Jeanne M. Fahey

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.

Induction of P‐glycoprotein expression and activity by ritonavir in bovine brain microvessel endothelial cells

Extended treatment with human immunodeficiency virus (HIV) protease inhibitors (HPIs) is standard in HIV/AIDS therapy. While these drugs have helped decrease the overall incidence of AIDS defining illnesses, the relative prevalence of HIV/AIDS dementia has increased. HPIs may cause induction of blood‐brain barrier (BBB) drug transporters (P‐glycoprotein; P‐gp) and thereby limit entry of HPIs into brain tissue, increasing the probability that the brain could become an HIV sanctuary site. Using bovine brain microvessel endothelial cells (BMEC) as an in‐vitro model of the BBB, the potential for the HIV protease inhibitor ritonavir to cause induction of P‐gp activity and expression was examined. BMEC were isolated from fresh cow brain by enzymatic digest and density centrifugation. Primary culture BMEC were co‐incubated with ritonavir or vehicle control for 120 h. Quantitative drug accumulation of rhodamine 123 (Rh123) and fluorescence microscopy were used as measures of P‐gp activity. P‐gp expression was assessed using quantitative Western blotting. Ritonavir decreased Rh123 cell accumulation and increased P‐gp immunoreactive protein in a concentration‐dependent manner. Fluorescent microscopy mirrored Rh123 quantitative studies. In BMEC pretreated with 30 μM ritonavir, Rh123 accumulation was decreased 40% and immunoreactive P‐gp protein increased 2‐fold. Collectively, a strong correlation between decreased Rh123 BMEC accumulation and increased P‐gp immunoreactive protein was observed (Spearman r2 = 0.77, P < 0.0001). Thus extended exposure of BMEC to ritonavir caused a concentration‐dependent increase in P‐gp activity and expression. Similar findings may occur at the clinical level with prolonged HIV protease inhibitor use, giving insight into the central nervous system as an HIV sanctuary site and eventual development of HIV dementia.

Interleukin‐1 Modulates GABAergic and Glutamatergic Function in Braina

Interleukin-1 is a polypeptide cytokine involved in the acute-phase response. We examined the effects of IL-1 on the major inhibitory and excitatory neurotransmitter systems in brain mediated by GABA and glutamate, respectively. IL-1 enhanced the effects of GABA as determined by GABA-dependent chloride uptake at 1 and 10 ng/ml in cortical synaptoneurosomes. This effect was attenuated by pretreatment with the IL-1 receptor antagonist. Neither IL-6 nor TNF was effective in this system. IL-1 decreased the effect of NMDA and glycine on intracellular calcium concentrations in cultured chick cortical neurons in both the presence and absence of the endogenous polyamine spermine. This effect was attenuated by pretreatment with the AMPA/kainate antagonist CNQX. These data indicate that IL-1 modulates both inhibitory and excitatory neurotransmitter function in brain.

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.

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.

Acute zolpidem administration produces pharmacodynamic and receptor occupancy changes at similar doses.

Zolpidem is chemically unrelated to classical benzodiazepines but has demonstrated relatively high affinity binding to the alpha(1) GABA(A) receptor. To assess pharmacodynamic and neurochemical effects of zolpidem, open-field behavior, pentylenetetrazole-induced seizure threshold and benzodiazepine receptor binding in vitro were evaluated in the same animal following a single dose of zolpidem. Zolpidem (2, 5 and 10 mg/kg), lorazepam (2 mg/kg) or vehicle was administered intraperitoneally in male CD-1 mice. Behavioral activity, assessed by three open-field parameters, was decreased following the two highest doses of zolpidem (5 and 10 mg/kg), and reached significance at the 10 mg/kg dose. Locomotor activity was also decreased significantly by lorazepam as expected. Pentylenetetrazole-induced seizure threshold was increased with the administration of 2 and 10 mg/kg zolpidem as well as with lorazepam. Apparent affinity (K(D)) of [3H]flunitrazepam, a non-selective ligand, for the benzodiazepine receptor in cortical membrane preparations was not significantly changed, while receptor number (Bmax) was decreased at all doses of zolpidem, reaching significance at the 10 mg/kg dose. These results confirm that the behavioral effects of zolpidem are similar to those of classical benzodiazepines. In addition, zolpidem had no significant effect on the affinity of the benzodiazepine receptor for [3H]flunitrazepam, but did decrease the density of receptor binding sites.

Gaboxadol - a different hypnotic profile with no tolerance to sleep EEG and sedative effects after repeated daily dosing

Gaboxadol, a selective extra synaptic GABA(A) receptor agonist, has been in clinical development for the treatment of insomnia. Development of tolerance to therapeutic effects (e.g. hypnotic and anticonvulsant and sedative) and withdrawal symptoms (e.g. REM sleep rebound and reduced seizure threshold) upon treatment discontinuation is reported for GABA(A) receptor allosteric modulators acting via the benzodiazepine binding site, e.g. zolpidem and indiplon. We conducted a head to head comparison in rats of the hypnotic (sleep EEG after 21 daily doses and 24 and 48 h after the last dose) and seizure threshold modifying (bicuculline assay 24 h after 28 daily doses) effects of gaboxadol and benzodiazepine ligands. Furthermore, we investigated in further details a previously reported apparent rapid development of tolerance to gaboxadols effects in a rat rotarod motor coordination assay and related this effect to CNS exposure levels and in vitro potency at extra synaptic GABA(A) receptors. Sleep EEG studies demonstrated lack of tolerance and withdrawal effects after 28 daily doses with gaboxadol, whereas zolpidem produced both tolerance and withdrawal effects under a similar dosing regimen. Daily dosing with gaboxadol, zolpidem or indiplon for 28 days and acute discontinuation of treatment left the threshold to bicuculline-induced seizures unchanged. The rapidly attenuated effect of repeated gaboxadol dosing was confirmed in the rotarod model. However, re-challenge of gaboxadol insensitive animals with gaboxadol produced a maximum response, ruling out that receptor desensitisation accounts for these effects. By comparing CNS exposure at rotarod responses and concentration response relation at cloned GABA(A) receptors expressed in Xenopus oocytes it appears that the decline in response in the rotarod model coincides with the steep part of the concentration response curve for gaboxadol at extra synaptic GABA(A) receptors. In conclusion, rat sleep EEG repeated dose studies of gaboxadol confirm a hypnotic-like profile and no withdrawal effects, whereas tolerance and withdrawal effects were shown with zolpidem. Withdrawal from gaboxadol, zolpidem and indiplon did not affect the seizure threshold to bicuculline. Gaboxadols apparent rapid development of tolerance in the rotarod assay appears to be kinetically determined.

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.

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.