dizocilpine-maleate and pregnenolone-sulfate

Evidence from clinical and preclinical studies implicates dysfunction of

Topics: Animals; Antipsychotic Agents; Behavior, Animal; Bridged Bicyclo Compounds, Heterocyclic; Disease Models, Animal; Dizocilpine Maleate; Elevated Plus Maze Test; HEK293 Cells; Humans; Male; Pregnenolone; Rats, Long-Evans; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Reflex, Startle; Schizophrenia; Steroids

Neurosteroids are produced de novo in neuronal and glial cells, which begin to express steroidogenic enzymes early in development. Studies suggest that neurosteroids may play important roles in neuronal circuit maturation via autocrine and/or paracrine actions. However, the mechanism of action of these agents is not fully understood. We report here that the excitatory neurosteroid pregnenolone sulfate induces a long-lasting strengthening of AMPA receptor-mediated synaptic transmission in rat hippocampal neurons during a restricted developmental period. Using the acute hippocampal slice preparation and patch-clamp electrophysiological techniques, we found that pregnenolone sulfate increases the frequency of AMPA-mediated miniature excitatory postsynaptic currents in CA1 pyramidal neurons. This effect could not be observed in slices from rats older than postnatal day 5. The mechanism of action of pregnenolone sulfate involved a short-term increase in the probability of glutamate release, and this effect is likely mediated by presynaptic NMDA receptors containing the NR2D subunit, which is transiently expressed in the hippocampus. The increase in glutamate release triggered a long-term enhancement of AMPA receptor function that requires activation of postsynaptic NMDA receptors containing NR2B subunits. Importantly, synaptic strengthening could also be triggered by postsynaptic neuron depolarization, and an anti-pregnenolone sulfate antibody scavenger blocked this effect. This finding indicates that a pregnenolone sulfate-like neurosteroid is a previously unrecognized retrograde messenger that is released in an activity-dependent manner during development.

Topics: Age Factors; Animals; Animals, Newborn; Antibodies; Calcium; Calcium Channel Blockers; Chelating Agents; Dizocilpine Maleate; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Drug Interactions; Egtazic Acid; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Membrane Potentials; Neuronal Plasticity; Patch-Clamp Techniques; Piperidines; Pregnenolone; Presynaptic Terminals; Quinolinic Acids; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Sodium Channel Blockers; Synapses; Synaptic Transmission; Tetrodotoxin; Time Factors

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 gamma-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 sigma1-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 sigma1-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 sigma1-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 sigma1-like receptor to enhance short-term presynaptic facilitation onto adult hippocampal CA1 neurons.

Topics: Animals; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GTP-Binding Protein alpha Subunits, Gi-Go; Hippocampus; In Vitro Techniques; Male; Morpholines; Neurons; Patch-Clamp Techniques; Pertussis Toxin; Piperazines; Pregnenolone; Rats; Rats, Sprague-Dawley; Receptors, sigma; Sigma-1 Receptor; Synapses; Synaptic Transmission; Time Factors

This work reports the first demonstration that corticosterone (CORT) has a rapid and transient effect on NMDA receptor-mediated Ca2+ signaling in cultured rat hippocampal neurons. Using single cell Ca2+ imaging, CORT and agonists of glucocorticoid receptors were observed to modulate the NMDA receptor-mediated Ca2+ signals in a completely different fashion from pregnenolone sulfate. In the absence of steroids, 100 micro m NMDA induced a transient Ca2+ signal that lasted for 30-70 s in 86.1% of the neurons prepared from postnatal rats (3-5 days old). After pre-treatment with 0.1-100 micro m CORT for 10-20 min, NMDA induced extremely prolonged Ca2+ elevation. This prolonged Ca2+ elevation was terminated by the application of MK-801 and followed by washing out of CORT. The proportion of CORT-modulated neurons within the NMDA-responsive cells increased from 25.1 to 95.5% when the concentration of CORT was raised from 0.1 to 50 micro m. Substitution of BSA-conjugated CORT produced essentially the same results. When hippocampal neurons were preincubated with 10 micro m cortisol and 1 micro m dexamethasone for 20 min, a very prolonged Ca2+ elevation was also observed upon NMDA stimulation. The CORT-prolonged Ca2+ elevation caused a long-lasting depolarization of the mitochondrial membrane, as observed with rhodamine 123. In contrast, incubation with 100 micro m pregnenolone sulfate did not considerably alter the time duration of NMDA-induced transient Ca2+ elevation, but caused a significant increase in the peak amplitude of Ca2+ elevation in hippocampal neurons. These results imply that high levels of CORT induce a rapid and non-genomic prolongation of NMDA receptor-mediated Ca2+ elevation, probably via putative membrane surface receptors for CORT in the hippocampal neurons.

