TH fiber density is sex-specific. This sex-specific loss during aging is in agreement with studies that have quantified other neuroanatomical measures in the rat mPFC. For example, male, but not female, rats lose neurons in the mPFC during aging and males lose more dendritic spines and branches than females [9, 28]. The only study that has examined the effects of aging on the prefrontal dopaminergic system in both sexes did not find a decrease in dopamine levels during aging in either sex [31], which is in contrast to studies that have examined the male and female mPFC separately [12, 34]. However, several studies have observed sexually dimorphic changes in the striatal dopaminergic system during aging [30–33].

No sex differences were found in the density of TH fibers of adult rats in the present study, but an overall difference in the number of fibers is possible. Previous work from our laboratory found that adult males had 18% greater volume in the mPFC than adult female rats of the same strain [28]. Since only the density of TH fibers was analyzed in the current study, it is possible that a sex difference in the total amount of TH fibers in the mPFC would be observed if the volume of the structure had been included in the analysis. It is important to note that the volume would not likely change the sex difference observed in the density of TH fibers during aging because a sex difference in volume was not observed in aged animals in the aforementioned study [28].

Gonadal hormones are known to alter the structure of the PFC. In adult female non-human primates, ovariectomy reduces the number of TH fibers and estrogen restores the fibers to control levels [36, 37]. There is evidence that estrogen often influences the aged female mPFC in a similar manner as in young animals when it is administered close to the loss of ovarian secretions. For example, recent work from our laboratory found that aged female rats receiving estrogen treatment had a greater density and volume of TH fibers in the mPFC than ovariectomized animals [39]. Also, treatment with estradiol increased apical and basal dendritic spine density in the PFC of aged female rhesus monkeys [52, 53], and long-term treatment with estradiol and medroxyprogesterone acetate resulted in a greater number of synaptophysin labeled boutons in the mPFC of aging female rats [54]. These studies are consistent with the hypothesis that low levels of circulating ovarian hormones in intact aging females protect them from the age-related losses found in males. Because a decrease in testosterone levels in male rats would result in less aromatization of testosterone to estrogen during aging, the lower levels of estrogen might also be responsible for the loss of TH fibers observed in the current study.

It is less clear whether the declining levels of testosterone in aging male rats play a role in the decreased density of TH fibers. Removing the testes in adult male rats results in an increase in TH fibers in the mPFC, and treatment with the testosterone restores TH levels to those of control animals [35–37]. However, androgen receptors decrease during aging in the cortex of male mice [55], and studies suggest that the aging brain may respond differently to testosterone than the adult brain [55–57]. Although no studies have examined the effects of testosterone depletion or replacement on dopamine in the aged PFC, research on the substantia nigra suggests that a decline in testosterone during aging could result in an impaired dopaminergic system. For example, TH and dopamine transporter immunoreactivity was reduced in the substantia nigra of aged male rats and testosterone treatment restored this to adult levels [58]. Therefore the reduction in TH fibers observed in the current study during aging in male rats may be related to a decreased level of testosterone and future studies need to examine whether the aging PFC would respond to testosterone in a similar manner.

Speculation on how the sex difference in the density of TH fibers arises during aging is further complicated by the presence of gonadal hormone receptors in adult rats in the ventral tegmental area (VTA), a region containing dopamine neurons that project to the mPFC [43, 59]. Provided that the aging VTA continues to respond to gonadal hormones, the effects of hormones on the dopaminergic system in the mPFC could result from hormone actions on dopaminergic cells in the VTA. However, the VTA also receives input from pyramidal cells in the mPFC [60], and recent work as shown that whereas only one quarter of the dopaminergic cells that project from the VTA to the mPFC contain androgen receptors, approximately half of the projections from the mPFC to the VTA were androgen receptor immunopositive [43]. The pyramidal cells projecting to the VTA selectively synapse on dopaminergic neurons that project back to the mPFC and thereby influence the dopamine system in the mPFC [60, 61]. Based on these observations it is possible that gonadal hormones are acting through hormone receptors in the mPFC to indirectly influence the prefrontal dopaminergic system.

