Expression Pattern of the LacZ Reporter in Secretogranin III Gene-trapped Mice

Gene Trapping and SgIII-gtKO Mice

The gene trapping used in this study has been published elsewhere.^20,21 The structure of the gene-trapping cassette, its genomic insertion site, and the expression of a fusion gene of the LacZ reporter and neomycin-resistance genes (β-Geo) in SgIII-gtKO mice (B6;129S-Scg3^tm1Lex strain; The Jackson Laboratory, Bar Harbor, ME) are shown in Fig. 1. The SgIII-gtKO and WT mice were generated in the genetic background of the C57BL/6J strain by the intercrossing of heterozygous mice. The genotypes of the offspring were determined by amplifying the SgIII alleles and the gene-trapping cassette by polymerase chain reaction using the appropriate primers, as previously reported. ¹⁸ The mice were maintained on a 12-hr light/dark cycle and given free access to tap water and standard chow (CRF-1; Oriental Yeast, Osaka, Japan). All animal experiments were performed in compliance with relevant laws and the guidelines of the Care and Use of Laboratory Animals of the Research Council of Akita Prefectural University (approval number 17-03 and 18-02).

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Figure 1.

Structure of the SgIII gene trap vector and genomic insertion. (A) The gene trap vector was inserted on mouse chromosome 9, between exons 11 and 12 of the SgIII genome. ¹⁸ The gene-trapping cassette contains a splice-acceptor site (SA) positioned at the 5′ end of a promoterless reporter region, β-geo (a fusion of neomycin phosphotransferase and β-galactosidase), allowing the vector to be spliced to the endogenous gene with an internal ribosomal re-entry site (IRES) between the SA site and the reporter gene sequence. The puromycin resistance gene (Puro) is also expressed when the gene trap vector is inserted into an intron under the control of a constitutive phosphoglycerate kinase gene (Pgk) promoter, which is thought to trap a gene to be transcriptionally silent in mouse embryonic stem cells. As a result, the LacZ-reporter gene can be translated even when it is not fused to the trapped gene. ²¹ The black triangles in the cassette indicate the loxP sites. Abbreviations: LTR, retroviral long terminal repeat; SD, splice donor site; SgIII, secretogranin III. (B) Expression of β-Geo detected by immunoblotting using an anti-β-galactosidase antibody (upper panels). Equal amounts of protein were extracted from each tissue (20 μg) of WT or SgIII-gtKO mice. Loading controls were provided by α-tubulin immunoblotting (lower panels).

LacZ Staining

For LacZ staining, tissue processing was performed as described previously.^22,23 Five-month-old male mice (n=5) were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital (0.1 mg/g body weight) and fixed with 50 ml of ice-cold 3.8% formaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) via cardiac perfusion for 10 min after washing out the blood with physiological saline perfusion for 3 min. The brain, spinal cord, and peripheral nervous and endocrine tissues were removed and postfixed in 1.9% formaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) at 4C overnight. The isolated islets (as described below) were also fixed by immersion in 3.8% formaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) at 4C overnight. Fixed brains were embedded in 2% agarose in 0.05 M sodium phosphate buffer (pH 7.4) and sliced coronally on a Microslicer DTK-1000 instrument (Dosaka EM; Kyoto, Japan) at a thickness of 400 μm. Slices were rinsed in 0.05 M sodium phosphate buffer (pH 8.0) 3× and reacted in 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) solution (0.1 M sodium phosphate buffer [pH 8.0], 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, 2 mM MgCl₂, and 1 mg/ml X-gal) for 12 hr at 37C with gentle shaking, as described elsewhere. ²⁴ After the LacZ staining, tissues were postfixed in 3.8% formaldehyde in 0.1 M sodium phosphate buffer (pH 8.0). Images were acquired using an MZ75 stereomicroscope (Leica Microsystems; Wetzlar, Germany) equipped with a DP72 CCD camera and a KL1600 LED lighting apparatus (Olympus; Tokyo, Japan). Part of the LacZ-stained tissue was embedded in paraffin for histological analysis.

Isolation of Pancreatic Islets

Pancreatic islets were isolated by collagenase digestion methods from 5-month-old male mice (n=4) as described previously. ²⁵ Briefly, 6 ml of collagenase solution (500 U/ml) (C-7657, type XI; Sigma-Aldrich, Inc. St. Louis, MO) was injected from the duodenal papilla of the pancreatic duct, and the pancreas was excised from the abdomen and digested at 37C for 30 min. After digestion, the tissue was dispersed by gentle pipetting and islets were isolated from exocrine tissues. The islets were handpicked under a stereomicroscope and used for LacZ staining and immunoblotting.

