Bojan Batinić

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.

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.

Magnesium Supplementation Diminishes Peripheral Blood Lymphocyte DNA Oxidative Damage in Athletes and Sedentary Young Man

Sedentary lifestyle is highly associated with increased risk of cardiovascular disease, obesity, and type 2 diabetes. It is known that regular physical activity has positive effects on health; however several studies have shown that acute and strenuous exercise can induce oxidative stress and lead to DNA damage. As magnesium is essential in maintaining DNA integrity, the aim of this study was to determine whether four-week-long magnesium supplementation in students with sedentary lifestyle and rugby players could prevent or diminish impairment of DNA. By using the comet assay, our study demonstrated that the number of peripheral blood lymphocytes (PBL) with basal endogenous DNA damage is significantly higher in rugby players compared to students with sedentary lifestyle. On the other hand, magnesium supplementation significantly decreased the number of cells with high DNA damage, in the presence of exogenous H2O2, in PBL from both students and rugby players, and markedly reduced the number of cells with medium DNA damage in rugby players compared to corresponding control nonsupplemented group. Accordingly, the results of our study suggest that four-week-long magnesium supplementation has marked effects in protecting the DNA from oxidative damage in both rugby players and in young men with sedentary lifestyle. Clinical trial is registered at ANZCTR Trial Id: ACTRN12615001237572.

Lipopolysaccharide exposure during late embryogenesis results in diminished locomotor activity and amphetamine response in females and spatial cognition impairment in males in adult, but not adolescent rat offspring.

Numerous basic and epidemiological studies have connected prenatal maternal immune activation with the occurrence of schizophrenia and/or autism. Depending on subtle differences in protocols of the used animal model, a variety of behavioral abnormalities has been reported. This study investigated behavioral differences in Wistar rat offspring of both genders, exposed to the 100 μg/kg per day dose of lipopolysaccharide (LPS) in late embryogenesis (embryonic days 15 and 16), while tested at their adolescent and young adult age (postnatal days 40 and 60, respectively). Immune activation was confirmed by detecting high levels of TNF-α and IL-6 in dam blood withdrawn 2h after the first dose of LPS. The animals were assessed in three consecutive trials of locomotor activity (novelty exploration, response to i.p. saline injection and challenge with 0.5mg/kg amphetamine), Morris water maze and social interaction tests. Overt behavioral dysfunction was perceived in adult rats only, and these changes were gender-distinctive. When compared with control rats, LPS females displayed baseline hypolocomotion and a decreased reactivity to amphetamine, while LPS males exhibited spatial learning (acquisition trials) and memory (probe trial) impairments. Prenatal treatment did not affect the time spent in social interaction. As maternal exposure to LPS in late gestation resulted in behavioral changes in offspring in early adulthood, it may model schizophrenia-like, but not autism-like endophenotypes. However, lack of a potentiated response to amphetamine testified that this model could not mimic positive symptoms, but rather certain traits of cognitive dysfunction and deficit symptoms, in males and females, respectively.

Sh-I-048A, an in vitro non-selective super-agonist at the benzodiazepine site of GABAA receptors: The approximated activation of receptor subtypes may explain behavioral effects

Enormous progress in understanding the role of four populations of benzodiazepine-sensitive GABAA receptors was paralleled by the puzzling findings suggesting that substantial separation of behavioral effects may be accomplished by apparently non-selective modulators. We report on SH-I-048A, a newly synthesized chiral positive modulator of GABAA receptors characterized by exceptional subnanomolar affinity, high efficacy and non-selectivity. Its influence on behavior was assessed in Wistar rats and contrasted to that obtained with 2mg/kg diazepam. SH-I-048A reached micromolar concentrations in brain tissue, while the unbound fraction in brain homogenate was around 1.5%. The approximated electrophysiological responses, which estimated free concentrations of SH-I-048A or diazepam are able to elicit, suggested a similarity between the 10mg/kg dose of the novel ligand and 2mg/kg diazepam; however, SH-I-048A was relatively more active at α1- and α5-containing GABAA receptors. Behaviorally, SH-I-048A induced sedative, muscle relaxant and ataxic effects, reversed mechanical hyperalgesia 24h after injury, while it was devoid of clear anxiolytic actions and did not affect water-maze performance. While lack of clear anxiolytic actions may be connected with an enhanced potentiation at α1-containing GABAA receptors, the observed behavior in the rotarod, water maze and peripheral nerve injury tests was possibly affected by its prominent action at receptors containing the α5 subunit. The current results encourage further innovative approaches aimed at linking in vitro and in vivo data in order to help define fine-tuning mechanisms at four sensitive receptor populations that underlie subtle differences in behavioral profiles of benzodiazepine site ligands.

Pituitary-Gonadal, Pituitary-Adrenocortical Hormones and IL-6 Levels Following Long-Term Magnesium Supplementation in Male Students

Abstract Background: Sleep deprivation, malnutrition and lack of physical activity are contemporary stress-related factors present in the student population. Stress activates the HPA and often suppresses the HPG axis, but also influences cytokine synthesis and consequently regulates immune response. Since magnesium deficiency facilitates negative pathophysiological consequences, a reasonable question imposes, wheth er Mg supplementation might correct the adrenal/go - n adal hormone balance and immuno-endocrine function. Methods: Fifteen male students were given 2 × 250 mg Mg for four weeks. Serum levels of FSH, LH, testosterone (T), ACTH and cortisol (C) were measured before and after supplementation and the T/C ratio was calculated. Furthermore, IL-6, red blood cells (RBC), hemoglobin (Hb), white blood cells (WBC) and the WBC differential were measured. Results: Despite no change in the serum level of ACTH, a statistically significant (p<0.05) decrease in the serum cortisol level appeared, accompanied with an IL-6 level reduction (p<0.05) after Mg supplementation. Analysis of the pituitarygonadal axis hormones showed an increasing trend of the FSH level (p=0.087), and a significant increase (p<0.05) in the T/C ratio. An RBC count increase (p<0.001) was found, along with a decrease in the percentage of neutrophils (p< 0.05), and a trend toward a lymphocyte percentage increase. Conclusions: The results suggest that chronic oral magnesium supplementation in male students improves the balance of pituitary-gonadal and pituitary-adrenal hormones and is involved in the regulation of the basal IL-6 level. Kratak sadržaj Uvod: Nedostatak sna, fizičke aktivnosti kao i neadekvatna ishrana su savremeni faktori stresa u studentskoj populaciji. Stres aktivira hormone osovine hipotalamus-hipofiza-nad- bubreg i često suprimira aktivnost osovine hipotalamus- hipofiza-gonade, a takođe utiče i na sintezu citokina, posred- no regulišući imuni odgovor. Kako nedostatak magnezijuma (Mg) može inicirati patofiziološke posledice, postavlja se pitanje da li njegov dodatak ishrani može povoljno uticati na ravnotežu hormona nadbubrežne žlezde i gonada kao i neuro-endokrinu funkciju. Metode: U ovoj studiji petnaest studenata muškog pola dobi- jało je 2 x 250 mg Mg tokom četiri nedelje a nivoi FSH, LH, testosterona (T), ACTH i kortizola (C) mereni su u kivi pre i nakon suplementacije. Takođe, određivani su i nivoi IL-6, eri- trocita, hemoglobina, leukocita kao i leukocitarna formula. Rezultati: Uprkos tome što nije bilo promene u nivou ACTH, naši rezultati su pokazali statistički značajno smanjenje nivoa kortizola (p<0,05), koje je bilo praćeno smanjenjem nivoa IL-6 (p<0,05) nakon dodatka Mg. Analiza hipofizno-gonad- nih hormona pokazala je trend u povećanju FSH (p=0,087) i značajno povećanje T/C količnika. Uočeno je značajno povećanje broja eritrocita (p<0,001), smanjenje procenta neutrofila (p<0,05) i trend povećanja procenta limfocita. Zaključak: Rezultati studije sugerišu da dugotrajni dodatak Mg ishrani studenata doprinosi održavanju ravnoteže hipofizno-gonadnih i hipofizno-adrenokortikalnih hormona i uldjučen je u regulaciju bazalnog nivoa citokina IL-6.

Positive modulation of α5 GABAA receptors in preadolescence prevents reduced locomotor response to amphetamine in adult female but not male rats prenatally exposed to lipopolysaccharide

We previously demonstrated that lipopolysaccharide (LPS) administered intraperitoneally (i.p.) to pregnant Wistar rat dams, at embryonic days 15 and 16 (E15/16), induced a decrease of baseline locomotor activity and diminished reactivity to amphetamine in adult female offspring. In the present study we aimed to assess the duration of LPS‐induced maternal immune activation (MIA) and investigate possible changes in levels of main neurotransmitters in fetal brain during MIA. We hypothesized that the observed behavioral changes may be linked with MIA‐induced disturbance of prenatal GABAergic system development, especially with α5 GABAA receptors (α5GABAARs), expression of which takes place between E14 and E17. Thereafter, we set to investigate if later potentiation of α5GABAARs in offsprings preadolescence (from postnatal day 22–28) could prevent the deficit in locomotor reactivity to amphetamine observed in adulthood, at postnatal day P60. The elevation of IL‐6 in amniotic fluid 6 h after LPS treatment (100 μg/kg, i.p.) at E15 was concurrent with a significant increase of GABA and decrease of glutamate concentration in fetal brain. Moreover, repeated administration of MP‐III‐022, a selective positive allosteric modulator of α5GABAARs, at a dose (2 mg/kg daily, i.p.) derived from a separate pharmacokinetic study, prevented the LPS‐induced decrease in locomotor reactivity to amphetamine (0.5 mg/kg, i.p.) in adult females. These results were not mirrored in the parallel set of experiments with male offspring from LPS‐treated rats. The results suggest that pharmacological potentiation of α5GABAARs activity in preadolescence may ameliorate at least some of adverse consequences of exposure to MIA in utero.

Acth-induced model of depression resistant to tricyclic antidepressants: Neuroendocrine and behavioral changes and influence of long-term magnesium administration

ABSTRACT Magnesium (Mg), is not only a modulator of the glutamatergic NMDA receptors’ affinity, it also prevents HPA axis hyperactivity, thus possibly being implicated in neurobiological features of mood disorders. Further uncovering of molecular mechanisms underlying magnesiums proposed effects is needed due to the recent shift in research of treatment resistant depression (TRD) towards glutamatergic pathways. Here, we applied Mg via drinking water for 28days (50mg/kg/day), in ACTH‐treated rats, an established animal model of depression resistant to tricyclic antidepressants. Using this model in male rats we measured (1) changes in hippocampal neurogenesis and behavioral alterations, (2) adrenal hormones response to acute stress challenge and (3) levels of biometals involved in regulation of monoamines turnover in rat prefrontal cortex. Our results support beneficial behavioral impact of Mg in TRD model together with increased hippocampal neurogenesis and BDNF expression. Furthermore, Mg prevented ACTH‐induced disruption in HPA axis function, by normalizing the levels of plasma ACTH, corticosterone and interleukin‐6, and by increasing the peripheral release of adrenaline, noradrenaline and serotonin after the acute stress challenge. Finally, the influence on copper/zinc ratio suggested probable magnesiums involvement in monoamine turnover in PFC. Our findings provide further insights into the possible pathways implicated in the behavioral modulation effects of Mg, as well as its central and peripheral effects in ACTH‐induced TRD model. Thus, further investigation of molecular signaling related to the glutamatergic transmission and role of Mg, could reveal prospects to novel treatment strategies that could be of particular importance for patients suffering from TRD. HIGHLIGHTSIn ACTH model of treatment‐resistant depression Mg alleviates behavioral changes.HPA axis hyperactivity induced by ACTH was attenuated in Mg‐administered rats.Study suggests involvement of Mg in central and peripheral turnover of monoamines.In ACTH model of treatment‐resistant depression Mg increases neurodevelopment.

Combined use of biocompatible nanoemulsions and solid microneedles to improve transport of a model NSAID across the skin: In vitro and in vivo studies

&NA; This study aimed to investigate the potential of lecithin‐based nanoemulsions costabilized by sucrose esters, with and without skin pretreatment with stainless steel microneedles, to improve delivery of aceclofenac, as a model drug, into/across the skin. The characterization revealed favorable droplet size (about 180 nm), narrow size distribution (<0.15), high surface charge (about −40 mV) and satisfying long‐term stability (one year at 4 ± 1 °C) of the formulation costabilized by sucrose palmitate, demonstrating a similar trend observed for the reference stabilized by widely used lecithin/polysorbate 80 combination. In vitro release/permeation testing and differential stripping on the porcine ear proved the superiority of the sucrose ester‐ over polysorbate‐based nanoemulsion. However, in vitro findings were not fully indicative of the in vivo performances – no significant differences were observed between investigated formulations in pharmacokinetic profile and total amount of aceclofenac deposited in the rat skin 24 h after dosing, simultaneously pointing to delayed aceclofenac delivery into the systemic circulation. In addition, the ratio of plasma concentrations of aceclofenac and its major metabolite in rats, diclofenac, was remarkably changed after topical application of tested nanoemulsions compared to intravenous administration of aceclofenac solution. Finally, skin pretreatment with microneedles improved aceclofenac delivery into/across the rat skin from tested formulations, resulting in 1.4–2.1‐fold increased bioavailability and 1.2–1.7‐fold enhanced level of aceclofenac retained in the skin, as measured 24 h after administration. Moreover, the plasma concentrations of aceclofenac 24 h after application of tested formulations (lecithin/sucrose palmitate vs. lecithin/polysorbate 80) combined with microneedles (173.37 ± 40.50 ng/ml vs. 259.23 ± 73.18 ng/ml) were significantly higher than those obtained through intact skin (105.69 ± 19.53 ng/ml vs. 88.38 ± 14.46 ng/ml). However, obtained results suggest that combination of microneedles and sucrose palmitate‐costabilized nanoemulsion could be useful to attain higher skin concentration, while combination of microneedles with polysorbate 80‐costabilized nanoemulsion could be a preferable option for enhancing drug delivery into the bloodstream.

Attaining in vivo selectivity of positive modulation of α3βγ2 GABAA receptors in rats: A hard task!

It is unclear whether GABAA receptors (GABAARs) that contain the α3-subunit are substantially involved in the anxiolytic effects of benzodiazepines (BDZs). In the present study, we tested YT-III-31, a newer BDZ ligand with functional preference for α3βγ2 GABAARs, in two paradigms of unconditioned anxiety, the open field and elevated plus maze in rats. The effective dose of YT-III-31 (2 mg/kg) displayed a clear anxiolytic-like profile, unhampered by sedative action, in both tests. At a higher dose (10 mg/kg), YT-III-31 induced ataxia in the rotarod and sedation in spontaneous locomotor activity test. The latter effect was preventable by flumazenil and βCCt, the non-selective and α1βγ2 GABAAR affinity-selective antagonist, respectively, demonstrating that sedative properties of YT-III-31, when attained, are mediated by the α1γ2 site. To elucidate the receptor substrate of subtle behavioral differences between YT-III-31 and diazepam, we approximated in vivo receptor potentiation for both ligands, based on estimated unbound concentrations in rat brains. Far different from diazepam, YT-III-31 has significantly lower affinity for the α1γ2 over other BDZ-sensitive sites, and at lower doses (1-2 mg/kg) was devoid of potentiation at α1βγ2 GABAARs. The approximation approach revealed a modest selectivity of YT-III-31 for α3γ2- in comparison to α2γ2 and α5γ2 binding sites, suggesting that its anxiolytic-like activity may not necessarily or predominantly reflect potentiation at α3βγ2 GABAARs. Nonetheless, as the anxiolytic effects are achievable at a dose devoid of any sedative potential, and having favorable safety (cytotoxicity) and metabolic stability profile, YT-III-31 represents a valuable candidate for further translational research.