Prior research has reported hemispheric asymmetries in the prefrontal dopaminergic system of adult animals in which the left hemisphere in both sexes has greater levels of dopamine than the right hemisphere [62, 63]. The current study examined the density of tyrosine hydroxylase in the right hemisphere of all subjects, and it is unknown if the results from the right hemisphere would generalize to the left in either sex. Future research should investigate the possibility of hemispheric differences in this measure during aging. In addition, the loss in tyrosine hydroxylase fibers in males was specific to layers 2/3 of the prefrontal cortex. Prior research has shown differences in dopamine fiber and D1 receptor density between the superficial and deeper layers of the mPFC in adult animals with greater densities found in the deeper layers [64, 65]. However, few studies have examined the individual layers of the mPFC and it is unknown if the age-related changes in dopaminergic function previously discussed were layer specific.

Furthermore, because hormones are thought to be influencing the density of TH fibers during aging, the localization and density of hormone receptors during aging may also influence the pattern of loss observed in the current study. For example, although research indicates that androgen receptors decrease during aging in the cortex of male mice [55], it is currently unknown if this loss occurs at the same rate in each of the layers in the rat mPFC. Therefore it is possible that the layer specificity of the loss in TH fibers observed in the current study relates to layer specific changes in dopamine or gonadal hormone receptors during aging.

5. Conclusions

The age-related behavioral deficits and the decreases in TH found in the current study support the hypothesis that changes in dopaminergic functioning in the mPFC contribute to cognitive decline during aging. Furthermore, they suggest that the prefrontal dopaminergic system and behaviors mediated by the PFC are less affected by aging in female rats than in males. The mechanisms underlying these sex differences in aging, most likely mediated by gonadal hormones, need further investigation.

Figure 7.

Figure 7

Open in a new tab

The density of TH fibers in layers 5/6 (mean+ SEM). In skeletonized images (A), there was an effect of sex (p < 0.05). In binary images (B), there was a trend for an effect of sex (p < 0.07).

Highlights.

The effects of sex and age on delayed alternation and dopamine fibers were examined.

Behavioral sex differences present in adult animals were reversed during aging.

Males, but not females, experienced a cognitive decline during aging.

Males, but not females, lost tyrosine hydroxylase fibers in the mPFC during aging.

Aged females had greater tyrosine hydroxylase fiber densities than aged males.

Acknowledgements

We would like to thank Pul Park and Jin Sung Yoon, as well as the staff in the Microscopy Suite in the Beckman Institute. Supported by NIA AG 022499

