Our observation of fairly abundant ERα immunoreactivity in cortical neurones of the mammalian PFC challenges the notion that ERα levels in this region are minimal or non-existent. The high density of ERα immunolabelled cells found in our study contrasts with the overall sparse ERα protein distribution found in some studies of rodent cortex (3
). However, our results are consistent with other immunolocalisation studies in rat cortex that find abundant and widespread ERα protein in neurones (4
). Furthermore, the detection of ERα protein in the human frontal cortex supports our previous demonstration of ERα mRNA in the human DLPFC by northern blotting, in situ
hybridisation, and nested RT-PCR followed by direct sequencing (16
). We demonstrated that ERα mRNA is commonly found in both pyramidal and nonpyramidal cortical neurones by in situ
). Although low to negligible levels of ERα mRNA have been reported in the rat medial PFC (17
), Pau et al.
), showed ERα mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR) in the adult frontal cortex of rhesus macaques. In addition, expression of ERα mRNA and protein in BA46 of ovarectomised and oestrogen replaced female rhesus macaques was detected by in situ
hybridisation and immunohistochemistry by members of our group (2
). Thus, although published reports differ regarding the distribution of ERα protein and mRNA in mammalian brain, many studies suggest that ERα protein present in the cortex.
The discrepancy between our finding of robust ERα protein and those of previous studies showing low levels of ERα mRNA and protein in cortex may result from methodological differences such as antibody specificity or concentration, riboprobe sensitivity, fixative choice and tissue preparation. For example, the concentration of primary antibody used may be critical in optimising ERα detection in fresh frozen tissue. Another methodological point to consider is that our immunohistochemical signal is not completely blocked by pre-incubation with ERα, suggesting that other proteins similar to ERα may contribute to the immunoreactive signal. Alternatively, incubating the primary antibody with high levels of the full-length ERα protein may artificially increase the background immunohistochemical signal. The ERα component of the anti-ERα: ERα protein complexes formed during preabsorption could be binding to other ERα binding partners such as steroid receptor coactivators in the tissue slice (11
) and this could increase the immunohistochemical signal at higher concentrations of ERα.
In the present study, we find robust ERα immunolabelling in the nucleus and cytoplasm. These results concur with earlier and recent reports of both nuclear and cytoplasmic ERα immunolabelling that can extend into the dendrites of brain neurones (2
). González et al.
) reported ERα immunoreactivity in nucleus and cytoplasm of pyramidal neurones in all layers of human adult temporal cortex and Mukai et al.
) showed that ERα was located in nuclei and cytoplasm within hippocampal neurones of rats. Consistent with other studies of rodent cortex (4
), we found prominent nuclear distribution of ERα in rat frontal cortex. Cytoplasmic expression of ERα protein has been previously observed in a number of species, including humans (28
), monkey (2
), and rat (30
). Experimental studies in rodents suggest that not only is ERα found in the spine heads and synaptic terminals of neurones, but also that this cytoplasmic subcellular localisation is influenced by the age and the oestrogen status of the animal (31
). Additionally, some variant forms of ERα mRNA have been detected in the human and monkey DLPFC by splice variant specific RT-PCR (18
). Some of these brain-detected variants are unable to translocate to the nucleus in transfected cells and would be expected to be exclusively cytoplasmic (19
). Taken together, our results suggest that ERα protein can be found in both the nucleus and cytoplasm in mammalian cortex and that the ERα protein distribution may vary from neurone to neurone within the cortex.
Our western blot analysis reveals multiple ERα immunoreactive bands with molecular weights that are consistent across the three species. In addition to the approximately 65 kDa ERα, immunopositive bands of molecular weight above and below the full-length ERα were also detected. The smaller immunoreactive bands at 49 and 55 kDa found in the frontal cortex of these three species are similar to that found in lysates from the MCF7 cell lines and normal tissue known to express splice variants of ERα mRNA (18
). It could be argued that the appearance of smaller molecular weight bands in human DLPFC are the result of protein degradation during extended post-mortem intervals. We believe that this is not the case in the present study because the monkey and rat cortical tissues were taken quickly after euthanasia and the same ERα immunoreactive smaller-sized bands were also recognised. Furthermore, smaller sized ERα bands were seen in the cell lysate preparations with no post-mortem interval. The immunoreactivity observed in the present study therefore may represent the expression of not only the classical ERα, but also smaller variant isoforms of ERα (32
). Larger ERα immunoreactive bands between 80 and 112 kDa have also been previously identified (28
). Various explanations may be offered for the appearance of these bands in western blots, including longer splice variants (34
), formation of homo- or heterodimers (35
), phosphorylation or ubiquitination (33
) and complexes of ERα with any of its possible binding partners (11
). Thus, we may expect to see not only the classical ERα 66 kDa immunoreactive band, but also additional isoforms.