Primary Antibodies

The rabbit polyclonal anti-SgIII antibody raised against rat SgIII peptides 373–471 ⁷ was used for immunohistochemistry and immunoblotting (1:2000 dilution). The mouse monoclonal anti-β-galactosidase antibody (1:2000 dilution, Z3781; Promega, Madison, WI) was used for immunoblotting to analyze the tissue expression of LacZ. The mouse monoclonal anti–glial fibrillary acidic protein (GFAP) antibodies (1:200 dilution, GF5, ab10062; Abcam, Cambridge, UK and 1:60 dilution, GF12.24; Progen Biotechnik GmbH, Heidelberg, Germany) and rabbit polyclonal anti–glutamine synthetase (GS) antibody (1:20,000 dilution, ab49873; Abcam) were used for immunohistochemistry and immunoblotting. The mouse monoclonal anti-α-tubulin antibody (1:3000 dilution, T5168; Sigma-Aldrich, Inc.), anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:500 dilution, ab9484; Abcam), and anti-β-actin antibody (1:5000 dilution, A5316; Sigma-Aldrich, Inc.) were used for loading control in the immunoblot analysis.

Immunohistochemistry

Tissues of 4- to 6-month-old male mice (n=5) were excised and fixed in Bouin’s fixative for 3 days. Tissue blocks were then dehydrated and embedded in paraffin. Five-µm-thick sections were mounted on silane-coated glass slides (Muto Pure Chemicals; Tokyo, Japan) and processed for immunohistochemistry as described previously. ¹⁷ For SgIII immunohistochemistry, deparaffinized sections were immersed in 0.01 M sodium citrate buffer (pH 6.0) and processed using microwave antigen retrieval. Subsequently, the sections were treated with Dako REAL Peroxidase-Blocking Solution (Dako; Glostrup, Denmark) and blocked with 5% normal goat serum (Vector Laboratories; Burlingame, CA) in PBS. After incubation with primary antibodies overnight at 4C, the sections were rinsed in PBS and incubated with secondary antibodies using EnVision Dual Link System horseradish peroxidase (HRP)-labeled polymer reagent (Dako). The sections were then visualized using a peroxidase substrate kit (SK-4100; Vector Laboratories) and counterstained with Mayer’s hematoxylin. The control experiment in the immunohistochemistry followed the literature which is describing its guidelines. ²⁶ As a negative control, sections were incubated with normal rabbit or mouse IgG (Santa Cruz; sc-2027 or sc-2025, 1 μg/mL) instead of the primary antibodies; we confirmed that there were no significant immunosignals in this case. Images were acquired using a BX51 microscope equipped with a DP72 CCD camera and digitized with the DP2-BSW software (Olympus).

Dual Staining of LacZ Expression and Immunohistochemistry

LacZ-stained tissues were embedded in 0.4% Agarose L (317-01182; Nippon Gene Co. Ltd., Tokyo, Japan) and 1% gelatin (16605-42; Nacalai Tesque, Kyoto, Japan) in 0.05 M sodium phosphate buffer (pH 7.4) and refixed in Bouin’s fixative for 24 hr. The tissue blocks were dehydrated and embedded in paraffin. Subsequently, deparaffinized sections were processed by antigen retrieval, reacted with primary antibodies, and incubated with secondary antibodies using the HRP-labeled polymer technique, as mentioned above.

Immunoblotting

For immunoblot analysis, tissue samples from 5-month-old male mice (n=6) were collected, frozen on dry ice, and stored at −80C until use. Frozen tissues were thawed and homogenized with lysis buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 2.5 mM MgCl₂, 1 mM EGTA, 1.0% Triton X-100, and a protease inhibitor cocktail (Complete Mini; Roche Diagnostics, Mannheim, Germany). The homogenates were spun at 20,000 × g for 10 min, the supernatants were collected, and the protein concentration was determined using a Coomassie brilliant blue protein assay reagent (Nacalai Tesque Inc.), as described previously. ¹⁷ Each protein extract was normalized for total protein content (5−40 µg), subjected to SDS-PAGE, and transferred to an Immobilon-P transfer membrane (Millipore; Billerica, MA). After blocking with 5% skimmed milk in Tris-buffered saline (pH7.4) supplemented with 0.05% Tween 20 for 1 hr, the membrane was incubated with primary antibodies. The peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratory; West Grove, PA), anti-rabbit IgG (cat. no. 111-035-144), and anti-mouse IgG (cat. no. 111-035-146) were diluted (1:5000) and used. Signal detection was performed using enhanced chemiluminescent Western blotting detection reagents (PRN2106; GE Healthcare Bio-Sciences KK, Tokyo, Japan).

