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• W1989888504 abstract "Pregnenolone sulfate (PREGS), one of the most abundantly produced neurosteroids in the mammalian brain, improves cognitive performance in rodents. The mechanism of this effect has been attributed to its allosteric modulatory actions on glutamate- and γ-aminobutyric acid-gated ion channels. Here we report a novel effect of PREGS that could also mediate some of its actions in the nervous system. We found that PREGS induces a robust potentiation of the frequency but not the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors in cultured hippocampal neurons. PREGS also decreased paired pulse facilitation of autaptic EPSCs evoked by depolarization, indicating that it modulates glutamate release probability presynaptically. PREGS potentiation of mEPSCs was mimicked by dehydroepiandrosterone sulfate and (+)-pentazocine but not by (−)-pentazocine, the synthetic (−)-enantiomer of PREGS or the inactive steroid isopregnanolone. The ς receptor antagonists, haloperidol and BD-1063, blocked the effect of PREGS on mEPSCs, as did pertussis toxin and the membrane-permeable Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl) ester. These results suggest that PREGS increases spontaneous glutamate release via activation of a presynaptic Gi/o-coupled ς receptor and an elevation in intracellular Ca2+ levels. We postulate that presynaptic actions of neurosteroids have a role in the maturation and/or maintenance of synaptic networks and the processing of information in the central nervous system. Pregnenolone sulfate (PREGS), one of the most abundantly produced neurosteroids in the mammalian brain, improves cognitive performance in rodents. The mechanism of this effect has been attributed to its allosteric modulatory actions on glutamate- and γ-aminobutyric acid-gated ion channels. Here we report a novel effect of PREGS that could also mediate some of its actions in the nervous system. We found that PREGS induces a robust potentiation of the frequency but not the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors in cultured hippocampal neurons. PREGS also decreased paired pulse facilitation of autaptic EPSCs evoked by depolarization, indicating that it modulates glutamate release probability presynaptically. PREGS potentiation of mEPSCs was mimicked by dehydroepiandrosterone sulfate and (+)-pentazocine but not by (−)-pentazocine, the synthetic (−)-enantiomer of PREGS or the inactive steroid isopregnanolone. The ς receptor antagonists, haloperidol and BD-1063, blocked the effect of PREGS on mEPSCs, as did pertussis toxin and the membrane-permeable Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl) ester. These results suggest that PREGS increases spontaneous glutamate release via activation of a presynaptic Gi/o-coupled ς receptor and an elevation in intracellular Ca2+ levels. We postulate that presynaptic actions of neurosteroids have a role in the maturation and/or maintenance of synaptic networks and the processing of information in the central nervous system. pregnenolone sulfate γ-aminobutyric acid N-methyl-d-aspartate NMDA receptor endoplasmic reticulum miniature excitatory postsynaptic current paired pulse facilitation excitatory postsynaptic current 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl) ester (−)-enantiomer of PREGS α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor guanosine 5′-3-O-(thio)triphosphate phospholipase C inositol 1,4,5-trisphosphate receptor In the 1980s, Baulieu and collaborators (reviewed in Ref. 1Baulieu E.E. Psychoneuroendocrinology. 1998; 23: 963-987Crossref PubMed Scopus (516) Google Scholar) made the important discovery that certain steroids are synthesized in the central and peripheral nervous systems. These compounds, known as neurosteroids, are produced locally in glial and neuronal cells and can exert important modulatory actions in the nervous system. A particularly abundant neurosteroid in the central nervous system is pregnenolone sulfate (PREGS)1 (1Baulieu E.E. Psychoneuroendocrinology. 1998; 23: 963-987Crossref PubMed Scopus (516) Google Scholar, 2Liere P. Akwa Y. Weill-Engerer S. Eychenne B. Pianos A. Robel P. Sjovall J. Schumacher M. Baulieu E.E. J. Chromatogr. B Biomed. Sci. Appl. 2000; 739: 301-312Crossref PubMed Scopus (148) Google Scholar). Although the neurophysiological role of endogenous PREGS has yet to be conclusively established, experiments involving exogenous administration of this compound suggest that it has a promnesic effect (3Flood J.F. Morley J.E. Roberts E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1567-1571Crossref PubMed Scopus (446) Google Scholar, 4Flood J.F. Morley J.E. Roberts E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10806-10810Crossref PubMed Scopus (170) Google Scholar, 5Vallee M. Mayo W. Darnaudery M. Corpechot C. Young J. Koehl M. Le Moal M. Baulieu E.E. Robel P. Simon H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14865-14870Crossref PubMed Scopus (292) Google Scholar, 6Akwa Y. Ladurelle N. Covey D.F. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14033-14037Crossref PubMed Scopus (122) Google Scholar, 7Vallee M. Shen W. Heinrichs S.C. Zorumski C.F. Covey D.F. Koob G.F. Purdy R.H. Eur. J. Neurosci. 2001; 14: 2003-2010Crossref PubMed Google Scholar). For example, post-training injection of PREGS into the hippocampus and amygdala of mice improves retention for foot shock active avoidance training (4Flood J.F. Morley J.E. Roberts E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10806-10810Crossref PubMed Scopus (170) Google Scholar). In addition, low levels of PREGS in the hippocampus were found to be correlated with a deficiency in cognitive performance in aged rats, which could be ameliorated by intrahippocampal injections of this neurosteroid (5Vallee M. Mayo W. Darnaudery M. Corpechot C. Young J. Koehl M. Le Moal M. Baulieu E.E. Robel P. Simon H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14865-14870Crossref PubMed Scopus (292) Google Scholar). More recently, it was demonstrated that PREGS attenuates amyloid peptide-induced amnesia in mice (8Maurice T. Su T.P. Privat A. Neuroscience. 1998; 83: 413-428Crossref PubMed Scopus (213) Google Scholar). Thus, PREGS or its analogs could potentially be used for the treatment of Alzheimer's disease and other neuropsychiatric disorders. Although the mechanisms by which PREGS produces cognitive effects are not fully understood, numerous studies suggest that this agent modulates several neuronal ion channels (9Rupprecht R. Holsboer F. Trends Neurosci. 1999; 22: 410-416Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). For instance, PREGS has been shown to inhibit γ-aminobutyric acid-type A (GABAA) receptors (10Lambert J.J. Belelli D. Hill-Venning C. Callachan H. Peters J.A. Cell Mol. Neurobiol. 1996; 16: 155-174Crossref PubMed Scopus (89) Google Scholar), to potentiateN-methyl-d-aspartate (NMDA) receptors (11Irwin R.P. Maragakis N.J. Rogawski M.A. Purdy R.H. Farb D.H. Paul S.M. Neurosci. Lett. 1992; 141: 30-34Crossref PubMed Scopus (156) Google Scholar, 12Wu F.S. Gibbs T.T. Farb D.H. Mol. Pharmacol. 1991; 40: 333-336PubMed Google Scholar, 13Park-Chung M. Wu F.S. Purdy R.H. Malayev A.A. Gibbs T.T. Farb D.H. Mol. Pharmacol. 1997; 52: 1113-1123Crossref PubMed Scopus (196) Google Scholar, 14Bowlby M.R. Mol. Pharmacol. 1993; 43: 813-819PubMed Google Scholar), and to inhibit voltage-gated Ca2+ channels (15ffrench-Mullen J.M. Danks P. Spence K.T. J. Neurosci. 1994; 14: 1963-1977Crossref PubMed Google Scholar). In addition to ion channels, PREGS has also been shown to target metabotropic receptors, such as ς receptors. These receptors were initially thought to belong to the opioid family of receptors, but they are now categorized separately (16Maurice T. Phan V.L. Urani A. Kamei H. Noda Y. Nabeshima T. Jpn. J. Pharmacol. 1999; 81: 125-155Crossref PubMed Scopus (189) Google Scholar). Two classes of pharmacologically defined ς receptors are widely accepted and are denoted as the ς1 and ς2 subtypes (16Maurice T. Phan V.L. Urani A. Kamei H. Noda Y. Nabeshima T. Jpn. J. Pharmacol. 1999; 81: 125-155Crossref PubMed Scopus (189) Google Scholar). ς1receptors bind (+)-benzomorphans and haloperidol with high affinity. In contrast, ς2 receptors bind haloperidol and (+)-benzomorphans with low affinity, and they also bind benzomorphans without enantioselectivity. ς ligand binding sites can be detected both intracellularly on the endoplasmic reticulum (ER) and extracellularly on the plasma membrane (17McCann D.J. Su T.P. Eur. J. Pharmacol. 1990; 188: 211-218Crossref PubMed Scopus (66) Google Scholar). A ς ligand-binding protein has recently been cloned from a number of tissues, including brain (18Moebius F.F. Striessnig J. Glossmann H. Trends Pharmacol. Sci. 1997; 18: 67-70Abstract Full Text PDF PubMed Scopus (107) Google Scholar). This binding protein exhibits the pharmacological profile of ς1 receptors and is expressed in the ER (19Kekuda R. Prasad P.D. Fei Y.J. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1996; 229: 553-558Crossref PubMed Scopus (396) Google Scholar, 20Seth P. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1997; 241: 535-540Crossref PubMed Scopus (188) Google Scholar, 21Seth P. Fei Y.J. Li H.W. Huang W. Leibach F.H. Ganapathy V. J. Neurochem. 1998; 70: 922-931Crossref PubMed Scopus (281) Google Scholar, 22Hanner M. Moebius F.F. Flandorfer A. Knaus H.G. Striessnig J. Kempner E. Glossmann H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8072-8077Crossref PubMed Scopus (842) Google Scholar, 23Mei J. Pasternak G.W. Biochem. Pharmacol. 2001; 62: 349-355Crossref PubMed Scopus (113) Google Scholar). Although its sequence has no known homology to other mammalian proteins, it shows similarity to the yeast sterol C8-C7 isomerase involved in ergosterol biosynthesis (24Moebius F.F. Bermoser K. Reiter R.J. Hanner M. Glossmann H. Biochemistry. 1996; 35: 16871-16878Crossref PubMed Scopus (61) Google Scholar). Plasma membrane ς receptors can be activated by PREGS and other neurosteroids but, in contrast to ς ligand binding proteins expressed in the ER, appear to be directly coupled to pertussis-sensitive G proteins (25Ueda H. Yoshida A. Tokuyama S. Mizuno K. Maruo J. Matsuno K. Mita S. Neurosci. Res. 2001; 41: 33-40Crossref PubMed Scopus (45) Google Scholar, 26Tokuyama S. Hirata K. Ide A. Ueda H. Neurosci. Lett. 1997; 233: 141-144Crossref PubMed Scopus (22) Google Scholar, 27Tokuyama S. Hirata K. Yoshida A. Maruo J. Matsuno K. Mita S. Ueda H. Neurosci. Lett. 1999; 268: 85-88Crossref PubMed Scopus (19) Google Scholar, 28Monnet F.P. Mahe V. Robel P. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3774-3778Crossref PubMed Scopus (419) Google Scholar). Both the cDNA sequence and physiological role of these G protein-coupled metabotropic ς receptors have yet to be fully characterized. In this paper, we report a novel effect of PREGS on glutamate release that depends on activation of plasma membrane ς receptors. Specifically, we measured the effects of this neurosteroid on miniature excitatory postsynaptic currents (mEPSCs) mediated by the α-amino-3-hydroxy-5-methylisoxazole-4-propionate subtype of ionotropic glutamate receptors. Miniature synaptic currents are the most elementary forms of synaptic transmission, representing the postsynaptic responses to action potential-independent spontaneous release of single presynaptic vesicles. It is well established that when a modulator affects presynaptic neurotransmitter release, it produces a change in the frequency but not in the amplitude of miniature synaptic events (for instance, see Ref. 29Li Y.X. Zhang Y. Lester H.A. Schuman E.M. Davidson N. J. Neurosci. 1998; 18: 10231-10240Crossref PubMed Google Scholar). We found that PREGS selectively induces a robust increase in the frequency of mEPSCs, indicating that it enhances the probability of glutamate release from presynaptic terminals. Moreover, we show that this effect depends on an elevation in intracellular Ca2+ levels triggered by activation of presynaptic Gi/o protein-coupled ς1-like receptors. Animal procedures were approved by the Institutional Animal Care and Use Committee of the University of New Mexico Health Sciences Center and conform to National Institutes of Health guidelines. Neuronal cultures were prepared in all cases from postnatal day 3–4 Sprague-Dawley rats. These experiments utilized either mixed hippocampal cell cultures (prepared as described previously (30Costa E.T. Olivera D.S. Meyer D.A. Ferreira V.M. Soto E.E. Frausto S. Savage D.D. Browning M.D. Valenzuela C.F. J. Biol. Chem. 2000; 275: 38268-38274Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar)) or autaptic neuronal cultures grown on glial cells attached to collagen-on-agarose microislands (prepared as described elsewhere (31Segal M.M. Furshpan E.J. J. Neurophysiol. 1990; 64: 1390-1399Crossref PubMed Scopus (131) Google Scholar)). Neurons grown on microislands were used for the studies of paired pulse facilitation (PPF). Neurons were used for electrophysiological experiments 8–14 days after culture. Whole-cell patch clamp experiments were performed using instrumentation and software previously described (30Costa E.T. Olivera D.S. Meyer D.A. Ferreira V.M. Soto E.E. Frausto S. Savage D.D. Browning M.D. Valenzuela C.F. J. Biol. Chem. 2000; 275: 38268-38274Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), with the exception that mEPSCs were first recorded on digital audiotape and then digitized by using a Digidata 1200 interface and pClamp 7 software (Axon Laboratories, Foster City, CA). Miniature EPSCs were analyzed using the Mini Analysis Program from Synaptosoft (Decatur, GA). We recorded from pyramidal-like neurons that had large somas and well defined dendritic processes. Neurons were clamped at −70 mV for most experiments and, when indicated, at −90 mV. The external solution contained 130 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm Hepes, 11 mm glucose, and 0.02 mm bicuculine methiodide (pH 7.4, ∼320 mosmol). For mEPSC recordings, this solution also contained 600 nmtetrodotoxin. For some recordings of autaptic currents, the concentration of Mg2+ was increased to 2 mm to favor PPF (32Mennerick S. Zorumski C.F. J. Physiol. 1995; 488: 85-101Crossref PubMed Scopus (129) Google Scholar). For recordings of mEPSCs, the internal solution contained 5 mm CsCl, 140 mmCsCH3SO3, 10 mm EGTA, 10 mm Hepes, pH 7.4, ∼300 mosmol. To record autaptic currents, the composition of the internal solution was 4 mmNaCl, 0.5 mm CaCl2, 5 mm EGTA, 10 mm Hepes, 140 mm potassium gluconate, pH 7.25, ∼280 mosmol. Patch pipette electrodes had resistances ranging from 3 to 7 megaohms. Autaptic EPSCs were generated by a 1.5- or 2-ms depolarizing pulse (from −70 mV to +20 mV); for studies of PPF, two pulses separated by 50 or 60 ms were delivered at a frequency of 0.05 Hz. Compounds were dissolved in Me2SO before dilution into external solution, and equal volumes of Me2SO were added to control external solutions. Me2SO concentrations never exceeded 0.05%. Tetrodotoxin and 6-cyano-7-nitroquinoxaline-2,3-dione were from Alexis Biochemicals (San Diego, CA); 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl) ester (BAPTA-AM) and pertussis toxin were fromCalbiochem; PREGS was from Steraloids (Newport, RI); BD-1063 anddl-2-amino-5-phosphonovalerate were from Tocris (Ellisville, MO). Synthesis of the (−)-enantiomer of PREGS (ent-PREGS) has been described elsewhere (33Nilsson K.R. Zorumski C.F. Covey D.F. J. Med. Chem. 1998; 41: 2604-2613Crossref PubMed Scopus (66) Google Scholar). (+)-Pentazocine succinate was generously provided by Kevin Gormley (National Institute on Drug Abuse). All other chemicals were from Sigma or Fluka (St. Louis, MO). The effects of all compounds were quantified with respect to the average of control and washout responses. The Kolmogorov-Smirnov test was used initially to test for significant differences between treatments in individual cells and to determine whether data followed a Gaussian distribution. Statistical comparisons of pooled data were performed by one-way analysis of variance followed by Bonferroni's post hoc test or by Student's t test. In all cases, a p< 0.05 was considered to indicate statistical significance. Statistical analyses were performed with the Mini Analysis program or Prism (GraphPad, San Diego, CA). Data are presented as mean ± S.E. in all cases. We first measured the effect of PREGS on mEPSCs to determine whether it modulated spontaneous glutamate release. To isolate AMPAR-mediated events, we recorded mEPSCs at −70 mV in 1 mm Mg2+-containing external solution. Under these conditions, 6-cyano-7-nitroquinoxaline-2,3-dione (20 μm) reduced the frequency of events by 96 ± 3% (n = 7) with respect to control (data not shown). As illustrated in Fig. 1, PREGS caused a robust, concentration-dependent, increase in mEPSC frequency. The effect of PREGS was significant at concentrations of ≥10 μm (Fig. 1 D). The onset of this effect occurred within ∼1 min after bath application of the neurosteroid and was fully reversible within ∼2–4 min after washout. The increase in mEPSC frequency was observed at both −70 and −90 mV. At these membrane potentials, PREGS (50 μm) increased mEPSC frequency by 494 ± 184% (n = 6) and 488 ± 192% (n = 7), respectively (data not shown). This finding indicates that the effect of PREGS is not due simply to an increase in the detection of subthreshold mEPSCs. The effect of PREGS was also not due to NMDAR activation, because recording in Mg2+-free external solution containing the NMDAR antagonistdl-2-amino-5-phosphonovalerate (50 μm) had no effect on the action of this neurosteroid. PREGS (50 μm) increased mEPSC frequency by 448 ± 151% (n = 4) or 485 ± 182% (n = 9) with respect to control in the presence of dl-2-amino-5-phosphonovalerate or Mg2+-containing external solution, respectively (data not shown). We did not detect any effect of PREGS on mEPSC amplitude at any of the concentrations examined; mEPSC amplitudes were changed by 1.6 ± 2.8, 0.55 ± 3.6, −1.6 ± 4.4, and −1.1 ± 3.5% with respect to control in the presence of 1, 10, 20, and 50 μm PREGS, respectively (see Fig. 1 C for an illustration of a lack of an effect of 20 μm PREGS). To eliminate the possibility of a nonspecific action and to determine the enantioselectivity of the PREGS effect, we tested the effect of the inactive neurosteroid, isopregnanolone, and the (−)-enantiomer of PREGS, ent-PREGS, respectively. As shown (Fig.1 D), these steroids did not induce a change in the frequency of mEPSCs. Since ent-PREGS has been shown to exert more potent inverted U-shaped effects than PREGS under some experimental conditions (6Akwa Y. Ladurelle N. Covey D.F. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14033-14037Crossref PubMed Scopus (122) Google Scholar), we also tested its effect at a 1 μmconcentration and found that it does not affect mEPSC frequency (2.5 ± 0.9% change with respect to control; n = 3). To confirm that PREGS increases the probability of glutamate release, we measured its effect on PPF of autaptic AMPAR-mediated EPSCs evoked by depolarizing pulses (Fig. 2). It is well established that manipulations that enhance the basal probability of release increase the impact of the first action potential (i.e. deplete synaptic vesicles), resulting in a reduction in PPF (For an example, see Ref. 34Sullivan J.M. J. Neurophysiol. 1999; 82: 1286-1294Crossref PubMed Scopus (150) Google Scholar). Accordingly, we found that treatment with PREGS (50 μm) increases the amplitude of the first EPSC by 43 ± 11% (n = 9;p < 0.01 by one-sample t testversus a theoretical mean of zero; Fig. 2 A) and also reduces PPF (Fig. 2 B), confirming that PREGS increases the probability of presynaptic glutamate release. PREGS has been shown to inhibit NMDA receptor-dependent [3H]norepinephrine release in hippocampal slices by a mechanism that involves ς1 receptors (28Monnet F.P. Mahe V. Robel P. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3774-3778Crossref PubMed Scopus (419) Google Scholar). We therefore examined the role of these receptors in the effects of PREGS on glutamate release. As shown in Fig. 3, the ς1 receptor antagonists, haloperidol and BD-1063 (preincubation for 30–45 min at 37 °C), blocked the PREGS-induced increase in mEPSC frequency. Moreover, pretreatment with haloperidol or BD1063 did not affect the average basal frequency of mEPSCs (Fig. 3,B1 and C1). This finding indicates that the decrease in PREGS efficacy in the presence of these two compounds is not due to an overall decline in the spontaneous release probability. We next determined whether the effect of PREGS could be mimicked by other ς receptor agonists. We tested the effect of DHEAS, another neurosteroid that activates ς1-like receptors in the brain (25Ueda H. Yoshida A. Tokuyama S. Mizuno K. Maruo J. Matsuno K. Mita S. Neurosci. Res. 2001; 41: 33-40Crossref PubMed Scopus (45) Google Scholar), and of (+)-pentazocine, the prototypical ς1receptor agonist. As shown in Fig. 4, these compounds produced a similar increase in mEPSC frequency to that produced by PREGS (Fig. 1 D). Conversely, (−)-pentazocine did not increase mEPSC frequency (Fig. 4 C). None of these compounds significantly affected mEPSC amplitude (data not shown).Figure 4ς1 receptor agonists DHEAS and (+)-pentazocine (PTZ) mimic the PREGS-dependent enhancement of mEPSC frequency. A, sample traces of mEPSC recordings obtained before (Control) and during administration of DHEAS (0.1 and 1.0 μm) and after washout of both concentrations from a single representative neuron (scale bars, 41 pA and 256 ms). B, sample traces of mEPSC recordings obtained before (Control) and during administration of (+)-pentazocine (50 μm) and after washout from a single representative neuron (scale bars, 41 pA and 1.3 s). C, summary graph of average percentage change in mEPSC frequency obtained with 0.1 (n = 6) and 1.0 μm (n = 6) DHEAS. Also shown are the effects of 0.5 (n = 2), 5 (n = 6), and 50 μm (+)-pentazocine (n = 5) and 50 μm (−)-pentazocine (n = 5). Eachbar represents the average ± S.E. (*,p < 0.05 by one-sample t testversus a theoretical mean of zero)View Large Image Figure ViewerDownload (PPT) Plasma membrane ς receptors have been shown to be coupled to Gi/o proteins (25Ueda H. Yoshida A. Tokuyama S. Mizuno K. Maruo J. Matsuno K. Mita S. Neurosci. Res. 2001; 41: 33-40Crossref PubMed Scopus (45) Google Scholar, 26Tokuyama S. Hirata K. Ide A. Ueda H. Neurosci. Lett. 1997; 233: 141-144Crossref PubMed Scopus (22) Google Scholar, 27Tokuyama S. Hirata K. Yoshida A. Maruo J. Matsuno K. Mita S. Ueda H. Neurosci. Lett. 1999; 268: 85-88Crossref PubMed Scopus (19) Google Scholar, 28Monnet F.P. Mahe V. Robel P. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3774-3778Crossref PubMed Scopus (419) Google Scholar). Therefore, we tested the effect of pertussis toxin treatment on the PREGS-induced increase in mEPSC frequency. As illustrated in Fig. 5, incubation of neurons for 36–48 h at 37 °C with 50 ng/ml pertussis toxin significantly reduced the effect of PREGS on mEPSC frequency. This result indicates that the effect of PREGS requires activation of the Gi/o subtype of G proteins. As also shown, pretreatment with pertussis toxin did not affect basal frequency of mEPSCs (Fig.5 B). This suggests that the decrease in PREGS efficacy in the presence of pertussis toxin is not due to an overall decline in the spontaneous release probability and is most likely due to a direct effect of pertussis toxin on the PREGS-mediated second messenger cascades that results in increased spontaneous glutamate release. ς receptors have been shown to regulate intracellular calcium (16Maurice T. Phan V.L. Urani A. Kamei H. Noda Y. Nabeshima T. Jpn. J. Pharmacol. 1999; 81: 125-155Crossref PubMed Scopus (189) Google Scholar,35Hayashi T. Maurice T. Su T.P. J. Pharmacol. Exp. Ther. 2000; 293: 788-798PubMed Google Scholar); therefore, we examined its role in the mechanism of action of PREGS. The results of these experiments are shown in Fig.6. As a control, neurons were first exposed to 50 μm PREGS, which produced the expected increase in mEPSC frequency. After washout, the same neurons were incubated for 15–20 min with the membrane-permeable Ca2+chelator, BAPTA-AM (10 or 20 μm). A subsequent exposure to 50 μm PREGS failed to induce an elevation in mEPSC frequency (Fig. 6, A and B). This result indicates that an elevation in intracellular Ca2+ levels is required for the presynaptic actions of PREGS. To eliminate the possibility that the effect of BAPTA-AM was an artifact due to run down of the effect of PREGS, we applied PREGS twice under control conditions (Fig. 6 C). As shown, the effect of a second application of PREGS closely reproduced that of the first application (same result seen in three additional neurons). In this paper, we report that PREGS induces a robust increase in the frequency but not the amplitude of AMPAR-mediated mEPSCs in hippocampal neurons cultured from neonatal rats. Moreover, we found that PREGS reduces PPF of AMPAR-mediated synaptic responses, indicating that this neurosteroid increases the probability of glutamate release at the presynaptic level. To the best of our knowledge, this is the first report of a modulatory effect of PREGS on the basal probability of glutamate release in central nervous system neurons. It is noteworthy, however, that we previously found that PREGS exerts presynaptic modulatory actions on glutamatergic terminals in the rat hippocampus. Specifically, we demonstrated that PREGS enhances PPF of NMDAR- and AMPAR-mediated EPSPs in CA1 pyramidal neurons in hippocampal slices from adult rats (36Partridge L.D. Valenzuela C.F. Neurosci. Lett. 2001; 301: 103-106Crossref PubMed Scopus (42) Google Scholar). Importantly, we did not find evidence indicating that PREGS affects the basal probability of glutamate release in these slices (36Partridge L.D. Valenzuela C.F. Neurosci. Lett. 2001; 301: 103-106Crossref PubMed Scopus (42) Google Scholar). Thus, PREGS appears to exert distinct effects on glutamate release depending on either the neuronal developmental stage or the type of neuronal preparation being used (i.e. it increases the probability of spontaneous glutamate release in hippocampal neurons cultured from neonatal rats and increases facilitation of evoked glutamate release in CA1 pyramidal neurons in hippocampal slices from adult rats). Our finding that PREGS affects glutamate release contributes to the growing evidence that this neurosteroid has important regulatory actions on the release of a number of neurotransmitters. In agreement with the results of our study, in vivo microdialysis experiments have demonstrated that PREGS increases basal acetylcholine release in the hippocampus and cortex of rats (37Rhodes M.E. Li P.K. Flood J.F. Johnson D.A. Brain Res. 1996; 733: 284-286Crossref PubMed Scopus (75) Google Scholar, 38Darnaudery M. Koehl M. Pallares M. Le Moal M. Mayo W. J. Neurochem. 1998; 71: 2018-2022Crossref PubMed Scopus (39) Google Scholar, 39Darnaudery M. Koehl M. Piazza P.V. Le Moal M. Mayo W. Brain Res. 2000; 852: 173-179Crossref PubMed Scopus (62) Google Scholar) and that it also increases basal dopamine release in the rat nucleus accumbens (40Barrot M. Vallee M. Gingras M.A. Le Moal M. Mayo W. Piazza P.V. Eur. J. Neurosci. 1999; 11: 3757-3760Crossref PubMed Scopus (45) Google Scholar). Not all studies, however, have shown a potentiating effect of PREGS on basal neurotransmitter release. Teschemacher et al. (41Teschemacher A. Kasparov S. Kravitz E.A. Rahamimoff R. Brain Res. 1997; 772: 226-232Crossref PubMed Scopus (26) Google Scholar) found that PREGS (1–50 μm) reduces the probability of GABA release in cultured hippocampal neurons. Taken together with our finding that PREGS increases glutamate release in the same type of neurons, the results of Teschemacher et al. (41Teschemacher A. Kasparov S. Kravitz E.A. Rahamimoff R. Brain Res. 1997; 772: 226-232Crossref PubMed Scopus (26) Google Scholar) indicate that this neurosteroid differentially regulates basal neurotransmitter release from GABAergic versus glutamatergic axonal terminals. It should also be emphasized that another study found that PREGS (10 nm to 3 μm) does not affect basal [3H]norepinephrine release in hippocampal slices from adult rats (28Monnet F.P. Mahe V. Robel P. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3774-3778Crossref PubMed Scopus (419) Google Scholar). Thus, the effects of PREGS appear to depend on the neurotransmitter specificity of a particular presynaptic terminal. Monnet et al. (28Monnet F.P. Mahe V. Robel P. Baulieu E.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3774-3778Crossref PubMed Scopus (419) Google Scholar) determined that PREGS decreases NMDA-evoked overflow of [3H]norepinephrine in hippocampal slices of adult rats and that ς receptor antagonists block this effect. This finding prompted us to evaluate the role of ς receptors in the mechanism of the PREGS-induced increase in spontaneous quantal glutamate release. We found that the ς receptor antagonists, haloperidol and BD-1063, block the effect of PREGS on the probability of glutamate release. In addition, the effect of PREGS was mimicked by DHEAS, another neurosteroid that binds to ς receptors. Importantly, (+)-pentazocine, the prototypical ς1receptor agonist, also mimicked the effects of PREGS, albeit at higher concentrations (5–50 μm) than expected; lower concentrations of (+)-pentazocine (0.1–10 μm) have been shown to activate metabotropic ς1" @default.
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• W1989888504 date "2002-08-01" @default.
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• W1989888504 title "Neurosteroids Enhance Spontaneous Glutamate Release in Hippocampal Neurons" @default.
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• W1989888504 doi "https://doi.org/10.1074/jbc.m202592200" @default.
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