Adjustment mechanism of the neural steroidal substances on the central nervous system glycine receptor and functional studies
|School||University of Science and Technology of China|
|Keywords||Cultured hippocampal and spinal neurons of rat Native and recombinant GlyRs Glycineric mIPSCs Neurosteroids Pregnanolone Estrogen 17-β-estradiol Nociception Development Whole-cell patch-clamp recording|
Steroid hormones exert the functions by binding to their respective intracellular receptors that can act as transcription factors and regulate gene expression. Limited by the rate of protein biosynthesis, the action of steroids at genome requires a time period that lasts from minutes to hours. Since the 1980’s, researchers have focused on the modulatory effects of steroid hormones in the central nervous system （CNS）. Considerable evidence has emerged that, during a millisecond to second time window, some steroids alter neuronal excitability via the cell surface through the interaction with specific neurotransmitter receptors or G-protein coupled receptors. The term, ’neuroactive steroids’, has been adopted for steroids with these particular properties. Furthermore, some neuroactive steroids were found to be present in higher concentrations in local tissue from the CNS than in the plasma. The steroidogenic enzymes for the synthesis of steroids were demonstrated to be expressed in neurons and glia at different locations in the CNS. Thus, the brain is a steroidogenic tissue and some steroids can be synthesized de novo in the CNS. In 1997, Baulieu named these steroids as ’neurosteroids’. In general, through the genomic and nongenomic effects within the CNS, neurosteroids exert a broad spectrum of action on neuronal function and plasticity.In the adult animal CNS, GABAA receptors （GABAARs） is a important inhibitory receptor, mediating the fast inhibitory synaptic neurotransmission. A majority of previous studies has focused on the modulatory effects of the neurosteroids on GABAergic machinery. For examples, the best-studied series of sedative-hypnotic pregnane steroids are potent endogenous allosteric enhancers of GABAARs. Also, estrogen can alter the neuronal activity through suppressing the inhibitory GABAergic synaptic transmission. However, little information is available up to now about the effects of neurosteroids on another inhibitoy receptor, glycine receptors （GlyRs）. It is now well known that GlyRs is extensively expressed in the CNS. In spinal cord, GlyRs also participates in mediating the fast inhibitory synaptic neurotransmission and is involved in sensory information processing （including nociceptive signal transduction）. Particularly, the GlyRα3 subunit is the essential target for inflammatory pain sensitization. In hippocampus, GlyRs is widely expressed, but no glycinergic synaptic transmission was recorded. Accordingly, the study of hippocampal GlyRs has been largely ignored. Several recent studies suggest that the tonic activation of GlyRs in hippocampus contributes to the activity in the synaptic network, the cross-inhibition of GABAARs, and the short-term plasticity. Additionally, GlyRs is functionally implicated in the development of cortex, retina and spinal cord. Thus, we wonder whether and how the neurosteroids modulate the GlyRs in central neurons. In the present studies, using the cultured hippocampal and spinal neurons, plasmid transform/transfection techniques and whole-cell patch clamp recording, we investigate the neurosteroids pregnanlone （PGN; 5β-pregnan-3α-ol-20-one） and 17-β-estradiol （E2） effects on the function of GlyRs and explore the possible physiological significance of the modulatory effects.1. Modulation of PGN on glycinergic response in cultured spinal dorsal horn neurons of ratIn the present study, we examined the effects of PGN and its three isomers on GlyRs by using whole-cell patch-clamp technique. Our results showed that PGN reversibly inhibited the amplitude of glycine-induced current (IGly) mediated by native GlyRs and recombinantα1-,α2-,α3- andα1β-GlyR. In cultured spinal dorsal horn neurons, PGN inhibited the IGly in dose-dependent manner, with an IC50 of 1.0±0.3μM. The inhibitory effect of PGN on IGly was voltage-independent and PGN shifted the concentration-response curve for IGly rightward in a parallel manner without altering the maximal value and Hill coefficient. The isomer of PGN, allopregnanolone slightly enhanced IGly, whereas iso-pregnanolone and iso-allopregnanolone did not affect the IGly significantly in cultured spinal dorsal horn neurons. Thus, our results suggest that the inhibitory effect of PGN on IGly is of a competitive type and depends on the stereo structure of PGN. Furthermore, PGN decreased the amplitude and frequency of the glycinergic miniature inhibitory postsynaptic currents （mIPSCs）. Through modulating the glycinergic inhibitory neurotransmission, PGN may affect the nociceptive sensory processing under physiological and pathological conditions.2. Modulation of 17-β-estradiol on GlyRs in cultured CNS neurons of ratIn cultured hippocampal neurons, we firstly examined the effects of E2 on GlyRs by using whole-cell patch-clamp technique, and found that E2 rapidly and reversibly inhibited the peak amplitude of IGly. The inhibitory effect of E2 on IGly was voltage-independent, and the inhibition was glycine concentration-independent, suggesting a noncompetitive mechanism of E2 effect. In the presence of calcium chelator （BAPTA）, protein kinase inhibitor （staurosporine）, classical estrogen receptor antagonist, and the G-protein inhibitor GDP-β-S, the extent of inhibition was not changed, suggesting that E2 directly inhibited GlyRs not by classical estrogen receptors （ERs） or other intracellular signal pathways activated by E2. Additionally, in vitro expression of GlyR subunits in recombination demonstrates that E2 selectively inhibited the peak amplitude of IGly mediated byα2 andα2β-GlyR. Furthermore, consistent with the decline ofα2 expression in spinal cord during development, the extent of E2 inhibition on IGly is significantly declined with time in cultured rat spinal neurons; while in cultured hippocampal neurons, the inhibition is independent of the days in culture. Thus, we suggest that GlyRs is a molecular target of E2 which directly binds to and noncompetitively modulates GlyRs, leading to the alteration of neuronal excitability and contributing to the estrogenic effects in hippocampus and the early CNS development.There are several novelties residing in the present studies: 1) Previous studies reported the effects of PGN on recombinant GlyRs. However, the effects of PGN on native GlyRs remain unclear. In this study, we examined the modulatory effects of PGN on native GlyRs and glycinergic mIPSC in cultured spinal dorsal horn neurons, which may reflect the effects of the endogenous PGN on GlyRs in vivo. In addition, we also investigated the mechanisms underlying the effects of PGN on glycinergic response. 2) Generally, estrogens exert the estrogenic effects in the CNS primarily by activating the estrogen receptors. Here, we demonstrate that E2 may directly affect the functioning of inhibitory GlyR channels, independent of the well known signaling pathways of E2 and classical ERs. Thus, this study might add a new molecular target of E2 and helpfully elucidate the multifaced estrogenic effects in the CNS.