Glial Cell Lines and Cell Culture

The rat-glioma-derived C6 (resource no. RCB2854) and RGC-6 (resource no. RCB2783) cell lines were provided by RIKEN BRC through the National BioResource Project of the MEXT, Japan. The C6 cell line was maintained in RPMI1640 (11875-093; Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS; S1780-500, Japan Bioserum, Tokyo, Japan), and the RGC-6 cell line was maintained in MEM (M4655; Sigma-Aldrich Inc.) supplemented with 10% FBS. These cell lines were cultured at 37C in a humidified atmosphere of 5% CO₂.

To evaluate astrocytic SgIII expression, activation of glioma cells was performed as follows. C6 and RGC-6 glioma cell lines grown in six-well plates to 80% confluency were stimulated with l-glutamic acid (13012-92; Nacalai Tesque Inc., concentrations of 1 or 2 mM) and cultured for 24 hr. ²⁷ After stimulation, cells were lysed and solubilized in 20 mM HEPES (pH 7.4) containing 0.1 M NaCl, 1 mM EDTA, 1% Triton X-100, 0.4 mM phenylmethanesulfonyl fluoride, and a protease inhibitor cocktail (Nacalai Tesque Inc.). The cell lysates were centrifuged at 11,000 × g for 10 min at 4C to obtain protein sample solutions. Aliquots of the soluble extracts were assessed by SDS-PAGE followed by immunoblotting. The quantities of SgIII were estimated via densitometry of the immunoblot bands with an appropriate molecular weight (70 kDa) using the LAS4000 (General Electric Company; Fairfield, CT) software.

Statistical Analysis

Statistical significance was evaluated using Student’s t-tests, and p<0.05 was considered significant. Statistical significance is indicated in each figure legend as follows: *p<0.05, **p<0.01.

Expression of SgIII/LacZ in Canonical Endocrine Tissues

The gene trap vector was inserted between exons 11 and 12 of the SgIII genome on chromosome 9 (Fig. 1A). ¹⁸ We first examined the SgIII/LacZ expression in SgIII-gtKO mice by immunoblotting using anti-SgIII and anti-β-galactosidase antibodies in the canonical endocrine tissues, where the SgIII expression had previously been established.^6,13–15,17 The expression of β-galactosidase was detected in the brain, pituitary gland, and pancreatic islets in SgIII-gtKO, but not in WT mice (Fig. 1B). In WT mice, positive signals for SgIII were detected in endocrine tissues, whereas no significant signal was detected in those of SgIII-gtKO animals (Fig. 2A). To determine whether the gene inserted in the SgIII genomic locus is expressed similarly as the endogenous SgIII, we then examined the consistency between the SgIII and LacZ expression by immunohistochemistry and histochemical X-gal staining. In immunohistochemical staining, a moderate-to-strong positive immunoreaction was observed in the glandular (anterior and intermediate lobes) and neural (posterior lobe) pituitary, adrenal medulla, and pancreatic islets in WT mice, whereas no immunoreaction was detected in SgIII-gtKO mice (Fig. 2B). Equivalently, LacZ expression in these tissues was detected in SgIII-gtKO mice (Fig. 3). The expression pattern in these endocrine tissues correlated well with that of SgIII determined by immunohistochemistry. Thus, it was revealed that the gene-trapping cassettes were well controlled by endogenous SgIII-promoter activity in SgIII-gtKO mice, and that this mouse strain is useful for the sensitive examination of SgIII expression.

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Figure 2.

