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, 103 (39), 14602-7

Characterization of Brain Neurons That Express Enzymes Mediating Neurosteroid Biosynthesis

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Characterization of Brain Neurons That Express Enzymes Mediating Neurosteroid Biosynthesis

Roberto C Agís-Balboa et al. Proc Natl Acad Sci U S A.

Abstract

Allopregnanolone (ALLO) and tetrahydrodeoxycorticosterone (THDOC) are potent positive allosteric modulators of GABA action at GABA(A) receptors. ALLO and THDOC are synthesized in the brain from progesterone or deoxycorticosterone, respectively, by the sequential action of two enzymes: 5alpha-reductase (5alpha-R) type I and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). This study evaluates 5alpha-R type I and 3alpha-HSD mRNA expression level in mouse brain by using in situ hybridization combined with glutamic acid decarboxylase 67/65, vesicular glutamate transporter 2, glial fibrillary acidic protein, and S100beta immunohistochemistry. We demonstrate that 5alpha-R type I and 3alpha-HSD colocalize in cortical, hippocampal, and olfactory bulb glutamatergic principal neurons and in some output neurons of the amygdala and thalamus. Neither 5alpha-R type I nor 3alpha-HSD mRNAs are expressed in S100beta- or glial fibrillary acidic protein-positive glial cells. Using glutamic acid decarboxylase 67/65 antibodies to mark GABAergic neurons, we failed to detect 5alpha-R type I and 3alpha-HSD in cortical and hippocampal GABAergic interneurons. However, 5alpha-R type I and 3alpha-HSD are significantly expressed in principal GABAergic output neurons, such as striatal medium spiny, reticular thalamic nucleus, and cerebellar Purkinje neurons. A similar distribution and cellular location of neurosteroidogenic enzymes was observed in rat brain. Taken together, these data suggest that ALLO and THDOC, which can be synthesized in principal output neurons, modulate GABA action at GABA(A) receptors, either with an autocrine or a paracrine mechanism or by reaching GABA(A) receptor intracellular sites through lateral membrane diffusion.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Mouse brain distribution of 5α-R type I (a) and 3α-HSD (b) mRNAs. (aA and bA) Somatosensory cortex, coronal section at bregma +1.10 mm (49). I to V identify the cortical layers. (Fig. 1 aB and bB) Hippocampus, coronal section at bregma −2.46 mm (49). CA, cornus ammonis; DG, dentate gyrus; O, stratum oriens; R, stratum radiatum; LM, layer lacunosum moleculare; M, molecular layer dentate gyrus; H, hilus. (aC and bC) Olfactory bulb, coronal section at bregma +3.92 mm (49). M, mitral; PG, periglomerular; G, granular cell layers. (aD and bD) Cerebellum. The Purkinje cell layer is indicated by arrowheads (▴). G, granule cell layer; M, molecular layer. (aE and bE) Striatal medium spiny neurons, coronal section as in A. Shown is unstained striatal fiber tract (stf). (aF and bF) Piriform cortex and amygdala, coronal section at bregma −1.58 mm (49). BLA, basolateral anterior amygdaloid nucleus; BM, basomedial amygdaloid nucleus; Ce, central amygdaloid nucleus; Pir, piriform cortex. (aG and bG) Thalamic nuclei, coronal section as in F. VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus. [Scale bars, 50 μm (aA and bA) and 100 μm (aB and bBaG and bG).]
Fig. 2.
Fig. 2.
5α-R type I and 3α-HSD colocalize in glutamatergic (pyramidal) neurons but not in GABAergic interneurons of somatosensory cortex. (A) 5α-R type I protein colocalizes with 3α-HSD mRNA in somatosensory cortical layer II. 3α-HSD mRNA, green. (A2) 5α-R type I protein, red. (A3) Merge of A1 and A2. Note that 3α-HSD mRNA and 5α-R type I protein cellular staining is absent in cortical layer I. (B) 5α-R type I colocalizes with VGLUT1 mRNA. (B1) VGLUT1 mRNA, green. (B2) 5α-R type I protein, red. (B3) Merge of B1 and B2. (C) 5α-R type I antiserum specificity. Western blot of mouse brain extract incubated with 5α-R type I antibody (C1), and 5α-R type I antibody preabsorbed with the 5α-R type I immunizing antigen (see Materials and Methods). Molecular markers are indicated at the right. (D) The 3α-HSD mRNA is not expressed with GAD67/65 in somatosensory layer I (LI) and layer II (LII) cortical interneurons. (D1) 3α-HSD mRNA, green. (D2) GAD67/65 protein, red. (D3) Merge of D1 and D2. [Scale bars, 40 μm (A) and 20 μm (BD).]
Fig. 3.
Fig. 3.
5α-R type I is expressed in pyramidal neurons of the hippocampus. (A) 5α-R type I mRNA colocalizes with VGLUT2 in CA1 pyramidal layer. (A1) 5α-R type I mRNA, green. (A2) VGLUT2 protein, red. (A3) Merge of A1 and A2. (B) VGLUT2 antiserum specificity. Western blot of mouse brain extract incubated with VGLUT2 antibody (B1) and VGLUT2 antibody preabsorbed with VGLUT2 immunizing antigen (B2) (see Materials and Methods). Molecular markers are indicated at the right. (C) 5α-R type I mRNA does not colocalize with S100β in the CA1 area of the hippocampus. (C1) 5α-R type I mRNA, green. (C2) S-100β protein, red. (C3) Merge of C1 and C2. O, stratum oriens; R, stratum radiatum. (Scale bars, 20 μm.)
Fig. 4.
Fig. 4.
Neuronal expression of 5α-R type I mRNA in olfactory bulb (OB), striatum, and RtN. (A) In OB, 5α-R type I mRNA colocalizes with VGLUT2 protein in mitral (M) cells. (A1) 5α-R type I mRNA, green. (A2) VGLUT2 protein, red. (A3) Merge of A1 and A2. Note the intense 5α-R type I mRNA staining in M cells (glutamatergic) and the weak 5α-R type I mRNA staining in granule (G) cells (GABAergic). (B) In the striatum, 5α-R type I mRNA colocalizes with GAD67/65. (B1) 5α-R type I mRNA, green. (B2) GAD67/65 protein, red. (B3) Merge of B1 and B2. (C) In RtN, 5α-R type I mRNA colocalizes with GAD67/65. (C1) 5α-R type I mRNA, green. (C2) GAD67/65 protein, red. (C3) Merge of (C1) and (C2). (Scale bars, 20 μm.)

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