Modification of NO-cGMP Pathway Differentially Affects Diazepam- and Flunitrazepam-Induced Spatial and Recognition Memory Impairments in Rodents.

This study investigated the influence of sildenafil and methylene blue (MB), two modulators of the nitric oxide (NO)-cyclic guanosine-3',5'-monophosphate (cGMP) pathway on amnesic effects of two benzodiazepines (BZs) (diazepam (DZ) and flunitrazepam (FNZ)), in rodents-mice and rats. In the modified elevated plus maze (mEPM) and novel object recognition (NOR) tests, MB given ip at a dose of 5 mg/kg 5 min prior to DZ administration (0.25 or 1 mg/kg, sc) enhanced/induced memory impairment caused by DZ. When MB (2.5, 5, and 10 mg/kg) was applied 5 min prior to FNZ administration (0.05 and 0.1 mg/kg), an effect was opposite and memory impairment induced by FNZ was reduced. When sildenafil (2.5 and 5 mg/kg, ip) was applied 5 min prior to DZ, we observed a reduction of DZ-induced memory deficiency in the mEPM test. A similar effect of sildenafil was shown in the NOR test when the drug was applied at doses of 1.25, 2.5, and 5 mg/kg prior to DZ. In the mEPM test, sildenafil at abovementioned doses had no effects on FNZ-induced memory impairment. In turns, sildenafil administered at doses of 2.5 and 5 mg/kg increased the effect of FNZ on memory impairment in the NOR test. In conclusion, the NO-cGMP pathway is involved differentially into BZs-induced spatial and recognition memory impairments assessed using the NOR and mEPM tests. Modulators of the NO-cGMP pathway affect animal behavior in these tests in a different way depending on what benzodiazepine is applied.

Flumazenil-insensitive benzodiazepine binding sites in GABAA receptors contribute to benzodiazepine-induced immobility in zebrafish larvae.

AIMS: Benzodiazepines (BZDs) produce various pharmacological actions by binding to and allosterically regulating GABAA receptors. Several in vitro studies have demonstrated diazepam, the prototypic BZD, produces a high-dose action that cannot be countered with the classical BZD-binding site antagonist flumazenil. Here, we investigate the existence and behavioral relevance of non-classical BZD binding sites in zebrafish larvae. MAIN METHODS: Zebrafish larvae were treated with a series of BZDs alone or combined with flumazenil, bicuculline (a non-selective GABAA receptor antagonist), or RO 15-4513 (a general BZD antagonist and a proposed antagonist interacting with α+/ß- interfaces in α4/6/ß3δ receptors), and their locomotor activities and behavioral phenotypes were recorded. KEY FINDINGS: Diazepam-induced hypolocomotion (sedation-like state) at low doses (10 and 20 mg L-1) was effectively antagonized by flumazenil or bicuculline, while diazepam-induced immobility (anesthesia-like state) at higher dose (30 mg L-1) was prevented by bicuculline (3 mg L-1) but not flumazenil, even at doses up to 150 mg L-1. Ro 15-4513 also failed to efficiently antagonize diazepam-induced immobility. Immobility induced by high dose of another 1,4-BZD, clonazepam, was also resistant to flumazenil. SIGNIFICANCE: These results provide direct in vivo evidence for non-classical BZD-binding sites, which may be located at the second transmembrane domain of GABAA receptors and contribute to BZD-induced anesthesia.

HPLC-based activity profiling for pharmacologically and toxicologically relevant natural products - principles and recent examples.

CONTEXT: Discovery of pharmacologically active natural products as starting points for drug development remains important and, for reasons of consumer safety, the identification of toxicologically relevant compounds in herbal drugs. OBJECTIVE: To explain, with the aid of relevant examples from our own research, how these goals can be achieved. METHODS: An in-house technology platform comprising pre-formatted extract libraries in 96-well format, miniaturized tracking of activity in extracts via HPLC-activity profiling, structure elucidation with microprobe NMR, and in vitro and in vivo pharmacological methods were used. RESULTS: Piperine was identified as a new scaffold for allosteric GABAA receptor modulators with in vivo activity that interacts at a benzodiazepine-independent binding site. Selectivity and potency were improved by iterative optimization towards synthetic piperine analogues. Dehydroevodiamine and hortiamine from the traditional Chinese herbal drug Evodiae fructus were identified as potent hERG channel blockers in vitro. The compounds induced torsades de pointes arrhythmia in animal models. CONCLUSIONS: The allosteric binding site for piperine analogues remains to be characterized and cardiac risks of herbal drugs need to be further evaluated to ensure consumer safety.

