Reversal of neurosteroid effects at α4β2δ GABAA receptors triggers anxiety at puberty

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Abstract

Puberty is characterized by mood swings and anxiety, which are often produced by stress. Here we show that THP (allopregnanolone), a steroid that is released as a result of stress, increases anxiety in pubertal female mice, in contrast to its anxiety-reducing effect in adults. Anxiety is regulated by GABAergic inhibition in limbic circuits. Although this inhibition is increased by THP administration before puberty and in adults, during puberty THP reduces the tonic inhibition of pyramidal cells in hippocampal region CA1, leading to increased excitability. This paradoxical effect of THP results from inhibition of α4βδ GABAA receptors. These receptors are normally expressed at very low levels, but at puberty, their expression is increased in hippocampal area CA1, where they generate outward currents. THP also decreases the outward current at recombinant α4β2δ receptors, and this effect depends on arginine 353 in the α4 subunit, a putative site for modulation by Cl. Therefore, inhibition of α4β2δ GABAA receptors by THP provides a mechanism for the generation of anxiety at puberty.

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Figure 1: The neurosteroid THP decreases outward current gated by α4β2δ GABAA receptors.
Figure 2: Arginine 353 in the α4 subunit is necessary for the direction-sensitive inhibition of α4β2δ GABAA receptors by THP.
Figure 3: Increased expression of α4 and δ subunits on dendrites of CA1 hippocampal pyramidal cells at the onset of puberty.
Figure 4: THP inhibits tonic GABAergic current recorded from hippocampal slices at puberty.
Figure 5: THP increases excitability of hippocampal pyramidal cells at the onset of puberty.
Figure 6: THP lowers the current threshold for spiking of pyramidal cells at the onset of puberty.
Figure 7: THP paradoxically increases anxiety after the onset of puberty.

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Acknowledgements

We thank C.A. Frye for performing steroid assays on hippocampal tissue, W. Sieghart for supplying the δ antibody, G. Homanics for supplying and genotyping the δ−/− mice, K. Perkins and D.H. Smith for discussions, and C. McBain and J. Celentano for reading the manuscript. The work in this study was supported by grants from the US National Institutes of Health (to S.S.S., C.A. and K.W.) and from the US National Institute of Alcoholism and Alcohol Abuse (to S.S.S.).

Author information

H.S. (slice physiology) and Q.H.G. (recombinant receptors) performed the electrophysiology experiments, C.A. performed the immunohistochemical experiments, M.Y. conducted the western blot and molecular studies, Y.R. conducted the behavioral experiments, M.D. did the transfections, K.W. supervised the molecular studies and contributed to the writing and S.S.S. supervised the study, performed the mutagenesis studies and wrote the paper.

Correspondence to Sheryl S Smith.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

GABA concentration-response relationships and THP administration. (PDF 70 kb)

Supplementary Fig. 2

THP inhibition of outward current is dependent upon GABA concentration. (PDF 72 kb)

Supplementary Fig. 3

Pharmacological changes in the tonic inhibitory current of CA1 hippocampus are consistent with increased expression of α4βδ receptors after the onset of puberty. (PDF 128 kb)

Supplementary Fig. 4

THP increases the input resistance of hippocampal pyramidal cells at the onset of puberty. (PDF 30 kb)

Supplementary Table 1

ANOVA – F Tables – recombinant receptor experiments. (PDF 50 kb)

Supplementary Table 2

Mutations. (PDF 36 kb)

Supplementary Table 3

Tukey's test—planned post-hoc comparisons. Mutations, 1 μM GABA. (PDF 48 kb)

Supplementary Table 4

Tukey's test—planned post-hoc comparisons. Mutations, 10 μM GABA. (PDF 41 kb)

Supplementary Table 5

Western blot. (PDF 40 kb)

Supplementary Table 6

Slice pharmacology. (PDF 35 kb)

Supplementary Table 7

Tonic current. (PDF 32 kb)

Supplementary Table 8

Slice physiology. (PDF 32 kb)

Supplementary Table 9

Tukey's post-hoc comparisons. (PDF 43 kb)

Supplementary Table 10

Neuronal excitability. (PDF 35 kb)

Supplementary Table 11

Cell-attached spiking, Tukey's test—planned post-hoc comparisons. (PDF 42 kb)

Supplementary Table 12

Two-way ANOVA. (PDF 35 kb)

Supplementary Table 13

Holm-Sidak post-hoc comparison. (PDF 52 kb)

Supplementary Table 14

ANOVA, elevated plus maze. (PDF 33 kb)

Supplementary Table 15

Least significant difference planned post-hoc comparisons. (PDF 55 kb)

Supplementary Methods (PDF 248 kb)

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