As noted above, the identification of other isoforms or splice variants of ERα in native mammalian tissue is not a novel finding. Studies characterising the ERα gene report the production of several different mRNA transcripts in normal tissue (18
). The relevance of these alternate transcripts is suggested by observations that several of the proteins they encoded have been found in rat uterus and pituitary (36
). Some of the protein isoforms found in rat uterus and pituitary are of particular interest because they are within the size range of the ERα-like proteins identified in the present study. Furthermore, studies investigating the function of variant ERα isoforms in vitro
) suggest that the ERα-like proteins may be physiologically meaningful and therefore, may be relevant to oestrogenic action in PFC.
In the present study, we describe ERα immunoreactivity in males; however, we may expect similar findings in females. Indeed, cortical ERα immunoreactivity appears to be similar in male and female rodents (3
); sex differences were not found in cell number, intensity or distribution of ERα immunoreactivity in rodent hippocampus or in adult human temporal cortex (28
). Additionally, our previous ERα mRNA studies in human PFC found no gender difference in ERα mRNA levels (16
). Similarly, ERα mRNA expression in male and female rhesus macaque cortex were comparable (15
) and cortical ERα mRNA levels do not differ by gender using real-time PCR across the first month of postnatal life in mice (39
). Behaviourally, oestrogen or selective oestrogen receptor modulators appear to alter brain activation and/or enhance task performance during PFC-influenced behavioural paradigms in male and female humans (6
Although behavioural and anatomical studies have provided a basis for inferring direct oestrogen-mediated action in the PFC, the molecular mechanism for these actions (e.g. genomic versus nongenomic actions) is not fully understood. Our results suggest that neuronal ERα constitutes a viable molecular mediator of oestrogen action in the frontal cortex of mammals. ERα protein was found in all layers of mammalian prefrontal cortex, prominent in neurones (both pyramidal and nonpyramidal) and not clearly identified in astrocytes. Using parallel methodology, we have identified prominent ERα immunoreactivity in the DLPFC of humans and monkeys as well as in the medial PFC of rats. Our observation confirms an earlier report that also detected robust ERα immunolabelling throughout the cortex and highlights the possibility that differences in methodology impact ERα detection in brain (4
Although the effects of oestrogen can be mediated throughout oestrogen-sensitive neurotransmitter systems that innervate the PFC such as serotonin and dopamine (40
), our detection of prominent ERα suggests that oestrogen can have direct effects on cortical neurones. Indeed, oestrogen can alter spine number and spine morphology in frontal cortical pyramidal neurones (41
). However, the ER responsible for mediating oestrogen action in pyramidal neurones in the primate frontal cortex was not determined in that study. The classical mechanism of action for oestrogen is via its known intracellular receptors ERα and ERβ; either or both may be involved. In the rodent PFC, ERβ mRNA expression is detectable and could be expressed at higher levels than ERα mRNA (17
), although the absolute molar abundance of ERα and ERβ mRNA or protein in the mammalian frontal cortex is unknown. Thus, ERα and ERβ may co-contribute to oestrogen binding, although we cannot exclude the possibility that a novel ER transcript such as ER-X (43
) or GPR30 (44
) may play a role, particularly in nongenomic signalling.
The localisation of ERα in PFC suggests that oestrogen may work via its classical intracellular receptor (full-length and/or variant isoforms). The widespread subcellular distribution of these ERα proteins in neurones (i.e. localised in both the cytoplasm and nucleus) suggests diverse mechanisms of action. Expression in the nucleus suggests that ERα could serve in the classical sense, as a transcription factor that directly modulates gene expression activity at oestrogen response elements or indirectly modulates gene activation via interaction with other transcription factors. Localisation of ERα outside the nucleus suggests that ERα may be mediating some oestrogen-induced nongenomic efforts; for example, via the cAMP or similar pathway (35
). Moreover, the identification of possible ERα isoforms in the present study may confer very different biological effects from the full-length protein, as demonstrated in clonal cell lines (19
). Future studies aimed at examining the molecular mechanism of cortical ERα action in various mammalian species is expected to contribute significantly to our understanding of oestrogenic action in the regulation of cognition and affect in humans.