Topics: Androstanols; Animals; Calcium; Calcium Signaling; Cells, Cultured; Corticosterone; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Hippocampus; Hormone Antagonists; Membrane Potentials; Mifepristone; Mitochondria; N-Methylaspartate; Neurons; Pregnenolone; Rats; Rats, Wistar; Receptors, Glucocorticoid; Receptors, N-Methyl-D-Aspartate

Our recent study demonstrated that neurosteroids might either facilitate or block chronic tolerance to the incoordinating effects of ethanol. The present study investigated the effects of neurosteroids on the development of rapid tolerance to ethanol-induced motor impairment using the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine [(+)-MK-801] or the gamma-aminobutyric acid (GABA) type A (GABA(A)) receptor agonist muscimol. Male Swiss mice were pretreated with pregnenolone sulfate (0.03 to 0.15 mg/kg) or dehydroepiandrosterone sulfate (0.05 to 0.20 mg/kg) before administration of ethanol (1.9 or 2.25 g/kg) and tested with the rota-rod apparatus. Twenty-four hours later, all animals were re-tested with the rota-rod after receiving the same dose of ethanol. Pretreatment with pregnenolone sulfate or with dehydroepiandrosterone sulfate significantly facilitated the acquisition of tolerance. However, the administration of (+)-MK-801 reversed the stimulatory action of pregnenolone sulfate but did not affect the actions of dehydroepiandrosterone sulfate on ethanol tolerance. Pretreatment with pregnenolone sulfate or dehydroepiandrosterone sulfate prevented the inhibitory action of muscimol on tolerance development. Taken together, our results suggest that neurosteroids may stimulate the development of rapid tolerance to ethanol and that GABA(A) and NMDA receptor systems may be involved in these actions.

Topics: Animals; Dehydroepiandrosterone Sulfate; Dizocilpine Maleate; Drug Interactions; Drug Tolerance; Ethanol; Male; Mice; Motor Activity; Muscimol; Pregnenolone; Steroids

1. The sigma(1) (sigma(1)) receptor cDNA was cloned in several animal species. Molecular tools are now available to identify its endogenous effectors, such as neuroactive steroids, and to establish its precise physiological role. In particular, the sigma(1) receptor is involved in memory processes, as observed in pharmacological and pathological rodent models of amnesia. 2. In order to establish the involvement of sigma(1) receptors in memory, a 16-mer oligodeoxynucleotide antisense to the sigma(1) receptor cDNA (aODN), and its mismatched control (mODN) were prepared and centrally administered into the mouse brain. The anti-amnesic effects induced by the selective sigma(1) agonist PRE-084 and the steroid dehydroepiandrosterone (DHEA) sulphate or pregnenolone sulphate were examined in ODN-treated animals. 3. The aODN treatment failed to affect the dissociation constant (K(d)) but significantly decreased the number of sigma(1) sites (B(max)) labelled with [(3)H]-(+)-SKF-10,047 in the hippocampus and cortex. In these structures, the in vivo binding levels were also diminished, according to the dose and number of injections, as compared with control animals injected with saline or mODN. 4. Cannulation and injections failed to affect the open-field behaviour of the animals. However, the anti-amnesic effects of PRE-084 and DHEA sulphate against the dizocilpine-induced impairments were blocked after aODN treatment in the short- and long-term memory tests. The anti-amnesic effects of pregnenolone sulphate remained unchanged. 5. These observations bring a molecular basis to the modulatory role of sigma(1) receptors in memory, and reveal that the anti-amnesic action of neuroactive steroids may not similarly involve an interaction with sigma(1) receptors.

