Intrinsic expression of transcortin in neural cells of the mouse brain: a histochemical and molecular study

Most systemic glucocorticoids (GCs) are bound to corticosteroid-binding globulin (CBG), also referred to as transcortin, a member of the SERPIN family of proteins (for recent reviews, see Moisan, 2010; Henley and Lightman, 2011). CBG is a glycoprotein with a molecular mass of ~55 kDa (Westphal, 1986; Westphal, 1971; Hammond, 1990). CBG-bound corticosteroid transport into the brain has been shown to be insignificant (Pardridge and Mietus, 1979), presumably because the size of the glycated CBG prevents it from crossing the blood–brain barrier. In addition to being a buffer for bioavailability of adrenal steroids, plasma CBG acts as a reservoir of GC hormones and allows GC delivery to target tissues (reviewed by Rosner, 1991; Hammond, 1995), particularly in sites of inflammation, where CBG–GC complexes are cleaved by elastase, thereby releasing GCs and creating high concentrations of GCs at the target cell. Finally, according to some authors, CBG may be directly involved in membrane effects of GCs, with the existence of a CBG receptor (Orchinik et al., 1997). In a recent study, we showed the endogenous expression of CBG in a human astrocytoma cell line (Pusch et al., 2009). In this culture system, CBG secretion could be rapidly induced after corticosterone stimulation in a dose- and time-dependent manner whereas dexamethasone, a potent GC agonist that is not bound by CBG, was ineffective (Pusch et al., 2009). Previously, we described CBG in human magnocellular hypothalamic neurons as well as in neurons of the rat hypothalamo-hypophyseal system, partially co-localized with the two important peptides of stress response – vasopressin and oxytocin (Sivukhina et al., 2006; Möpert et al., 2006; Jirikowski et al., 2007). With immunohistochemistry and in situ hybridization methods, CBG has also been observed in a portion of the periventricular neurons, the ependymal cells lining the third ventricle and in the choroid plexus (Möpert et al., 2006; Jirikowski et al., 2007). However, detailed morphological analysis of CBG distribution in the different populations of brain cells has not been performed, and the brain Cbg structure has not been reported.

Recently, we successfully developed a mouse model of full CBG deficiency by specific deletion of the gene encoding CBG, i.e. Cbg, also called SerpinA6 gene (Richard et al., 2010). These knockout mice display the features of the HPA axis regulation observed in very rare CBG-deficient patients (for review, see Gagliardi et al., 2010; Henley and Lightman, 2011), i.e. very low total circulating levels of GC hormones but normal free active GC levels in basal conditions. After stress exposure, CBG-deficient mice (Cbg^−/−) demonstrated insufficient free GC levels, leading to insufficient GC signaling and inappropriate behavioral responses such as despair-like behaviors (Richard et al., 2010). The availability of these Cbg knockout mice provides a unique opportunity to evaluate the importance of CBG within the brain.

In the current study, we examined CBG expression in various brain regions of wild-type and CBG-deficient mice using immunohistochemistry and reverse-transcription polymerase chain reaction (RT-PCR). Double immunostaining for neuronal and glial markers followed by confocal laser scanning microscopy were used for further characterization of CBG-positive cells. RT-PCR of the full-length cDNA was then used to confirm the local synthesis of CBG in the brain and to identify its cDNA structure. Finally, real-time quantitative PCR was employed to evaluate the relative expression of Cbg mRNA in various brain regions and liver.

The generation of Cbg^−/− mice has been previously described (Richard et al., 2010). These mice bred and developed normally and showed no gross anatomical abnormalities. Three to 6-month-old Cbg^−/− mice and their wild-type littermates (later referred to as Cbg^+/+) were obtained by breeding Cbg^+/− males and females and selecting Cbg^−/− and Cbg^+/+ by specific genotyping from tail biopsies.

All mice of the study were males and had a C57BL/6J genetic background exceeding 98%. Mice were kept in an animal room (23°C) with a 12 h:12 h light:dark cycle (lights on at 07:00 h) and with ad libitum access to chow and water. All experiments were conducted in strict compliance with current European Conventions and approved by the Institutional Committee (protocol validated on 25 February 2011) as well by the Regional veterinary services (agreement no. A33-063-920).

For detection of Cbg mRNA, total RNA was extracted from brains, pituitaries and portions of liver (used as a positive control) of Cbg^−/− (N=5) and Cbg^+/+ mice (N=5) collected from mice killed between 19:00 h and 20:00 h (at the onset of the dark phase), frozen immediately on dry ice and stored at −80°C. Dissection of brain regions (cortex, amygdala, hippocampus and hypothalamus) was performed bilaterally from the whole frozen brains using a ‘Micro Punch’ (1.2 mm; Harris, Saint-Quentin Fallavier, France) in a RNA-free environment at −20°C. Total RNA was isolated with TRIzol® reagent (Invitrogen, Cergy Pontoise, France) according to the manufacturer's protocol, and RNA quality was evaluated by spectrophotometry (Thermo Scientific, Wilmington, DE, USA) at 260 nm. RNA was considered of high quality at a 260/280 nm ratio in the range 1.8–2.0. Total RNA was reverse transcribed with Superscript III (Invitrogen) in 13 μl of reaction mixture containing 2 μg of RNA, 1 μl (50 ng ml⁻¹) of random hexamers, 1 μl (10 mmol l⁻¹) of desoxynucleoside triphosphates (dNTPs) and diethylpyrocarbonate (DEPC) water to volume. This mixture was heated in a water bath at 65°C for 5 min and then cooled on ice. 4 μl of first strand buffer (5×), 1 μl (0.1 mol l⁻¹) dithiothreitol (DTT), 1 μl RNaseOUT Inhibitor (Invitrogen) and 1 μl reverse transcriptase (Superscript III Reverse Transcriptase; Invitrogen) were added. The mixture was incubated for 55 min at 50°C in a water bath and, finally, the reaction was stopped by heating the mixture at 75°C for 10 min.

cDNA was obtained after the reverse transcription of total RNA isolated from the different brain regions, pituitaries and liver of Cbg^−/− and Cbg^+/+ mice as described above. The PCR consisted of 2 μl cDNA, 5 μl of the sequence-specific primers mix (2 μmol l⁻¹), 10 μl PCR buffer mix 2× (GoTaq Flexi Buffer 2×, 3 mmol l⁻¹ MgCl₂ 0.8 mmol l⁻¹ dNTPs), 0.1 μl GoTaq Hotstart Polymerase (Invitrogen), made up to 20 μl with PCR water.

The oligonucleotides used for specific amplification are listed in Table 1. PCR was carried out using a Thermal Cycler 2720 (Applied Biosystems, Villebon-sur-Yvette, France). PCR was programmed to conduct one cycle at 94°C (5 min), followed by 40 cycles at 94°C (30 s), including annealing (58°C for 1 min) and extension (72°C for 1.5 min). The final extension was performed at 72°C for 10 min. PCR products were separated by electrophoresis in 2% agarose gel in a Tris–Borate–EDTA buffer stained with ethidium bromide. The cDNA bands were visualized under ultraviolet light.

For the sequencing analysis, in order to obtain sufficient cDNA material, nested PCR using the Platinum® Taq DNA Polymerase High Fidelity (Invitrogen) and specific primers sets (Table 1) was performed under the same PCR conditions, using as template the same reverse-transcription products as above. PCR products obtained by nested PCR for hypothalamus and liver were first gel-purified on a spin-column using a QIAquick PCR Purification Kit (Qiagen, Courtaboeuf, France). The sequencing reactions were then performed using the BigDye Terminator v. 3.1 kit (Applied Biosystems) and migrated on a ABI 3130xl 16-capillary sequencer (Applied Biosystems).

Primer sequences were designed using Primer Express software (Applied Biosystems).

In order to establish a comparative quantification of Cbg mRNA in the mouse brain, we applied real-time quantitative PCR from reverse-transcribed total RNA as described previously (Richard et al., 2010). Total RNA isolated from the hypothalamus, pituitary, prefrontal cortex, amygdala and hippocampus from Cbg^−/− and Cbg^+/+ mice was reverse transcribed into cDNA as described above. Then, 5 μl of cDNA (diluted 1:20) was amplified using 10 μl of Mesagreen qPCR master mix (Eurogentec, Seraing, Belgium) and 5 μl of primer mix at 300 nmol l⁻¹ into a total volume of 20 μl onto an ABI7500 thermocycler (Applied Biosystems). The PCR program consisted of 40 cycles, each at 95°C (15 s) and 60°C (1 min). The primers amplified a 50 bp fragment of mouse Cbg (Table 1). Specificity of the PCR reaction was validated by a melting-curve analysis and evaluation of PCR efficiency using five dilution points of the calibrator sample. mRNA levels of target genes were normalized using a 18S RNA expression for each sample (cDNA diluted 1:2000). 18S proved to be the best internal control in our experiments, compared to cyclophilin and β2 microglobulin genes. Relative quantification of normalized mRNA levels was calculated using SDS2.1 software (Applied Biosystems).

For morphological investigations, intact Cbg^−/− (N=3) and Cbg^+/+ mice (N=3) were anesthetized with a rapid isoflurane exposure (Aerrane; Baxter SA, Maurepas, France) and sacrificed by transcardiac perfusion with 4% paraformaldehyde in isotonic phosphate-buffered saline solution (PBS, pH 7.4). These mice were sacrificed between 19:00 h and 20:00 h (at the onset of the dark phase). After fixation, brains, pituitaries and liver (used as a positive control) were removed and post-fixed in the same fixative at 4°C. Samples were embedded in 6% agarose and sectioned on vibratome (VT1000E; Leica Instruments, Nußloch, Germany). Brains were cut coronally, pituitaries and liver horizontally (30–50 μm thick) and collected in PBS until immunostaining was performed.

The following commercially available antibodies were used at optimal dilutions: anti-Serpin A6 (R&D Systems, Minneapolis, MN, USA; goat polyclonal, 1:100), anti-neuronal nuclear antigen (NeuN, Chemicon, Merck Millipore, Billerica, MA, USA; mouse monoclonal, clone A60, 1:100), anti-cyclic nucleotide phosphodiesterase (CNPase, Sigma-Aldrich, St Louis, MO, USA; mouse monoclonal, clone 11-5B, 1:1000), anti-glial fibrillary acidic protein (GFAP, Merck Millipore; rabbit polyclonal, 1:2000), anti-ionized calcium binding adaptor molecule 1 (Iba1, Biocare Medical, Concord, CA, USA; rabbit polyclonal, 1:600). Prior to the immunofluorescence staining, blocking of nonspecific binding sites was performed for 1.5 h at room temperature using PBS (pH 7.4), containing 1% Triton X-100 (Roth, Karlsruhe, Germany) and 10% normal donkey serum (Sigma-Aldrich). For double staining, the mixture of primary antibodies, diluted in PBS containing 0.1% Aurion BSA-c (Aurion Immuno Gold Reagents & Accessories, Wageningen, The Netherlands) was used. Incubation with the primary antibody was carried out for 3 days at 4°C (Irintchev et al., 2005; Hoffmann et al., 2009). After washing in PBS, the mixture of appropriate secondary antibody conjugated with Alexa Fluor® 488 Dye and Alexa Fluor® 568 Dye (Invitrogen), diluted 1:300 in PBS containing 0.1% Aurion BSA-c, was applied for 2.5 h at room temperature. Finally, after subsequent washes in PBS, sections were affixed onto microscopic slides and mounted with an anti-fading medium (Fluoromount G; Southern Biotechnology Associates, Biozol, Eching, Germany). For immunohistochemical controls, primary antibodies were omitted and were devoid of specific staining.

Immunostained sections were examined under a confocal laser-scanning microscope TCS SP5 (Leica, Mannheim, Germany). Digital images were analyzed using Adobe Photoshop (Adobe Systems, v. 8.0.1). Anatomical identification of labeled structures was based on the cytoarchitectonic descriptions of the mouse brain (Franklin and Paxinos, 2007).

Values were expressed as means ± s.e.m. Statistics were performed using the non-parametric Mann–Whitney U-test. The level of significance was set at P<0.05. On graphs, * indicates P<0.05 and ** indicates P<0.01.

Slight to moderate specific CBG immunoreactivity was observed as a granulated reaction product in the perinuclear cytoplasm of neurons in the different brain regions of Cbg^+/+ mice (Fig. 1A–G,L,N) and was absent in Cbg^−/− (Fig. 1J,K,M represents cerebral cortex, paraventricular and supraoptic hypothalamic nuclei, respectively). In the cerebellum of Cbg^+/+ mice, CBG immunoreactivity was observed in large neurons within the granule cell layer (Fig. 1A, arrows), and many Purkinje cells and their processes were also stained for CBG (Fig. 1A, arrowhead). Some CBG-stained cells were distributed throughout the amygdala complex and the hypothalamic periventricular region (Fig. 1B, arrowheads). In the hippocampus, CBG immunoreactivity was observed in the cytoplasm of granule cells (Fig. 1C, arrowhead) and of some interneurons (Fig. 1C, arrow). Various regions of the cerebral cortex contained CBG-positive perykariya, some of which had a pyramidal cell-like morphology (Fig. 1D). CBG-positive cells were also found in brain areas known to be associated with stress response and regulation of the HPA axis: throughout the hypothalamus, CBG immunoreactivity was observed in cell bodies (Fig. 1E, arrowheads; Fig. 1L) as well as their processes within the paraventricular nucleus (Fig. 1E, arrow). Intense CBG immunoreactivity was observed in some endocrine cells of the anterior pituitary lobe (Fig. 1F, arrowheads). Numerous magnocellular CBG-immunoreactive cells were also present in the supraoptic nucleus (Fig. 1G, arrowheads; Fig. 1N). CBG immunoreactivity was found in axon terminals within the median eminence and in Herring bodies of the posterior pituitary lobe. Compared to the liver (Fig. 1H), the level of CBG staining within the brain was low. Immunohistochemistry for CBG in the liver revealed strong staining in numerous hepatocytes throughout the liver parenchyma (Fig. 1H, arrowheads) as well as in the close vicinity of sinusoids (Fig. 1H, arrow) in Cbg^+/+ mice. No specific staining was observed in the liver of Cbg^−/− animals (Fig. 1I).

In order to analyze cellular localization of CBG within the brain, double immunofluorescence labeling with well-known neuronal and glial markers was performed. CBG was expressed in neurons, as demonstrated by double staining with NeuN in amygdala (Fig. 2A–A″), but was also observed in other populations of brain cells. CBG immunoreactivity was also found in a few glial fibrillary acidic protein (GFAP)-positive cells in the periventricular hypothalamic region (Fig. 2B–B″). Staining for CBG was also observed in oligodendrocyte cell bodies and processes, as demonstrated by double labeling with 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) in cerebellum (Fig. 2C–C″) and cerebral cortex (Fig. 2D–D″). Double immunohistochemistry with ionized calcium binding adaptor molecule 1 (Iba1) showed that microglial cells were devoid of specific CBG signal in any of the regions studied (Fig. 2E–E″).

To answer the question concerning intrinsic expression of Cbg in the brain, RT-PCR was applied to amplify the full-length Cbg cDNA in Cbg^+/+ and Cbg^−/− mice using two sets of overlapping primers (Fig. 3, Table 1), which produced a 718 bp 5′ cDNA fragment and a 689 bp 3′ cDNA fragment.

As illustrated in Fig. 4, specific bands of the expected size were seen in the pituitary, prefrontal cortex, hypothalamus, hippocampus and liver, while in the amygdala this band was very weak or absent. No amplification of the 689 bp and 718 bp bands was detected in Cbg^−/− mice (data not shown).

To identify the structure of Cbg cDNA in the brain, a sequencing analysis of the full-length cDNA obtained from hypothalamic and liver mRNA of a wild-type mice was performed. We first applied nested PCR, using primers within the first PCR products in order to increase the amount of Cbg cDNA (Fig. 3, Table 1). Sequencing of both strands of a 633 bp corresponding to the 5′ half and of a 621 bp fragment corresponding to the 3′ half of Cbg cDNA revealed a 100% homology with the liver Cbg cDNA.

In order to estimate the relative level of Cbg mRNA expression in the various brain regions and in the liver, primers amplifying a short fragment (Table 1) were used for quantitative real-time PCR (Fig. 5). As expected based on the immunohistochemical study, relative mRNA expression of Cbg in the brain was significantly lower when compared to its expression in the liver; about 200 times less expressed in the pituitary and hippocampus when compared to the liver. However, in every tissue examined, the level of Cbg mRNA was significantly higher in wild-type when compared with Cbg^−/− mice (P<0.05 for each tissue). Moreover, the analysis of the melting curve of PCR products showed a single specific product in each tissue of Cbg^+/+ mice that was absent in Cbg^−/− animals.

The expression of CBG in the brain has been reported before by various authors in rats (de Kloet et al., 1984; Perrot-Applanat et al., 1984; Möpert et al., 2006; Jirikowski et al., 2007) and humans (Sivukhina et al., 2006). Our previous findings using immunohistochemistry, in situ hybridization, western blot (Möpert et al., 2006) and RT-PCR (Jirikowski et al., 2007) suggested coexpression of the steroid-binding globulin with the classical neuroendocrine peptides within the rat hypothalamus as well as intrinsic synthesis of Cbg mRNA in various brain regions. Here, we extend the data obtained previously by the use of a mouse model in which expression of the Cbg gene is totally abolished in each tissue of the animal. We present data refining CBG localization within brain regions; we show that the same gene encodes brain and liver CBG and we confirm that Cbg mRNA is produced within cerebral regions and thus not transported from blood. These data suggest a specific role for CBG protein in brain.

First, we located CBG in the hypothalamic magnocellular perikarya, in axonal varicosities in the periventricular nucleus, in the internal zone of the median eminence, and in Herring bodies of the posterior pituitary lobe, which shows that magnocelluar CBG in mouse is subject to axonal transport and terminal release in the respective neurohemal organs. In a former immunoelectron microscopical study, we observed a similar morphology for sex hormone-binding globulin (SHBG) and oxytocin in the rat hypothalamo-neurohypophyseal system, where they were colocalized in secretory vesicles (Herbert et al., 2006). We thus conclude that there may be a similar situation for CBG in mice.

Our colocalization studies with neuronal marker protein (NeuN) showed that many neuronal systems other than the hypothalamic neuroendocrine nuclei contained CBG-immunostaining. Such neurons occurred in functionally quite diverse cerebral regions including the amygdala, the prefrontal cortex and the hippocampus, implicated in an interconnected way in the central stress response (McEwen, 2007; Joëls and Baram, 2009). Interestingly, phenotypic analysis of Cbg^−/− mice demonstrated that these mice show brain-specific deficit in situations of stress (Richard et al., 2010). In particular, despair-like behavior and altered memory response during stress has been demonstrated in these mice. These brain-specific alterations have been linked to the lack of plasma CBG although a possible contribution of brain CBG cannot be excluded (Minni et al., 2012). Thus, the specific involvement of brain CBG compared to plasma CBG has yet to be defined. The production and analysis of brain specific knockout for the Cbg gene would be very useful in this respect.

Finally, positive staining was found in the granule and Purkinje cells of the mouse cerebellum. Little is known about the effects of steroids on cerebellar structures; however, data show that postnatal exposure to clinically routine doses of hydrocortisone or dexamethasone was associated with impaired cerebellar growth (Tam et al., 2011). In another study, data showed that GCs increase cerebellar cell death and induce apoptosis in immature granule neurons via a non-genomic mechanism (Aden et al., 2008).

While our earlier results showed the presence of CBG in several regions of the brain, we have not provided a detailed analysis of cellular distribution of CBG throughout the brain. Double immunostaining for CBG and for glial markers GFAP and CNPase showed that a considerable portion of non-neuronal cells in the brain contains CBG. Immunostaining for GFAP and CBG was found in many of the ependymal cells, lining the third ventricle, in tanycytes, in epithelial cells of the choroid plexus and in small fractions of fibrillary and protoplasmic astrocytes. All these cell types are known to be derived from astroglia, and therefore contain GFAP. Astrocytes are known as supportive cells that interact with neurons and are able to transfer ions to other astrocytes acting as a sufficient ‘buffer’ for the glutamate. GCs stimulate an increase of glutamate, with initial reversible remodeling and eventual cell death (Campbell and Macqueen, 2004). This could indicate that CBG may be important as a buffer in this cell fraction if local CBG levels were high enough for such a function. The low levels of Cbg mRNA found in this study do not favor this hypothesis but these levels might be upregulated in stress conditions. A portion of the oligodendrocytes also contained CBG, as shown by double immunostaining with CNPase. The complex role of GCs in the maturation of oligodendrocytes and myelinogenesis has been demonstrated (Preston and McMorris, 1984; Raschke et al., 2008) and could be explained by documented expression of GC receptors in oligodendrocyte progenitor cells (Bohn et al., 1991). Currently, high doses of GCs are widely used in the treatment of multiple sclerosis, a well-described disease characterized by oligodendrocyte degeneration. However, the potentially negative influence of GCs on remyelination has also been demonstrated (Clarner et al., 2011), and patients with multiple sclerosis show decreased GC receptor affinity and sensitivity when compared to controls (Ysrraelit et al., 2008). Altogether, it might be a further indication for a possible dysregulation of the intracellular CBG expression. Microglia cells were, in all cases, CBG negative in Cbg^+/+ animals. Given the fact that microglia cells are resident macrophages in the brain and thus form the immunoreactive cell fraction, their lack of CBG underlines their specific role. In a former study, we investigated the CBG expression and stimulus-dependent liberation of CBG in a human glioblastoma cell line (Pusch et al., 2009). Here, we found clear evidence for the expression of the steroid-binding globulin in glia-like non-neuronal cells. The functional properties of the different glial cell types are diverse. While astrocytes and tanycytes are involved in blood–brain barrier functions and may somehow take up CBG from serum, ependymal cells line the ventricles and may accumulate CBG from the cerebrospinal fluid CSF (Schwarz and Pohl, 1992; Predine et al., 1984; Orchinik et al., 1997) in addition to possible intrinsic biosynthesis.

Previous RT-PCR of a Cbg cDNA fragment suggested the presence of Cbg mRNA in the respective brain regions, indicating intrinsic expression of CBG within the brain (Jirikowski et al., 2007). Although CBG has been shown to be expressed in several organs (Miska et al., 2004; Misao et al., 1994; Misao et al., 1999; Scrocchi et al., 1993; del Mar Grasa et al., 2001; de Kloet and McEwen, 1976), the majority of systemic CBG is thought to be of hepatic origin (Hammond et al., 1987; Scrocchi et al., 1993). The absence of CBG protein and mRNA expression in brain and liver of the CBG-deficient mice obtained by disruption of the Cbg gene proves, for the first time, that the same gene encodes brain and liver CBG. The full-length sequencing of hypothalamic Cbg revealed a 100% homology with liver Cbg cDNA, which confirms that the cDNA and thus the protein sequences are identical in liver and hypothalamus.

A quantitative analysis of brain and liver Cbg mRNA had not been performed before in any of the studied species. Liver Cbg mRNA is about 200 times more abundant than pituitary or hippocampal Cbg mRNA. In hypothalamus, cortex and amygdala, the presence of Cbg mRNA appeared to be very low. The low levels of brain mRNA expression compared with liver levels question a putative role of CBG as an efficient GC buffer in brain. Possibly, brain CBG levels may be upregulated in some conditions such as after an intense stress. Also, our quantification by quantitative PCR on the large brain region might underestimate CBG levels within particular cell nuclei. Nevertheless, the immunohistochemical study suggests a role that could be locally important. As mentioned above, the analysis of brain-specific knockout mice would be very useful in order to discriminate between the role of CBG in the brain versus in the plasma.

Our findings suggest that CBG in mouse brain is expressed in many different regions and in different cell types. Clearly, the multiple locations imply multiple functional properties that are yet to be determined. Most probably, the importance of brain CBG exceeds the function of a mere steroid transporter. The specific role of CBG within the brain awaits the availability of a brain-specific knockout for the Cbg gene. Nevertheless, the data provided in this study enhance the fact that brain CBG may have a role and should not be neglected.

Real-time PCR experiments and sequencing analysis were performed at the Genotyping and Sequencing facility of Bordeaux. We express our thanks to Sabine Hitschke (University of Jena, Germany) for her excellent technical assistance, to Dr Hartmut Oehring (University of Jena, Germany) for helpful suggestions, to Dr Valery Grinevich (MPI for Medical Research, Heidelberg, Germany) for his critical discussion and helpful comments and to Martine Schuler for revising the writing of the manuscript.

 
• BSA-c

  bovine serum albumin acetylated

 
• CBG

  corticosteroid-binding globulin

 
• cDNA

  complementary deoxyribonucleic acid

 
• CNPase

  cyclic nucleotide phosphodiesterase

 
• dNTPs

  desoxynucleoside triphosphates

 
• DTT

  dithiothreitol

 
• EDTA

  ethylenediaminetetraacetic acid

 
• GC

  glucocorticoid

 
• GFAP

  glial fibrillary acidic protein

 
• HPA axis

  hypothalamo–pituitary–adrenal axis

 
• Iba1

  ionized calcium binding adaptor molecule 1

 
• mRNA

  messenger ribonucleic acid

 
• NeuN

  neuronal nuclear antigen

 
• PBS

  phosphate-buffered saline

 
• RNA

  ribonucleic acid

 
• RNAse

  ribonuclease

 
• RT-PCR

  reverse-transcription polymerase chain reaction