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The Expression Pattern of the Na(+) Sensor, Na(X) in the Hydromineral Homeostatic Network: A Comparative Study between the Rat and Mouse - PubMed

  • ️Sun Jan 01 2012

The Expression Pattern of the Na(+) Sensor, Na(X) in the Hydromineral Homeostatic Network: A Comparative Study between the Rat and Mouse

Benjamin Nehmé et al. Front Neuroanat. 2012.

Abstract

The Scn7a gene encodes for the specific sodium channel Na(X), which is considered a primary determinant of sodium sensing in the brain. Only partial data exist describing the Na(X) distribution pattern and the cell types that express Na(X) in both the rat and mouse brain. To generate a global view of the sodium detection mechanisms in the two rodent brains, we combined Na(X) immunofluorescence with fluorescent cell markers to map and identify the Na(X)-expressing cell populations throughout the network involved in hydromineral homeostasis. Here, we designed an anti-Na(X) antibody targeting the interdomain 2-3 region of the Na(X) channel's α-subunit. In both the rat and mouse, Na(X) immunostaining was colocalized with vimentin positive cells in the median eminence and with magnocellular neurons immunopositive for neurophysin associated with oxytocin or vasopressin in both the supraoptic and paraventricular nuclei. Na(X) immunostaining was also detected in neurons of the area postrema. In addition to this common Na(X) expression pattern, several differences in Na(X) immunostaining for certain structures and cell types were found between the rat and mouse. Na(X) was present in both NeuN and vimentin positive cells in the subfornical organ and the vascular organ of the lamina terminalis of the rat whereas Na(X) was only colocalized with vimentin positive cells in the mouse circumventricular organs. In addition, Na(X) immunostaining was specifically observed in NeuN immunopositive cells in the median preoptic nucleus of the rat. Overall, this study characterized the Na(X)-expressing cell types in the network controlling hydromineral homeostasis of the rat and mouse. Na(X) expression pattern was clearly different in the nuclei of the lamina terminalis of the rat and mouse, indicating that the mechanisms involved in systemic and central Na(+) sensing are specific to each rodent species.

Keywords: NaX channel; Scn7a; circumventricular organs; magnocellular neurons; sodium channel; sodium homeostasis.

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Figures

Figure 1
Figure 1

Synthetic peptide used to produce the anti-NaX antibody. (A) Representation of the partial nucleotide sequence of the NaX-ID2–3. The nucleotide sequence corresponds to the nucleic acid targeted for RT-PCR experiment carried out with the primers MOU66 and MOU59. The sequence of the synthetic peptide used for the rabbit immunization is highlighted in gray. (B) 12% acrylamide SDS-PAGE analysis characterizes the purification of the NaX-GST fusion protein. Lanes contain non-induced crude lysate (1), 0.1 mM IPTG-induced crude lysate (2) and purified 49 kDa NaX-GST fusion protein (3). M, protein size marker.

Figure 2
Figure 2

Specificity and validation of the anti-NaX antibody. (A) The immune serum collected from the rabbits was assessed by ELISA and revealed the presence of the NaX protein and not the GST tag. The microplate was coated with the NaX peptide (1 μg/well), incubated with pre-immune serum (dotted line), or double purified immune serum (black line: antibody titer >>1/512,000). To control purification efficiency, ELISA was also performed on GST protein with double purified immune serum (dashed line). The rabbit antibodies were revealed with goat anti-rabbit IgG, HRP conjugated secondary antibody and the absorbance was read at 450 nm. (B) The purified anti-NaX antibody (antibody concentration 1/250) was tested for immunohistochemical staining without (−) or with (+) preabsorption with the antigen peptide (50 μg/ml). Fluorescent immunostaining in the MnPO nucleus served as a positive control, whereas absence of immunostaining in the BST nucleus is a negative control. Scale bar: 200 μm (low magnification), 10 μm (high magnification). (C) The immunohistochemical staining was correlated with the presence of NaX mRNA revealed by RT-PCR experiment carried out on micropunched region of the rat MnPO and BST. Note that the expected size of amplified products was 883 bp for NaX and 200 bp for GAPDH (housekeeping gene). Commercial rat brain total RNA (Clontech Laboratories Inc.) was used as a positive control.

Figure 3
Figure 3

Distribution of NaX immunostaining in the median eminence of the rat and mouse. Schematic illustration of the median eminence (ME) of the rat (A1) and of the mouse (B1). ME was filled in red for better identification. f, fornix; 3V, third ventricle. Representative picture of the fluorescent NaX immunostaining (green) was obtained from the rat (A2) and from the mouse (B2). The cell type expressing NaX was identified using anti-GFAP and anti-vimentin fluorescent immunostaining (red). Scale bar: 100 μm. The arrow head points the top-right, the bottom-right, and the bottom-left corner of the inset, which represents a high magnification zone of the ME in the rat (A3) and in the mouse (B3); scale bar: 20 μm. Note that NaX staining is present in vimentin positive cells.

Figure 4
Figure 4

Distribution of NaX immunostaining in the vascular organ of the lamina terminalis of the rat. (A) Schematic illustration of the vascular organ of the lamina terminalis (OVLT) in the ventral forebrain. This small structure is filled in red for better visualization. ac, anterior commissure; CPu, caudate putamen; HDB, horizontal limb of the diagonal band; LV, lateral ventricle; MS, medial septum; ox, optic chiasm; 3V, third ventricle. (B) Representative picture of the fluorescent NaX immunostaining (green) was obtained from the rat. The OVLT region is delimited by the dashed line. 3V, third ventricle. The cell type expressing NaX was identified using anti-NeuN, anti-GFAP, and anti-vimentin fluorescent immunostaining (red); scale bar: 100 μm. (C) High magnification pictures of the staining merge; scale bar: 20 μm. The arrow head points the top-right corner (left panel) or the top-left corner (middle and right panel) of the enlarged area. Note that NaX staining is present in NeuN expressing cells and in vimentin positive cells.

Figure 5
Figure 5

Distribution of NaX immunostaining in the vascular organ of the lamina terminalis of the mouse. (A) Schematic illustration of the vascular organ of the lamina terminalis (OVLT) in the ventral forebrain of the mouse. The OVLT appears in red for better visualization. ac, anterior commissure; CPu, caudate putamen; HDB, horizontal limb of the diagonal band; LV, lateral ventricle; MS, medial septum; 3V, third ventricle. (B) Representative picture of the fluorescent NaX immunostaining (green) was obtained from the mouse. The OVLT region is delimited by the dashed line. 3V, third ventricle. The cell type expressing NaX was identified using anti-NeuN, anti-GFAP, and anti-vimentin fluorescent immunostaining (red); scale bar: 50 μm. (C) High magnification pictures of the staining merge; scale bar: 20 μm. The arrow head points the top-left corner of the enlarged zone. NaX staining is only present in vimentin positive cells.

Figure 6
Figure 6

Distribution of NaX immunostaining in the subfornical organ of the rat. (A) Schematic illustration of the subfornical organ (SFO) in the rat brain. The SFO appears in red for better visualization. cc, corpus callosum; CPu, caudate putamen; fi, fimbria hippocampus; ic, internal capsule; LV, lateral ventricle; 3V, third ventricle. (B) Representative picture of the fluorescent NaX immunostaining (green) was obtained from the rat. The cell type expressing NaX was identified using anti-NeuN, anti-GFAP, and anti-vimentin fluorescent immunostaining (red); scale bars: 50 μm. (C) Inset representing high magnification area of the SFO; scale bar: 20 μm. The enlarged zone is pointed by the arrow head (top-left corner of the inset). NaX staining is present in NeuN positive cells in the core of the SFO and in vimentin positive cells in the cell layer boarding the third ventricle.

