BACKGROUND
nerve cells require highly complex mechanisms of ca2+ regulation since a precise intracellular ca2+ concentration is needed for the appropriate development and function of these cells.
plasma membrane ca2+-atpases  are major components of this regulation.
they hydrolyze atp in order to get the energy to transport ca2+ from the cytoplasm to the extracellular media across the plasma membrane.
the presence of four main isoforms  encoded by four genes has been shown widely distributed in most eukaryotic cells  <cit> .
therefore, a strong selective pressure has existed to keep all four isoforms through evolution.
conserved domains among pmca isoforms correspond to essential transport functions, whereas high diversity domains concern regulatory and functional specialization of each isoform  <cit> .
the association between tissue expression and specific cellular functions seems to be the key of this diversity.
during neural development, the maintenance of resting cytosolic ca2+ at nm levels is critical for correct differentiation, dendritic growing and neural maturation  <cit> , and pmcas are major players in this function.
we have reported in a previous study the specific distribution of pmca isoforms linked to specific cell types and maturation cell stages during prenatal development in chick cerebellum  <cit> .
however, the widespread use of mice as a reference laboratory animal, which presents a mainly postnatal brain development, motivated us to investigate the functional and spatiotemporal expression patterns of these proteins in the mouse brain.
these aims contribute to fully understand the ca2+ metabolism in mouse brain during development.
although the mrna distribution of pmca isoforms in developing mouse has been reported by in situ hybridization  <cit> , a study at the protein level as well as at the functional level in different areas of developing mouse brain was still lacking.
in this work, we have analyzed the atpase activity and isoforms expression of pmca during postnatal neural development in the mouse.
these results will contribute to a better understanding of the specific role of these proteins in critical moments of ca2+ homeostasis during brain development.
RESULTS
functional profile of pmca in different brain areas during development
the pmca protein showed the capacity to catalyze atp-hydrolysis in a ca2+-dependent manner from the first stage in all areas .
besides, the pmca activity during postnatal development followed two patterns, reaching the vmax at p <dig> in the cortex and at p <dig> stage in hippocampus and cerebellum.
although the values of vmax were similar in all regions  the increase of activity was more pronounced  in cortex and hippocampus than in cerebellum .
protein expression levels of pmca isoforms in developing cortex, hippocampus and cerebellum
the expression of pmca isoforms during development was examined in the mv by western blot using isoform-specific antibodies .
the anti-pmca <dig> antibody stained two protein bands whose expression progressively increased with development until p <dig>  in the three analyzed regions.
the intensity of the lower band was higher in cortex and hippocampus, while in cerebellum both variants were similarly expressed.
these bands seem to correspond to pmca1a  and pmca1b  variants, according to filoteo et al.
<cit> .
the pmca <dig> antibody stained a spectrum of bands around 126- <dig> kda in the three regions, whose intensity progressively increased with developmental stage.
this was previously observed in developing chick cerebellum  <cit> .
the anti-pmca <dig> antibody recognized three main variants whose expression increased with development.
the anti-pmca <dig> reaction was very strong in the three regions and increased during development, detecting mainly a protein band that could correspond to variant pmca4b  and faintly the variant 4a  in hippocampus and cerebellum, according to filoteo et al.
<cit> .
immunoreaction with β-tubulin  was used as protein loading control for quantification .
overall, the largest increase in the expression of total pmca appeared at p <dig> stage in cortex and at p <dig> in the other regions , being in good agreement with the kinetic data.
isoform-specific localization of pmca pumps on sagittal sections of developing mouse brain
in order to address if changes in activity and expression levels of pmcas during development were associated with the maturation of specific cell types, we analyzed their localization in developing brain regions by immunohistochemistry .
the observed distribution was generally consistent with the temporal pattern given by western blotting.
in cerebral cortex , all pmca isoforms were expressed in neural precursors at p <dig>  but a more restricted distribution was observed with cell differentiation.
the pmca <dig> expression was comparatively lower at all stages.
at p <dig> stage, the four isoforms were highly expressed in the neuropil and also in the plasma membrane of the soma of pyramidal cells.
the cytoplasm of bodies and dendritic trunks were always pmca-negative.
in the hippocampal ammon's horn , the four isoforms were weakly stained in poor-defined structures at the earliest stages.
at p <dig>  cellular layers are already established and pmca <dig> and  <dig> were the highest expressed isoforms.