Topics: Amnesia; Animals; Avoidance Learning; Behavior, Animal; Binding Sites; Blotting, Western; Brain; Cerebral Cortex; Dehydroepiandrosterone; Dizocilpine Maleate; Dose-Response Relationship, Drug; Hippocampus; In Vitro Techniques; Learning Disabilities; Male; Maze Learning; Memory; Mice; Morpholines; Oligonucleotides, Antisense; Phenazocine; Pregnenolone; Receptors, sigma; Sigma-1 Receptor

This study examined the effects of sigma(1) receptor agonist SA4503 and neuroactive steroids dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate (PREGS) and progesterone (PROG) on spatial working and reference memory in a radial arm maze task in rats. The insertion of a 6-min delay between the 2nd and 3rd choices caused a specific decline in working memory, but had no effect on reference memory. This decline in working memory was improved by SA4503, but not by DHEAS, PREGS or PROG. A non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine significantly impaired both working and reference memory in the presence or absence of a delay. The dizocilpine-induced impairments in the presence of a 6-min delay were ameliorated by SA4503, DHEAS and PREGS, whereas PROG had no effect. The beneficial effects of SA4503, DHEAS and PREGS were antagonized by treatment with sigma(1) receptor antagonist N, N-dipropyl-2-(4-methoxy-3-(2-phenylethoxy)phenyl)-ethylamine hydrochloride (NE-100). Furthermore, PROG attenuated the ameliorating effects of SA4503, DHEAS and PREGS on dizocilpine-induced memory deficits. These results suggest that sigma(1) receptors play a significant role in short-term working memory. Furthermore, it is suggested that DHEAS and PREGS ameliorate dizocilpine-induced memory impairments by acting as sigma(1) receptor agonists, while PROG antagonizes their effects by acting as a sigma(1) receptor antagonist.

Topics: Animals; Anisoles; Antipsychotic Agents; Dehydroepiandrosterone Sulfate; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Male; Maze Learning; Memory; Nootropic Agents; Piperazines; Pregnenolone; Progesterone; Propylamines; Psychomotor Performance; Rats; Rats, Wistar; Receptors, sigma; Steroids; Time Factors

The sigma(1)-receptor is a one-transmembrane endoplasmic reticulum protein that binds neurosteroids and dextrorotatory benzomorphans. The roles of sigma(1)-receptors in regulating intracellular Ca(2+) in NG108 cells were examined in this study. sigma(1)-Ligands pregnenolone sulfate, (+)-pentazocine, and 2-(4-morpholino)ethyl-1-phenylcyclohexane-1-carboxylate hydrochloride modulate Ca(2+) signaling in NG108 cells via two modes of action. First, nanomolar concentrations of the ligands, without effect by themselves, potentiated the bradykinin-induced increase of the cytosolic free Ca(2+) concentration in a bell-shaped manner. This effect of sigma(1)-ligands was unaffected by depletion of Ca(2+) from perfusion buffer and was blocked by a 21-mer antisense oligodeoxynucleotide against the cloned sigma(1)-receptors. Second, after the cells were depleted of the endoplasmic reticulum Ca(2+) stores, the depolarization (75 mM KCl)-induced increase in cytosolic free Ca(2+) was potentiated by 2-(4-morpholino)ethyl-1-phenylcyclohexane-1-carboxylate hydrochloride, whereas it was inhibited by pregnenolone sulfate and (+)-pentazocine. These effects, albeit opposite in direction, were blocked by both the 21-mer antisense oligodeoxynucleotide and pertussis toxin. Western blotting indicates that sigma(1)-receptors are increased on the plasma membrane and the nuclear membrane in the presence of sigma(1)-ligand. These results suggest that Ca(2+) signaling via sigma(1)-receptors may represent a novel mechanism that affects intracellular Ca(2+) concentrations.

Topics: Animals; Bradykinin; Calcium; Calcium Signaling; Dizocilpine Maleate; Morpholines; Nifedipine; Oligonucleotides, Antisense; Pentazocine; Potassium Chloride; Pregnenolone; Rabbits; Receptors, sigma; Tumor Cells, Cultured

The ability of the nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine methyl ester (L-NAME), to modulate the attenuating effects of neurosteroids on the aging- and NMDA receptor antagonist dizocilpine-induced learning impairment, was tested in mice using two different behavioral models of long-term memory. The performance of aged mice (16 months old) in step-down type of passive-avoidance and elevated plus-maze paradigms was significantly impaired compared to that of young mice (3 months old). Neurosteroids pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS), at 1-20 mg/kg, s.c., significantly improved the passive-avoidance and plus-maze performances in aged mice. Neurosteroids PS and DHEAS, at doses 1-20 mg/kg, s.c., significantly attenuated dizocilpine (0.1 mg/kg, i.p.)-induced amnesia, without producing any promnestic effects alone in adult mice. In both cognitive tasks, the effects exhibited by the neurosteroids tested had a bell-shaped curve. Preadministration of L-NAME (10 and 20 mg/kg, i.p.), at doses that did not disrupt cognition alone in either young or aged mice, significantly blocked the beneficial and antiamnesic effects of neurosteroids PS (5 mg/kg) and DHEAS (10 mg/kg). A selective action of L-NAME on the effects of neurosteroids was indicated, since the effects of L-NAME were completely reversed by L-arginine (300 mg/kg, i.p.), a competitive substrate for NO synthase. Neither L-NAME nor L-arginine alone affected the antinociception, locomotor activity or rota-rod performance of young or aged mice. These observations suggest that a NO-dependent mechanism may be involved in the beneficial and antiamnesic effects of neurosteroids PS and DHEAS on the aging- and dizocilpine-induced impairment of learning and memory processes.

Topics: Aging; Amnesia; Animals; Avoidance Learning; Dehydroepiandrosterone Sulfate; Dizocilpine Maleate; Enzyme Inhibitors; Learning Disabilities; Male; Maze Learning; Memory; Mice; Mice, Inbred Strains; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nootropic Agents; Pregnenolone

Pregnenolone sulfate (PS) acts as a positive allosteric modulator of N-methyl-D-aspartate (NMDA) receptor-mediated responses. In the retina, we previously observed that the synthesis of pregnenolone and PS increases after stimulation of NMDA receptors and blockade of the synthesis reduces retinal cell death. This study was carried out to explore in the isolated and intact retina the possible role of PS in NMDA-induced excitotoxicity. Lactate dehydrogenase (LDH) measurements and morphological analysis revealed that a 90-min exogenous application of PS at 0.1-500 microM concentrations potentiated NMDA-induced cell death and at 50-500 microM concentrations caused cytotoxicity. After 45 min, either NMDA or PS caused no significant LDH release; but their co-application resulted in a high degree of toxicity. In addition, we found that a mild NMDA insult developed into serious damage when even low PS concentrations (0.1-10 microM) were used. Toxicity-inducing and -potentiating effects were specific to PS modulatory action on NMDA receptors, in that they were blocked by 4-(3-phosphonopropyl)2-piperazinecarboxylic acid (CPP) and MK-801 but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and neither dehydroepiandrosterone sulfate nor pregnenolone caused LDH release. Prevention of degenerative signs was seen in retinae pretreated with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, thus indicating a Na+/Cl--dependent acute mode of excitotoxic cell death responsible for PS toxicity. The positive interaction between the neurosteroid and NMDA receptors was further proved by a PS dose-dependent increase in NMDA-induced stimulation of [3H] MK-801 binding to retinal membranes. The results suggest a crucial role of PS in retinal vulnerability and propose the toxicity-potentiating effects as an important key in linking NMDA-induced endogenous synthesis to acute excitotoxicity.