Figure 7
Figure 7

Distribution of NaX immunostaining in the subfornical organ of the mouse. (A) Schematic illustration of the subfornical organ (SFO) in the mouse brain (red filling). CPu, caudate putamen; fi, fimbria hippocampus; ic, internal capsule; LV, lateral ventricle; 3V, third ventricle. (B) Representative picture of the fluorescent NaX immunostaining (green) was obtained from the mouse. The cell type expressing NaX was identified using anti-NeuN, anti-GFAP, and anti-vimentin fluorescent immunostaining (red); scale bars: 50 μm. (C) Inset representing high magnification zone of the SFO; scale bar: 20 μm. The detailed zone is pointed by the arrow head (bottom-left corner of the inset). NaX staining is only present in the vimentin positive cells boarding the third ventricle.

Figure 8
Figure 8

NaX immunostaining was restricted to the median preoptic nucleus of the rat. (A1) Schematic illustration of the median preoptic nucleus (MnPO) of the rat. The nucleus appears in red for better visualization. Note that the ventral part is separated from the dorsal part of the nucleus by the anterior commissure (ac). f, fornix; HDB, horizontal limb of the diagonal band; ic, internal capsule; LV, lateral ventricle; ox, optic chiasm; 3V, third ventricle. (A2) Representative picture of fluorescent NaX immunostaining (green) was obtained from the ventral part of the rat MnPO; scale bar: 50 μm. The cell type expressing NaX was identified using anti-NeuN fluorescent immunostaining (red). The arrow head points the top-right corner of the inset that represents a high magnification zone of the ventral MnPO; scale bar: 20 μm. Note the colocalization of the fluorescent NaX and NeuN immunostaining. (B1) Schematic illustration of the mouse MnPO (red filling). (B2) Representative picture showing that fluorescent NaX immunostaining was restricted to the cell layer boarding the third ventricle in the mouse; scale bar: 50 μm. The arrow head points the top-right corner of the inset that represents a high magnification zone of the ventral MnPO in the rat (A3) and in the mouse (B3); scale bar: 20 μm.

Figure 9
Figure 9

NaX is expressed in the magnocellular neuroendocrine cells of the rat and mouse supraoptic nucleus. (A) NaX mRNA expression was revealed by RT-PCR experiment carried out on micropunched region of the SON and PVN. Note that the expected size of the amplified products was 883 bp for NaX and 200 bp for GAPDH (housekeeping gene). Schematic illustration of the SON in the rat (B1) and mouse (C1). The SON appears in red for better visualization. Representative distribution of fluorescent NaX immunostaining (green) in the magnocellular neurons of the SON obtained from the rat (B2) and mouse (C2). The neurochemical content of the cells expressing NaX was identified with the anti-oxytocin neurophysin and the anti-vasopressin neurophysin fluorescent immunostaining (red); scale bars: 50 μm. The arrow head points the top-right or the bottom-left corner of the inset, which represents a high magnification zone of the rat SON (B3) and the mouse SON (C3); scale bar: 20 μm. Not that NaX immunostaining was present in both vasopressinergic and oxytocinergic magnocellular cells in the rat and mouse.

Figure 10
Figure 10

NaX is expressed in the magnocellular and parvocellular neuroendocrine cells of the rat and mouse paraventricular nucleus. Schematic illustration of the representative PVN sections in the rat (A1) and in the mouse (B1). The PVN divisions appear in red for better visualization. Representative distribution of fluorescent NaX immunostaining (green) in the magnocellular part (PaM) and in the parvocellular part (PaP) of the PVN obtained from the rat (A2) and mouse (B2). The neurochemical content of the cells expressing NaX was identified with the anti-oxytocin neurophysin and the anti-vasopressin neurophysin fluorescent immunostaining (red); scale bars: 50 μm. The arrow head points the top-right, the bottom-right, and the bottom-left corner of the inset, which represents a high magnification zone of the PVN in the rat (A3) and in the mouse (B3); scale bar: 20 μm. Not that NaX immunostaining was present in both vasopressinergic and oxytocinergic magnocellular cells in the rat and mouse.

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