they were located in the plasma membrane of the soma of developing pyramidal neurons and showed an intense punctuate staining throughout the neuropil of stratum oriens and radiatus.
at p <dig>  all isoforms appeared with similar expression levels and distribution.
in contrast to the other regions, the developing cerebellum revealed clear differences of distribution among pmca isoforms .
thus at p <dig>  pmca <dig> and  <dig> showed a lower expression in cerebellar neural precursors than pmca <dig> and  <dig>  from p <dig> on, pmca <dig> and  <dig> were observed as intense punctuate staining in the neuropil within molecular layer.
pmca <dig> was distributed in similar cellular regions as pmca <dig> and  <dig>  being clearly located in the plasma membrane of the soma and dendritic tree of purkinje cells.
by contrast, pmca <dig> at p <dig> stage was mostly detected in the soma and primary dendritic trunk of purkinje cells.
in the granular layer, mature granule cells were pmca1- and pmca3-positives, while all isoforms seem to be expressed in cerebellar synaptic glomeruli.
to analyze in detail if pmca distribution in the neuropil and cerebellar glomeruli corresponds with a synapse distribution, double immunohistochemistry assays were performed in the three regions at p <dig>  using anti-pmca <dig> or anti-pmca <dig> antibodies  as template and the synaptic marker synaptophysin .
a clear and specific co-localization of pmcas with synaptophysin was observed, thus confirming the presence of these pumps in synaptic areas.
discussion
in this work we report the functional presence and immunolocalization of pmca isoforms during postnatal development of mouse cortex, hippocampus and cerebellum, three major regions implicated in processes of learning and stereotype movements.
the functional analysis of pmca activity revealed that these pumps are active from birth and they are upregulated during brain development.
the pmca activity was comparatively higher than the activities of the two intracellular ca2+ pumps, spca  <cit>  and serca  in developing mouse, pointing up the high contribution of pmcas to the atp-dependent ca2+ transport throughout development.
this early necessity of ca2+ regulation in neural precursors is in good agreement with that found in chick cerebellum during prenatal development  <cit> , proving the importance of ca2+ signaling in early events of neural development.
as in chick, pmca activity profile in mouse was closely related with the time-dependence of isoform expression.
we observed in cerebral cortex a fast increase of pmca activity during the first postnatal week, which is associated with a similar temporal augment of pmca1- <dig> isoform expression.
however, in hippocampus and cerebellum a slow increase in pmca activity was observed, also related to a later upregulation of pmca isoforms expression.
these differences reflect special ca2+ requirements during development of different neural regions.
the increase of pmca activity is accompanied by changes in isoform-expression and distribution, in correlation with functional cell maturation.
in fact, the major increase of pmca activity mainly occurs during the period of greatest synaptic development.
the presence of different pmcas in the neuropil from early developmental stages and the co-localization with the synaptophysin protein, point us to the contribution of these ca2+ pumps in the formation and maturity of synapses.
this synaptic distribution of pmcas has been also observed in developing and adult rat  <cit> , adult pig  <cit>  and developing chick  <cit> , indicating that their localization is conserved among species.
the expression of all pmca isoforms was quite similar in cortex and hippocampus, but differs from that in cerebellum.
thus, the mouse cerebellar pmca <dig> and  <dig> showed the same distribution pattern as in adult rat  <cit> , but differ from that found in developing chick  <cit>  where they are restricted to the soma and primary dendritic trunk of purkinje cells.
the cerebellar pmca <dig> was likewise localized in different species  <cit> , while pmca <dig> showed similar distribution to that observed for pmca <dig> and  <dig> in chick cerebellum  <cit> .
furthermore, the localization of pmca <dig> in developing mouse hippocampus differs from that reported in developing rat, where it was confined to the soma and dendrites of few scattered cells in ca <dig>  <cit> .
the mentioned differences could be due to species-dependent expression, or variation between pre- and post-natal neural development.
besides, recent studies in rat cerebellum report a postsynaptic localization for pmca <dig> and a presynatic location for pmca <dig>  <cit> , which support our results.
the precise targeting of each pmca isoform  to definite cellular areas and their overlapping localization in others, albeit at diverse expression levels, can be explained by different roles of pmca isoforms in neural ca2+ homeostasis.
in fact, specific spatio-temporal patterns of pmca isoforms have been also reported in other excitable tissues with different tasks, as developing retina  <cit> , and cochlea  <cit> .
on the other hand, pmca isoforms differ in their kinetics and affinity for ca2+ and calmodulin  <cit> , and are also differentially regulated by phosphorylation  <cit> , proteolysis  <cit> , or acidic phospholipids  <cit> .
thus, the expression in a cell type or subcellular region does not necessarily imply functional redundancy, since diverse ca2+ regulation exists in different compartments of the cell during development.
besides, other extrusion systems expressed in pmca-positive areas as the na+/ca2+ exchanger  <cit> , serca  <cit>  or spca  <cit> , also show upregulation during development.
moreover, each pmca isoform may be integrated in multiprotein complexes that include pmca-regulatory proteins, and that are involved in specific and localized ca2+ signals, e.g.