Expression of SgIII in canonical endocrine tissues. (A) Immunoblot analysis. Equal amounts of the protein extracts from the pituitary glands (10 μg), adrenal glands (10 μg), and pancreatic islets (5 μg) of WT or SgIII-gtKO mice were used for SDS-PAGE, followed by immunoblotting using anti-SgIII and anti-GAPDH antibodies. (B) Immunohistochemical analysis. Paraffin sections of pituitary glands, adrenal glands and pancreases from SgIII-gtKO and WT mice were immunostained with anti-SgIII antibody. Arrowheads indicate pancreatic islets. Sections were counterstained with hematoxylin. Scale, 50 μm. Abbreviations: ant, anterior pituitary lobe; int, intermediate pituitary lobe; post, posterior pituitary lobe; co, adrenal cortex; md, adrenal medulla; SgIII, secretogranin III; WT, wild-type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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Figure 3.

LacZ expression in endocrine tissues. Whole-mount LacZ staining of the pituitary glands (A, D), adrenal glands (B, E), and pancreases (C, F) from SgIII-gtKO (A–C) and WT (D–F) mice. Arrowheads in (C) and (F) indicate the pancreatic islets, and the inset panels show the isolated islets. Scale, 0.5 mm. Abbreviations: ant, anterior pituitary lobe; int, intermediate pituitary lobe; post, posterior pituitary lobe; co, adrenal cortex; md, adrenal medulla; SgIII, secretogranin III; WT, wild-type.

LacZ Expression in the Brain and Spinal Cord

To determine the LacZ expression in the CNS, we then conducted a comprehensive analysis using coronal tissue slices from SgIII-gtKO mice. The β-galactosidase activity was detected in various brain regions and the spinal cord, including the olfactory bulb, cerebral cortex, caudate putamen, hypothalamus, hippocampus, thalamus, amygdala, midbrain, cerebellum, pons, medulla oblongata, and gray matter of the spinal cord of SgIII-gtKO mice (Fig. 4A–E and K–O), whereas no β-galactosidase activity was detected in control WT mice (Fig. 4F–J and P–T). LacZ expression was strong in the cerebral cortex, hippocampus, and molecular layer of the cerebellum and moderate in the caudate putamen, hypothalamus, amygdala, midbrain, medulla oblongata, and dorsal (posterior) horn of the spinal cord.

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Figure 4.

LacZ expression in slice preparations of the brain and spinal cord. The panels from (A) to (E) and (K) to (O) of SgIII-gtKO or (F) to (J) and (P) to (T) of WT mice are arranged from the rostral to the caudal. Scale, 1 mm. Abbreviations: ac, anterior colliculi; am, amygdaloid nuclei; cb, cerebellar cortex; cg, central gray; ci, cingulate cortex; cn, cochlear nuclei; cp, caudate-putamen; fr, frontal cortex; hp, hippocampus; hy, hypothalamus; lv, lateral ventricle; md, medulla oblongata; ob, olfactory bulb; oc, occipital cortex; pa, parietal cortex; pc, piriform cortex; sn, substantia nigra; sg, gray matter of the cervical spinal cord; th, thalamus; 3v, third ventricle; 4v, fourth ventricle; SgIII, secretogranin III; WT, wild-type.

In the slices of cerebral cortex and hippocampus, the cortical layers II and III and the dentate gyrus (DG) exhibited a stronger expression of LacZ (Fig. 5A and B). A moderate β-galactosidase activity was detected in other cortical areas and hippocampal cornu ammonis (CA) fields. The paraventricular nuclei (Fig. 5C and D), dorsomedial nuclei, and arcuate nuclei (Fig. 5E and F) of the rostral and intermediate hypothalamus showed a moderate expression of LacZ. Moreover, the molecular layer and piriform neuron layer of the cerebellar cortex showed a stronger expression of LacZ (Fig. 5G and H).

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Figure 5.

LacZ expression in brain tissues of SgIII-gtKO mice. Strong LacZ expression was observed in the slices of the cerebral cortex and hippocampus (A), rostral hypothalamus (C), intermediate hypothalamus (E), and cerebellum (G). The panels on the right (B, D, F, and H) are enlarged images of the rectangles enclosed by the line in the images on the left. Scale, 250 μm. Abbreviations: arc, arcuate nuclei; ca, cornu ammonis; dg, dentate gyrus; dm, dorsomedial hypothalamic nuclei; gr, granular layer of the cerebellar cortex; mo, molecular layer of the cerebellar cortex; pv, paraventricular hypothalamic nuclei; II/III, layers II and III of the cerebral cortex; 3v, third ventricle; SgIII, secretogranin III.

