Evaluation of in vivo interactions in mice between flurazepam and two neuroactive steroids.

The development of neuroactive steroids as anticonvulsant medications may be useful both as a primary treatment and as an adjuvant to other anticonvulsants. They may be limited, however, by sedative and ataxic side effects. In the current study, 3 alpha-hydroxy-5 beta-pregnan-20-one and alfaxalone, two prototypic neuroactive steroids, were shown to potentiate the ability of flurazepam to antagonize electrically precipitated tonic hindlimb extension in mice at doses that by themselves had little antiseizure efficacy. While alfaxalone alone lacked motor incoordinating effects at a dose (18.0 mg/kg) that potentiated the antiseizure efficacy of flurazepam, the same dose of 3 alpha-hydroxy-5 beta-pregnan-20-one possessed both the ability to potentiate flurazepam's anticonvulsant effect and disrupt mouse rotorod performance. The data suggest that allosteric interactions that have been described in vitro between neuroactive steroids and other modulators of the GABAA receptor complex may have relevance for the intact animal. Finally, the data also suggest that neuroactive steroids could be developed as short-lived adjuvant antiseizure medications in certain critical situations (e.g., medication-refractory status epilepticus). However, the motor incoordinating effects resulting from the combination of neuroactive steroids and flurazepam suggest that their usefulness as adjuvant medications in the chronic therapy of seizure disorders may be limited.

7-Nitroindazole and methylene blue, inhibitors of neuronal nitric oxide synthase and NO-stimulated guanylate cyclase, block MK-801-elicited behaviors in mice.

We examined the abilities of 7-nitroindazole and methylene blue, inhibitors of the neuronal isoform of nitric oxide synthase (NOS) and nitric oxide-stimulated guanylate cyclase activity respectively, to attenuate explosive episodic jumping behavior(s) ("popping") elicited by MK-801 in mice. MK-801, like phencyclidine (PCP), is a high-affinity, noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. We have postulated that MK-801-elicited popping behavior in mice represents an animal model of schizophrenia, because popping behavior is markedly inhibited/antagonized by both typical and atypical antipsychotic drugs. In the present study, popping behavior induced by MK-801 was measured using an automated detection system that quantifies vertical displacements on the testing platform. 7-Nitroindazole (100 mg/kg) and methylene blue (32 and 100 mg/kg) significantly reduced the number and force of MK-801-elicited popping behavior. Mouse rotorod performance did not differ between animals receiving 7-nitroindazole, methylene blue, or their respective vehicles, suggesting that attenuation of MK-801-elicited popping behavior was not due to either sedation or ataxia caused by 7-nitroindazole or methylene blue. Our findings suggest that nitric oxide may, in part, mediate behaviors induced by NMDA receptor antagonists, like MK-801, and that inhibitors of NOS may have antipsychotic actions.

Computerized measurement of MK-801-elicited popping and hyperactivity in mice.

MK-801, a high-affinity phencyclidine (PCP) analogue, is a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) subclass of glutamate receptor that elicits hyperactivity, stereotypic behaviors, and "popping," an explosive episodic jumping behavior, in mice. The schizophreniform psychosis precipitated by PCP in humans has stimulated interest in studying MK-801-elicited mouse behaviors for their potential development as animal models of idiopathic psychosis. We describe a computerized method for measuring popping and hyperactivity elicited by MK-801 in mice, based on vertical displacements of a platform. This computerized procedure allows for the automatic measurement of discrete "pops" per individual episode of popping behavior, the force of each one of the explosive jumps, and the duration of discrete episodes of popping; these latter measures could not be easily ascertained by visual inspection alone. Moreover, the computerized measurements facilitate quantitative evaluation of the effects of pharmacological manipulations on MK-801-elicited popping. For example, the antipsychotic haloperidol was shown to reduce significantly both MK-801-induced popping and hyperactivity. Ideally, MK-801-elicited mouse popping and hyperactivity will serve as a useful preclinical screening paradigm for potential antipsychotic medications. Additionally, it is hoped that the use of this automated system will contribute to a greater understanding of the mechanisms of MK-801-induced popping and hyperactivity.

MK-801 alters the GABAA receptor complex and potentiates flurazepam's antiseizure efficacy.

MK-801 is an uncompetitive allosteric antagonist that interferes with glutamate-gated calcium ion conductance through the NMDA receptor-associated ionophore. In an outbred strain of mouse, MK-801 elicits episodes of explosive "popping" behaviors that may serve as a preclinical screening paradigm for novel antipsychotic medications. This investigation examined the effects of MK-801, at doses associated with the elicitation of popping, on the GABAA receptor complex in cerebral cortex, and flurazepam's ability to antagonize electrically precipitated seizures. Twenty four hours after MK-801 administration, there was an increased density of the radiolabeled antagonist-preferring conformation of the central benzodiazepine binding site and a potentiation of flurazepam's antiseizure efficacy. The data show that interference with NMDA receptor-mediated calcium ion conductance is associated with a relatively selective change in the GABAA receptor complex in cerebral cortex, and has functional behavioral consequences. Moreover, the data provide additional evidence for a delicate balance between GABAergic and glutamatergic transmission. Disturbance of this balance can have behavioral consequences for the animal.

Interference with nitric oxide production and action potentiates the antiseizure efficacy of flurazepam.

The effect of inhibiting "downstream" consequences of NMDA receptor stimulation with 7-nitroindazole, an inhibitor of the neuronal form of nitric oxide synthase (NOS), and methylene blue, an inhibitor of the nitric oxide (NO)-sensitive soluble guanylyl cyclase, on electrically precipitated tonic hindlimb extension in mice was studied. Moreover, the abilities of these compounds to potentiate the antiseizure efficacy of flurazepam were also examined. When administered alone, 7-nitroindazole (10.0-100 mg/kg) and methylene blue (1.0-100 mg/kg) did not share the ability of MK-801 (0.1 to 1.0 mg/kg) to antagonize electrically precipitated tonic hindlimb extension. However, doses of MK-801 (0.18 mg/kg), 7-nitroindazole (100 mg/kg), and methylene blue (10.0 and 100 mg/kg) that were devoid of apparent antiseizure efficacy by themselves potentiated the ability of flurazepam to antagonize electrically precipitated seizures. NMDA receptor antagonists cause neuronal toxicity, interfere with acquisition of spatial memory and induction of long-term potentiation in the hippocampal CA1 region, and precipitate psychoses in susceptible individuals. Thus, the development of both open-channel blockers of the NMDA receptor complex that can be administered in lower doses, and inhibitors of the "downstream" consequences of NMDA receptor-gated transient elevations of intraneuronal calcium ions as potential adjunctive antiseizure medications should be considered. Moreover, administration of these compounds with benzodiazepines may attenuate some of the neurotoxicity that may result from NMDA receptor antagonism.

A colposcopical lesion of the uterine cervix frequently associated with papillomavirus type 16 as detected by in situ and Southern blot hybridization: a cytohistological correlation study.

A convoluted aceto-white lesion was observed by colposcopy on the uterine cervix of 19 patients. A search for specific types of papillomavirus was undertaken in these lesions which proved histologically to be a vertical association of CIN in the lower strata of the squamous epithelium and typical condyloma in the uppermost layers. Previous cell samples correlated with histology in only 11 out of the 19 cases, and were not therefore an accurate predictor of histologic findings. However 16 out of our 19 cases had sufficient cellular changes to warrant colposcopy and biopsy. Papillomavirus capsid antigen was detected by peroxidase-antiperoxidase staining technique in 12 out of the 19 cases (63.1%). In situ hybridization using biotinylated HPV-16 DNA probes was positive in 13 out of 18 cases (72.2%). Southern blot hybridization gave the typical PstI pattern of HPV-DNA in 8 out of 9 cases examined. The detection of HPV-DNA and HPV capsid antigen was particularly high in this "convoluted" cervical lesion, suggesting that the virus remains biologically active. It may therefore be assumed that this lesion represents a particular phase of the disease process in which early gene function and late gene expression of the viral genome are markedly manifested. Furthermore, this lesion may well represent a link between the early manifestation of HPV infection (condyloma) and CIN, which constitute a morphologic continuum initiated by papillomaviruses.