Phosphorylation influences neurosteroid modulation of synaptic GABAA receptors in rat CA1 and dentate gyrus neurones

doi:10.1016/S0028-3908(03)00251-X

Neuropharmacology

Volume 45, Issue 6, November 2003, Pages 873-883

https://doi.org/10.1016/S0028-3908(03)00251-X Get rights and content

Abstract

The neurosteroid 5β-pregnan-3α-ol-20-one (5β3α) is a potent, endogenous, positive allosteric modulator of the GABA_A receptor. Relatively low concentrations of 5β3α (10–100 nM), thought to occur physiologically, caused a concentration-dependent slowing of the decay of GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs) recorded from hippocampal CA1 pyramidal neurones. However, much greater concentrations of this neurosteroid (≥300 nM) were required to similarly influence dentate granule cell mIPSCs. By contrast, the allosteric modulators pentobarbitone and flunitrazepam were equi-effective in prolonging mIPSCs in both neuronal types. Hence, the neurosteroid selectively differentiates between the synaptic GABA_A receptors of these hippocampal neurones. Inhibition of either protein kinase A, or C, greatly reduced the sensitivity of CA1 synaptic GABA_A receptors to 5β3α, but not pentobarbitone, whereas stimulation of PKC had no effect on steroid sensitivity. However, in dentate gyrus granule cells, activation of PKC made mIPSCs sensitive to a previously ineffective concentration of 5β3α. Collectively, these results suggest that the GABA-modulatory effects of physiological levels of the neurosteroid will not be uniformly experienced throughout the central nervous system, or even within the same brain region such as the hippocampus, but will be neurone-specific and will be dependent on the phosphorylation status of the GABA_A receptor, or associated proteins.

Introduction

Certain endogenous steroids such as 5α-pregnan-3α-ol-20-one (5α3α) and 5β-pregnan-3α-ol-20-one (5β3α) act in a rapid, non-genomic manner to allosterically enhance GABA_A receptor function (Lambert et al., 2001). As a consequence these compounds are anticonvulsant, anxiolytic, sedative, hypnotic and at higher doses induce general anesthesia (Gasior et al., 1999). These observations, coupled with the demonstration that such steroids derive not only from endocrine glands (e.g. ovaries and adrenals; i.e. neuroactive steroids), but may additionally be synthesized in the central nervous system (CNS; i.e. neurosteroids), by glia and certain neurones (Paul and Purdy, 1992, Reddy, 2003), has led to speculation that the activity of the brain’s major inhibitory neurotransmitter receptor may be subject to both remote, endocrine and local, paracrine “fine-tuning” by these endogenous regulators. In support of this concept, inhibition of brain 5α3α synthesis (by the administration of a 5α-reductase inhibitor), is coincident with a reduction of both the behavioural and electrophysiological effects of the GABA_A receptor agonist muscimol (Pinna et al., 2000) and this treatment reduces the decay of GABA-mediated inhibitory post-synaptic currents, suggesting that in untreated animals there is an endogenous neurosteroid tone (Puia et al., 2003). However, although the pharmacological effects of administered neurosteroids are well documented (Gasior et al., 1999), their putative physiological/pathophysiological role(s) remains equivocal. In part, this derives from a paucity of information on the influence of physiologically-relevant concentrations of these steroids on inhibitory synaptic transmission in the brain.

Here we have investigated the interaction of both physiological (low nM) and pharmacological (high nM–low μM) concentrations (Lambert et al., 1995) of 5β3α on GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs) recorded from CA1 and dentate granule neurones of the rat hippocampus. The study clearly demonstrates that physiological concentrations of 5β3α selectively prolong the decay of mIPSCs recorded from CA1 neurones, but that greater concentrations of the neurosteroid are required to similarly influence dentate gyrus granule cell mIPSCs, a selectivity that seems in part to be dependent upon the relative activity of endogenous kinases.

Our observations, coupled with neurosteroid synthesis being brain region-dependent (Mellon and Vaudry, 2001, Melcangi et al., 2001), suggest that the physiological influence of this endogenous modulator on neuronal excitability will not be globally experienced throughout the CNS, but will be brain region- and indeed neurone-specific.

Section snippets

Hippocampal slice preparation

Hippocampal slices were prepared from Sprague Dawley rats, of either sex, aged 16–22 days as described previously (Dale et al., 2000). Animals were killed by cervical dislocation in accordance with Schedule 1 of the UK Government Animals (Scientific Procedures) Act 1986. Following decapitation, the brain was quickly removed and placed in high-Mg²⁺ ice-cold artificial cerebro-spinal fluid (aCSF) containing (in mM): NaCl, 124; KCl, 3; NaHCO₃, 26; NaH₂PO₄, 1.25; CaCl₂, 2; MgSO₄, 11; d-glucose, 10,

General properties of CA1 pyramidal neurone and dentate granule cell mIPSCs

Miniature IPSCs recorded from CA1 pyramidal and dentate gyrus granule neurones (–60 mV; 35 °C), were reversibly inhibited by bicuculline (10 μM) and reversed in direction at a holding potential of ~0 mV (in symmetrical chloride recording solutions), consistent with these currents being mediated by GABA_A receptors. The 10–90% rise time, mIPSC peak amplitude and weighted decay time constant for CA1 pyramidal neurones and dentate gyrus granule cells were similar (Table 1). The decay phase of

Discussion

Endogenous neurosteroid levels are not static, but change in response to a variety of physiological, or pathophysiological stimuli, for example, being elevated in acute stress and during pregnancy; subject to cyclical fluctuations (e.g. during the menstrual cycle) and altered in certain psychiatric disorders and by drugs including fluoxetine and alcohol (Paul and Purdy, 1992, Lambert et al., 1995, Uzunova et al., 1998, Rupprecht et al., 2001, Morrow et al., 2001). To appreciate how

Acknowledgements

The work reported here was supported by an EC Bioscience and Health Grant BMH4-CT97-2359, the Commission of the European Communities RTD program “Quality of Life and Management of Living Resources” QLK1-CT-2000-00179, The Anonymous Trust (Dundee) and by Tenovus Tayside. S.C.H. and B.G.F. were supported by an MRC studentship and a Caledonian Research Foundation Fellowship, respectively. We are grateful to Dr John Dempster for help with data acquisition and analysis and to Dr Delia Belelli for

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Citation Excerpt :
For example, GABAAR δ-subunit KO mice display reduced sensitivity to neurosteroids, which could be restored by activation of PKC with phorbol esters (Vicini et al., 2002). Further, inhibition of either PKA or PKC decreased the sensitivity of GABAARs to neurosteroids in hippocampal CA1 pyramidal neurons, whereas, in contrast, activation of PKC had no effect (Harney et al., 2003). Interestingly, and in the same preparation, PKC activation did enhance the sensitivity of GABAARs to neurosteroid in dentate gyrus granule cells.
γ-Aminobutyric acid type A receptors (GABA_ARs) are the principal mediators of fast synaptic inhibition in the brain as well as the low persistent extrasynaptic inhibition, both of which are fundamental to proper brain function. Thus unsurprisingly, deficits in GABA_ARs are implicated in a number of neurological disorders and diseases. The complexity of GABA_AR regulation is determined not only by the heterogeneity of these receptors but also by its posttranslational modifications, the foremost, and best characterized of which is phosphorylation. This review will explore the details of this dynamic process, our understanding of which has barely scratched the surface. GABA_ARs are regulated by a number of kinases and phosphatases, and its phosphorylation plays an important role in governing its trafficking, expression, and interaction partners. Here, we summarize the progress in understanding the role phosphorylation plays in the regulation of GABA_ARs. This includes how phosphorylation can affect the allosteric modulation of GABA_ARs, as well as signaling pathways that affect GABA_AR phosphorylation. Finally, we discuss the dysregulation of GABA_AR phosphorylation and its implication in disease processes.
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Phosphorylation influences neurosteroid modulation of synaptic GABAA receptors in rat CA1 and dentate gyrus neurones

Abstract

Introduction

Section snippets

Hippocampal slice preparation

General properties of CA1 pyramidal neurone and dentate granule cell mIPSCs

Discussion

Acknowledgements

Neuropharmacology

Neuron

J. Biol. Chem.

Trends Pharmacol. Sci.

Neuropharmacology

Trends Neurosci.

Brain Res. Brain Res. Rev.

Trends Pharmacol. Sci.

Brain Res. Mol. Brain Res.

Neuropharmacology

Int. Rev. Neurobiol.

Brain Res. Brain Res. Rev.

Neuron

Neuropharmacology

Neuroscience

Neuropharmacology

Trends Pharmacol. Sci.

Brain Res. Brain Res. Rev.

The synaptic basis of GABAA, slow

J. Neurosci.

International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function

Pharmacol. Rev.

Gamma-aminobutyric acid A receptor subunit expression predicts functional changes in hippocampal dentate granule cells during postnatal development

J. Neurochem.

Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABAA receptors

Br. J. Pharmacol.

Effects of a naturally occurring neurosteroid on GABAA IPSCs during development in rat hippocampal or cerebellar slices

J. Physiol.

Direct measurement of adenosine release during hypoxia in the CA1 region of the rat hippocampal slice

J. Physiol.

Phosphorylation influences neurosteroid modulation of synaptic GABA_A receptors in rat CA1 and dentate gyrus neurones

The synaptic basis of GABA_A, slow

Pharmacological characterization of a novel cell line expressing human alpha(4)beta(3)delta GABA_A receptors

Effects of a naturally occurring neurosteroid on GABA_A IPSCs during development in rat hippocampal or cerebellar slices