In the section-level analysis, GFAP-positive astrocytes in the hippocampal DG and cerebral cortex were the main cellular elements that expressed LacZ (Fig. 6A–D). In the hippocampus, CA pyramidal neurons and DG granular cells did not exhibit β-galactosidase activity (Fig. 6B). Conversely, some of the pyramidal neurons of the cerebral cortex expressed LacZ (Fig. 6C). The perivascular structure showed an overlapping of the LacZ blue staining and GFAP-positive perivascular astrocytes (Fig. 6D). In the cerebellar cortex, Purkinje cells, Bergmann glia (which are a GFAP-positive cerebellar-specific subtype of astrocytes), and a limited number of cells located in the granular layer near the piriform neuron layer expressed LacZ (Fig. 6E–G). In addition to these cells, LacZ expression was observed in GFAP-positive astrocytes localized in the medulla oblongata (data not shown). In the lateral ventricle, ependymal cells expressed LacZ (Fig. 6H).

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Figure 6.

LacZ expression in cellular elements in brain tissues of SgIII-gtKO mice. After LacZ staining, paraffin sections were prepared and counterstained with hematoxylin (A, C, E, G, and H) or reacted with anti-GFAP antibodies (B, D, and F). The panels show the hippocampal DG (A and B), cerebral cortex (C and D), cerebellum (E–G), and lateral ventricle and ependyma (H). The insets in (C), (E), and (F) are enlarged images of the rectangles enclosed by the line. The inset in (C) shows large pyramidal neurons in layer V, and that in (F) shows Purkinje cells and GFAP-immunolabeled Bergmann glia and their radial processes. The asterisks on the insets in (E) and (F) indicate Purkinje cells. The open arrowheads in (F) and (G) indicate LacZ-expression-positive cells in the granular layer. Scale, 20 μm. Abbreviations: Bg, Bergmann glia; cp, choroid plexus; Ep, ependymal cell; gr, granular layer; lv, lateral ventricle; mo, molecular layer; pAs, perivascular astrocyte; po, polymorphic layer; Py, pyramidal neuron; wm, white matter; SgIII, secretogranin III; GFAP, glial fibrillary acidic protein; DG, dentate gyrus.

LacZ Expression in the Peripheral Nervous Tissues

To assess the LacZ expression in the peripheral nervous tissues, we analyzed the retina, optic nerve, and trigeminal ganglion. In the retina, LacZ expression was detected in the ganglionic cell layer, inner granular layer, and outer granular layer (Fig. 7A–C). A weak expression of LacZ was observed in the optic nerves (Fig. 7D), and expression in glial cells was inferred from transverse section images (Fig. 7E). In the trigeminal ganglion, a moderate β-galactosidase activity was detected in the assumed ganglion cells and ganglionic glial cells (Fig. 7F). No β-galactosidase activity was detected in these tissues of WT mice (Fig. 7G–I). To identify the LacZ-expressing cellular elements, we then performed a section-level analysis. In the optic nerve, GFAP-positive astrocytes expressed LacZ (Fig. 7J and K). In the trigeminal ganglion, LacZ expression was observed in some ganglion cells and surrounding satellite cells (Fig. 7L and M).

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Figure 7.

LacZ expression in the peripheral nervous tissues of SgIII-gtKO mice. In the eye, LacZ expression was observed in the retina. The panels show a horizontal slice of the eye (A), a flat-mounted retinal preparation (B), and a transverse retinal section (C). LacZ is expressed in the ganglion cell layer and inner and outer granular layers. In the peripheral nervous system, LacZ expression was observed in the optic nerves (D and E) and trigeminal ganglion (F). Panels (D) and (E) show the shape and a transverse section of the optic nerve. Panels (G)–(I) show the negative control of LacZ staining in WT mice tissues: a flat-mounted retina (G), optic nerves (H), and trigeminal ganglion (I). Panels (J) and (K) show GFAP immunolabeling of the optic nerve transverse section and (K) shows an enlarged image of the rectangle enclosed by the line in (J). The arrows indicate LacZ-positive signals in glial cells in (E) and GFAP-immunolabeled astrocytes in (K). The trigeminal ganglion sections were counterstained with hematoxylin (L) or reacted with an anti-GS antibody (M). Scales, 500 μm (A, B, D, F, and G–I), 100 μm (C and E), and 20 μm (J to M). Abbreviations: ch, choroid; ci, ciliary body; ga, ganglionic cell layer; ig, inner granular layer; od, optic disk; og, outer granular layer; re, retina; sc, sclera; St, satellite cells; Tg, trigeminal ganglion neuron; SgIII, secretogranin III; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase.