Topics: Animals; Dizocilpine Maleate; Drug Synergism; L-Lactate Dehydrogenase; Male; N-Methylaspartate; Neuroprotective Agents; Neurotoxins; Pregnenolone; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Retina

Pregnenolone sulfate (PS) has been reported to selectively augment glutamate-induced depolarizations mediated by the NMDA subtype of the glutamate receptor. The present study examines the ability of this neuroactive steroid to potentiate NMDA-mediated increases in intracellular calcium in cultured chick cortical neurons using the fluorescent dye Fura2. PS, in the absence of NMDA and glycine, significantly elevated intracellular calcium at 250 and 500 microM. This increase in free calcium was significantly attenuated at 250 microM PS by the prior addition of 50 microM CNQX, 10 microM dizocilpine or 1 microM nimodipine. NMDA and glycine, when added to the cells in saturating concentrations of 500 and 50 microM, respectively, consistently increased intracellular free calcium over baseline levels. In the presence of NMDA and glycine, both 50 and 100 microM PS produced a further significant rise in intracellular free calcium. The prior addition of CNQX, dizocilpine or both compounds together significantly inhibited this elevation in free calcium. The application of the endogenous polyamine spermine (250 microM) significantly potentiated the response of chick cortical neuronal cells to NMDA and glycine. PS, in the presence of NMDA, glycine and spermine, produced a further increase in intracellular free calcium at concentrations of 50 and 100 microM. The prior application of CNQX, dizocilpine or both compounds together significantly attenuated this rise in free calcium. These data confirm that PS is a positive allosteric modulator of the NMDA receptor and provide evidence that this neurosteroid does not interact with the polyamine modulatory site.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Cells, Cultured; Chick Embryo; Dizocilpine Maleate; Fura-2; Glycine; N-Methylaspartate; Nimodipine; Pregnenolone; Spermine

Ammonia and benzodiazepines are thought to be involved in the pathogenesis of hepatic encephalopathy. The present study was undertaken to evaluate the effect of various benzodiazepine-receptor ligands and neurosteroids on ammonia toxicity in mice. Administration of ammonium acetate (8-15 mmole/kg; i.p.) to Swiss Webster mice resulted in a dose-dependent increase in mortality. Pretreatment with the central benzodiazepine receptor agonist clonazepam or the antagonist Ro15-1788 (7 mg/kg each; i.p.) had no significant effect on the lethal response to 10 mmole/kg ammonium acetate. However, pretreatment with the putative antagonist of the peripheral-type benzodiazepine receptor, PK 11195 (10 mg/kg; i.p.), reduced mortality from 50 to 10%. Ro5-4864 (10 mg/kg; i.p.), an agonist of the peripheral-type benzodiazepine receptors, had no effect on ammonia toxicity. The neurosteroid, pregnenolone sulfate (20 mg/kg; i.p.) reduced mortality from 50 to 25%. The non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (2 mg/kg; i.p.), had no effect on the lethal response to ammonium acetate. The results from the present study suggest a role for peripheral-type benzodiazepine receptors and specific neurosteroids in the alleviation of ammonia toxicity in mice.

Topics: Ammonia; Animals; Clonazepam; Dizocilpine Maleate; Dose-Response Relationship, Drug; Isoquinolines; Male; Mice; Pregnenolone