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Pardo JV, Lee JT, Sheikh SA, Surerus-Johnson C, Shah H, Munch KR, Carlis JV, Lewis SM, Kuskowski MA, Dysken MW. Where the brain grows old: Decline in anterior cingulate and medial prefrontal function with normal aging. Neuroimage. 2007;35:1231–1237. doi: 10.1016/j.neuroimage.2006.12.044. doi: S1053-8119(07)00002-X [pii]; 10.1016/j.neuroimage.2006.12.044 [doi]. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Raz N, Lindenberger U, Rodrigue KM, Kennedy KM, Head D, Williamson A, Dahle C, Gerstorf D, Acker JD. Regional brain changes in aging healthy adults: general trends, individual differences and modifiers. Cereb Cortex. 2005;15:1676–1689. doi: 10.1093/cercor/bhi044. doi: 10.1093/cercor/bhi044. [DOI] [PubMed] [Google Scholar]
  • 3.Resnick SM, Pham DL, Kraut MA, Zonderman AB, Davatzikos C. Longitudinal magnetic resonance imaging studies of older adults: a shrinking brain. J Neurosci. 2003;23:3295–3301. doi: 10.1523/JNEUROSCI.23-08-03295.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.de Brabander JM, Kramers RJ, Uylings HB. Layer-specific dendritic regression of pyramidal cells with ageing in the human prefrontal cortex. Eur J Neurosci. 1998;10:1261–1269. doi: 10.1046/j.1460-9568.1998.00137.x. [DOI] [PubMed] [Google Scholar]
  • 5.Huttenlocher PR. Synaptic density in human frontal cortex - developmental changes and effects of aging. Brain Res. 1979;163:195–205. doi: 10.1016/0006-8993(79)90349-4. [DOI] [PubMed] [Google Scholar]
  • 6.Jacobs B, Driscoll L, Schall M. Life-span dendritic and spine changes in areas 10 and 18 of human cortex: a quantitative Golgi study. J Comp Neurol. 1997;386:661–680. [PubMed] [Google Scholar]
  • 7.Masliah E, Mallory M, Hansen L, DeTeresa R, Terry RD. Quantitative synaptic alterations in the human neocortex during normal aging. Neurology. 1993;43:192–197. doi: 10.1212/wnl.43.1_part_1.192. [DOI] [PubMed] [Google Scholar]
  • 8.Grill JD, Riddle DR. Age-related and laminar-specific dendritic changes in the medial frontal cortex of the rat. Brain Res. 2002;937:8–21. doi: 10.1016/s0006-8993(02)02457-5. [DOI] [PubMed] [Google Scholar]
  • 9.Markham JA, Juraska JM. Aging and sex influence the anatomy of the rat anterior cingulate cortex. Neurobiol Aging. 2002;23:579–588. doi: 10.1016/s0197-4580(02)00004-0. [DOI] [PubMed] [Google Scholar]
  • 10.Wallace M, Frankfurt M, Arellanos A, Inagaki T, Luine V. Impaired recognition memory and decreased prefrontal cortex spine density in aged female rats. Ann N Y Acad Sci. 2007;1097:54–57. doi: 10.1196/annals.1379.026. doi: 1097/1/54 [pii]; 10.1196/annals.1379.026 [doi] [DOI] [PubMed] [Google Scholar]
  • 11.Stuss DT, Benson DF. Neuropsychological studies of the frontal lobes. Psychol Bull. 1984;95:3–28. [PubMed] [Google Scholar]
  • 12.Mizoguchi K, Shoji H, Tanaka Y, Maruyama W, Tabira T. Age-related spatial working memory impairment is caused by prefrontal cortical dopaminergic dysfunction in rats. Neuroscience. 2009 doi: 10.1016/j.neuroscience.2009.05.023. doi: 10.1016/j.neuroscience.2009.05.023. [DOI] [PubMed] [Google Scholar]
  • 13.Ando S, Ohashi Y. Longitudinal study on age-related changes of working and reference memory in the rat. Neurosci Lett. 1991;128:17–20. doi: 10.1016/0304-3940(91)90750-n. doi: 0304-3940(91)90750-N [pii] [DOI] [PubMed] [Google Scholar]
