The results of the current experiments suggest that acute BPA exposure at a dose below the U.S.E.P.A. reference safe daily limit (U.S.E.P.A., 1993
) antagonizes memory consolidation in adult male rats. BPA impaired memory in both the OR and OP tasks, and was associated with lower densities of dendritic spines, from approximately 10-25%, in both CA1 and the mPFC. This is the first report showing that acute BPA administration in adult males induces memory deficits and is associated with reduced dendritic spine density. These results are in agreement with, and add to, previous studies that demonstrated BPA-induced memory impairment during development (Carr et al., 2003
; Goncalves et al., 2010
; Xu et al., 2010
) and adulthood (Inagaki et al., 2007
), and a blocking effect of BPA on estradiol and testosterone dependent formation of dendritic spine synapses in male and female rodents (MacLusky et al., 2005
; Leranth et al., 2008a
). We also identified a decrease in PSD-95 in the post synaptic density in the hippocampus and an increase in cytosolic pCREB in the mPFC in BPA treated males.
In this study we examined BPA effects on memory consolidation, a process that is believed to take place in the first two hours after acquisition of new knowledge and involves many cellular signaling pathways that result in synaptic changes. The association between memory and spine synapses has been supported in several studies (Leuner, Falduto & Shors, 2003
; Luine, Attalla, Mohan, Costa & Frankfurt, 2006
; Wallace, Luine, Arellanos & Frankfurt 2006
; Luine & Frankfurt, 2011
); however, the specific role of these morphological changes in memory consolidation processes is less clear. It has been shown that both estrogens and androgens can facilitate a rapid enhancement in synaptic density, in both the CA1 region of the hippocampus and mPFC in rodents (Hajszan, MacLusky, Johansen, Jordan & Leranth, 2007
; Leranth, Petnehazy & MacLusky, 2003
; Leranth, Hajszan & MacLusky, 2004
; MacLusky, Luine, Hajszan & Leranth, 2005
; Inagaki et al., 2007
), as well as in non-human primates (Hao et al., 2003
; Leranth, Shanabrough & Redmond 2002
). Moreover, it has been demonstrated that immediate post-training treatment with estrogens, but not a delayed treatment (1-4h post-training), can enhance memory across different tasks (Luine et al., 2003
; McGaugh, 2000
; Packard and Theather 1997a
; Rhodes and Frye, 2004
). Fewer studies have assessed gonadal hormone influences on male memory, but castration is associated with declines in spatial working memory, and both estrogens and androgens can restore performance in some, but not all, memory tasks (Luine, 2008
). Taken together, these studies strongly suggest that gonadal hormones are involved in memory consolidation processes by increasing synaptic density in CA1 and mPFC. In relation to BPA effects on spine synapses, a previous study showed that 300 μg/kg of BPA, given for 4 days, reduced spine synapses in the PFC and CA1 area of adult male rats by approximately 50% (Leranth et al, 2008b
). Moreover, MacLusky et al (2005)
, using a dose and time course of BPA treatment which is more similar to our current study (40 ug/kg of BPA given at the same time as 60 ug/kg of estradiol), showed a significant antagonism in estrogen-dependent spine synapse formation 30 min later in OVX female rats. Our study provides further understanding of the role of synaptic plasticity in memory consolidation processes. When memory consolidation was involved (experiment 1), dendritic spine density values were higher than when memory was not involved (experiment 2). These values were also higher than previously reported in morphological studies from our lab (Frankfurt et al., 2009
; Frankfurt et al., 2011
; Luine and Frankfurt, 2011
) in which memory consolidation was not involved. Thus, our study demonstrates, for the first time, that memory consolidation is consistent with higher dendritic spine density in both CA1 and mPFC, and that BPA can interfere with this increase. However, it is not clear whether the current behavioral and morphological changes involve action of BPA on estrogen or androgen receptors since BPA can antagonize hormone actions at both receptors (See Leranth et al, 2008b
; Wolstenholme, et al, 2011
for further discussion).
Another goal of this study was to elucidate possible mechanisms underlying the effects of BPA on dendritic spine density in the hippocampus and mPFC that may alter memory processes. Poimenova, Markaki, Rahiotis and Kitraki (2010)
demonstrated that BPA exposure during development led to increased corticosterone levels in mid-adolescent male rats following performance of the Y-maze task and impaired memory. One possible explanation for our results, therefore, is that the subjects had increased corticosterone levels following BPA exposure and memory task conditions, and therefore performed poorly at the memory challenge (T2). Indeed, several studies have shown that acute stress, and the resulting elevation in corticosterone levels, can impair memory in young adult male rats (Diamond et al, 1996
; Diamond, Park, Heman & Rose, 1999
; Diamond et al., 2006
; Woodson, Macintosh, Fleshner & Diamond 2003
; Sandi et al., 2005
; Park et al., 2006
; Park, Zoladz, Conrad, Fleshner & Diamond, 2008
), and block enhancement of dendritic spine density in the hippocampus (Diamond et al., 2006
). However, measurement of corticosterone levels in our study revealed no significant difference between the groups, ruling out the possibility that BPA impaired memory indirectly by elevating the levels of this stress-hormone.
A remaining caveat of the study is that BPA treatment led to increased exploration during T2 in the object recognition test and decreased exploration during T2 in the object placement test (). There were, however, no group differences in exploration during T1 which might influence performance in T2 (data not shown). It should be noted that BPA treatment was alternated among the subjects so that BPA treated subjects in the object recognition test were the controls for the object placement test. Thus, the exploration difference appears to be due to more exploration in half of the cohort and not to a BPA effect on exploration. In support, preliminary experiments for this study, conducted in other subjects, did not show effects of BPA on exploration in either T1 or T2 (Eilam-Stock and Luine, unpublished observation).
The rapid effect of gonadal hormones on memory and neural plasticity strongly suggest that these effects involve non-genomic mechanisms. Recent research revealed many possible pathways by which gonadal hormones might affects these processes, including interactions with pCREB (Abraham & Herbison, 2005
; Szego et al., 2006
; Wade & Dorsa, 2003
), BDNF (Scharfman & MacLusky, 2006
; Solum & Handa, 2002
), NMDAR-2b (Adams, Fink, Janssen, Shah & Morrison, 2004
; d’Anglemont de Tassigny et al., 2007
) and PSD-95 (Akama & McEwen, 2003
; d’Anglemont de Tassigny et al., 2007
; Li et al., 2004
). Since BPA is an endocrine disruptor with anti-estrogen/androgen properties, it might impair memory and synaptic plasticity processes by blocking proteins that are activated in cell-signaling pathways and memory consolidation. Data regarding the involvement of BPA in such mechanisms are very limited, though some studies suggest that BPA effects pCREB (Canesi, Betti, Lorusso, Ciacci & Gallo, 2005
; Quesada et al., 2002
), and NMDA receptor subunit NR2B (Xu et al., 2010a, b). In our experiment, we examined the levels of these two proteins, in addition to other memory process related proteins, in the hippocampus and the mPFC. No significant differences between groups in the levels of NMDA-R2B, BDNF, PKMζ, and AMPA-GluR2, in either the hippocampus or the PFC were found in the present study. However, significantly higher levels of PSD-95 in the post synaptic density fraction of the hippocampus of control subjects were found as compared to BPA-treated subjects. This finding is consistent with the BPA-dependent decrease in spine density and relevant to the memory impairments by BPA, since PSD-95 is believed to play an important role in synaptic plasticity by facilitating the binding of receptors and channels to the post-synaptic membrane (see Kim & Sheng, 2004
; van Zundert, Yoshii & Constantine-Paton, 2004
, for review). In addition, we found lower nuclear (not statistically significant) and higher cytosolic levels of pCREB in the PFC of BPA treated subjects. Since CREB is a transcription factor that is active in the cell’s nucleus when phosphorylated, it might be hypothesized that BPA is preventing translocation of pCREB to the nucleus and may also be secondarily altering transcription factor functions. The absence of significant effects on nuclear pCREB and other memory consolidation related proteins in this study, may be related to the specific time of sacrifice (40 minutes post injections and sample trials, T1, of the memory task). It is possible that some of these proteins are activated earlier or later in the memory consolidation process and that other BPA effects would be observed at other sacrifice times. Future studies employing a more detailed time course following the sample trial are needed in order to further clarify the underlying mechanisms of BPA’s actions.
The results of the present study suggest a relationship between memory performance and dendritic spine density in the hippocampus and mPFC. However, since animals were sacrificed 11 days after the last memory test, neuroanalysis cannot be directly correlated to memory performance. Even though we found altered neuronal processes after BPA exposure in animals that displayed BPA-induced memory impairment a few days prior to sacrifice, we cannot determine whether BPA actually blocked the formation of a new memory on the day of sacrifice. The rational for this design was our hypothesis that BPA interrupts memory consolidation processes rather than memory retrieval. Additionally, it is important to note that on the day of sacrifice, all subjects explored the objects for well over the 10 second threshold, making it likely that the animals indeed formed a new memory. This issue requires further investigation in order to determine whether the observed memory impairment was due to BPA interference with memory consolidation processes or memory retrieval.
Since BPA was administered subcutaneously, rather than orally, in the present study, one might argue that our results are not relevant to the actual risks of human exposure to BPA. In a preliminary study, however, Hajszan & Leranth (2010)
reported no differences between SC and oral administration of BPA on the percent of reduction in spine synapses in both the hippocampus and the PFC, and Taylor, Welshons & VomSaal (2008)
found no differences in circulating BPA following oral or SC dosing in neonatal mice. Therefore, SC injections seem to be viable and relevant for modeling potential effects of BPA in humans.
In conclusion, our findings demonstrate, for the first time, that BPA can impair memory in adult male rats and block synaptic plasticity processes. Moreover, our results suggest novel evidence for the underlying biomolecular mechanisms of BPA’s actions in memory-related areas of the brain. These results have significant clinical implications, since the BPA dose used in our experiments was 20% lower than the U.S. E.P.A. recommended safe daily limit (50 ug/kg/day). BPA is produced at an estimated rate of 6.5 billion pounds a year, and people are exposed to it on a daily basis. In Canada and the European Union, BPA has been eliminated from use in baby products, where its effects are most profound. In the United States, however, no such steps have been taken to reduce human exposure to BPA, and the status of BPA as an environmental hazard is still under debate. Our study affords a better understanding of the effects BPA exerts on memory, neural plasticity and their biochemical mechanisms, as well as its level of potential danger to society at large.