Posttraumatic stress two years after the Oklahoma City bombing in youths geographically distant from the explosion.

This article describes Posttraumatic Stress Disorder (PTSD) symptomatology in 69 sixth-grade youths who resided within 100 miles of Oklahoma City at the time of the 1995 bombing of the Alfred P. Murrah Federal Building. These youths neither had any direct physical exposure nor personally knew anyone killed or injured in the explosion. A survey conducted two years after the bombing assessed exposure, PTSD symptoms, and functioning. PTSD symptom frequency was measured with the Impact of Event Scale--Revised. Our BCD criteria for defining PTSD caseness was modeled after DSM-IV B, C, and D criteria requiring one reexperiencing, three avoidance/numbing, and two arousal symptoms for diagnosis. Those who met our BCD criteria had significantly higher PTSD symptom scores than those who did not. Both increased mean PTSD symptom score and meeting our caseness definition were associated with increased functioning difficulties. Media exposure and indirect interpersonal exposure (having a friend who knew someone killed or injured) were significant predictors of symptomatology. These findings suggest that children geographically distant from disaster who have not directly experienced an interpersonal loss report PTSD symptoms and functional impairment associated with increased media exposure and indirect loss.

Design and synthesis of isoxazole containing bioisosteres of epibatidine as potent nicotinic acetylcholine receptor agonists.

An efficient synthesis of isoxazole containing isosteres of epibatidine is described. The synthesis proceeded from N-tert-butoxycarbonyl (Boc)-exo-2-(methoxycarbonyl)-7-azabicyclo[2.2.1]heptane (9). Compound 9 was reacted with the dilithium salt of an appropriately substituted oxime in tetrahydrofuran (THF). Cyclodehydration of the resultant beta-keto oxime and deprotection of the N-Boc group in 5 N aqueous HCl afforded the isoxazole containing isosteres of epibatidine (6-8). The binding affinities of these compounds were determined at the nicotinic acetylcholine receptor for the displacement of [3H]cystisine. The unsubstituted isoxazole containing isostere (6) showed the lower binding potency compared to the 3'-methylisoxazole isostere (7). Substitution with a phenyl group at the 3'-position of the isoxazole significantly reduced the binding potency. The in vivo toxicological studies of these analogs were also performed. The LD50 of the analogs ranged in the order: Me > H > Ph.

Effects of S-21007, a potent 5-HT3 partial agonist, in mouse anxiety.

AIM: To study the effect of S-21007, a 5-HT3 partial agonist in different animal models of anxiety in mice. METHODS: S-21007 effects were evaluated in the behavior tests after intraperitoneal and oral acute treatment or in the light/dark test after both acute and chronic treatments. RESULTS: S-21007 presented anxiolytic-like properties after acute administration in the light/dark box test, the mirrored chamber test, and the elevated plus-maze at low doses 10 ng.kg(-1)-100 micrograms.kg-1, 1-100 micrograms.kg-1 and 10-100 micrograms.kg-1, respectively. In the light/dark box test, S-21007 was active orally after acute treatment at 100 ng.kg(-1)-10 mg.kg-1 and after chronic treatment (14 d) at 1-10 micrograms.kg-1. S-21007 was devoid of sedative or stimulatory effects. CONCLUSION: S-21007 exhibited anxiolytic-like properties. The mechanism of action may be a desensitization of 5-HT3 receptor or an antagonist activity on the 5-HT3 receptors.

2beta-Substituted analogues of 4'-iodococaine: synthesis and dopamine transporter binding potencies.

A series of 2beta-substituted analogues of 4'-iodococaine (3) was synthesized and evaluated in an in vitro dopamine transporter (DAT) binding assay. Selective hydrolysis at the 2beta-position of 3 gave the carboxylic acid 15 that served as the intermediate for the synthesis of compounds 4, 5, and 6-11. The 2beta-alkyl derivatives were obtained from ecgonine methyl ester (17) through a series of reactions leading to the aldehyde 20. Wittig reaction of 20 with methyltriphenylphosphorane followed by hydrogenation and benzoylation gave the products 12 and 13. The binding affinity of 4'-iodococaine (3) was 10-fold less than that of cocaine. The hydroxymethane, acetate, amide, benzyl ester, oxidazole, and ethane derivatives of 3 exhibited decreased binding while the vinyl, phenyl, and ethyl esters showed a moderate increase in binding affinity. Only the isopropyl derivative 8 exhibited a 2-fold increase in binding affinity compared with 4'-iodococaine (3). Hydroxylation of 8 at the 2'-position gave 14 which enhanced not only the binding potency at the DAT by another 2-fold but also the selectivity at the DAT over the norepinephrine and serotonin transporters. Compound 14 failed to stimulate locomotor activity in C57BL/6J mice over a wide dose range and blocked cocaine-induced locomotor stimulant action.

Genotypic differences between C57BL/6 and A inbred mice in anxiolytic and sedative actions of diazepam.

The role of genotype in susceptibility to the behavioral actions of benzodiazepines is not well characterized. To develop a model for such studies, we have characterized the anxiolytic and sedative activities of diazepam in C57BL/6J and A/J inbred mice. C57BL/6J mice were more responsive than A/J mice to diazepam-induced anxiolytic-like activity in the mirrored chamber aversion assay and the elevated plus-maze assay. Basal activity of the two strains did not differ in either assay. In contrast, the two strains were equally responsive to the anxiolytic effects of the 5-HT1A receptor partial agonist, buspirone. C57BL/6J mice were also more susceptible to the sedative effects of diazepam than were A/J mice. Flumazenil blocked the effects of diazepam in these behavioral assays. Measurement of diazepam and nordiazepam in blood and brain suggested that the response differences are of a pharmacodynamic rather than a pharmacokinetic nature. Taken together, these findings indicate that C57BL/6J and A/J mice provide a valuable tool for behavioral genetic studies of the mechanisms underlying the pharmacological actions of benzodiazepines.

Inherited, selective hypoanalgesic response to cytisine in the tail-flick test in CF-1 mice.

This study extends the pharmacological characterization of the genotype- dependent difference in analgesic responsiveness to neuronal nicotinic agonists between CD-1 and CF-1 strains of mice. Acute analgesic potency of cytisine measured by the tail-flick assay differed by > 3200-fold between CD-1 and CF-1 outbred strains of mice. Analgesic non-responsiveness of the CF-1 strain was pharmacologically selective. Morphine produced a dose-dependent analgesic response of similar magnitude in both strains. Other pharmacological actions of cytisine, including inhibition of locomotor activity, induction of seizures and lethality, did not differ between these strains. Hyporesponsiveness to the analgesic action of both nicotine and cytisine was observed in two different CF-1 sublines. Biodistribution of [3H]cytisine in blood did not differ between the CF-1 and CD-1 strains. These pharmacological characteristics indicate that the CD-1-CF-1 strain pair provides a useful pharmacogenetic tool for investigating the mechanistic bases of analgesia induced by nicotinic cholinergic agonists.

Synthesis and ligand binding studies of 4'-iodobenzoyl esters of tropanes and piperidines at the dopamine transporter.

Four analogs and two homologs of cocaine, designed as potent cocaine antagonists, were synthesized. The SN2 reaction between ecgonine methyl ester (13) or appropriately substituted piperidinol (19, 21) and appropriately substituted 4-iodobenzoyl chloride gave 4-iodobenzoyl esters of tropanes and piperidines (5-8). 2'-Hydroxycocaine (9) was obtained from 2'-acetoxycocaine (12) by selective transesterification with MeOH saturated with dry HCl gas. 2'-Acetoxycocaine (12) was synthesized from acetylsalicyloyl chloride (23) and ecgonine methyl ester (13). The binding affinities of these compounds were determined at the dopamine transporter for the displacement of [3H]WIN-35428. An iodo group substitution at the 4'-position of cocaine decreased dopamine transporter binding potency, while a hydroxy or acetoxy group at the 2'-position exhibited increased binding potency for the dopamine transporter compared to cocaine (10- and 3.58-fold, respectively). 2'-Hydroxylation also enhanced the bidning potency of 4'-iodococaine (5) by 10-fold. Replacement of the tropane ring with piperidine led to poor binding affinities.

The GABAA receptor alpha 6 subunit gene (Gabra6) is tightly linked to the alpha 1-gamma 2 subunit cluster on mouse chromosome 11.

We have established that the GABAA receptor alpha 6 (Gabra6) and alpha 1 (Gabra 1) subunit genes are tightly linked on mouse chromosome 11 by analysing the strain distribution patterns of RFLPs for the two genes and microsatellite markers flanking these genes in 26 BXD recombinant inbred strains. These results further demonstrate clustering of the GABAA receptor subunit genes on mouse chromosomes and the synteny for these clusters between the mouse and human genomes.

2'-Substitution of cocaine selectively enhances dopamine and norepinephrine transporter binding.

Few studies have characterized the effect of substituents at the 2'-position of cocaine on transporter binding potency and selectivity. We synthesized 2'-OH-, 2'-F- and 2'-acetoxy-cocaines and compared their binding potencies for rat dopamine, norepinephrine and 5-hydroxytryptamine transporters to cocaine, 3'-OH-, 4'-OH-, 2'-OH,4'-I-cocaine derivatives, and to the transporter selective ligands WIN 35,428, nisoxetine and paroxetine. Unlike most substitutions, 2'-OH- and 2'-acetoxy-groups increased cocaine's binding potency for the dopamine transporter (10- and 4-fold, respectively). These substituents also enhanced binding to the norepinephrine transporter (52- and 35-fold, respectively) but had less effect on 5-hydroxytryptamine transporter binding. 2'-Hydroxylation also enhanced binding of 4'-I cocaine, an analog with low DA binding potency. The ability of 2'-substituents to substantially increase cocaine binding potency and to alter selectivity for brain transporters indicates the potential importance of the 2'-position in transporter binding.

Inherited, selective hyporesponsiveness to the analgesic action of nicotine in mice.

The acute dose-dependent analgesic activity of nicotine, as measured by the tail-flick assay, differed significantly between CD-1 and CF-1 outbred strains of mice. Differing responsiveness to the tail-flick stimulus did not explain this pharmacological effect. The inherent analgesic hyporesponsiveness of CF-1 mice was pharmacologically selective. Xilocaine and morphine produced an analgesic response of large magnitude in CF-1 mice. Reduced efficacy of nicotine in the CF-1 analgesia assay was not observed in its action on locomotor activity or in the induction of seizures and lethality. These findings have practical significance in identifying the importance of genotype in choice of strain for preclinical pharmacological studies of nicotine-induced analgesia and indicate that genetic analysis may provide a valuable tool for investigating the mechanism underlying the analgesic action of nicotine.

Anxiolytics by ethanol, diazepam and buspirone in a novel murine behavioral assay.

This study extends the pharmacological characterization of a novel quantitative murine behavioral method, the mirrored chamber aversion assay, which appears to be selectively sensitive to anxiolytic agents. Behavioral effects of acute diazepam administration were compared with those of the 5-HT1A anxiolytic buspirone and those of ethanol in C57BL/6J mice. These known anxiolytics produced a dose-dependent reduction in avoidance behavior of large magnitude, as evidenced by statistically significant decreases in latency to enter and increases in time spent in the mirrored chamber. Anxiolytic-like effects of these compounds in the mirrored chamber assay differed from those observed by the elevated plus-maze method. The behavioral effects of the test compounds were not due to alteration of locomotor activity. These findings indicate that the murine mirrored chamber assay responds to several agents known to be anxiolytic in man but differs from the plus-maze in the pharmacological spectrum of the anxiolytics to which it is sensitive.

Selective behavioral alterations on addition of a 4'-phenyl group to cocaine.

We synthesized a cocaine analog in which a phenyl group was added at the para-position of the benzene ring of cocaine. This substitution caused a modest reduction (four-fold compared with cocaine) in binding potency for the primate (Papio) dopamine transporter as judged by displacement of [3H]WIN 35,428 binding from caudate/putamen membranes. Behavioral effects of this structural modification in the mouse were complex and selective, comprising absence of stimulation of locomotor activity, enhanced inhibition of locomotion and reduced lethal potency. Convulsant potency was unaltered. Substituents at the 4'-position of cocaine are important in its actions. Simple changes in the chemical structure of this drug may produce complex and selective changes in its neurochemical and behavioral actions.

Generation of polyclonal catalytic antibodies against cocaine using transition state analogs of cocaine conjugated to diphtheria toxoid.

Six novel transition state analogs (TSAs) of cocaine (10-14 and 17) and one non-cocaine, p-aminophenylphosphonyl ester of cyclohexanol (19), were synthesized and characterized by 1H- and 13C-NMR and FAB-MS. (1R)-ecgonine methyl ester or cyclohexanol were subjected to phenylphosphonylation in the presence of dicyclohexyl carbodiimde (DCC) and 4-N,N-dimethyl aminopyridine (4-DMAP). TSA-IV (10), however, was synthesized from norcocaine which was protected with dibromoethane to yield 4 before acid hydrolysis, esterification and phenylphosphonylation were carried out. TSA-III (11) TSA-I (12) and (19), using various length spacer arms, were coupled with the immunogenic protein, diphtheria toxoid (DT). The TSAs coupled with DT were used to immunize mice and after appropriate boosts their sera were tested for the presence and titer of anti-TSA polyclonal antibodies using ELISA. Preliminary results show that the mice immunized with these TSAs produced high titers of polyclonal catalytic antibodies, except for (19), with the ability to hydrolyze the substrate 125I-4'-iodococaine in an in vitro assay, even in the presence of noncatalytic anti-TSA antibodies.

Establishment of chronic intravenous drug self-administration in the C57BL/6J mouse.

A wide variety of drugs that have significant human abuse potential have been demonstrated to function as positive reinforcers in animals. The present study was designed to characterize a new mouse model of chronic intravenous drug self-administration. Adult male C57BL/6J mice, implanted with external jugular infusion catheters, were given access to response-contingent injections. They did not initiate responding for saline delivery, whereas the C57BL/6J mice initiated morphine, cocaine, methamphetamine and pentobarbital self-administration. Drug-maintained responding was consistently and significantly higher for each compound than for saline responding. In contrast to C57BL/6J mice, DBA/2J mice failed to initiate cocaine self-administration. Thus, chronic intravenous drug self-administration procedures can be adapted to the inbred mouse.

Genetic determinants of susceptibility to the rewarding and other behavioral actions of cocaine.

The role of genotype as a determinant of biologically based inter-individual differences in vulnerability to substance abuse has received little systematic investigation except in the case of alcohol. This report describes the use of an animal model, the inbred mouse, to identify and to characterize variants with inherently altered susceptibilities to the rewarding and other behavioral actions of cocaine. Among a battery of nine inbred strains chosen solely for their genetic diversity, genetic polymorphisms commonly occurred which altered the potency and/or efficacy of cocaine to induce conditioned place preference, oral self-administration, motor activity activation, seizures and lethality. These changes in cocaine sensitivity generally were of a behavior-specific and pharmacodynamic nature. One strain, DBA/2J, found to be markedly hyporesponsive to the rewarding action of cocaine, also was hyporesponsive to the rewarding effects of amphetamine, etonitazene, phencyclidine, caffeine and procaine. We speculate that this strain has an inherent generalized appetitive defect. The frequent occurrence and large magnitude of inherent phenotypic changes in cocaine responsiveness which we have identified among inbred mouse strains now permits an analytical genetic study of processes underlying cocaine-mediated reinforcement.

Issues surrounding the assessment of the genetic determinants of drugs as reinforcing stimuli.

A wide variety of drugs used in medicine and on a non-prescription basis have abuse liability. However, not all psychoactive drugs have demonstrable reinforcing properties either in animal models or in humans. In order to predict the abuse liability of a compound or to understand determinants of the abuse liability, several animal testing procedures have been developed. These procedures include tests for physical dependence, tolerance, disruption of ongoing behaviors, discriminative stimulus properties, and the direct or indirect assessment of the reinforcement. While tests for the production of tolerance and physical dependence may not predict abuse liability, they would significantly modify the abuse risk. This paper will focus on the methods available for the assessment of the reinforcing properties and the advantages of using genetically defined mice to better understand the determinants of the reinforcing properties and to isolate phenotypes that are hyper-responsive and hypo-responsive to the reinforcing properties of drugs under study. Examples of genetically determined differences are provided and areas of inadequate information are discussed.

Latency to enter a mirrored chamber: a novel behavioral assay for anxiolytic agents.

Many animal species exhibit approach-avoidance responses upon the novel placement of a mirror into an individual animal's environment. With a view toward identifying new behavioral measures with qualitatively or quantitatively different responses to anxiolytic agents, we developed a mirrored chamber apparatus for which adult male BALB/cByJ mice showed an extended latency to enter. Administration of diazepam significantly reduced this latency to enter a mirrored chamber in a dosage-dependent manner. The psychomotor stimulant, methylphenidate, had no effect on latency to enter the mirrored chamber at a dose which stimulated locomotor activity to the same extent as diazepam. Thus, the decreased latency to enter the mirrored chamber brought about by diazepam seems unlikely to reflect the motor effects of this benzodiazepine. The potency of diazepam was significantly lower in the mirrored chamber assay than it was on three other measures of exploratory activity--"head-dipping" performance, plus-maze performance and locomotor activity stimulation. The findings of our study indicate that the mirrored chamber method is simple to carry out, nonpunishing, rapid and quantitative and that it possesses pharmacological attributes which distinguish its response to anxiolytics from other assays of exploratory behavior.

Fetal and newborn lambs differ in their cardiopulmonary responsiveness to adenosine agonists.

We investigated cardiorespiratory responses to adenosine analogs in the pre- and postnatal periods in an unanesthetized chronic animal preparation. Heart rate, blood pressure, breathing movements and blood gases were measured in 8 fetal lambs (117-130 days gestation) and 9 newborn lambs (4-30 days). Various doses of analogs were given by intravenous infusion. L-N6-5'-Phenylisopropyladenosine (L-PIA), 5-N-ethyl carboxamidoadenosine (NECA) and cyclohexyladenosine (CHA) were studied. All analogs produced dose-dependent bradycardia and hypotension in the fetus. However, in the newborn, NECA produced a dose-dependent tachycardia, whereas PIA and CHA produced a dose-dependent bradycardia. Fetal breathing movements were interrupted by all the analogs, but they did not produce apnea in the newborn. No changes in blood gases were observed. The actions of the adenosine agonists were blocked by caffeine.

Familial gigantiform cementoma: classification and presentation of a large pedigree.

Very few cases of gigantiform cementoma have been reported, and those associated with a positive family history are especially rare. Confusion exists about the relationship of gigantiform cementoma to florid osseous dysplasia, cementifying fibroma, and diffuse chronic sclerosing osteomyelitis. It has been unclear whether gigantiform cementoma should be accorded the status of a separate entity. In this article, we report our findings on a family that, over five generations, has exhibited clinical, radiographic, and/or histologic findings consistent with the designation familial gigantiform cementoma. This pedigree consists of 55 members. Significant heterogeneity in expression of this trait was noted. The pattern of occurrence of the trait is consistent with an autosomal dominant mode of inheritance with variable expressivity of the phenotype. We suggest that familial gigantiform cementoma should be recognized as a separate entity.

Effects of caffeine, cocaine and their combination on fixed-interval behavior in rats.

The effects of the central nervous system stimulants, caffeine and cocaine, on schedule-controlled behavior were determined in rats trained to perform a fixed-interval (FI) 5-minute task. When given alone caffeine produced a doubling of FI response rate at a dose of 10 mg/kg and reduced responding at a dose of 32 mg/kg. Cocaine, which was also expected to increase FI responding, did not increase response rate at doses of 3.2 or 10 mg/kg and decreased the rate of responding at a dose of 32 mg/kg. Caffeine had minimal effects on quarter life and appeared to increase local rates of responding across the interval. Cocaine decreased quarter life dramatically at a dose that had no effect on overall response rate. Local rates of responding were increased early in the interval and decreased in the later segments. The effects of both drugs were found to be rate-dependent. When these compounds were given in combination the results obtained appeared to be related to the rate of responding that caffeine alone would produce.