  • 14.Barense MD, Fox MT, Baxter MG. Aged rats are impaired on an attentional set-shifting task sensitive to medial frontal cortex damage in young rats. Learn Mem. 2002;9:191–201. doi: 10.1101/lm.48602. doi: 10.1101/lm.48602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Backman L, Lindenberger U, Li SC, Nyberg L. Linking cognitive aging to alterations in dopamine neurotransmitter functioning: recent data and future avenues. Neurosci Biobehav Rev. 2010;34:670–677. doi: 10.1016/j.neubiorev.2009.12.008. doi: 10.1016/j.neubiorev.2009.12.008. [DOI] [PubMed] [Google Scholar]
  • 16.Juraska JM, Lowry NC. Neuroanatomical changes associated with cognitive aging. Curr Top Behav Neurosci. 2012;10:137–162. doi: 10.1007/7854_2011_137. doi: 10.1007/7854_2011_137. [DOI] [PubMed] [Google Scholar]
  • 17.Suhara T, Fukuda H, Inoue O, Itoh T, Suzuki K, Yamasaki T, Tateno Y. Age-related changes in human D1 dopamine receptors measured by positron emission tomography. Psychopharmacology (Berl) 1991;103:41–45. doi: 10.1007/BF02244071. [DOI] [PubMed] [Google Scholar]
  • 18.de Keyser J, De Backer JP, Vauquelin G, Ebinger G. The effect of aging on the D1 dopamine receptors in human frontal cortex. Brain Res. 1990;528:308–310. doi: 10.1016/0006-8993(90)91672-4. [DOI] [PubMed] [Google Scholar]
  • 19.Inoue M, Suhara T, Sudo Y, Okubo Y, Yasuno F, Kishimoto T, Yoshikawa K, Tanada S. Age-related reduction of extrastriatal dopamine D2 receptor measured by PET. Life Sci. 2001;69:1079–1084. doi: 10.1016/s0024-3205(01)01205-x. [DOI] [PubMed] [Google Scholar]
  • 20.Kaasinen V, Kemppainen N, Nagren K, Helenius H, Kurki T, Rinne JO. Age-related loss of extrastriatal dopamine D(2) -like receptors in women. J Neurochem. 2002;81:1005–1010. doi: 10.1046/j.1471-4159.2002.00895.x. [DOI] [PubMed] [Google Scholar]
  • 21.Kaasinen V, Vilkman H, Hietala J, Nagren K, Helenius H, Olsson H, Farde L, Rinne J. Age-related dopamine D2/D3 receptor loss in extrastriatal regions of the human brain. Neurobiol Aging. 2000;21:683–688. doi: 10.1016/s0197-4580(00)00149-4. [DOI] [PubMed] [Google Scholar]
  • 22.Ota M, Yasuno F, Ito H, Seki C, Nozaki S, Asada T, Suhara T. Age-related decline of dopamine synthesis in the living human brain measured by positron emission tomography with L-[beta-11C]DOPA. Life Sci. 2006;79:730–736. doi: 10.1016/j.lfs.2006.02.017. doi: 10.1016/j.lfs.2006.02.017. [DOI] [PubMed] [Google Scholar]
  • 23.Uylings HB, Groenewegen HJ, Kolb B. Do rats have a prefrontal cortex? Behav Brain Res. 2003;146:3–17. doi: 10.1016/j.bbr.2003.09.028. [DOI] [PubMed] [Google Scholar]
  • 24.Goldman-Rakic PS, Brown RM. Regional changes of monoamines in cerebral cortex and subcortical structures of aging rhesus monkeys. Neuroscience. 1981;6:177–187. doi: 10.1016/0306-4522(81)90053-1. doi: 0306-4522(81)90053-1 [pii] [DOI] [PubMed] [Google Scholar]
  • 25.Lacreuse A, Kim CB, Rosene DL, Killiany RJ, Moss MB, Moore TL, Chennareddi L, Herndon JG. Sex age, and training modulate spatial memory in the rhesus monkey (Macaca mulatta) Behav Neurosci. 2005;119:118–126. doi: 10.1037/0735-7044.119.1.118. doi: 10.1037/0735-7044.119.1.118. [DOI] [PubMed] [Google Scholar]
  • 26.Lacreuse A, Herndon JG, Killiany RJ, Rosene DL, Moss MB. Spatial cognition in rhesus monkeys: male superiority declines with age. Horm Behav. 1999;36:70–76. doi: 10.1006/hbeh.1999.1532. doi: 10.1006/hbeh.1999.1532. [DOI] [PubMed] [Google Scholar]
  • 27.Lukoyanov NV, Andrade JP, Dulce Madeira M, Paula-Barbosa MM. Effects of age and sex on the water maze performance and hippocampal cholinergic fibers in rats. Neurosci Lett. 1999;269:141–144. doi: 10.1016/s0304-3940(99)00442-5. [DOI] [PubMed] [Google Scholar]
  • 28.Yates MA, Markham JA, Anderson SE, Morris JR, Juraska JM. Regional variability in age-related loss of neurons from the primary visual cortex and medial prefrontal cortex of male and female rats. Brain Res. 2008;1218:1–12. doi: 10.1016/j.brainres.2008.04.055. doi: 10.1016/j.brainres.2008.04.055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Frick KM, Burlingame LA, Delaney SS, Berger-Sweeney J. Sex differences in neurochemical markers that correlate with behavior in aging mice. Neurobiol Aging. 2002;23:145–158. doi: 10.1016/s0197-4580(01)00237-8. [DOI] [PubMed] [Google Scholar]
  • 30.Luine VN, Renner KJ, Heady S, Jones KJ. Age and sex-dependent decreases in ChAT in basal forebrain nuclei. Neurobiol Aging. 1986;7:193–198. doi: 10.1016/0197-4580(86)90042-4. [DOI] [PubMed] [Google Scholar]
  • 31.Tanila H, Taira T, Piepponen TP, Honkanen A. Effect of sex and age on brain monoamines and spatial learning in rats. Neurobiol Aging. 1994;15:733–741. doi: 10.1016/0197-4580(94)90056-6. [DOI] [PubMed] [Google Scholar]
  • 32.Veng LM, Granholm AC, Rose GM. Age-related sex differences in spatial learning and basal forebrain cholinergic neurons in F344 rats. Physiol Behav. 2003;80:27–36. doi: 10.1016/s0031-9384(03)00219-1. [DOI] [PubMed] [Google Scholar]
  • 33.McDermott JL, Dluzen DE. Aging and sex differences in striatal dopaminergic function. Neuroscience. 2007;149:401–408. doi: 10.1016/j.neuroscience.2007.06.058. doi: 10.1016/j.neuroscience.2007.06.058. [DOI] [PubMed] [Google Scholar]
  • 34.Luine V, Bowling D, Hearns M. Spatial memory deficits in aged rats: contributions of monoaminergic systems. Brain Res. 1990;537:271–278. doi: 10.1016/0006-8993(90)90368-l. doi: 0006-8993(90)90368-L [pii] [DOI] [PubMed] [Google Scholar]
  • 35.Kritzer MF. Long-term gonadectomy affects the density of tyrosine hydroxylase-but not dopamine-beta-hydroxylase-, choline acetyltransferase- or serotonin-immunoreactive axons in the medial prefrontal cortices of adult male rats. Cereb Cortex. 2003;13:282–296. doi: 10.1093/cercor/13.3.282. [DOI] [PubMed] [Google Scholar]
  • 36.Kritzer MF, Kohama SG. Ovarian hormones differentially influence immunoreactivity for dopamine beta-hydroxylase, choline acetyltransferase, and serotonin in the dorsolateral prefrontal cortex of adult rhesus monkeys. J Comp Neurol. 1999;409:438–451. doi: 10.1002/(sici)1096-9861(19990705)409:3<438::aid-cne8>3.0.co;2-5. [DOI] [PubMed] [Google Scholar]
  • 37.Kritzer MF, Kohama SG. Ovarian hormones influence the morphology, distribution, and density of tyrosine hydroxylase immunoreactive axons in the dorsolateral prefrontal cortex of adult rhesus monkeys. J Comp Neurol. 1998;395:1–17. doi: 10.1002/(SICI)1096-9861(19980525)395:1<1::AID-CNE1>3.0.CO;2-4 [pii] [PubMed] [Google Scholar]
  • 38.Wang C, Leung A, Sinha-Hikim AP. Reproductive aging in the male brown-Norway rat: a model for the human. Endocrinology. 1993;133:2773–2781. doi: 10.1210/endo.133.6.8243304. [DOI] [PubMed] [Google Scholar]
  • 39.Chisholm NC, Packard AR, Koss WA, Juraska JM. The effects of long-term treatment with estradiol and medroxyprogesterone acetate on tyrosine hydroxylase fibers and neuron number in the medial prefrontal cortex of aged female rats. 2012;153 doi: 10.1210/en.2012-1412. doi: 10.1210/en.2012-1412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Dudley SD. Responsiveness to estradiol in central nervous system of aging female rats. Neurosci Biobehav Rev. 1982;6:39–45. doi: 10.1016/0149-7634(82)90005-7. [DOI] [PubMed] [Google Scholar]
  • 41.Shughrue PJ, Lane MV, Merchenthaler I. Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. J Comp Neurol. 1997;388:507–525. doi: 10.1002/(sici)1096-9861(19971201)388:4<507::aid-cne1>3.0.co;2-6. [DOI] [PubMed] [Google Scholar]