Expression of SgIII in the Cerebellum

Although there are reports that SgIII is expressed at several sites in the CNS,^11,16,19 knowledge about cerebellar SgIII expression has not been fully obtained. Therefore, we further analyzed the cerebellar expression of SgIII in WT and SgIII-gtKO mice. An immunoblotting analysis revealed the expression of three fragmented bands of SgIII in the cerebellum of WT mice (Fig. 8A), suggesting a similar proteolytic processing from the SgIII precursor as that observed in the adrenal gland, compared with other endocrine tissues (Fig. 2A).

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Figure 8.

Expression of SgIII in the cerebellum. (A) Cerebellar tissue lysates (40 μg of protein extract) from WT and SgIII-gtKO mice were used for SDS-PAGE, followed by immunoblotting using anti-SgIII and control anti-GAPDH antibodies. Three SgIII-specific bands were detected (arrowheads), and two bands near 37 and 20 kDa were nonspecific. Immunohistochemistry using anti-SgIII (B, C, and E) and anti-GS (D) antibodies. The asterisks indicate Purkinje cells, and the large black arrowheads indicate Bergmann glia in the piriform neuron layer. The open arrowheads indicate basket-like cells. The large arrows indicate Golgi-like cells that do not express GS, and the small arrows indicate the neuropil surrounding the granule cell perikaryon (considered to be the cerebellar glomerulus). The small black arrowheads indicate GS-positive astrocytes in which SgIII expression was not detected in the granular layer. Scale, 20 μm. Abbreviations: gr, granular layer; mo, molecular layer; wm, white matter; SgIII, secretogranin III; GFAP, glial fibrillary acidic protein; GS, glutamine synthetase; WT, wild-type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

We then analyzed the cellular elements that express SgIII in the cerebellum by immunohistochemistry (Fig. 8B). Purkinje cells and Bergmann glia in the piriform neuron layer and Golgi-like cells showing negative immunoreactivity of GS and localizing in the granular layer expressed SgIII strongly. The molecular layer, basket-like cells, and neuropil around the granule cells showed a moderate level of SgIII expression. Slightly stronger expression in the granular layer was observed in the assumed cerebellar glomerulus, an intertwined mass of nerve fibers consisting of granule cell dendrites and Golgi cell axon terminals surrounding mossy fiber terminals. Conversely, GS immunoreactivity-positive gray matter astrocytes in the granular layer and fibrous astrocytes located in the white matter exhibited negligible SgIII expression.

Glial Expression and Reactive Induction of SgIII

SgIII expression in glial cells, mostly interpreted as astrocytes in the CNS, has been reported. ¹⁶ Our LacZ-reporter-based analysis of SgIII expression revealed that cortical and hippocampal astrocytes and cerebellar Bergmann glia constitutively expressed SgIII. In contrast, it was extremely difficult to detect SgIII expression in cerebellar gray matter astrocytes in the granular layer and fibrous astrocytes in the white matter. Among the roles of astrocytes, synaptic glutamate clearance is one of the critical functions in regulating neurotransmitter homeostasis. ²⁸ Therefore, we treated two rat glioma-derived cell lines with similar properties, C6 and RGC-6, with glutamate to investigate the relationship between the changes in SgIII expression level and glutamate stimulation. Although the basal level of SgIII expression was very low in these glioma cells compared with that detected in the control pituitary tissue (Fig. 9A), SgIII expression was significantly induced (1.5−2.0-fold) by glutamate treatment (Fig. 9B).

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Figure 9.

Expression of SgIII in C6 and RGC-6 glioma cells cultured with glutamate. (A) Immunoblot analysis of SgIII (70 kDa) and β-actin in the total cell lysates (20 μg) from C6 and RGC-6 cells cultured with the indicated concentration of glutamate for 24 hr and in rat pituitary lysates (2 μg). (B) Quantification of the immunoblot of SgIII in C6 and RGC-6 cells. The intensities of the immunoreactive bands on the immunoblots presented in (A) were quantified by densitometric analysis. The expression levels of each protein were normalized to that of β-actin and are expressed as arbitrary units, whereas the mean levels in 0 mM glutamate are defined as 1.0. Data are presented as mean ± SEM for each group (n=6 per group: six sample preparations from six independent experiments). *p<0.05; **p<0.01. Abbreviation: SgIII, secretogranin III.