Marija Milic

The role of α1 and α5 subunit-containing GABAA receptors in motor impairment induced by benzodiazepines in rats.

Benzodiazepines negatively affect motor coordination and balance and produce myorelaxation. The aim of the present study was to examine the extent to which populations of &ggr;-aminobutyric acid A (GABAA) receptors containing &agr;1 and &agr;5 subunits contribute to these motor-impairing effects in rats. We used the nonselective agonist diazepam and the &agr;1-selective agonist zolpidem, as well as nonselective, &agr;1-subunit and &agr;5-subunit-selective antagonists flumazenil, &bgr;CCt, and XLi093, respectively. Ataxia and muscle relaxation were assessed by rotarod and grip strength tests performed 20 min after intraperitoneal treatment. Diazepam (2 mg/kg) induced significant ataxia and muscle relaxation, which were completely prevented by pretreatment with flumazenil (10 mg/kg) and &bgr;CCt (20 mg/kg). XLi093 antagonized the myorelaxant, but not the ataxic actions of diazepam. All three doses of zolpidem (1, 2, and 5 mg/kg) produced ataxia, but only the highest dose (5 mg/kg) significantly decreased the grip strength. These effects of zolpidem were reversed by &bgr;CCt at doses of 5 and 10 mg/kg, respectively. The present study demonstrates that &agr;1 GABAA receptors mediate ataxia and indirectly contribute to myorelaxation in rats, whereas &agr;5 GABAA receptors contribute significantly, although not dominantly, to muscle relaxation but not ataxia.

PWZ-029, AN INVERSE AGONIST SELECTIVE FOR α5 GABAA RECEPTORS, IMPROVES OBJECT RECOGNITION, BUT NOT WATER-MAZE MEMORY IN NORMAL AND SCOPOLAMINE-TREATED RATS

Inverse agonism at the benzodiazepine site of α(5) subunit-containing GABA(A) receptors is an attractive approach for the development of putative cognition-enhancing compounds, which are still far from clinical application. Several ligands with binding and/or functional selectivity for α(5) GABA(A) receptors have been synthesized and tested in a few animal models. PWZ-029 is an α(5) GABA(A) selective inverse agonist whose memory enhancing effects were demonstrated in the passive avoidance task in rats and in Pavlovian fear conditioning in mice. In the present study we investigated the effects of PWZ-029 administration in novel object recognition test and Morris water maze, in normal and scopolamine-treated rats. All the three doses of PWZ-029 (2, 5 and 10 mg/kg) improved object recognition after the 24-h delay period, as shown by significant differences between the exploration times of the novel and old object, and the respective discrimination indices. PWZ-029 (2 mg/kg) also successfully reversed the 0.3 mg/kg scopolamine-induced deficit in recognition memory after the 1-h delay. In the Morris water maze test, PWZ-029 (5, 10 and 15 mg/kg) did not significantly influence swim patterns, either during five acquisition days or during the treatment-free probe trial. PWZ-029 (2, 5 and 10 mg/kg) also proved to be ineffective in the reversal of the 1mg/kg scopolamine-induced memory impairment in the water maze. The present mixed results encourage use of a variety of tests and experimental conditions in order to increase the predictability of preclinical testing of selective α(5) GABA(A) inverse agonists.

Midazolam impairs acquisition and retrieval, but not consolidation of reference memory in the Morris water maze.

Amnesia is one of the most discussed properties of the benzodiazepine class of drugs. The effects of benzodiazepines on human memory are usually anterograde, while changes in retrograde memory functions were seldom reported. Such inconsistent findings have prompted numerous animal studies investigating the influences of these positive modulators of inhibitory neurotransmission on different stages of memory. Among the benzodiazepines, memory effects of midazolam are of special interest due to its many and varied clinical applications. The present Morris water maze study in adult male Wistar rats was performed in three experiments in which midazolam was administered at doses of 0.5, 1 and 2 mg/kg intraperitoneally, before or immediately after each of five daily learning sessions, with two trials in a session, as well as before the probe test. Midazolam impaired acquisition and subsequent retention of spatial learning of the position of the hidden platform even at a pre-training dose of 0.5 mg/kg. This low dose was not associated with impairment of the procedural component of learning, manifested by increased time spent in the periphery of the pool. The lack of midazolam effect on consolidation has not been confounded by the observed below-chance performance of the control group since our additional experiment using diazepam also administered immediately after each of five learning sessions has revealed a similar pattern of results. Finally, midazolam administered before the probe test impaired retrieval of reference memory at all tested doses. Hence, induction of retrograde, besides anterograde amnesia should be kept in mind as a possibility when midazolam is used in clinical settings.

Tolerance liability of diazepam is dependent on the dose used for protracted treatment.

BACKGROUND Behavioral effects of benzodiazepines following repeated exposure vary according to the intrinsic efficacy of the benzodiazepine studied, treatment schedule and the behavioral parameters evaluated. METHODS We applied the behavioral paradigms of spontaneous locomotor activity, elevated plus maze and grip strength to investigate the sedative, anxiolytic and myorelaxant effect of acute challenge with 2 mg/kg diazepam administered after 14 days of protracted treatment with 0.5, 2 or 10 mg/kg of diazepam. In addition, we studied the effects of everyday handling and intraperitoneal (ip) administration on animal behavior. RESULTS Tolerance to the sedative effect of 2 mg/kg diazepam ensued after 14 days of protracted treatment with 2 and 10 mg/kg of diazepam. In contrast, treatment with the lowest dose (0.5 mg/kg) of diazepam resulted in potentiation of the sedative effect of acute challenge with 2 mg/kg diazepam thus confounding the detection of the anxiolytic effect of diazepam. Asensitization-like response to the anxiolytic action of 2 mg/kg diazepam was seen after protracted treatment with the intermediate dose (2 mg/kg); however, anxiolytic effect was absent after protracted administration of the highest dose. Partial tolerance to the muscle relaxant effect of 2 mg/kg diazepam ensued after protracted treatment with diazepam regardless of the dose. Daily handling or ip administration did not alter the behavioral response to acute challenge with 2 mg/kg diazepam in all the three behavioral paradigms studied. CONCLUSION The presented results showed that behavioral effects of acute challenge with diazepam were differently affected by the dose administered during protracted treatment.

Duration of treatment and activation of α1-containing GABAA receptors variably affect the level of anxiety and seizure susceptibility after diazepam withdrawal in rats

Long-term use of benzodiazepine-type drugs may lead to physical dependence, manifested by withdrawal syndrome after abrupt cessation of treatment. The aim of the present study was to investigate the influence of duration of treatment, as well as the role of α1-containing GABAA receptors, in development of physical dependence to diazepam, assessed through the level of anxiety and susceptibility to pentylenetetrazole (PTZ)-induced seizures, 24h after withdrawal from protracted treatment in rats. Withdrawal of 2mg/kg diazepam after 28, but not after 14 or 21 days of administration led to an anxiety-like behavior in the elevated plus maze. Antagonism of the diazepam effects at α1-containing GABAA receptors, achieved by daily administration of the neutral modulator βCCt (5mg/kg), did not affect the anxiety level during withdrawal. An increased susceptibility to PTZ-induced seizures was observed during diazepam withdrawal after 21 and 28 days of treatment. Daily co-administration of βCCt further decreased the PTZ-seizure threshold after 21 days of treatment, whilst it prevented the diazepam withdrawal-elicited decrease of the PTZ threshold after 28 days of treatment. In conclusion, the current study suggests that the role of α1-containing GABAA receptors in mediating the development of physical dependence may vary based on the effect being studied and duration of protracted treatment. Moreover, the present data supports previous findings that the lack of activity at α1-containing GABAA receptors is not sufficient to eliminate physical dependence liability of ligands of the benzodiazepine type.

βCCT, an antagonist selective for α1GABAA receptors, reverses diazepam withdrawal-induced anxiety in rats

The abrupt discontinuation of prolonged benzodiazepine treatment elicits a withdrawal syndrome with increased anxiety as a major symptom. The neural mechanisms underlying benzodiazepine physical dependence are still insufficiently understood. Flumazenil, the non-selective antagonist of the benzodiazepine binding site of GABA(A) receptors was capable of preventing and reversing the increased anxiety during benzodiazepine withdrawal in animals and humans in some, but not all studies. On the other hand, a number of data suggest that GABA(A) receptors containing α(1) subunits are critically involved in processes developing during prolonged use of benzodiazepines, such are tolerance to sedative effects, liability to physical dependence and addiction. Hence, we investigated in the elevated plus maze the level of anxiety 24 h following 21 days of diazepam treatment and the influence of flumazenil or a preferential α(1)-subunit selective antagonist βCCt on diazepam withdrawal syndrome in rats. Abrupt cessation of protracted once-daily intraperitoneal administration of 2 mg/kg diazepam induced a withdrawal syndrome, measured by increased anxiety-like behavior in the elevated plus maze 24 h after treatment cessation. Acute challenge with either flumazenil (10mg/kg) or βCCt (1.25, 5 and 20 mg/kg) alleviated the diazepam withdrawal-induced anxiety. Moreover, both antagonists induced an anxiolytic-like response close, though not identical, to that seen with acute administration of diazepam. These findings imply that the mechanism by which antagonism at GABA(A) receptors may reverse the withdrawal-induced anxiety involves the α(1) subunit and prompt further studies aimed at linking the changes in behavior with possible adaptive changes in subunit expression and function of GABA(A) receptors.

Trends in incidence of non-melanoma and melanoma skin cancers in central Serbia

Online first: January 30, 2018 SUMMARY Introduction/Objective The incidence of both melanoma and non-melanoma skin cancers (NMSC) has been increasing over the past decades worldwide. NMSC is the most common cancer in white population and melanoma is one of the deadliest cancers today. The objective of the paper was to determine trends in age-standardized incidence rates of NMSC and melanoma in central Serbia from 1999 to 2013. Method A descriptive epidemiological study was done. Data about incidence for NMSC and melanoma were obtained from the Serbian Cancer Registry and data about population originating from 1991, 2001, and 2011 censuses. Crude incidence rates were calculated per 100,000 inhabitants. Direct method of standardization was performed with the world population as the standard. Trend lines were estimated using linear regression. Results During a 15-year period, the total number of new NMSC cases was 41,719 [21,690 (52%) in men and 20,029 (48%) in women]. There were 5,781 new cases of melanoma [2,969 (51.4%) in men and 2,812 (48.6%) in women]. A significantly increasing incidence trend for NMSC both in men (y = 0.617x + 24.29, R2 = 0.500) and women (y = 0.672x + 0.670, R2 = 0.670) was determined. In the same period, a statistically significant increase of incidence trend for melanoma was determined in men (y = 0.111x + 3.708, R2 = 0.384) and in women (y = 0.098x + 3.375, R2 = 0.409). NMSC was registered in persons of all ages. NMSC incidence increased rapidly in persons older than 50 years. Melanoma predominates in children and adolescents and is registered more frequently than NMSC in persons bellow 60 years of age. Conclusion Our findings showed significantly increasing trend of age-standardized incidence rates for both NMCC and melanoma. In the observed period, there were 7.2 times more new cases of NMSC than melanoma in the population of central Serbia. There were more registered new cases of NMSC and melanoma in men than in women. Screening of skin cancers and earlier diagnosis may improve treatment and prognosis.

Illict drug use and academia in North Kosovo: Prevalence, patterns, predictors and health-related quality of life

Purpose The purpose of this study were to estimate the prevalence and patterns of illicit drug use in a sample of University students from North Kosovo, to assess factors associated with illicit drug use and to assess health-related quality of life (HRQoL) among students according to illicit drug use. Methods A cross-sectional study was conducted at the Student Public Health Center, where 514 University students were enrolled from April to June 2015 in North Kosovo. Participants completed the general socio-demographic and behavioral questionnaire, Beck Depression Inventory (BDI) and the SF-36 questionnaire for HRQoL assessment. Data on lifetime illicit drug use were self-reported. Results As much as 16.0% of students reported ever illicit drug use. The most frequently used drugs were marijuana (9.3%) and bromazepam (7.6%). Factors associated with ever illicit drug use were: being smoker and alcohol user, having chronic diseases and having higher depressive symptoms score. Ever illicit drug users reported all domains of HRQoL as worse. Conclusion These results could serve as a tool for implementation of preventive strategies and University policies to promote healthy lifestyles and behaviors. Measurement of HRQoL could also be used as indicator of the effect of interventions designed to reduce and/or prevent illicit drug use at institutions of higher education.