  • 42.Maggi A, Zucchi I. Sexual differentiation of mammalian frontal cortex. Life Sci. 1987;40:1155–1160. doi: 10.1016/0024-3205(87)90233-5. [DOI] [PubMed] [Google Scholar]
  • 43.Aubele T, Kritzer MF. Androgen influence on prefrontal dopamine systems in adult male rats: localization of cognate intracellular receptors in medial prefrontal projections to the ventral tegmental area and effects of gonadectomy and hormone replacement on glutamate-stimulated extracellular dopamine level. Cereb Cortex. 2012;22:1799–1812. doi: 10.1093/cercor/bhr258. doi: 10.1093/cercor/bhr258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Kritzer MF, McLaughlin PJ, Smirlis T, Robinson JK. Gonadectomy impairs T-maze acquisition in adult male rats. Horm Behav. 2001;39:167–174. doi: 10.1006/hbeh.2001.1645. doi: 10.1006/hbeh.2001.1645 [doi]: S0018-506X(01)91645-9 [pii] [DOI] [PubMed] [Google Scholar]
  • 45.Gibbs RB, Johnson DA. Sex-specific effects of gonadectomy and hormone treatment on acquisition of a 12-arm radial maze task by Sprague Dawley rats. Endocrinology. 2008;149:3176–3183. doi: 10.1210/en.2007-1645. doi: 10.1210/en.2007-1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Bimonte-Nelson HA, Singleton RS, Nelson ME, Eckman CB, Barber J, Scott TY, Granholm AC. Testosterone, but not nonaromatizable dihydrotestosterone, improves working memory and alters nerve growth factor levels in aged male rats. Exp Neurol. 2003;181:301–312. doi: 10.1016/s0014-4886(03)00061-x. doi: S001448860300061X [pii]. [DOI] [PubMed] [Google Scholar]
  • 47.Frick KM. Estrogens and age-related memory decline in rodents: what have we learned and where do we go from here? Horm Behav. 2009;55:2–23. doi: 10.1016/j.yhbeh.2008.08.015. doi: 10.1016/j.yhbeh.2008.08.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Gibbs RB. Long-term treatment with estrogen and progesterone enhances acquisition of a spatial memory task by ovariectomized aged rats. Neurobiol Aging. 2000;21:107–116. doi: 10.1016/s0197-4580(00)00103-2. doi: S0197-4580(00)00103-2 [pii]. [DOI] [PubMed] [Google Scholar]
  • 49.Miller MM, Hyder SM, Assayag R, Panarella SR, Tousignant P, Franklin KB. Estrogen modulates spontaneous alternation and the cholinergic phenotype in the basal forebrain. Neuroscience. 1999;91:1143–1153. doi: 10.1016/s0306-4522(98)00690-3. [DOI] [PubMed] [Google Scholar]
  • 50.Chisholm NC, Juraska JM. Long-term replacement of estrogen in combination with medroxyprogesterone acetate improves acquisition of an alternation task in middle-aged female rats. Behav Neurosci. 2012;126:128–136. doi: 10.1037/a0026461. doi: 10.1037/a0026461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Floresco SB, Magyar O. Mesocortical dopamine modulation of executive functions: beyond working memory. Psychopharmacology (Berl) 2006;188:567–585. doi: 10.1007/s00213-006-0404-5. doi: 10.1007/s00213-006-0404-5. [DOI] [PubMed] [Google Scholar]
  • 52.Hao J, Rapp PR, Leffler AE, Leffler SR, Janssen WG, Lou W, McKay H, Roberts JA, Wearne SL, Hof PR, Morrison JH. Estrogen alters spine number and morphology in prefrontal cortex of aged female rhesus monkeys. J Neurosci. 2006;26:2571–2578. doi: 10.1523/JNEUROSCI.3440-05.2006. doi: 26/9/2571 [pii]: 10.1523/JNEUROSCI.3440-05.2006 [doi] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Rapp PR, Morrison JH, Roberts JA. Cyclic estrogen replacement improves cognitive function in aged ovariectomized rhesus monkeys. J Neurosci. 