Neurosteroid replacement therapy using the allopregnanolone-analogue ganaxolone following preterm birth in male guinea pigs.

BACKGROUND: Children born preterm, especially boys, are at increased risk of developing attention deficit hyperactivity disorder (ADHD) and learning difficulties. We propose that neurosteroid-replacement therapy with ganaxolone (GNX) following preterm birth may mitigate preterm-associated neurodevelopmental impairment. METHODS: Time-mated sows were delivered preterm (d62) or at term (d69). Male preterm pups were randomized to ganaxolone (Prem-GNX; 2.5 mg/kg subcutaneously twice daily until term equivalence), or preterm control (Prem-CON). Surviving male juvenile pups underwent behavioural testing at d25-corrected postnatal age (CPNA). Brain tissue was collected at CPNA28 and mature myelinating oligodendrocytes of the hippocampus and subcortical white matter were quantified by immunostaining of myelin basic protein (MBP). RESULTS: Ganaxolone treatment returned the hyperactive behavioural phenotype of preterm-born juvenile males to a term-born phenotype. Deficits in MBP immunostaining of the preterm hippocampus and subcortical white matter were also ameliorated in animals receiving ganaxolone. However, during the treatment period weight gain was poor, and pups were sedated, ultimately increasing the neonatal mortality rate. CONCLUSION: Ganaxolone improved neurobehavioural outcomes in males suggesting that neonatal treatment may be an option for reducing preterm-associated neurodevelopmental impairment. However, dosing studies are required to reduce the burden of unwanted side effects.

Design, Synthesis, and Docking Study of Pyrimidine-Triazine Hybrids for GABA Estimation in Animal Epilepsy Models.

A series of new pyrimidine-triazine hybrids (4a-t) was designed and synthesized, from which potent anticonvulsant agents were identified. Most of the compounds exhibited promising anticonvulsant activity against the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests, along with minimal motor impairment with higher safety compared to the standard drugs, phenytoin and carbamazepine. In the series, 5-(4-(4-fluorophenyl)-6-(4-hydroxyphenyl)-2-thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-3(6H)-one (4o) and 5-(6-(4-hydroxy-3-methoxyphenyl)-4-(4-hydroxyphenyl)-2-thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-3(6H)-one (4s) emerged as most potent anticonvulsant agents with median doses of 22.54 and 29.40 mg/kg (MES ED50 ), 285.02 and 293.42 mg/kg (scPTZ ED50 ), and 389.11 and 412.16 mg/kg (TD50 ), respectively. Docking studies were also performed for all synthesized compounds to get insight into the binding pattern toward the GABAA receptor as a possible mechanism of their anticonvulsant action, and in silico ADME studies were carried out to predict the safety and stability of the molecules. The increased GABA level in the experimental animals in the neurochemical estimation assay confirmed their GABAergic modulating activity. The most potent compounds were also evaluated for their neurotoxic and hepatotoxic effects. Fortunately, they did not show any sign of neurotoxicity or hepatotoxicity, suggesting that they have a broad spectrum of anticonvulsant activity with a large safety margin. Together, this research suggested that 4o and 4s may serve as leads in the discovery and development of new anticonvulsant drugs.

Disruption of brain MEK-ERK sequential phosphorylation and activation during midazolam-induced hypnosis in mice: Roles of GABAA receptor, MEK1 inactivation, and phosphatase MKP-3.

Midazolam is a positive allosteric modulator at GABAA receptor that induces a short hypnosis and neuroplasticity, in which the sequential phosphorylation of MEK1/2 and ERK1/2 was shown to play a role. This study investigated the parallel activation of p-MEK and p-ERK and regulatory mechanisms induced by midazolam through the stimulation of GABAA receptors in the mouse brain. During the time course of midazolam (60mg/kg)-induced sleep in mice (lasting for about 2h) p-Ser217/221 MEK1/2 was increased (+146% to +258%) whereas, unexpectedly, p-Tyr204/Thr202 ERK1/2 was found decreased (-16% to -38%), revealing uncoupling of MEK to ERK signals in various brain regions. Midazolam-induced p-MEK1/2 upregulation was prevented by pretreatment (30min) with flumazenil (10mg/kg), indicating the involvement of GABAA receptors. Also unexpectedly, midazolam-induced p-ERK1/2 downregulation was not prevented by flumazenil (10 or 30mg/kg). Notably, during midazolam-induced sleep the content of inactivated p-Thr286 MEK1, which can dampen ERK1/2 activation, was increased (+33% to +149%) through a mechanism sensitive to flumazenil (10mg/kg). Midazolam also increased MKP-3 (+13% to +73%) content and this upregulation was prevented by flumazenil (10mg/kg); an effect suggesting ERK inactivation because MKP-3 is the phosphatase selective for ERK1/2 dephosphorylation. The results indicate that during midazolam-induced sleep in mice there is an uncoupling of p-MEK (increased) to p-ERK (decreased) signals. p-ERK1/2 downregulation (not involving GABAA receptors) is the result of increased inactivated MEK1 and phosphatase MKP-3 (both effects involving GABAA receptors). These findings are relevant for the neurobiology and clinical use of benzodiazepines.

Design, Synthesis and Evaluation of the Antidepressant and Anticonvulsant Activities of Triazole-Containing Benzo[d]oxazoles.

BACKGROUND: Epilepsy and depression are two of the common diseases seriously threatening life and health of human. A shared neurobiological substrate led to the bidirectional relationship and high comorbid occurrence of the two disorders. Recently, an increasing number of patients with epilepsy (PWE) require some form of antidepressant medication. However, most of the available antidepressants are inadequate for PWE for some reasons. So, the search for novel and increasingly effective drugs with anticonvulsant and antidepressant activities is necessary. METHODS: A series of 2-substituted-6-(4H-1,2,4-triazol-4-yl)benzo[d]oxazoles (5a-p) were designed and synthesized. Their anticonvulsant activities were evaluated using maximal electroshock shock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure models in mice. Their antidepressant activities were screened with the forced swimming test (FST). RESULTS: All the compounds showed anti-MES activities in different degree, among which 5g and 5j were the most promising one with ED50 value of 31.7 and 12.7 mg/kg, respectively. What's more, 5g and 5j also exhibited nice anti-scPTZ activities and low neurotoxicity. Interestingly, these compounds also showed good antidepressant activities in FST. And the efficacy of 5g were also confirmed by a tail suspension test and a open field test. The pretreatment of thiosemicarbazide (an inhibitor of γ- aminobutyric acid synthesis enzyme) significantly increased the ED50 of 5g in MES and reversed the reductions in the immobility time of 5g in FST. CONCLUSION: Triazole-containing benzo[d]oxazole is a good skeleton to develop compounds with both anticonvulsant and antidepressant activities. We have got the compound 5g, which display remarkable antidepressant and anticonvulsant activities, and the GABAergic system was involved in the action mechanism of 5g.

Bioconcentration and endocrine disruption effects of diazepam in channel catfish, Ictalurus punctatus.

Recently, the detection of pharmaceuticals in surface waters has increased worldwide. Pharmaceuticals are typically found in the environment at concentrations well below therapeutic levels in humans; however, their mechanisms of action may be largely unknown in non-target organisms, such as teleost species. Thus, chronic exposure to these types of compounds warrants further investigation. The goal of this study was to examine the potential for diazepam, a model benzodiazepine drug, to bioconcentrate in tissues of channel catfish and to examine its ability to interact with the endocrine system through modulation of steroid hormones and/or steroidogenic genes. To investigate the bioconcentration potential of diazepam, channel catfish (Ictalurus punctatus) were exposed to 1 ng/mL diazepam for seven days, followed by clean water for another seven days, using an abbreviated OECD 305 Fish Bioconcentration Test study design. This concentration of diazepam is well below environmentally relevant concentrations of diazepam (ng/L). To evaluate steroidogenic effects, fish were exposed to 1 ng/mL diazepam for seven days only. Steroid hormone concentrations were analyzed for various tissues, as well as expression of selected steroidogenic genes. Calculated bioconcentration factors for diazepam were well below regulatory threshold values in all tissues analyzed. No changes in steroid hormone concentration were detected in any tissue analyzed; however, the steroidogenic gene cytochrome P450 side chain cleavage (P450scc) was significantly down-regulated at day 5 and 3ß-hydroxy steroid dehydrogenase (3ß-HSD) was significantly down-regulated at day 7 in the gonad. These results indicate that although diazepam does not significantly bioconcentrate, low-level chronic exposure to diazepam may have the potential to interact with endocrine function by altering gene expression.

Benzodiazepine-induced spatial learning deficits in rats are regulated by the degree of modulation of α1 GABA(A) receptors.

Despite significant advances in understanding the role of benzodiazepine (BZ)-sensitive populations of GABAA receptors, containing the α1, α2, α3 or α5 subunit, factual substrates of BZ-induced learning and memory deficits are not yet fully elucidated. It was shown that α1-subunit affinity-selective antagonist ß-CCt almost completely abolished spatial learning deficits induced by diazepam (DZP) in the Morris water maze. We examined a novel, highly (105 fold) α1-subunit selective ligand-WYS8 (0.2, 1 and 10 mg/kg), on its own and in combination with the non-selective agonist DZP (2 mg/kg) or ß-CCt (5 mg/kg) in the water maze in rats. The in vitro efficacy study revealed that WYS8 acts as α1-subtype selective weak partial positive modulator (40% potentiation at 100nM). Measurement of concentrations of WYS8 and DZP in rat serum and brain tissues suggested that they did not substantially cross-influence the respective disposition. In the water maze, DZP impaired spatial learning (acquisition trials) and memory (probe trial). WYS8 caused no effect per se, did not affect the overall influence of DZP on the water-maze performance and was devoid of any activity in this task when combined with ß-CCt. Nonetheless, an additional analysis of the latency to reach the platform and the total distance swam suggested that WYS8 addition attenuated the run-down of the spatial impairment induced by DZP at the end of acquisition trials. These results demonstrate a clear difference in the influence of an α1 subtype-selective antagonist and a partial agonist on the effects of DZP on the water-maze acquisition.

Long-lasting effects of neonatal pentobarbital administration on spatial learning and hippocampal synaptic plasticity.

Exposure of newborn rats to antiepileptics such as barbiturates has long-lasting detrimental effects on the hippocampus and hippocampus-dependent behavior. However, the long-term consequences of neonatal administration with barbiturates on the hippocampal synaptic plasticity remain unresolved. In this study, we investigated the long-lasting effects of a neonatal administration of pentobarbital on spatial memory, paired-pulse plasticity in the population spikes, and long-term potentiation (LTP) in the hippocampal CA1 region of rats in vivo. Eight weeks after administration of pentobarbital (10 or 20mg/kg) on the seventh postnatal day (P7), rats showed impaired induction in LTP. During paired-pulse stimulation, pentobarbital-treated rats exhibited a greater facilitation of the test pulse population spike, suggesting a disruption in the inhibitory GABAergic synaptic transmission. Spatial learning in hidden platform task of the Morris water maze was impaired in pentobarbital-treated rats. Our present findings indicate that neonatal treatment with pentobarbital causes alterations in function of the hippocampal inhibitory synaptic transmission that persist into adulthood, likely contributing to the long-lasting abnormalities in the hippocampal LTP as well as learning ability. We also demonstrated significant respiratory disturbances, i.e., severe hypoxia, hypercapnia, and extracellular acidosis, in rats treated with pentobarbital on P7. Given that extracellular acidosis can also modulate synaptic transmission in the developing hippocampus, this finding led us to speculate regarding the influence of respiratory disturbances in pentobarbital-induced long-lasting hippocampal dysfunctions.

Scopolamine- and diazepam-induced amnesia are blocked by systemic and intraseptal administration of substance P and choline chloride.

Systemic (IP) and/or intraseptal (IS) administration of scopolamine (SCP) and diazepam (DZP) induce amnesia, whereas IP injection of the neuropeptide substance P (SP) and choline chloride (ChCl) produce memory facilitation. The septohippocampal cholinergic system has been pointed out as a possible site of SCP and DZP-induced amnesia as well as for the mnemonic effects induced by SP and ChCl. We performed a series of experiments in order to investigate the interactions between cholinergic and GABA/benzodiazepine (GABA/BZD) systems with the SPergic system on inhibitory avoidance retention. Male Wistar rats were trained and tested in a step-down inhibitory avoidance task (1.0 mA footshock). Animals received, pre-training, IP (1.0 mg/kg) or IS (1.0 nM/0.5 microl) injection of DZP, SCP (SCP; 1.0 mg/kg - IP or 0.5 microM/0.5 microl--IS) or vehicle (VEH). Immediately after training they received an IP or IS injections of SP 1-11 (50 microg/kg--IP or 1.0 nM/0.5 microl--IS), SP 1-7 (167 microg/kg--IP or 1.0 nM/0.5 microl--IS), ChCl (20 mg/kg--IP or 0.3 microM/0.5 microl--IS) or VEH. Rats pretreated with SCP and DZP showed amnesia. Post-trial treatments with SP 1-11, SP 1-7 or ChCl blocked the amnesic effects of SCP and DZP. These findings suggest an interaction between SPergic and cholinergic mechanisms with GABAergic systems in the modulation of inhibitory avoidance retention and that the effects of these treatments are mediated, at least in part, by interactions in the septohippocampal pathway.

Relations between open-field, elevated plus-maze, and emergence tests in C57BL/6J and BALB/c mice injected with GABA- and 5HT-anxiolytic agents.

Two 5HT(1A) receptor agonists and chlordiazepoxide were examined in open-field, elevated plus maze, and emergence tests. At doses with no effect in the open-field, chlordiazepoxide increased open and open/total arm visits as well as open arm duration in the elevated plus maze, whereas 5HT(1A) receptor agonists showed an anxiolytic response on a single measure. The anxiolytic action of chlordiazepoxide was limited to the less active BALB/c strain. Unlike the 5HT(1A) receptor agonists, chlordiazepoxide was also anxiolytic in the emergence test, once again only in BALB/c and not C57BL/6J mice. Significant correlations were found between emergence latencies and specific indicators of anxiety in the elevated plus-maze in chlordiazepoxide-treated but not in mice treated with buspirone and 8-OH-DPAT. These results indicate that elevated plus-maze and emergence tests depend on benzodiazepine receptors. In contrast, 5HT(1A) receptor agonists were ineffective in the emergence test and no correlation was found between emergence latencies and specific indicators of anxiety in the elevated plus-maze. Though superficially similar, the emergence test seems to tap into a partially separate facet of anxiety.

Buprenorphine alters desmethylflunitrazepam disposition and flunitrazepam toxicity in rats.

High-dosage buprenorphine (BUP) consumed concomitantly with benzodiazepines (BZDs) including flunitrazepam (FZ) may cause life-threatening respiratory depression despite a BUP ceiling effect and BZDs' limited effects on ventilation. However, the mechanism of BUP/FZ interaction remains unknown. We hypothesized that BUP may alter the disposition of FZ active metabolites in vivo, contributing to respiratory toxicity. Plasma FZ, desmethylflunitrazepam (DMFZ), and 7-aminoflunitrazepam (7-AFZ) concentrations were measured using gas chromatography-mass spectrometry. Intravenous BUP 30 mg/kg pretreatment did not alter plasma FZ and 7-AFZ kinetics in Sprague-Dawley rats infused with 40 mg/kg FZ over 30 min, whereas resulting in a three-fold increase in the area under the curve (AUC) of DMFZ concentrations compared with control (p < 0.01). In contrast, BUP did not significantly modify plasma DMFZ concentrations after intravenous infusion of 7 mg/kg DMFZ, whereas resulting in a similar peak concentration to that generated from 40 mg/kg FZ administration. Regarding the effects on ventilation, BUP (30 mg/kg) as well as its combination with FZ (0.3 mg/kg) significantly increased PaCO(2), whereas only BUP/FZ combination decreased PaO(2) (p < 0.001). Interestingly, FZ (40 mg/kg) but not DMFZ (40 mg/kg) significantly increased PaCO(2) (p < 0.05), whereas DMFZ but not FZ decreased PaO(2) (p < 0.05). Thus, decrease in PaO(2) appears related to BUP-mediated effects on DMFZ disposition, although increases in PaCO(2) relate to direct BUP/FZ additive or synergistic dynamic interactions. We conclude that combined high-dosage BUP and FZ is responsible for increased respiratory toxicity in which BUP-mediated alteration in DMFZ disposition may play a significant role.

Prenatal diazepam exposure alters respiratory control system and GABAA and adenosine receptor gene expression in newborn rats.

In experimental animals, prenatal diazepam exposure has clearly been associated with behavioral disturbances. Its impact on newborn breathing has not been documented despite potential deleterious consequences for later brain development. We addressed this issue in neonatal rats (0-2 d) born from dams, which consumed 2 mg/kg/d diazepam via drinking fluid throughout gestation. In vivo, prenatal diazepam exposure significantly altered the normoxic-breathing pattern, lowering breathing frequency (105 vs. 125 breaths/min) and increasing tidal volume (16.2 vs. 12.7 mL/kg), and the ventilatory response to hypoxia, inducing an immediate and marked decrease in tidal volume (-30%) absent in controls. In vitro, prenatal diazepam exposure significantly increased the respiratory-like frequency produced by pontomedullary and medullary preparations (+38% and +19%, respectively) and altered the respiratory-like response to application of nonoxygenated superfusate. Both in vivo and in vitro, the recovery from oxygen deprivation challenges was delayed by prenatal diazepam exposure. Finally, real-time PCR showed that prenatal diazepam exposure affected mRNA levels of alpha1 and alpha2 GABAA receptor subunits and of A1 and A2A adenosine receptors in the brainstem. These mRNA changes, which are region-specific, suggest that prenatal diazepam exposure interferes with developmental events whose impact on the respiratory system maturation deserves further studies.

Clinical and pathologic features of cynomolgus macaques (Macaca fascicularis) infected with aerosolized Yersinia pestis.

Since the anthrax attacks of 2001, the emphasis on developing animal models of aerosolized select agent pathogens has increased. Many scientists believe that nonhuman primate models are the most appropriate to evaluate pulmonary response to, vaccines for, and treatments for select agents such as Yersinia pestis (Y. pestis), the causative agent of plague. A recent symposium concluded that the cynomolgus macaque (Macaca fascicularis) plague model should be characterized more fully. To date, a well-characterized cynomolgus macaque model of pneumonic plague using reproducible bioaerosols of viable Y. pestis has not been published. In the current study, methods for creating reproducible bioaerosols of viable Y. pestis strain CO92 (YpCO92) and pneumonic plague models were evaluated in 22 Indonesian-origin cynomolgus macaques. Five macaques exposed to doses lower than 250 CFU remained free of any indication of plague infection. Fifteen macaques developed fever, lethargy, and anorexia indicative of clinical plague. The 2 remaining macaques died without overt clinical signs but were plague-positive on culture and demonstrated pathology consistent with plague. The lethal dose of plague in humans is reputedly less than 100 organisms; in this study, 66 CFU was the dose at which half of the macaques developed fever and clinical signs (ED(50)), The Indonesian cynomolgus macaque reproduces many aspects of human pneumonic plague and likely will provide an excellent model for studies that require a macaque model.

The effect of chronic lorazepam administration in aging mice.

