Gary A. Pritchard

Pregnenolone sulfate potentiation of NMDA-mediated increases in intracellular calcium in cultured chick cortical neurons

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.

Prenatal cocaine exposure: Decreased sensitization to cocaine and decreased striatal dopamine transporter binding in offspring

Pregnant mice were treated with cocaine, 10 mg/kg/day, during days 13 to 20 of gestation. Cocaine sensitization and dopamine transporter binding were evaluated in offspring at 6 weeks of age. Sensitization, defined as the increase in activity after 5 injections of cocaine compared to 1 injection, was reduced in cocaine-exposed mice. Dopamine transporter binding in striatum was also significantly reduced in cocaine-exposed mice compared to controls.

Rapid Communication Isolation and Characterization of the Promoter of the Human GABAA Receptor α1 Subunit Gene

Abstract: The GABAA receptor, as assessed by ligand binding and chloride flux measurement in vivo and in vitro, is down‐regulated in response to chronic benzodiazepine exposure. The mRNA levels of the α1 and γ2 subunits of the receptor are also reduced. We have isolated the promoter of the gene encoding the α1 subunit of the GABAA receptor to elucidate the regulatory mechanism of its expression. A DNA segment 650 bp long has been Isolated that includes 151 bp of untranslated 5’end of the cDNA sequence and 500 bp of potential promoter‐enhancer region. The transcriptional activity of this DNA segment linked to the firefly luciferase gene showed a strong orientation specificity. The promoter activity was localized to a 60‐bp segment by deletion mapping. Mobility shift binding assay results suggest that this segment may interact with one or more factors in HeLa cell nuclear extracts to form a transcriptional complex. Primary cultures of embryonic chick cortical cells transfected with the promoter‐luciferase construct were treated chronically with lorazepam. Transcriptional activity of this promoter construct was strongly repressed by chronic administration of lorazepam.

Pharmacodynamic and receptor binding changes during chronic lorazepam administration

To assess pharmacodynamic and neurochemical aspects of tolerance, lorazepam (2 mg/kg/day), or vehicle was administered chronically to male Crl: CD-1(ICR)BR mice via implantable osmotic pump. Open-field behavior, benzodiazepine receptor binding in vitro, receptor autoradiography, and muscimol-stimulated chloride uptake were examined at both 1 and 14 days. Open-field activity was depressed in lorazepam-treated animals on Day 1. On Day 14, open-field parameters were indistinguishable from those of vehicle-treated animals, indicating behavioral tolerance. Benzodiazepine binding, as determined by the specific binding of [125I]diazepam, was also decreased in cortex on Day 14. Hippocampal binding was unchanged following chronic lorazepam exposure. Apparent affinity in cortical membrane preparations was unchanged, indicating that altered ligand uptake was due to decreased receptor number. Muscimol-stimulated chloride uptake into cortical synaptoneurosomes from lorazepam-treated animals was not significantly different on Day 1 or Day 14 compared to vehicle-treated animals. These results confirm that down-regulation of benzodiazepine receptor binding is closely associated with behavioral tolerance to benzodiazepines. These observed changes in binding are not necessarily associated with robust changes in receptor function.

The NMDA Receptor Competitive Antagonist CPP Modulates Benzodiazepine Tolerance and Discontinuation

Benzodiazepine discontinuation is characterized by a syndrome of increased activity and reduced seizure threshold that is similar to effects mediated by the glutamatergic system. To elucidate the involvement of the glutamatergic system in benzodiazepine tolerance and discontinuation, we administered lorazepam, the NMDA antagonist CPP, and the combination of these compounds either concomitantly or consecutively to mice via osmotic pumps and evaluated pentylenetetrazole-induced seizure threshold, open-field activity, and benzodiazepine receptor binding during and after chronic administration. Animals receiving lorazepam alone developed partial tolerance at 7 days and complete tolerance at 14 days to the anticonvulsant effects of lorazepam. This effect was partly attenuated by CPP coadministration with lorazepam. This combination produced only partial tolerance. A reduction in seizure threshold was observed 4 days after discontinuation of lorazepam alone. This effect was abolished by coadministration of CPP with lorazepam and by CPP administration during the withdrawal period. Benzodiazepine binding in most structures examined was significantly reduced at 14 days during chronic lorazepam administration (versus 1 day), and coadministration of CPP did not alter this decrement. After lorazepam discontinuation, binding was increased at 4 and 7 days versus chronically treated animals and versus vehicle within the cerebral cortex. This effect was abolished by coadministration of CPP as well as by CPP administration during the lorazepam withdrawal period. These data support the involvement of the glutamatergic system in benzodiazepine tolerance and discontinuation.

Polyamine neurotoxicity is antagonized by dizocilpine in cultured chick cortical neurons

Release of endogenous polyamines may contribute to neuronal loss in ischemia and related conditions. Primary cortical neurons were exposed to spermine and spermidine and subsequently assayed for [3H]ouabain binding to quantify neuronal loss. Neuronal survival was significantly decreased in the presence of spermine at 24 h (500 microM), 48 h (250 microM and 500 microM) and 72 h (10-500 microM) relative to controls. Co-application of 250 microM spermine and 10 microM dizocilpine for 48 h completely inhibited the effect of spermine alone. Spermidine exposure (10-500 microM) did not alter neuronal survival at any of the time points. These data indicate that the polyamine spermine is toxic to neurons in vitro and that toxicity is prevented by the NMDA-associated channel antagonist dizocilpine.

Lorazepam Attenuates the Behavioral Effects of Dizocilpine

To characterize the potential interaction between the excitatory and inhibitory neurotransmitter systems, the effects of dizocilpine, CPP, and lorazepam on open-field behavior and pentylenetetrazol-induced seizures were evaluated in mice. Dizocilpine (0.01-0.1 mg/kg), CPP (1-10 mg/kg), or vehicle was administered intraperitoneally 15 min prior to lorazepam (0.2-2 mg/kg) or vehicle. Behavioral monitoring began 25 min after the lorazepam injection. Upon completion of testing, unrestrained mice were infused intravenously with pentylenetetrazole until the onset of a full tonic-clonic seizure. The highest dose of dizocilpine by itself significantly increased the average distance traveled, the number of rears, and the number of stereotypies during the test period. Lorazepam alone dose dependently decreased activity on all behavioral parameters. Lorazepam also completely antagonized the hyperactivity produced by dizocilpine when the two compounds were coadministered. This antagonism is most likely due to an interaction in the regulation of dopaminergic tone which underlies motor activity. Lorazepam exerted a dose-dependent anticonvulsant effect. Dizocilpine alone had no effect on seizure induction and did not potentiate the anticonvulsive effect of lorazepam when coadministered with lorazepam. CPP reduced the number of rears and the number of stereotypies during the test period. CPP did not alter the pentylenetetrazol-induced seizure threshold and did not influence the anticonvulsant effect of lorazepam.

Characterization of Ca2+-mobilizing excitatory amino acid receptors in cultured chick cortical cells

The effects of glutamate and other more selective excitatory amino acid (EAA) analogs on intracellular free calcium concentration ( [Ca2+]i) were examined in Fura 2-loaded cultured chick embryo cortical cells (90% neuronal). Four EAA receptors were evident in these studies: an N-methyl-D-aspartate (NMDA) receptor, a kainate receptor, and two quisqualate receptors. The [Ca2+]i response to NMDA was blocked or reversed by selective antagonists such as 2-amino-5-phosphonovalerate (APV), MK801 and ketamine, as well as by desmethylimipramine and dextromethorphan. Glycine potentiated the [Ca2+]i response to NMDA, and high concentrations of glycine selectively overcame blockade by kynurenic acid, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and cis-piperidine-2,3-dicarboxylic acid (PDA). The [(Ca2+]i response to kainate was generally larger than the NMDA response, and the kainate response desensitized slightly over the first minute. CNQX was more potent as an antagonist of the kainate response than of the NMDA response, even in the absence of added glycine; kynurenic acid and PDA conversely had little effect on the kainate response in these cells at concentrations which blocked the NMDA response. The desensitization of the [Ca2+]i response to kainate was greatly augmented by quisqualate and by the putative ionotropic quisqualate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). In the absence of kainate, both quisqualate and AMPA increased [Ca2+]i though less so than did NMDA or kainate. Quisqualate (and AMPA and glutamate) were not acting as partial agonists at the kainate receptor, since the potency of these agonists in reversing the kainate [Ca2+]i response was independent of kainate concentration. Quisqualate, but not AMPA, also produced a small increase in [Ca2+]i which preceded the negative effect of this agonist on the kainate response. This increase in [Ca2+]i could also be evoked by quisqualate or glutamate after inhibition of the kainate response by AMPA. Quisqualate and glutamate, but not the other EAA agonists, also increased [Ca2+]i after chelation of extracellular calcium with EGTA. This effect appears to be mediated by the metabotropic quisqualate receptor. These cells should provide a useful system for studying regulation and interactions of EAA receptors, and for screening drugs which might act at these receptors.

In situ hybridization histochemistry as a method to assess GABAAreceptor subunit mRNA expression following chronic alprazolam administration

Previous work in our laboratory has demonstrated region-specic effects for chronic alprazolam on binding and function at the GABAA receptor. The present study evaluated regional changes in mRNA expression of several subunits of the GABAA receptor following chronic alprazolam administration that might underlie these effects. Mice received alprazolam (2 mg/kg/day) or vehicle via subcutaneously implanted osmotic pumps for 1, 7, 14 or 28 days. In situ hybridization histochemistry was performed on tissue sections using [35S]dATP oligonucleotide probes corresponding to the a1 and g2 subunits of the GABAA receptor. Specic hybridization was clearly demonstrated and a1 subunit mRNA expression in frontoparietal cortex (layers IIIV) on day 1 of infusion was reduced in animals receiving alprazolam compared to vehicle. On subsequent days, there were no alterations in the levels of a1 subunit mRNA in the frontoparietal cortex, hippocampus or dentate gyrus. Expression of g2subunit mRNA was increased on day 1 in the frontoparietal cortex (layer VI), hippocampus and dentate gyrus. mRNA expression was also increased in the dentate gyrus on day 28 of infusion. Comparison of the present study with the results of chronic treatment with other benzodiazepines clearly demonstrates that the pattern of mRNA subunit alterations obtained is both treatment- and region-specic. This makes a de¢nitive conclusion regarding benzodiazepines and their interactions with GABAA receptors di¤cult at best.

Polyamine potentiation and inhibition of NMDA-mediated increases of intracellular free Ca2+ in cultured chick cortical neurons.

Polyamine potentiation and inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated Ca2+ changes was studied in cultured chick cortical neurons. Spermidine and spermine potentiated the effect of saturating concentrations of NMDA and glycine. No effect of spermidine or spermine was observed in the absence of NMDA or in the presence of either kainate or quisqualate. Similarly, antagonism of the NMDA receptor complex with dizocilpine (an open channel blocker), or with competitive antagonists to the NMDA or glycine binding sites greatly attenuated or completely abolished the combined effects of polyamines plus NMDA and glycine. N-Acetylspermine and N-acetylspermidine, in the presence or absence of NMDA and glycine, were without effect. These data strongly suggest that spermidine and spermine are potent and selective agonists at the polyamine binding site. Putrescine and diethylenetriamine were ineffective as antagonists of NMDA-mediated intracellular free Ca2+ increases in the presence or absence of added spermine or spermidine. Arcaine and 1,10-diaminodecane, however, antagonized NMDA-mediated intracellular free Ca2+ increases in the presence and absence of spermine and spermidine, and therefore appear to act either as inverse agonists at the polyamine binding site or as open channel blockers of the NMDA receptor.