2003;23:5708–5714. doi: 10.1523/JNEUROSCI.23-13-05708.2003. doi: 23/13/5708 [pii] [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Chisholm NC, Juraska JM. Effects of long-term treatment with estrogen and medroxyprogesterone acetate on synapse number in the medial prefrontal cortex of aged female rats. Menopause. 2012;19:804–811. doi: 10.1097/gme.0b013e31824d1fc4. doi: 10.1097/gme.0b013e31824d1fc4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Thakur MK, Asaithambi A, Mukherjee S. Synthesis and phosphorylation of androgen receptor of the mouse brain cortex and their regulation by sex steroids during aging. Mol Cell Biochem. 2000;203:95–101. doi: 10.1023/a:1007064307220. [DOI] [PubMed] [Google Scholar]
  • 56.Goudsmit E, Van de Poll NE, Swaab DF. Testosterone fails to reverse spatial memory decline in aged rats and impairs retention in young and middle-aged animals. Behav Neural Biol. 1990;53:6–20. doi: 10.1016/0163-1047(90)90729-p. [DOI] [PubMed] [Google Scholar]
  • 57.Chambers KC, Thornton JE, Roselli CE. Age-related deficits in brain androgen binding and metabolism, testosterone, and sexual behavior of male rats. Neurobiol Aging. 1991;12:123–130. doi: 10.1016/0197-4580(91)90050-t. [DOI] [PubMed] [Google Scholar]
  • 58.Cui R, Zhang G, Kang Y, Cheng Q, Tan H, Cui H, Shi G. Amelioratory effects of testosterone propionate supplement on behavioral, biochemical and morphological parameters in aged rats. Exp Gerontol. 2012;47:67–76. doi: 10.1016/j.exger.2011.10.009. doi: 10.1016/j.exger.2011.10.009. [DOI] [PubMed] [Google Scholar]
  • 59.Mitra SW, Hoskin E, Yudkovitz J, Pear L, Wilkinson HA, Hayashi S, Pfaff DW, Ogawa S, Rohrer SP, Schaeffer JM, McEwen BS, Alves SE. Immunolocalization of estrogen receptor beta in the mouse brain: comparison with estrogen receptor alpha. Endocrinology. 2003;144:2055–2067. doi: 10.1210/en.2002-221069. [DOI] [PubMed] [Google Scholar]
  • 60.Carr DB, Sesack SR. Projections from the rat prefrontal cortex to the ventral tegmental area: target specificity in the synaptic associations with mesoaccumbens and mesocortical neurons. J Neurosci. 2000;20:3864–3873. doi: 10.1523/JNEUROSCI.20-10-03864.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Gariano RF, Groves PM. Burst firing induced in midbrain dopamine neurons by stimulation of the medial prefrontal and anterior cingulate cortices. Brain Res. 1988;462:194–198. doi: 10.1016/0006-8993(88)90606-3. [DOI] [PubMed] [Google Scholar]
  • 62.Sullivan RM, Duchesne A, Hussain D, Waldron J, Laplante F. Effects of unilateral amygdala dopamine depletion on behaviour in the elevated plus maze: role of sex, hemisphere and retesting. Behav Brain Res. 2009;205:115–122. doi: 10.1016/j.bbr.2009.07.023. doi: 10.1016/j.bbr.2009.07.023. [DOI] [PubMed] [Google Scholar]
  • 63.Duchesne A, Dufresne MM, Sullivan RM. Sex differences in corticolimbic dopamine and serotonin systems in the rat and the effect of postnatal handling. Prog Neuropsychopharmacol Biol Psychiatry. 2009;33:251–261. doi: 10.1016/j.pnpbp.2008.11.012. doi: 10.1016/j.pnpbp.2008.11.012. [DOI] [PubMed] [Google Scholar]
  • 64.Lu XY, Churchill L, Kalivas PW. Expression of D1 receptor mRNA in projections from the forebrain to the ventral tegmental area. Synapse. 1997;25:205–214. doi: 10.1002/(SICI)1098-2396(199702)25:2<205::AID-SYN11>3.0.CO;2-X. doi: 2-X. [DOI] [PubMed] [Google Scholar]
  • 65.Van De Werd HJ, Rajkowska G, Evers P, Uylings HB. Cytoarchitectonic and chemoarchitectonic characterization of the prefrontal cortical areas in the mouse. Brain Struct Funct. 2010;214:339–353. doi: 10.1007/s00429-010-0247-z. doi: 10.1007/s00429-010-0247-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES