Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

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.


  1. Buchanan, C.M., Eccles, J.S. & Becker, J.B. Are adolescents the victims of raging hormones: evidence for activational effects of hormones on moods and behavior at adolescence. Psychol. Bull. 111, 62–107 (1992).

    Article  CAS  Google Scholar 

  2. Hayward, C. & Sanborn, K. Puberty and the emergence of gender differences in psychopathology. J. Adolesc. Health 30S, 49–58 (2002).

    Article  Google Scholar 

  3. Modesti, P.A. et al. Changes in blood pressure reactivity and 24-hour blood pressure profile occurring at puberty. Angiology 45, 443–450 (1994).

    Article  CAS  Google Scholar 

  4. Rudolph, U. et al. Benzodiazepine actions mediated by specific γ-aminobutyric acidA receptor subtypes. Nature 401, 796–800 (1999).

    Article  CAS  Google Scholar 

  5. Majewska, M.D., Harrison, N.L., Schwartz, R.D., Barker, J.L. & Paul, S.M. Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science 232, 1004–1007 (1986).

    Article  CAS  Google Scholar 

  6. Purdy, R.H., Morrow, A.L., Moore, P.H., Jr. & Paul, S.M. Stress-induced elevations of gamma-aminobutyric acid type A receptor-active steroids in the rat. Proc. Natl. Acad. Sci. USA 88, 4553–4557 (1991).

    Article  CAS  Google Scholar 

  7. Freeman, E.W., Purdy, R.H., Coutifaris, C., Rickels, K. & Paul, S.M. Anxiolytic metabolites of progesterone: correlation with mood and performance measures following oral progesterone administration to healthy female volunteers. Neuroendocrinology 58, 478–484 (1993).

    Article  CAS  Google Scholar 

  8. Bitran, D., Dugan, M., Renda, P., Ellis, R. & Foley, M. Anxiolytic effects of the neuroactive steroid pregnanolone (3alpha-OH-5beta-pregnan-20-one) after microinjection in the dorsal hippocampus and lateral septum. Brain Res. 850, 217–224 (1999).

    Article  CAS  Google Scholar 

  9. Chang, Y., Wang, R., Barot, S. & Weiss, D.S. Stoichiometry of a recombinant GABAA receptor. J. Neurosci. 16, 5415–5424 (1996).

    Article  CAS  Google Scholar 

  10. Wohlfarth, K.M., Bianchi, M.T. & Macdonald, R.L. Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit. J. Neurosci. 22, 1541–1549 (2002).

    Article  CAS  Google Scholar 

  11. Bianchi, M.T., Haas, K.F. & Macdonald, R.L. α1 and α6 subunits specify distinct desensitization, deactivation and neurosteroid modulation of GABAA receptors containing the δ subunit. Neuropharmacology 43, 492–502 (2002).

    Article  CAS  Google Scholar 

  12. Belelli, D., Casula, A., Ling, A. & Lambert, J.J. The influence of subunit composition on the interaction of neurosteroids with GABAA receptors. Neuropharmacology 43, 651–661 (2002).

    Article  CAS  Google Scholar 

  13. Wei, W., Zhang, N., Peng, Z., Houser, C.R. & Mody, I. Perisynaptic localization of delta subunit-containing GABA(A) receptors and their activation by GABA spillover in the mouse dentate gyrus. J. Neurosci. 23, 10650–10661 (2003).

    Article  CAS  Google Scholar 

  14. Stell, B.M., Brickley, S.G., Tang, C.Y., Farrant, M. & Mody, I. Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by delta subunit-containing GABA-A receptors. Proc. Natl. Acad. Sci. USA 100, 14439–14444 (2003).

    Article  CAS  Google Scholar 

  15. Corpechot, C. et al. Brain neurosteroids during the mouse oestrous cycle. Brain Res. 766, 276–280 (1997).

    Article  CAS  Google Scholar 

  16. Wisden, W., Laurie, D.J., Monyer, H. & Seeburg, P. The distribution of 13 GABA-A receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. J. Neurosci. 12, 1040–1062 (1992).

    Article  CAS  Google Scholar 

  17. Smith, S.S. et al. GABAA receptor α4 subunit suppression prevents withdrawal properties of an endogenous steroid. Nature 392, 926–929 (1998).

    Article  CAS  Google Scholar 

  18. Smith, S.S., Ruderman, Y., Frye, C.A., Homanics, G.E. & Yuan, M. Steroid withdrawal in the mouse results in anxiogenic effects of 3α,5β-THP: a possible model of premenstrual dysphoric disorder. Psychopharmacology (Berl.) 186, 323–333 (2006).

    Article  CAS  Google Scholar 

  19. Sundstrom-Poromaa, I. et al. Hormonally regulated α4β2δ GABAA receptors are a target for alcohol. Nat. Neurosci. 5, 721–722 (2002).

    Article  CAS  Google Scholar 

  20. Staley, K.J. & Mody, I. Shunting of excitatory input to dentate gyrus granule cells by a depolarizing GABA-A receptor-mediated postsynaptic conductance. J. Neurophysiol. 68, 197–212 (1992).

    Article  CAS  Google Scholar 

  21. Staley, K.J. & Proctor, W.R. Modulation of mammalian dendritic GABA(A) receptor function by the kinetics of Cl- and HCO3- transport. J. Physiol. (Lond.) 519, 693–712 (1999).

    Article  CAS  Google Scholar 

  22. Alger, B.E. & Nicoll, R.A. Pharmacological evidence for two kinds of GABA receptor on rat hippocampal pyramidal cells studied in vitro. J. Physiol. (Lond.) 328, 125–141 (1982).

    Article  CAS  Google Scholar 

  23. Lambert, N.A., Borroni, A.M., Grover, L.M. & Teyler, T.J. Hyperpolarizing and depolarizing GABA-A receptor-mediated dendritic inhibition in area CA1 of the rat hippocampus. J. Neurophysiol. 66, 1538–1548 (1991).

    Article  CAS  Google Scholar 

  24. Wu, Y., Wang, W. & Richerson, G. GABA transaminase inhibition induces spontaneous and enhances depolarization-evoked GABA efflux via reversal of the GABA transporter. J. Neurosci. 21, 2630–2639 (2001).

    Article  CAS  Google Scholar 

  25. Miyazawa, A., Fujiyoshi, Y. & Unwin, N. Structure and gating mechanism of the acetylcholine receptor pore. Nature 423, 949–955 (2003).

    Article  CAS  Google Scholar 

  26. Kelley, S.P., Dunlop, J.I., Kirkness, E., Lambert, J.J. & Peters, J.A. A cytoplasmic region determines single-channel conductance in 5-HT3 receptors. Nature 424, 321–324 (2003).

    Article  CAS  Google Scholar 

  27. Chen, J., Mitcheson, J.S., Lin, M. & Sanguinetti, M.C. Functional roles of charged residues in the putative voltage sensor of the HCN2 pacemaker channel. J. Biol. Chem. 275, 36465–36471 (2000).

    Article  CAS  Google Scholar 

  28. Fadalti, M. et al. Changes of serum allopregnanolone levels in the first 2 years of life and during pubertal development. Pediatr. Res. 46, 323–327 (1999).

    Article  CAS  Google Scholar 

  29. Caraiscos, V.B. et al. Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by alpha 5 subunit-containing gamma-aminobutyric acid type A receptors. Proc. Natl. Acad. Sci. USA 101, 3662–3667 (2004).

    Article  CAS  Google Scholar 

  30. Stell, B.M. & Mody, I. Receptors with different affinities mediate phasic and tonic GABA(A) conductances in hippocampal neurons. J. Neurosci. 22, RC223 (2002).

    Article  Google Scholar 

  31. Perkins, K.L. Cell-attached voltage-clamp and current-clamp recording and stimulation techniques in brain slices. J. Neurosci. Methods 154, 1–18 (2006).

    Article  CAS  Google Scholar 

  32. Belelli, D., Peden, D.R., Rosahl, T.W., Wafford, K. & Lambert, J.J. Extrasynaptic GABA-A receptors for thalamocortical neurons: A molecular target for hypnotics. J. Neurosci. 25, 11513–11520 (2005).

    Article  CAS  Google Scholar 

  33. Ulrich, D. & Huguenard, J.R. Nucleus-specific chloride homeostasis in rat thalamus. J. Neurosci. 17, 2348–2354 (1997).

    Article  CAS  Google Scholar 

  34. Girdler, S.S., Beth Mechlin, M., Light, K.C. & Morrow, A.L. Ethnic differences in allopregnanolone concentrations in women during rest and following mental stress. Psychophysiology 43, 331–336 (2006).

    Article  Google Scholar 

  35. McEwen, B.S. Stressed or stressed out: what is the difference? J. Psychiatry Neurosci. 30, 315–318 (2005).

    PubMed  PubMed Central  Google Scholar 

  36. Lundgren, P., Stromberg, J., Backstrom, T. & Wang, M. Allopregnanolone-stimulated GABA-mediated chloride ion flux is inhibited by 3beta-hydroxy-5alpha-pregnan-20-one (isoallopregnanolone). Brain Res. 982, 45–53 (2003).

    Article  CAS  Google Scholar 

  37. Ramsey, I.S., Moran, M.M., Chong, J.A. & Clapham, D.E. A voltage-gated proton-selective channel lacking the pore domain. Nature 440, 1213–1216 (2006).

    Article  CAS  Google Scholar 

  38. Olsen, R.W. & Snowman, A. Chloride-dependent enhancement by barbiturates of gamma-aminobutyric acid receptor binding. J. Neurosci. 2, 1812–1823 (1982).

    Article  CAS  Google Scholar 

  39. Turner, J.H. & Raymond, J.R. Interaction of calmodulin with the serotoin 5-hydroxytryptamine2A receptor. A putative regulator of G protein coupling and receptor phosphorylation by protein kinase C. J. Biol. Chem. 280, 30741–30750 (2005).

    Article  CAS  Google Scholar 

  40. Haas, K.F. & Macdonald, R.L. GABAA receptor subunit gamma2 and delta subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts. J. Physiol. (Lond.) 514, 27–45 (1999).

    Article  CAS  Google Scholar 

  41. Chen, X. et al. Inhibition of a background potassium channel by Gq protein alpha-subunits. Proc. Natl. Acad. Sci. USA 103, 3422–3427 (2006).

    Article  CAS  Google Scholar 

  42. Prescott, S.A. & de Koninck, Y. Gain control of firing rate by shunting inhibition: roles of synaptic noise and dendritic saturation. Proc. Natl. Acad. Sci. USA 100, 2076–2081 (2003).

    Article  CAS  Google Scholar 

  43. Bai, D. et al. Distinct functional and pharmacological properties of tonic and quantal inhibitory postsynaptic currents mediated by y-aminobutyric acid(A) receptors in hippocampal neurons. Mol. Pharmacol. 59, 814–824 (2000).

    Article  Google Scholar 

  44. Maguire, J.L., Stell, B.M., Rafizadeh, M. & Mody, I. Ovarian cycle-linked changes in GABA(A) receptors mediating tonic inhibition alter seizure susceptibility and anxiety. Nat. Neurosci. 8, 797–804 (2005).

    Article  CAS  Google Scholar 

  45. Romeo, R.D. Neuroendocrine and behavioral development during puberty: a tale of two axes. Vitam. Horm. 71, 1–25 (2005).

    Article  CAS  Google Scholar 

  46. Agis-Balboa, R.C. et al. Characterization of brain neurons that express enzymes mediating neurosteroid biosynthesis. Proc. Natl. Acad. Sci. USA 103, 14602–14607 (2006).

    Article  CAS  Google Scholar 

  47. Mele, P. et al. Increased expression of the gene for the Y1 receptor of the neuropeptde Y in the amygdala and paraventricular nucleus of Y1R/LacZ transgenic mice in response to restraint stress. J. Neurochem. 89, 1471–1478 (2004).

    Article  CAS  Google Scholar 

  48. Freeman, E.W., Frye, C.A., Rickels, K., Martin, P.A. & Smith, S.S. Allopregnanolone levels and symptom improvement in severe premenstrual syndrome. J. Clin. Psychopharmacol. 22, 516–520 (2002).

    Article  CAS  Google Scholar 

  49. Schmidt, P.J., Nieman, L.K., Danaceau, M.A., Adams, L.F. & Rubinow, D.R. Differential behavioral effects of gonadal steroids in women with premenstrual syndrome. N. Engl. J. Med. 338, 209–216 (1998).

    Article  CAS  Google Scholar 

  50. Andreen, L. et al. Relationship between allopregnanolone and negative mood in postmenopausal women taking sequential hormone replacement therapy with vaginal progesterone. Psychoneuroendocrinology 30, 212–224 (2004).

    Article  Google Scholar 

Download references


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

Authors and Affiliations



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.

Corresponding author

Correspondence to Sheryl S Smith.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Shen, H., Gong, Q., Aoki, C. et al. Reversal of neurosteroid effects at α4β2δ GABAA receptors triggers anxiety at puberty. Nat Neurosci 10, 469–477 (2007).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing