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Background and Purpose: GABA A receptors are regulated by numerous classes of allosteric modulators. However, regulation of receptor macroscopic desensitisation remains largely unexplored and may offer new therapeutic opportunities. Here, we report the emerging potential for modulating desensitisation with analogues of the endogenous inhibitory neurosteroid, pregnenolone sulfate., Experimental Approach: New pregnenolone sulfate analogues were synthesised incorporating various heterocyclic substitutions located at the C-21 position on ring D. The pharmacological profiles of these compounds were assessed using electrophysiology and recombinant GABA A receptors together with mutagenesis, molecular dynamics simulations, structural modelling and kinetic simulations., Key Results: All seven analogues retained a negative allosteric modulatory capability whilst exhibiting diverse potencies. Interestingly, we observed differential effects on GABA current decay by compounds incorporating either a six- (compound 5) or five-membered heterocyclic ring (compound 6) on C-21, which was independent of their potencies as inhibitors. We propose that differences in molecular charges, and the targeted binding of analogues to specific states of the GABA A receptor, are the most likely cause of the distinctive functional profiles., Conclusions and Implications: Our findings reveal that heterocyclic addition to inhibitory neurosteroids not only affected their potency and macroscopic efficacy but also affected innate receptor mechanisms that underlie desensitisation. Acute modulation of macroscopic desensitisation will determine the degree and duration of GABA inhibition, which are vital for the integration of neural circuit activity. Discovery of this form of modulation could present an opportunity for next-generation GABA A receptor drug design and development., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Background: Pregnenolone sulfate (PregS), an endogenous neurosteroid, which negatively and positively modulates gamma amino butyric acid subunit A (GABA A ) and N-methyl D-aspartate (NMDA) receptors (R) respectively, among other potential neuroplastic changes on synaptic processes, has shown some beneficial effects on treating cognitive impairment associated with schizophrenia (CIAS) and negative symptoms. Lurasidone (Lur), an atypical antipsychotic drug (AAPD), and tandospirone (Tan), a 5-HT 1A  R partial agonist, have also been reported to improve cognitive or negative symptoms, or both, in some schizophrenia patients., Methods: We tested whether PregS, by itself, and in combination with Lur or Tan could rescue persistent deficits produced by subchronic treatment with the NMDAR antagonist, phencyclidine (PCP)-in episodic memory, executive functioning, and social behavior, using novel object recognition (NOR), operant reversal learning (ORL), and social interaction (SI) tasks, in male C57BL/6 J mice., Results: PregS (10, but not 3 mg/kg) significantly rescued subchronic PCP-induced NOR and SI deficits. Co-administration of sub-effective doses (SEDs) of PregS (3 mg/kg) + Lur (0.1 mg/kg) or Tan (0.03 mg/kg) rescued scPCP-induced NOR and SI deficits. Further, PregS (30, but not 10 mg/kg) rescued PCP-induced ORL deficit, as did the combination of SED PregS (10 mg/kg) +SED Lur (1 mg/kg) or Tan (1 mg/kg)., Conclusion: PregS was effective alone and as adjunctive treatment for treating two types of cognitive impairments and negative symptoms in this schizophrenia model. Further study of the mechanisms by which PregS alone and in combination with AAPDs and 5-HT 1A  R partial agonists, rescues the deficits in cognition and SI in this preclinical model is indicated., (Copyright © 2018. Published by Elsevier B.V.)

Sulfonated steroids are increasingly recognized as a circulating reservoir of precursors for the local production of active steroids in certain target tissues. As an alternative to sulfonation of unconjugated steroids by cytosolic sulfotransferases, their direct formation from sulfonated precursors has been described. However, productivity and physiological relevance of this sulfate pathway of steroidogenesis are still widely unclear. Applying the porcine testis as a model, conversion of pregnenolone sulfate (P5S, sulfate pathway) by CYP17A1 was assessed in comparison to the parallel conversions of pregnenolone (P5, Δ5-pathway) and progesterone (P4, Δ4-pathway). To characterize conversions in the virtual absence of competing enzyme activities, in a first series of experiments porcine recombinant CYP17A1 was incubated with the respective substrate in the presence of bovine recombinant cytochrome P450 oxidoreductase (CPR) and cytochrome b5 (b5). Moreover, porcine testicular microsomal fractions were used as a source of homologous CYP17A1, CPR and b5. Invariably 17α-hydroxylation of P5S was, if at all, only minimal and no formation of dehydroepiandrosterone sulfate from P5S was detectable. Consistent with earlier studies porcine CYP17A1 efficiently metabolized P4 and P5 in both assay systems. Metabolism of P4 and P5 by testicular microsomal protein varied substantially between the five animals tested. In conclusion, a physiologically relevant sulfate pathway for the production of C19-steroids from P5S via CYP17A1 is very unlikely in the porcine testis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Patients affected by protocadherin 19 (PCDH19)-female limited epilepsy (PCDH19-FE) present a remarkable reduction in allopregnanolone blood levels. However, no information is available on other neuroactive steroids and the steroidogenic response to hormonal stimulation. For this reason, we evaluated allopregnanolone, pregnanolone, and pregnenolone sulfate by liquid chromatographic procedures coupled with electrospray tandem mass spectrometry in 12 unrelated patients and 15 age-matched controls. We also tested cortisol, estradiol, progesterone, and 17OH-progesterone using standard immunoassays. Apart from estradiol and progesterone, all the considered hormones were evaluated in basal condition and after stimulation with adrenocorticotropic hormone (ACTH). A generalized decrease in blood levels of almost all measured neuroactive steroids was found. When considering sexual development, cortisol and pregnenolone sulfate basal levels were significantly reduced in postpubertal girls affected by PCDH19-FE. Of interest, ACTH administration did not recover pregnenolone sulfate serum levels but restored cortisol to control levels. In prepubertal girls with PCDH19-FE, by challenging adrenal function with ACTH we disclosed defects in the production of cortisol, pregnenolone sulfate, and 17OH-progesterone, which were not apparent in basal condition. These findings point to multiple defects in peripheral steroidogenesis associated with and potentially relevant to PCDH19-FE. Some of these defects could be addressed by stimulating adrenocortical activity., (Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.)

Pregnenolone sulfate (PS) is a neuroactive steroid hormone produced in the brain. In this study, the effects of PS on synthesis and secretion of rat pituitary prolactin (PRL) were examined. To accomplish this, GH3 rat pituitary adenoma cells were treated with PS, which showed significantly increased mRNA and protein levels of PRL compared with the control. The mechanism of action responsible for the effects of PS on PRL synthesis and secretion was investigated by pretreating cells with inhibitors of traditional PRL- or the PS-related signaling pathway. PS-stimulated PRL transcription was significantly reduced by inhibitors of PKA, PKC and MAPK, but unchanged by GABAAR and NMDAR inhibitors. Western blotting analysis revealed that the total ERK1/2 level was upregulated in a time-dependent manner following PS treatment. An approximate 10% increase in GH3 cell proliferation was also observed in response to PS relative to the control. In the animal study, levels of PRL in the pituitary and in serum were elevated by PS. PS-stimulated PRL synthesis was also found to be associated with decreased expression of PRL target genes such as GNRH1, FSHB and LHB. These findings show that PS upregulates PRL synthesis and secretion in vivo and in vitro via MAPK signaling, suggesting that it has the potential for use as a therapeutic hormone to treat PRL-related disorders such as hypoprolactinemia and low milk supply., (© 2016 Society for Endocrinology.)

Increased amyloid beta (AB) peptide concentration is one of the initiating factors in the neurodegeneration process. It has been suggested that cholesterol induces the synthesis of AB peptide from amyloid precursor protein or facilitates the formation of amyloid plaque by lowering the aggregation threshold of the peptide. It is also shown that AB peptides may affect cholesterol metabolism and the synthesis of steroid hormones such as progesterone and estradiol. Pregnenolone (P) and pregnenolone sulfate (PS) are the major steroids produced from cholesterol in neural tissue. In toxicity conditions, the effect of AB peptides on P and PS levels has not yet been determined. Furthermore, it has not been clearly defined how changes in cellular P and PS levels affect neuronal cell survival. The aim of this study was to determine the effects of AB peptides on cellular changes in P and PS levels depending on the level of their main precursor, cholesterol. Cholesterol and toxic concentrations of AB fragments (AB 25-35, AB 1-40 and AB 1-42) were applied to PC-12 and SH-SY5Y cells. Changes in cellular cholesterol, P and PS levels were determined simultaneously in a dose-and time-dependent manner. The cell viability and cell death types were also evaluated. AB peptides affected both cell viability and P/PS levels. Steroid levels were altered depending on AB fragment type and the cholesterol content of the cells. Treatment with each of the AB fragments alone increased P levels by twofold. However, combined treatment with AB peptides and cholesterol increased P levels by approximately sixfold, while PS levels were increased only about 2.5 fold in both cell lines. P levels in the groups treated with AB 25-35 were higher than those in AB 1-40 and AB 1-42 groups. The cell viabilities were significantly low in the group treated by AB and cholesterol (9 mM). The effect of AB peptides on P levels might be a result of cellular self-defense. On the other hand, the rate of P increase might be playing a key role in the cell death mechanism of AB toxicity depending on cellular cholesterol levels.

Learning and memory impairment is one of the most challenging complications of cirrhosis and present treatments are unsatisfactory. The exact mechanism of cirrhosis cognitive dysfunction is unknown. Pregnenolone sulfate (PREGS) is an excitatory neurosteroid that acts as a N-methyl-D-aspartate (NMDA) receptor agonist and GABAA receptor antagonist. In this study we evaluated the effect of intra CA1 infusion of PREGS on cirrhotic rats' memory function using the Y-maze test. Hippocampal brain-derived neurotrophic factor (BDNF) mRNA expression was also evaluated. Three weeks after bile duct ligation (BDL) surgery, rats were under stereotaxic surgery for insertion of two guide cannulas in the CA1 region of the hippocampus. After 1-week of recovery, PREGS was administered through CA1 cannulas in cirrhotic rats, while control or sham groups received vehicle. For evaluation of NMDA receptor role in memory-enhancing effects of PREGS, DL-2-Amino-5-phosphonopentanoic acid (AP5) which is a potent and competitive antagonist of NMDA receptor, co-administered with PREGS and for assessment of hippocampal BDNF mRNA expression, quantitative Real-time reverse transcriptase-PCR (RT-PCR) was used. Results showed that 28 days after BDL, cirrhotic animals' memory significantly decreased in comparison with control and sham groups, while PREGS infusion could restore memory impairment (P<0.05). PREGS effects on memory of cirrhotic rats were antagonized by DAP5. RT-PCR findings have shown that hippocampal relative BDNF mRNA expression was up-regulated in PREGS-treated groups in comparison with the BDL group (P<0.001). Our findings suggest that PREGS has a memory-enhancing effect in cirrhosis memory deficit in acute therapy and this effect may be through NMDA (glutamate) receptor involvement and BDNF mRNA expression., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Conventionally, the concentration of steroidal sulfates was estimated by indirect or immuno‑based assays before the use of liquid-chromatography tandem mass spectrometry (LC-MS/MS). In the present study, a validated LC-MS/MS method is described for the simultaneous quantification of dehydroepiandrosterone sulfate (DHEA-S), estrone sulfate (E1‑S), androsterone sulfate (ADT‑S), pregnenolone sulfate (Preg‑S) and allopregnanolone sulfate (Allopreg‑S). E1‑S binding to serum proteins was observed, especially for the high concentration quality control serum samples, leading to -10 to -15% bias using a polymer-based SPE. This protein binding can be efficiently eliminated using a Waters Oasis™ WAX following the same extraction procedure. Most likely, the E1‑S binding elimination on Oasis™ WAX can be attributed to its different sorbent structure, where the benzeno group of E1-S can interact with the benzene of the backbone of Oasis™ WAX. With this improvement, the method has been fully validated according to the FDA guidelines. The low quantification limits (LLOQs) are 40ng/mL, 40pg/mL, 5ng/mL, 1.5ng/mL and 0.25ng/mL for DHEA‑S, E1-S, ADT‑S, Preg‑S and Allopreg-S, respectively. A good linearity is obtained with R>0.99 for all compounds within the appropriate calibration range. Accuracies of all levels of QCs are within the range of 10% for DHEA-S, E1‑S, ADT‑S and Preg‑S while for Allopreg‑S, the accuracy is within the 15% range. The interday coefficient variance is 5.5-9.5% for the low limits of quantification of all five compounds while values of 1.3-9.9% are found for higher levels of QCs of all five compounds. Recovery of the five compounds in stripped serum is equivalent to that in unstripped serum. The average recovery difference is less than 5% between stripped and unstripped serum for each compound. All results of other test parameters such as matrix, hemolysis and lipemic effects as well as stabilities meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In many tissues sulfonated steroids exceed the concentration of free steroids and recently they were also shown to fulfill important physiological functions. While it was previously demonstrated that cholesterol sulfate (CS) is converted by CYP11A1 to pregnenolone sulfate (PregS), further conversion of PregS has not been studied in detail. To investigate whether a steroidogenic pathway for sulfonated steroids exists similar to the one described for free steroids, we examined the interaction of PregS with CYP17A1 in a reconstituted in-vitro system. Difference spectroscopy revealed a Kd-value of 74.8±4.2μM for the CYP17A1-PregS complex, which is 2.5-fold higher compared to the CYP17A1-pregnenolone (Preg) complex. Mass spectrometry experiments proved for the first time that PregS is hydroxylated by CYP17A1 at position C17, identically to pregnenolone. A higher Km- and a lower kcat-value for CYP17A1 using PregS compared with Preg were observed, indicating a 40% reduced catalytic efficiency when using the sulfonated steroid. Furthermore, we analyzed whether the presence of cytochrome b5 (b5) has an influence on the CYP17A1 dependent conversion of PregS, as was demonstrated for Preg. Interestingly, with 17OH-PregS no scission of the 17,20-carbon-carbon bond occurs, when b5 is added to the reconstituted in-vitro system, while b5 promotes the formation of DHEA from 17OH-Preg. When using human SOAT-HEK293 cells expressing CYP17A1 and CPR, we could confirm that PregS is metabolized to 17OH-PregS, strengthening the potential physiological meaning of a pathway for sulfonated steroids., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background and Purpose: GABAA receptors are regulated by numerous classes of allosteric modulators. However, regulation of receptor macroscopic desensitisation remains largely unexplored and may offer new therapeutic opportunities. Here, we report the emerging potential for modulating desensitisation with analogues of the endogenous inhibitory neurosteroid, pregnenolone sulfate. Experimental Approach: New pregnenolone sulfate analogues were synthesised incorporating various heterocyclic substitutions located at the C‐21 position on ring D. The pharmacological profiles of these compounds were assessed using electrophysiology and recombinant GABAA receptors together with mutagenesis, molecular dynamics simulations, structural modelling and kinetic simulations. Key Results: All seven analogues retained a negative allosteric modulatory capability whilst exhibiting diverse potencies. Interestingly, we observed differential effects on GABA current decay by compounds incorporating either a six‐ (compound 5) or five‐membered heterocyclic ring (compound 6) on C‐21, which was independent of their potencies as inhibitors. We propose that differences in molecular charges, and the targeted binding of analogues to specific states of the GABAA receptor, are the most likely cause of the distinctive functional profiles. Conclusions and Implications: Our findings reveal that heterocyclic addition to inhibitory neurosteroids not only affected their potency and macroscopic efficacy but also affected innate receptor mechanisms that underlie desensitisation. Acute modulation of macroscopic desensitisation will determine the degree and duration of GABA inhibition, which are vital for the integration of neural circuit activity. Discovery of this form of modulation could present an opportunity for next‐generation GABAA receptor drug design and development. [ABSTRACT FROM AUTHOR]

Abstract Stimulation of transient receptor potential M3 (TRPM3) channels with the steroid pregnenolone sulfate increases the transcriptional activation potential of Elk-1, a transcription factor that regulates serum response element-mediated transcription. Here, we show that an influx of Ca2+ ions into the cells is essential for the activation of Elk-1 following stimulation of TRPM3. Using genetically encoded Ca2+ buffers, we show that a rise in cytoplasmic Ca2+ is required for the upregulation of the transcriptional activation potential of Elk-1, while buffering of Ca2+ in the nucleus had no inhibitory effect on the transcriptional activity of Elk-1. Pharmacological and genetic experiments showed that extracellular signal-regulated protein kinase (ERK1/2) functions as signal transducer connecting TRPM3 channels with the Elk-1 transcription factor. Accordingly, dephosphorylation of ERK1/2 in the nucleus by MAP kinase phosphatase attenuated TRPM3-mediated Elk-1 activation. Moreover, we show that the Ca2+/calmodulin-dependent protein phosphatase calcineurin is part of a shut-off-device for the signaling cascade connecting TRPM3 channels with the activation of Elk-1. The fact that TRPM3 channel stimulation activates Elk-1 connects TRPM3 with the biological functions of Elk-1, including the regulation of proliferation, differentiation, survival, transcription, and cell migration. [ABSTRACT FROM AUTHOR]

Abstract Positive modulators of NMDA receptors are important candidates for therapeutic development to treat psychiatric disorders including autism and schizophrenia. Sulfated neurosteroids have been studied as positive allosteric modulators of NMDA receptors for years, but we understand little about the cellular fate of these compounds, an important consideration for drug development. Here we focus on a visualizable sulfated neurosteroid analogue, KK-169. As expected of a pregnenolone sulfate analogue, the compound strongly potentiates NMDA receptor function, is an antagonist of GABA A receptors, exhibits occlusion with pregnenolone sulfate potentiation, and requires receptor domains important for pregnenolone sulfate potentiation. KK-169 exhibits somewhat higher potency than the natural parent, pregnenolone sulfate. The analogue contains a side-chain alkyne group, which we exploited for retrospective click labeling of neurons. Although the anionic sulfate group is expected to hinder cell entry, we detected significant accumulation of KK-169 in neurons with even brief incubations. Adding a photolabile diazirine group revealed that the expected plasma membrane localization of KK-169 is likely lost during fixation. Overall, our studies reveal new facets of the structure-activity relationship of neurosteroids at NMDA receptors, and their intracellular distribution suggests that sulfated neurosteroids could have unappreciated targets in addition to plasma membrane receptors. Highlights • KK-169 is a potent and efficacious positive allosteric modulator of NMDA receptors. • KK-169 is a clickable analogue of pregnenolone sulfate. • KK-169 reveals intracellular accumulation of sulfated steroids. [ABSTRACT FROM AUTHOR]

The transient receptor potential melastatin-3 (TRPM3) channel belongs to the family of transient receptor potential (TRP) cation channels that are expressed in a variety of tissues and cell types, including dorsal root ganglia, cardiomyocytes and pancreatic beta-cells. Although its natural ligands are currently unknown, TRPM3 channels can be activated by the neurosteroid pregnenolone sulfate, the synthetic ligand CIM0216, and by noxious heat. TRPM3 channels are regulated by phosphoinositides, and perhaps by calmodulin. Stimulation of TRPM3 induces an intracellular signaling cascade involving a rise in intracellular Ca 2+ , activation of the protein kinases Raf, ERK and JNK, and the activation of the stimulus-responsive transcription factors AP-1, CREB, Egr-1, and Elk-1. Functionally, stimulation of TRPM3 channels is connected with heat sensation by somatosensory neurons, insulin secretion by pancreatic beta-cells, regulation of neurotransmitter release, iris constriction, and tumor promotion. With the development of highly specific activators and inhibitors of TRPM3 channels, we expect that additional tissue-specific functions of TRPM3 channels will be discovered, establishing TRPM3 channels as a new therapeutic target. [ABSTRACT FROM AUTHOR]

Pregnenolone sulfate (PS) is a neuroactive steroid hormone produced in the brain. In this study, the effects of PS on synthesis and secretion of rat pituitary prolactin (PRL) were examined. To accomplish this, GH3 rat pituitary adenoma cells were treated with PS, which showed significantly increased mRNA and protein levels of PRL compared with the control. The mechanism of action responsible for the effects of PS on PRL synthesis and secretion was investigated by pretreating cells with inhibitors of traditional PRLor the PS-related signaling pathway. PS-stimulated PRL transcription was significantly reduced by inhibitors of PKA, PKC and MAPK, but unchanged by GABAAR and NMDAR inhibitors. Western blotting analysis revealed that the total ERK1/2 level was upregulated in a time-dependent manner following PS treatment. An approximate 10% increase in GH3 cell proliferation was also observed in response to PS relative to the control. In the animal study, levels of PRL in the pituitary and in serum were elevated by PS. PS-stimulated PRL synthesis was also found to be associated with decreased expression of PRL target genes such as GNRH1, FSHB and LHB. These findings show that PS upregulates PRL synthesis and secretion in vivo and in vitro via MAPK signaling, suggesting that it has the potential for use as a therapeutic hormone to treat PRLrelated disorders such as hypoprolactinemia and low milk supply. [ABSTRACT FROM AUTHOR]

The role of peripheral neurosteroids and their related mechanisms on nociception have not been thoroughly investigated. Based on emerging evidence in the literature indicating that neurosteroids and their main target receptors, i.e., sigma-1, GABA A and NMDA, affect P2X-induced changes in neuronal activity, this study was designed to investigate the effect of peripherally injected dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulfate (PREGS) on P2X receptor-mediated mechanical allodynia in rats. Intraplantar injection of either neurosteroids alone did not produced any detectable changes in paw withdrawal frequency to the innocuous mechanical stimulation in naïve rats. However, When DHEAS or PREGS were co-injected with a sub-effective dose of αβmeATP, mechanical allodynia was developed and this was dose dependently blocked by pre-injection of the P2X antagonist, TNP-ATP. These results demonstrates that DHEAS and PREGS potentiate the activity of P2X receptors which results in the enhancement of αβmeATP-induced mechanical allodynia. In order to investigate the potential role of peripheral sigma-1, GABA A and NMDA receptors in this facilitatory action, we pretreated animals with BD-1047 (a sigma-1 antagonist), muscimol (a GABA A agonist) or MK-801 (a NMDA antagonist) prior to DHEAS or PREGS + αβmeATP injection. Only BD-1047 effectively prevented the facilitatory effects induced by neurosteroids on αβmeATP-induced mechanical allodynia. Collectively, we have shown that peripheral neurosteroids potentiate P2X-induced mechanical allodynia and that this action is mediated by sigma-1, but not by GABA A nor NMDA, receptors. [ABSTRACT FROM AUTHOR]

Background and Purpose Transient receptor potential-3 ( TRPM3) channels function as Ca2+ permeable cation channels. While the natural ligands for these channels are still unknown, several compounds have been described that either activate or inhibit TRPM3 channel activity. Experimental Approach We assessed TRPM3-mediated gene transcription, which relies on the induction of intracellular signalling to the nucleus following activation of TRPM3 channels. Activator protein-1 ( AP-1) and Egr-1-responsive reporter genes were integrated into the chromatin of the cells. This strategy enabled us to analyse gene transcription of the AP-1 and Egr-1-responsive reporter genes that were packed into an ordered chromatin structure. Key Results The neurosteroid pregnenolone sulfate strikingly up-regulated AP-1 and Egr-1 transcriptional activity, while nifedipine and D-erythro-sphingosine, also putative activators of TRPM3 channels, exhibited either no or TRPM3-independent effects on gene transcription. In addition, pregnenolone sulfate robustly enhanced the transcriptional activation potential of the ternary complex factor Elk-1. Pregnenolone sulfate-induced activation of gene transcription was blocked by treatment with mefenamic acid and, to a lesser extent, by the polyphenol naringenin. In contrast, progesterone, pregnenolone and rosiglitazone reduced AP-1 activity in the cells, but had no inhibitory effect on Egr-1 activity in pregnenolone sulfate-stimulated cells. Conclusion and Implications Pregnenolone sulfate is a powerful activator of TRPM3-mediated gene transcription, while transcription is completely inhibited by mefenamic acid in cells expressing activated TRPM3 channels. Both compounds are valuable tools for further investigating the biological functions of TRPM3 channels. Linked Articles This article is part of a themed section on the pharmacology of TRP channels. To view the other articles in this section visit [ABSTRACT FROM AUTHOR]

Pregnenolone sulfate (PS), an endogenously occurring neurosteroid, has been shown to modulate the activity of several neurotransmitter-gated channels, including the NMDA receptor (NMDAR). NMDARs are glutamate-gated ion channels involved in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. In this study, we analyzed the effects of PS on calcium signaling in cultured hippocampal neurons and HEK293 cells expressing NMDAR. The cells were loaded with the Ca2+ sensor Fura-2. In agreement with previous electrophysiological experiments, PS potentiated the increases in intracellular Ca2+ induced by an exogenous application of glutamate; however, PS also increased intracellular Ca2+ in the absence of exogenous NMDA agonist. The agonist-independent effect of PS was induced in all neurons studied and in HEK293 cells expressing GluN1/GluN2A-B receptors in a neurosteroid-specific manner. We conclude that PS is an endogenous NMDA agonist that activates the GluN1/GluN2A-B receptors. [ABSTRACT FROM AUTHOR]

Pregnenolone sulfate is a steroid metabolite with a plethora of actions and functions. As a neurosteroid, pregnenolone sulfate modulates a variety of ion channels, transporters, and enzymes. Interestingly, as a sulfated steroid, pregnenolone sulfate is not the final- or waste-product of pregnenolone being sulfated via a phase II metabolism reaction and renally excreted, as one would presume from the pharmacology textbook knowledge. Pregnenolone sulfate is also the source and thereby the starting point for subsequent steroid synthesis pathways. Most recently, pregnenolone sulfate has been functionally "upgraded" from modulator of ion channels to an activating ion channel ligand. This review will focus on molecular aspects of the neurosteroid, pregnenolone sulfate, its metabolism, concentrations in serum and tissues and last not least will summarize the functional data. [ABSTRACT FROM AUTHOR]

Abstract: The neurosteroid pregnenolone sulfate (PS), a representative excitatory neuromodulator, has a variety of neuropharmacological actions, such as memory enhancement and convulsant effects. In this study, the effects of PS on glycinergic transmission, such as glycinergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs), were investigated in acutely isolated medullary dorsal horn neurons by use of a conventional whole-cell patch-clamp technique. PS significantly increased the frequency but decreased the amplitude of glycinergic mIPSCs in a concentration-dependent manner. PS also accelerated the decay time constant of glycinergic mIPSCs. The PS-induced decrease in mIPSC amplitude was due to the direct postsynaptic inhibition of glycine receptors because PS inhibited the glycine-induced Cl− currents in a noncompetitive manner. The PS-induced increase in mIPSC frequency was not due to the activation of α7 nicotinic acetylcholine, NMDA, σ1 receptors and voltage-dependent Ca2+ channels, which are known to be molecular targets of PS. On the other hand, the PS-induced increase in mIPSC frequency was completely attenuated either in the Ca2+-free external solution or in the presence of transient receptor potential (TRP) channel blockers, suggesting that PS elicits an increase in Ca2+ concentration within glycinergic nerve terminals via the activation of putative TRP channels. The PS-mediated modulation of glycinergic synaptic transmission, such as the enhancement of presynaptic glycine release and direct inhibition of postsynaptic glycine receptors, might have a broad impact on the excitability of medullary dorsal horn neurons and therefore affect the processing of nociceptive transmission from orofacial tissues. [Copyright &y& Elsevier]

Abstract: This study examined the role of forebrain n-methyl-d-aspartate receptors (NMDA-Rs) in the promnesiant effects of natural (+) pregnenolone sulfate (PREGS) and its synthetic (−) enantiomer ent-PREGS in young adult mice. Using the two-trial arm discrimination task in a Y-maze, PREGS and ent-PREGS administration to control mice increased memory performances. In mice with a knock-out of the NR1 subunit of NMDA-Rs in the forebrain, the promnesiant effect of ent-PREGS was maintained whereas the activity of PREGS was lost. Memory enhancement by PREGS involves the NMDA-R activity in the hippocampal CA1 area and possibly in some locations of the cortical layers, whereas ent-PREGS acts independently of NMDA-R function. [ABSTRACT FROM AUTHOR]

Abstract: Pregnenolone sulfate (PS) acts as an excitatory neuromodulator and has a variety of neuropharmacological actions, such as memory enhancement and convulsant effects. In the present study, we investigated the effect of PS on glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) in acutely isolated dentate gyrus (DG) hilar neurons by use of a conventional whole-cell patch-clamp technique. PS significantly increased sEPSC frequency in a concentration-dependent manner without affecting the current amplitude, suggesting that PS acts presynaptically to increase the probability of spontaneous glutamate release. However, known molecular targets of PS, such as α7 nicotinic ACh, NMDA, σ1 receptors and voltage-dependent Ca2+ channels, were not responsible for the PS-induced increase in sEPSC frequency. In contrast, the PS-induced increase in sEPSC frequency was completely occluded in a Ca2+-free external solution, and was significantly reduced by either the depletion of presynaptic Ca2+ stores or the blockade of ryanodine receptors, suggesting that PS elicits Ca2+-induced Ca2+ release (CICR) within glutamatergic nerve terminals. In addition, the PS-induced increase in sEPSC frequency was completely occluded by transient receptor potential (TRP) channel blockers. These data suggest that PS increases spontaneous glutamate release onto acutely isolated hilar neurons via presynaptic CICR, which was triggered by the influx of Ca2+ through presynaptic TRP channels. The PS-induced modulation of excitatory transmission onto hilar neurons could have a broad impact on the excitability of hilar neurons and affect the pathophysiological functions mediated by the hippocampus. [ABSTRACT FROM AUTHOR]

Abstract: Pregnenolone (P), the main precursor of the steroids, and its sulfate ester, pregnenolone sulfate (PS), are the major neurosteroids produced in the neural tissue. Many neuroendocrinological studies stressed the neuroprotective role of neurosteroids although it has been suggested that the inhibition of P and PS synthesis can delay neuronal cell death. The potential roles of P and PS in vital neuronal functions and in amyloid beta peptide (Aβ) toxicity are not clearly identified. This work aims to investigate the effects of P and PS on cell viability and Aβ peptide toxicity in a concentration and exposure time-dependent manner in rat PC-12 cells. The cells were treated with 20μM Aβ peptide 25–35 and variable concentrations of P and PS ranging from 0.5μM to 100μM. To examine the effects of steroid treatment on Aβ peptide toxicity, 0.5μM (low) and 50μM (high) neurosteroids were used. The cell viability and lactate dehydrogenase release of cells were evaluated after 24, 48 and 72h. Morphological changes of cells were also examined. The treatment with higher than 1μM concentrations of P and PS significantly decreased the cell viability comparing to untreated cells. At lower concentrations, P and PS had no toxic actions until 72h. The Aβ treatment resulted in a significant decrease in cell viability comparing to untreated cells. P showed a dose-dependent protective effect against Aβ peptide in PC-12 cells. But its sulfate ester did not have the same effect on Aβ peptide toxicity, even it significantly decreased cell viability in Aβ-treated cells. Consequently, the discrepant effects of P and PS on Aβ peptide toxicity may provide insight on the pathogenesis of Alzheimer’s disease. [Copyright &y& Elsevier]

Pregnenolone sulfate (PregS) is a neurosteroid that acts as a negative modulator of the GABAA receptor complex and a positive modulator of glutamate NMDA receptors. PregS improves learning and memory when centrally or systemically administered. However, systemic high doses of PregS, which facilitate the passive avoidance retention, have been shown to impair this learning when a high foot-shock punishment was used. Moreover, moderate or high PregS doses present a well documented anxiogenic-like profile in several animal models of anxiety. In previous experiments, we have shown that unilateral intrahippocampal 5 ng of PregS improves spatial recognition memory and reverses learning impairment induced by co-administration of alcohol and nicotine. In the present experiment, we analyzed the effects of bilateral intrahippocampal 5 ng of PregS on the passive avoidance task, using a high foot-shock punishment (0.5 mA), as well as on anxiety-like behaviour measured in the open field test in the same experimental subjects. As a control, we have injected the neurosteroid allopregnanolone (AlloP) into the hippocampus, which produces opposite behavioural and neurochemical profiles to those of PregS, by acting as a positive GABAA modulator and showing anxiolytic and sedative behavioural effects. Results showed that bilateral intrahippocampal 5 ng of PregS deteriorated passive avoidance retention but also presented an anxiogenic-like profile in the open field test, decreasing the locomotion and the time spent in the central zone without affecting either total activity or time spent in the periphery of the field (thigmotaxis). AlloP administration did not affect passive avoidance retention, and also showed a non anxiolytic-like profile, possibly related to fluctuations of endogenous AlloP concentrations induced by the stress generated by the high foot-shock punishment received before the anxiety tests. In conclusion, a post-training intrahippocampal anxiogenic dose of the neurosteroid PregS impairs passive avoidance retention. These results highlight the important role of the hippocampus in several behavioural effects of neurosteroids on learning, memory processes and anxiety. [ABSTRACT FROM AUTHOR]

Abstract: Pregnenolone sulfate (PREGS) has been shown, either at high nanomolar or at micromolar concentrations, to increase neuronal activity by inhibiting GABAergic and by stimulating glutamatergic neurotransmission. PREGS is also a potent modulator of sigma type 1 (σ1) receptors. It has been proposed that these actions of PREGS underlie its neuropharmacological effects, and in particular its influence on memory processes. On the other hand, the PREGS-mediated increase in neuronal excitability may become dangerous under particular conditions, for example in the case of excitotoxic stress or convulsions. However, the physiopathological significance of these observations has recently been put into question by the failure to detect significant levels of PREGS within the brain and plasma of rats and mice, either by direct analytical methods based on liquid chromatography/mass spectrometry (LC/MS) or enzyme linked immunosorbent assay (ELISA) with specific antibodies against PREGS, or by indirect gas chromatography/mass spectrometry (GC/MS) analysis with improved sample workup. These recent results have not come to the attention of a large number of neurobiologists interested in steroid sulfates. However, although available direct analytical methods have failed to detect levels of PREGS above 0.1–0.3ng/g in brain tissue, it may be premature to completely exclude the local formation of biologically active PREGS within specific and limited compartments of the nervous system. In contrast to the situation in rodents, significant levels of sulfated 3β-hydroxysteroids have been measured in human plasma and brain. Previous indirect measures of steroid sulfates by radioimmunoassays (RIA) or GC/MS had detected elevated levels of PREGS in rodent brain. The discrepancies between the results of different assay procedures have revealed the danger of indirect analysis of steroid sulfates. Indeed, PREGS must be solvolyzed/hydrolyzed prior to RIA or GC/MS analysis, and it is the released, unconjugated PREG which is then quantified. Extreme caution needs to be exercised during the preparation of samples for RIA or GC/MS analysis, because the fraction presumed to contain only steroid sulfates can be contaminated by nonpolar components from which PREG is generated by the solvolysis/hydrolysis/derivatization reactions. [Copyright &y& Elsevier]

The GABA(A) receptor is a target of endogenous and synthetic neurosteroids. Little is known about the residues required for neurosteroid action on GABA(A) receptors. We have investigated pregnenolone sulfate (PS) inhibition of the Caenorhabditis elegans UNC-49 GABA receptor, a close homolog of the mammalian GABA(A) receptor. The UNC-49 locus encodes two GABA receptor subunits, UNC-49B and UNC-49C. UNC-49C is sensitive to PS but UNC-49B is not sensitive. By analyzing chimeric receptors and receptors containing site-directed mutations, we identified two regions required for PS inhibition. Four residues in the first transmembrane domain are required for the majority of the sensitivity to PS, but a charged extracellular residue at the end of the M2 helix also plays a role. Strikingly, mutation of one additional M1 residue reverses the effect of PS from an inhibitor to an enhancer of receptor function. Mutating the M1 domain had little effect on sensitivity to the inhibitor picrotoxin, suggesting that these residues may mediate neurosteroid action specifically, and not allosteric regulation in general. [ABSTRACT FROM AUTHOR]

Abstract: N-methyl-d-aspartate receptors play a critical role in synaptogenesis, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits. The subunit composition determines the biophysical and pharmacological properties of the N-methyl-d-aspartate receptor channel complex. In this study, we report that responses mediated by recombinant rat N-methyl-d-aspartate receptors expressed in human embryonic kidney HEK293 cells are differentially affected by naturally occurring neurosteroid pregnenolone sulfate. We show that responses induced by 1mM glutamate in NR1-1a/NR2A and NR1-1a/NR2B receptors are potentiated five- to eight-fold more by pregnenolone sulfate than responses of NR1-1a/NR2C and NR1-1a/NR2D receptors with no differences in the concentration of pregnenolone sulfate that produced 50% potentiation. In addition to potentiation, pregnenolone sulfate also has an inhibitory effect at recombinant N-methyl-d-aspartate receptors, with values of the concentration of pregnenolone sulfate that produces 50% inhibition of NR1/NR2D=NR1/NR2C

Abstract: Pregnenolone sulfate is one of the most abundantly produced neurosteroids in the brain. The present paper studies the effect of pregnenolone sulfate on excitatory synaptic transmission in the pyramidal cells of the layer V–VI of the prelimbic cortex using whole-cell patch-clamp in slices. We found that pregnenolone sulfate inhibited stimulus-evoked excitatory postsynaptic currents (EPSC); the effect of pregnenolone sulfate was significant at concentration of 1 μM and increased with an increase in concentrations; pregnenolone sulfate had no effect on the amplitude and frequency of miniature excitatory spontaneous postsynaptic currents; pregnenolone sulfate significantly enhanced the paired-pulse facilitation (PPF) and inhibited dopamine and 5-HT-evoked increase in the frequency of spontaneous excitatory postsynaptic currents; the protein kinase A inhibitor H89 and Rp-cAMPS enhanced PPF and canceled the effect of pregnenolone sulfate on PPF; pregnenolone sulfate inhibited the protein kinase A agonist forskolin-evoked increase in the frequency of spontaneous excitatory postsynaptic currents; the Gi protein inhibitor N-ethylmaleimide canceled the effect of pregnenolone sulfate on PPF. These results suggest that pregnenolone sulfate inhibits stimulus-evoked EPSC via presynaptic inhibition of protein kinase A in rat prelimbic cortical neurons. [Copyright &y& Elsevier]

Abstract: Neurosteroids have been linked to cognitive performance, and their levels are altered in neuropsychiatric diseases. These neuromodulators are produced in the brain where they have important effects on synaptic transmission at postsynaptic γ-amino-butyric acid receptors and N-methyl-D-aspartate receptors and at presynaptic sites. We previously found, in cultured neonatal hippocampal neurons, that the neurosteroid, pregnenolone sulfate, acts presynaptically through a σ1-like receptor to modulate basal glutamate release. The present study was designed to test whether pregnenolone sulfate acts through a similar presynaptic receptor in adult hippocampal neurons. The σ1-receptor agonist, 2-(4-morpholino)ethyl-1-phenylcyclohexane-1-carboxylate, enhanced paired-pulse facilitation (PPF) by a similar extent to that which we had previously reported for pregnenolone sulfate. The σ1-receptor antagonists, 1-(4-Iodophenyl)-3-(2-adamantyl)guanidine and 1[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine, blocked the pregnenolone sulfate enhancement of PPF as did pretreatment of slices in pertussis toxin. We conclude that pregnenolone sulfate acts through a Gi/o-coupled σ1-like receptor to enhance short-term presynaptic facilitation onto adult hippocampal CA1 neurons. [Copyright &y& Elsevier]

Abstract: The two neurosteroids 3α-hydroxy-5α-pregnane-20-one (allopregnanolone; AlloP) and pregnenolone sulfate (PregS) affect neuronal GABAA receptors differently. While AlloP mainly potentiates the currents through GABAA receptors, PregS reduces such currents. The present study aimed at clarifying the interaction of AlloP and PregS at GABAA receptors in neurons from the medial preoptic nucleus of male rat. AlloP has previously been shown to dramatically prolong GABA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in these neurons. Here, by recording sIPSCs under voltage-clamp conditions with the perforated-patch technique, it was shown that PregS by itself did not significantly affect the amplitude or time course of such currents. However, PregS, in a concentration-dependent manner, reduced the AlloP-evoked prolongation of sIPSC decay when the two neurosteroids were applied together. In contrast to sIPSC amplitude and time course, sIPSC frequency was significantly reduced by 10 μM PregS alone. Further, although 1.0 μM AlloP alone induced a clear increase in sIPSC frequency, the frequency was not significantly different from control when 1.0 μM AlloP was applied in combination with 10 μM PregS. In addition to the effects on sIPSC parameters, PregS reduced the baseline current evoked by 1.0 μM AlloP in the absence of GABA application or synaptic activity. PregS by itself did not significantly affect the baseline current. The main effects of AlloP and PregS on the sIPSC time course were mimicked by a simplified model with AlloP assumed to reduce the rate of GABA unbinding from the receptor and PregS assumed to increase the rate of desensitization. [Copyright &y& Elsevier]

Pregnenolone sulfate (PS) is an endogenous neurosteroid synthesized by glial cells, which acts as a potent convulsant when injected intracerebroventricularly and intraperitoneally. PS is found in relatively high concentrations in the hippocampus. But its convulsant action in the hippocampus has not been characterized. A range of PS doses were infused directly into the right hippocampus of 42 rats, which were subsequently monitored for behavioral and electrographic seizures. At the highest dose (4 μmol), PS produced status epilepticus (SE) and severe behavioral convulsions. As the dose of PS was reduced, the fraction of rats having SE diminished (ED50 for SE=2.7 μmol). At doses lower than 300 nmol, PS infusion produced discrete electrographic seizures (ED50=68 nmol) associated with mild behavioral seizures. Both the behavioral seizure score (BSS) and the total number of seizures during the observation period changed in a dose-dependant manner. In separate experiments in cultured hippocampal neurons, PS enhanced NMDA-evoked whole-cell currents (EC50=16 μM). The results demonstrate that the hippocampus is highly sensitive to the convulsant effects of PS and that the enhancement of NMDA currents could contribute to the convulsant action of PS. [Copyright &y& Elsevier]

To characterize the direct effects of thyroid hormones on native γ-aminobutyric acidA (GABAA) receptors, rapid (5 s) actions of a series of iodothyronines on muscimol-stimulated uptake of 36Cl− were investigated in synaptoneurosomes prepared from rat brain. The results were correlated with molecular modeling of the active compounds. Dose–response curves for muscimol in the presence of 3,3′, 5-l-triiodothyronine (l-T3) indicated a noncompetitive inhibition of muscimol-stimulated 36Cl− uptake by the thyroid hormone. Synaptoneurosomes prepared from cerebellum were less sensitive to l-T3 than those from cerebral cortex, in terms of the potency of the hormone. The overall efficacy approached complete inhibition for both brain regions. Muscimol-stimulated 36Cl− uptake was inhibited differentially by iodothyronine derivatives. One group of compounds with IC50 values of 18–30 μM included l-thyroxine (l-T4), d-thyroxine (d-T4), 3,3′, 5,5′-tetraiodothyroacetic acid (Tetrac), and 3,3′, 5-triiodothyroacetic acid (Triac). A second group with values of 75–100 μM included 3,3′, 5′-l-triiodothyronine (reverse T3; r-T3), 3,3′-diiodo-l-thyronine (3,3′-l-T2) and 3,5-diiodo-l-thyronine (3,5-d-T2). A final group of inactive compounds with IC50 values greater than 100 μM included 3′,5′-diiodo-l-thyronine (3′,5′-l-T2), 3-iodo-l-thyronine (l-T1), 3′-iodo-l-thyronine (3′-l-T1), and l-thyronine (l-T0). Molecular modeling of the active iodothyronines using the Gaussian03 series of programs indicated close correspondences with models of the GABA-inhibitory neurosteroid pregnenolone sulfate (PREGS), suggesting common mechanisms of action at the GABAA receptor. [Copyright &y& Elsevier]

Alterations in neurosteroid levels may play a role in affective disorders including those related to changes in the levels of ovarian steroids. The effects of pregnancy and delivery on circulatory and brain levels of dehydroepiandrosterone (DHEA), pregnenolone (PN), their sulfate esters and the enzymatic activities of sulfatase and sulfotransferase were examined in rats. Our findings indicate an increase, not reflected in the brain cortex, in serum DHEA levels, at the end of pregnancy with a partial decrease following delivery. DHEA sulfate levels in the cortex and PN levels in both serum and cortex decreased following delivery with no changes in its sulfated form. Sulfatase levels were high both before and after delivery with no changes noted in sulfotransferase levels, compared to controls. We speculate that changes in the level or ratio of sulfated and free neurosteroids may play a role in postpartum behavioral disorders due to their antagonistic GABA[sub A] modulatory effect. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Prenatal exposure to diazepam (DZ), a positive allosteric modulator of the γ-aminobutyric acidA (GABAA) receptor complex, exerts profound effects that become more evident during puberty and in many cases are sex-specific, suggesting that such exposure interferes with the activity of steroid hormones. Apart from their well known effects on the genome, the reduced metabolites of many steroid hormones also interact directly with membrane receptors, including those for N-methyl-d-aspartate (NMDA). In this study, we compared the effects of several neurosteroids on NMDA receptors from normal rats and those exposed in utero to DZ (1.25 mg/kg per day) from the 14th through the 20th day of gestation.In superfused rat hippocampal synaptosomes, activation of the NMDA receptor stimulates the basal release of [3H]noradrenaline ([3H]NA), which was used in our study as an index of receptor function. [3H]NA release was evoked in a concentration-dependent manner by NMDA (100 μM) plus glycine (GLY). The maximal increase (68.23±3.86%) with respect to basal release was achieved with a GLY concentration of 10 μM, and the EC50 for GLY was 0.1 μM. Release stimulated by 100 μM NMDA+0.1μM GLY was not modified by any of the neurosteroids tested, with the exception of pregnenolone sulfate (PREG-S), which produced a 78.57±3.94% reduction in release at the maximal concentration used (0.3 μM). In synaptosomes from animals exposed in utero to DZ, the inhibitory effect of PREG-S was reduced by 46.55±2.33%.Given the important roles played by NMDA receptors in physiological and pathological processes within the central nervous system (CNS), characterization of NMDA receptor modulation is an important objective. The fact that this modulation can be altered by exposure in utero to DZ indicates that the behavioral abnormalities observed in exposed animals might be partially attributed to an altered sensitivity of NMDA receptors to the modulatory effects of neurosteroids. [Copyright &y& Elsevier]

To investigate developmental changes in neurosteroid modulation of GABAA receptors, whole-cell currents were elicited by applying GABA with allopregnanolone or pregnenolone sulfate (PS) to dentate granule cells (DGCs), acutely isolated from 7–14-day-old and adult rats. GABA evoked larger currents from dentate granule cells acutely isolated from adult rats (adult DGCs) than from neonatal DGCs, due to increased efficacy (1662±267 pA in adult DGCs versus 1094±198 pA in neonatal DGCs, P=0.004), and current density (0.072±0.01 pA/μm2 in neonatal rat DGCs to 0.178±0.02 pA/μm2 in adult DGCs), but unchanged potency (EC50 was 18.5±2 μM in adult DGCs, and 26.6±7.9 μM in neonatal DGCs, P=0.21). Allopregnanolone sensitivity of GABAA receptor currents increased during development due to an increased potency (21.1±4.7 nM in adult DGCs versus 94.6±9 nM in neonatal DGCs, P=0.0002). The potency and efficacy of PS inhibition of GABAA receptor currents were remained unchanged during development (13±6 μM and 13.2±5.9 μM, P=0.71 and 85.5%±3.5% and 83.6%±0.8%, P=0.29, respectively). To investigate possible mechanism of developmental changes in GABAA receptor properties, in situ hybridization for α1, α4 and γ2 subunit mRNAs was performed in dentate gyrus of the two age groups. Qualitatively, α1 subunit mRNA was expressed at low levels in neonatal rats while it was well expressed in adult rats. The α4 and γ2 subunits were well expressed in the dentate gyrus of adult and neonatal rats. Immunohistochemical staining for α1 subunit in hippocampal slices from neonatal and adult rats was examined under confocal laser scanning microscope. This demonstrated that cell bodies and dendrites of granule cells are moderately positive for the α1 staining in adult rats but weakly so in neonatal rats. Higher-magnification images demonstrate large number of clusters of α1-subunit in the cell bodies of dentate granule cells of adult rat but rare clusters in granule cells of neonatal rats.Maturation of GABAA receptors in DGCs is characterized by increased number of GABAA receptors that are more sensitive to endogenous neurosteroid allopregnanolone, which might be related to increased expression of α1 subunit. [Copyright &y& Elsevier]