Journal List > Korean J Physiol Pharmacol > v.15(3) > 1025735

Shin, Han, Lee, Han, Park, Erdelyi, Szabo, and Ryu: Direct Corticosteroid Modulation of GABAergic Neurons in the Anterior Hypothalamic Area of GAD65-eGFP Mice

Abstract

Corticosterone is known to modulate GABAergic synaptic transmission in the hypothalamic paraventricular nucleus. However, the underlying receptor mechanisms are largely unknown. In the anterior hypothalamic area (AHA), the sympathoinhibitory center that project GABAergic neurons onto the PVN, we examined the expression of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) of GABAergic neurons using intact GAD65-eGFP transgenic mice, and the effects of corticosterone on the burst firing using adrenalectomized transgenic mice. GR or MR immunoreactivity was detected from the subpopulations of GABAergic neurons in the AHA. The AHA GABAergic neurons expressed mRNA of GR (42%), MR (38%) or both (8%). In addition, in brain slices incubated with corticosterone together with RU486 (MR-dominant group), the proportion of neurons showing a burst firing pattern was significantly higher than those in the slices incubated with vehicle, corticosterone, or corticosterone with spironolactone (GR-dominant group; 64 vs. 11∼14%, p<0.01 by χ2-test). Taken together, the results show that the corticosteroid receptors are expressed on the GABAergic neurons in the AHA, and can mediate the corticosteroid-induced plasticity in the firing pattern of these neurons. This study newly provides the experimental evidence for the direct glucocorticoid modulation of GABAergic neurons in the AHA in the vicinity of the PVN.

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Fig. 1.
Single cell RT-PCR analyses for GR and MR mRNA transcripts in GABAergic neurons of the AHA. Each product represents the expression of GR or MR in 10 individual neurons. The number represents the expression of GR and MR in each cell. The expected size of the products of GR and MR is 221 bp and 157 bp, respectively. No product was amplified in the (–) RT control.
kjpp-15-163f1.tif
Fig. 2.
GR and MR protein expression in the AHA. (A∼F) GR-immunoreactivity in the AHA cells. GR-immunoreactivity is depicted in red (A, D) and the cell bodies of eGFP (+) neurons (B, E) in the AHA are shown in green. Co-localizations of GR-immunoreactivity on the cell bodies of eGFP (+) neurons are illustrated in yellow. (G∼L) MR-immunoreactivity in the AHA cells. MR-immunoreactivity is depicted in red (G, J) and the cell bodies of eGFP (+) neurons (H, K) in the AHA are shown in green. Co-localizations of MR-immunoreactivity on the cell bodies of eGFP (+) neurons are illustrated in yellow. Panel A∼C and G∼I show 400× magnification of fluorescent photomicroscopes in the AHA (scale bar=50 μm). Panel D∼F and J∼L are higher magnifications of the rectangular areas in the C and I (scale bar=10 μm). Colocalization of GR- or MR- immunoreactivity and eGFP expressing neurons are indicated by arrows (F, L, respectively).
kjpp-15-163f2.tif
Fig. 3.
Proportion of AHA GABAergic neurons showing the burst firing at selective activation of corticosteroid receptors in vitro. (A, B) GABAergic neurons in the AHA showing burst action (A) or non-burst action potentials (B). Burst firing represents a cluster of action potentials riding on a slow depolarizing hump (A). The train of the action potential was evoked by 20∼60 pA of depolarizing current pulses (500-ms duration) at a holding potential of ∼ –80 mV. (C) comparing the proportion of bursting neurons in each group. In response to depolarizing currents, the proportion of the burst firing neurons of the MR-dominant group (100 nM CORT+500 nM RU486) is significantly higher than that of the other three treatment groups (∗∗p<0.01 by χ2-test). The total number of recorded neurons in each group is shown in parenthesis. CTL, control; CORT, corticosteroid; Spiro, spironolactone. Scale bars in A and B are 150 ms and 50 mV.
kjpp-15-163f3.tif
Table 1.
Information on the primers used in the study
Gene Accession number Sequence (Forward/Reverse) Length (bp) Tm Product size (bp)
GAD65 NM_008078 GCTCATCGCGTTCACATCAG 20 63.0 300
    AGTAACCCTCCACCCCAAGC 20 62.5  
GAD67 NM_008077 GGCTGATTACCTCTACGCCAAG 22 62.3 320
    CCTGACCCAACCTCTCTATCTCC 23 62.9  
GR X04435 GCCCCAAGTGAAAACAGAGA 20 60.23 221
    GATCCTGCTGCTGAGAAAGG 20 60.1  
MR AJ311855 ATATGGAAAGGCGCTGGAGT 20 60.98 157
    TCTTTGCTGCTCCCTTGAGT 20 60.13  
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