: it has been described that pmca <dig> and pmca <dig> can bind to na+/h+ exchanger regulatory factor  <cit>  or proteins of the membrane-associated guanylate kinase family  <cit> , respectively.
on the other hand, they can be differentially integrated in membrane sub-domains implicated in signal transduction  <cit> .
the presence of pmca isoforms in mouse cortex, hippocampus and cerebellum, from the earliest post-natal stages, and their differences in localization and content may well be associated to isoform-dependent roles in ca2+ signaling during neural development.
this is supported by studies performed in pc <dig> neural-cell type, where suppression of pmca <dig> and  <dig> expression slowed neurite extension and reduced cell survival  <cit> .
furthermore, recent observations of pmca isoform-expression changes in breast cancer cell lines  <cit>  and during cancer cell differentiation  <cit>  also support the importance of pmcas in cell differentiation.
, weak; +, present; ++, strong; +++, very strong.
CONCLUSIONS
the findings reported here show an upregulation of pmca activity and pmca isoform expression during brain development, related with a general coexpression of isoforms in cortical and hippocampal regions and different localization in cerebellum.
this study provides insights into the specific implications of pmca isoforms in ca2+ homeostasis during morphological and functional neuronal maturation.

 nerve cells require highly complex mechanisms of ca2+ regulation since a precise intracellular ca2+ concentration is needed for the appropriate development and function of these cells. plasma membrane ca2+-atpases are major components of this regulation. they hydrolyze atp in order to get the energy to transport ca2+ from the cytoplasm to the extracellular media across the plasma membrane. the presence of four main isoforms encoded by four genes has been shown widely distributed in most eukaryotic cells. therefore, a strong selective pressure has existed to keep all four isoforms through evolution. conserved domains among pmca isoforms correspond to essential transport functions, whereas high diversity domains concern regulatory and functional specialization of each isoform. the association between tissue expression and specific cellular functions seems to be the key of this diversity. during neural development, the maintenance of resting cytosolic ca2+ at nm levels is critical for correct differentiation, dendritic growing and neural maturation, and pmcas are major players in this function. we have reported in a previous study the specific distribution of pmca isoforms linked to specific cell types and maturation cell stages during prenatal development in chick cerebellum. however, the widespread use of mice as a reference laboratory animal, which presents a mainly postnatal brain development, motivated us to investigate the functional and spatiotemporal expression patterns of these proteins in the mouse brain. these aims contribute to fully understand the ca2+ metabolism in mouse brain during development. although the mrna distribution of pmca isoforms in developing mouse has been reported by in situ hybridization, a study at the protein level as well as at the functional level in different areas of developing mouse brain was still lacking. in this work, we have analyzed the atpase activity and isoforms expression of pmca during postnatal neural development in the mouse. these results will contribute to a better understanding of the specific role of these proteins in critical moments of ca2+ homeostasis during brain development.  functional profile of pmca in different brain areas during development the pmca protein showed the capacity to catalyze atp-hydrolysis in a ca2+-dependent manner from the first stage in all areas. besides, the pmca activity during postnatal development followed two patterns, reaching the vmax at p in the cortex and at p stage in hippocampus and cerebellum. although the values of vmax were similar in all regions the increase of activity was more pronounced in cortex and hippocampus than in cerebellum. protein expression levels of pmca isoforms in developing cortex, hippocampus and cerebellum the expression of pmca isoforms during development was examined in the mv by western blot using isoform-specific antibodies. the anti-pmca antibody stained two protein bands whose expression progressively increased with development until p in the three analyzed regions. the intensity of the lower band was higher in cortex and hippocampus, while in cerebellum both variants were similarly expressed. these bands seem to correspond to pmca1a and pmca1b variants, according to filoteo et al.. the pmca antibody stained a spectrum of bands around 126- kda in the three regions, whose intensity progressively increased with developmental stage. this was previously observed in developing chick cerebellum. the anti-pmca antibody recognized three main variants whose expression increased with development. the anti-pmca reaction was very strong in the three regions and increased during development, detecting mainly a protein band that could correspond to variant pmca4b and faintly the variant 4a in hippocampus and cerebellum, according to filoteo et al.. immunoreaction with β-tubulin was used as protein loading control for quantification. overall, the largest increase in the expression of total pmca appeared at p stage in cortex and at p in the other regions, being in good agreement with the kinetic data. isoform-specific localization of pmca pumps on sagittal sections of developing mouse brain in order to address if changes in activity and expression levels of pmcas during development were associated with the maturation of specific cell types, we analyzed their localization in developing brain regions by immunohistochemistry. the observed distribution was generally consistent with the temporal pattern given by western blotting. in cerebral cortex, all pmca isoforms were expressed in neural precursors at p but a more restricted distribution was observed with cell differentiation. the pmca expression was comparatively lower at all stages. at p stage, the four isoforms were highly expressed in the neuropil and also in the plasma membrane of the soma of pyramidal cells. the cytoplasm of bodies and dendritic trunks were always pmca-negative. in the hippocampal ammon's horn, the four isoforms were weakly stained in poor-defined structures at the earliest stages. at p cellular layers are already established and pmca and were the highest expressed isoforms. they were located in the plasma membrane of the soma of developing pyramidal neurons and showed an intense punctuate staining throughout the neuropil of stratum oriens and radiatus. at p all isoforms appeared with similar expression levels and distribution. in contrast to the other regions, the developing cerebellum revealed clear differences of distribution among pmca isoforms. thus at p pmca and showed a lower expression in cerebellar neural precursors than pmca and from p on, pmca and were observed as intense punctuate staining in the neuropil within molecular layer. pmca was distributed in similar cellular regions as pmca and being clearly located in the plasma membrane of the soma and dendritic tree of purkinje cells. by contrast, pmca at p stage was mostly detected in the soma and primary dendritic trunk of purkinje cells. in the granular layer, mature granule cells were pmca1- and pmca3-positives, while all isoforms seem to be expressed in cerebellar synaptic glomeruli. to analyze in detail if pmca distribution in the neuropil and cerebellar glomeruli corresponds with a synapse distribution, double immunohistochemistry assays were performed in the three regions at p using anti-pmca or anti-pmca antibodies as template and the synaptic marker synaptophysin. a clear and specific co-localization of pmcas with synaptophysin was observed, thus confirming the presence of these pumps in synaptic areas. discussion in this work we report the functional presence and immunolocalization of pmca isoforms during postnatal development of mouse cortex, hippocampus and cerebellum, three major regions implicated in processes of learning and stereotype movements. the functional analysis of pmca activity revealed that these pumps are active from birth and they are upregulated during brain development. the pmca activity was comparatively higher than the activities of the two intracellular ca2+ pumps, spca and serca in developing mouse, pointing up the high contribution of pmcas to the atp-dependent ca2+ transport throughout development. this early necessity of ca2+ regulation in neural precursors is in good agreement with that found in chick cerebellum during prenatal development, proving the importance of ca2+ signaling in early events of neural development. as in chick, pmca activity profile in mouse was closely related with the time-dependence of isoform expression. we observed in cerebral cortex a fast increase of pmca activity during the first postnatal week, which is associated with a similar temporal augment of pmca1- isoform expression. however, in hippocampus and cerebellum a slow increase in pmca activity was observed, also related to a later upregulation of pmca isoforms expression. these differences reflect special ca2+ requirements during development of different neural regions. the increase of pmca activity is accompanied by changes in isoform-expression and distribution, in correlation with functional cell maturation. in fact, the major increase of pmca activity mainly occurs during the period of greatest synaptic development. the presence of different pmcas in the neuropil from early developmental stages and the co-localization with the synaptophysin protein, point us to the contribution of these ca2+ pumps in the formation and maturity of synapses. this synaptic distribution of pmcas has been also observed in developing and adult rat, adult pig and developing chick, indicating that their localization is conserved among species. the expression of all pmca isoforms was quite similar in cortex and hippocampus, but differs from that in cerebellum. thus, the mouse cerebellar pmca and showed the same distribution pattern as in adult rat, but differ from that found in developing chick where they are restricted to the soma and primary dendritic trunk of purkinje cells. the cerebellar pmca was likewise localized in different species, while pmca showed similar distribution to that observed for pmca and in chick cerebellum. furthermore, the localization of pmca in developing mouse hippocampus differs from that reported in developing rat, where it was confined to the soma and dendrites of few scattered cells in ca. the mentioned differences could be due to species-dependent expression, or variation between pre- and post-natal neural development. besides, recent studies in rat cerebellum report a postsynaptic localization for pmca and a presynatic location for pmca, which support our results. the precise targeting of each pmca isoform to definite cellular areas and their overlapping localization in others, albeit at diverse expression levels, can be explained by different roles of pmca isoforms in neural ca2+ homeostasis. in fact, specific spatio-temporal patterns of pmca isoforms have been also reported in other excitable tissues with different tasks, as developing retina, and cochlea. on the other hand, pmca isoforms differ in their kinetics and affinity for ca2+ and calmodulin, and are also differentially regulated by phosphorylation, proteolysis, or acidic phospholipids. thus, the expression in a cell type or subcellular region does not necessarily imply functional redundancy, since diverse ca2+ regulation exists in different compartments of the cell during development. besides, other extrusion systems expressed in pmca-positive areas as the na+/ca2+ exchanger, serca or spca, also show upregulation during development. moreover, each pmca isoform may be integrated in multiprotein complexes that include pmca-regulatory proteins, and that are involved in specific and localized ca2+ signals, e.g.: it has been described that pmca and pmca can bind to na+/h+ exchanger regulatory factor or proteins of the membrane-associated guanylate kinase family, respectively. on the other hand, they can be differentially integrated in membrane sub-domains implicated in signal transduction. the presence of pmca isoforms in mouse cortex, hippocampus and cerebellum, from the earliest post-natal stages, and their differences in localization and content may well be associated to isoform-dependent roles in ca2+ signaling during neural development. this is supported by studies performed in pc neural-cell type, where suppression of pmca and expression slowed neurite extension and reduced cell survival. furthermore, recent observations of pmca isoform-expression changes in breast cancer cell lines and during cancer cell differentiation also support the importance of pmcas in cell differentiation., weak; +, present; ++, strong; +++, very strong.  the findings reported here show an upregulation of pmca activity and pmca isoform expression during brain development, related with a general coexpression of isoforms in cortical and hippocampal regions and different localization in cerebellum. this study provides insights into the specific implications of pmca isoforms in ca2+ homeostasis during morphological and functional neuronal maturation. 
overall, our findings support a close relationship between the ontogeny of pmca isoforms and specific requirements of ca2+ during development of different brain areas. immunohistochemistry assays in cortex and hippocampus showed co-expression of all isoforms in the neuropil associated with synapses and in the plasma membrane of pyramidal cells soma, while cerebellum showed a more isoform-specific distribution pattern in purkinje cells. pmca activity was detected from birth, with a faster evolution in cortex than in hippocampus and cerebellum. these results show an upregulation of pmca activity and pmca isoforms expression during brain development in mouse, with specific localizations mainly in cerebellum. considering the critical role of ca2+ signalling in neuronal development and plasticity, we have analyzed pmca-mediated ca2+-atpase activity and pmca-isoform content in membranes from mouse cortex, hippocampus and cerebellum during postnatal development. plasma membrane ca2+-atpases are high affinity ca2+ transporters actively involved in intracellular ca2+ homeostasis.
 plasma membrane ca2+-atpases are high affinity ca2+ transporters actively involved in intracellular ca2+ homeostasis. considering the critical role of ca2+ signalling in neuronal development and plasticity, we have analyzed pmca-mediated ca2+-atpase activity and pmca-isoform content in membranes from mouse cortex, hippocampus and cerebellum during postnatal development.  pmca activity was detected from birth, with a faster evolution in cortex than in hippocampus and cerebellum. western blots revealed the presence of the four isoforms in all regions, with similar increase in their expression patterns as those seen for the activity profile. immunohistochemistry assays in cortex and hippocampus showed co-expression of all isoforms in the neuropil associated with synapses and in the plasma membrane of pyramidal cells soma, while cerebellum showed a more isoform-specific distribution pattern in purkinje cells.  these results show an upregulation of pmca activity and pmca isoforms expression during brain development in mouse, with specific localizations mainly in cerebellum. overall, our findings support a close relationship between the ontogeny of pmca isoforms and specific requirements of ca2+ during development of different brain areas. 