To assess benzodiazepine tolerance in aged animals, lorazepam or vehicle was administered chronically to male Crl: CD-1(ICR)BR mice. Pharmacodynamic and neurochemical endpoints were examined on days 1 and 14 of drug administration. There was no age-related significant difference in plasma lorazepam levels. Young and middle-aged animals demonstrated behavioral tolerance to lorazepam, while the aged animals showed a similar trend which failed to reach significance. In addition, aged animals also showed a trend toward tolerance to the anticonvulsant effects of lorazepam. There were no changes in alpha1 mRNA levels in cortex or hippocampus following administration of lorazepam when compared to vehicle-treated animals in any age group. Aged animals, however, had an initial increase in alpha1 mRNA expression in cortex and hippocampus on day 1 of vehicle treatment followed by decreased expression on day 14. These age-related changes were abolished by lorazepam administration. In summary, age-related sensitivity to the effects of lorazepam was not demonstrated in the present study. However, comparison of these data to other studies indicates that the effect of chronic benzodiazepine treatment may be specific to the benzodiazepine administered, the technique used to quantify mRNA expression changes, the subunits of the GABA(A) receptor investigated and the brain region analyzed. The phenomenon of benzodiazepine sensitivity in the elderly is an area of research which remains controversial and may well be compound specific. Determining benzodiazepines that do not produce pharmacodynamic sensitivity, such as lorazepam, may allow more careful prescribing and dosing of these drugs, and perhaps even the development of specific agents which could avoid this sensitivity.

Individual differences in aggressive responding to intravenous flumazenil administration in adult male parolees.

Nonhuman and human studies have shown that benzodiazepine (BZD) receptor agonists can modify aggressive behaviour. However, it is unknown whether flumazenil, a BZD receptor antagonist, enhances or inhibits aggressive behaviour. The present study was designed to investigate the effects of acute administrations of flumazenil on aggressive responding in adult humans. Six adult males with histories of childhood conduct disorder (DSM IV R) participated in experimental sessions. Aggression was measured using the Point Subtraction Aggression Paradigm (PSAP; Cherek 1992), which provided subjects with aggressive and monetary-reinforced response options. Acute doses of flumazenil (2 and 3mg) did not produce statistically significant changes in either monetary-reinforced responding or aggressive responding. The analysis of individual subjects data revealed that aggressive responses varied across subjects. The results are discussed in terms of individual differences based on the previous history of BZD abuse. Additional laboratory research is needed to better clarify the behavioural mechanisms by which BZD receptor antagonists modify human aggressive responding.

Potential of ketamine and midazolam, individually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain.

Recently, it was reported that anesthetizing infant rats for 6 h with a combination of anesthetic drugs (midazolam, nitrous oxide, isoflurane) caused widespread apoptotic neurodegeneration in the developing brain, followed by lifelong cognitive deficits. It has also been reported that ketamine triggers neuroapoptosis in the infant rat brain if administered repeatedly over a period of 9 h. The question arises whether less extreme exposure to anesthetic drugs can also trigger neuroapoptosis in the developing brain. To address this question we administered ketamine, midazolam or ketamine plus midazolam subcutaneously at various doses to infant mice and evaluated the rate of neuroapoptosis in various brain regions following either saline or these various drug treatments. Each drug was administered as a single one-time injection in a dose range that would be considered subanesthetic, and the brains were evaluated by unbiased stereology methods 5 h following drug treatment. Neuroapoptosis was detected by immunohistochemical staining for activated caspase-3. It was found that either ketamine or midazolam caused a dose-dependent, statistically significant increase in the rate of neuroapoptosis, and the two drugs combined caused a greater increase than either drug alone. The apoptotic nature of the neurodegenerative reaction was confirmed by electron microscopy. We conclude that relatively mild exposure to ketamine, midazolam or a combination of these drugs can trigger apoptotic neurodegeneration in the developing mouse brain.

Reversal of prenatal diazepam-induced deficit in a spatial-object learning task by brief, periodic maternal separation in adult rats.

In the rat, prenatal exposure to diazepam (DZ) induces a permanent reduction in GABA/BZ receptor (R) function and behavioural abnormalities. Environmental modifications during early stages of life can influence brain development and induce neurobiological and behavioural changes throughout adulthood. Indeed, a subtle, periodic, postnatal manipulation increases GABA/BZ R activity and produces facilitatory effects on neuroendocrine and behavioural responses. We here investigated the impact of prenatal treatment with DZ on learning performance in adult 3- and 8-month-old male rats and the influence of a brief, periodic maternal separation on the effects exerted by prenatal DZ exposure. Learning performance was examined employing a non-aversive spatial, visual and/or tactile task, the "Can test". Behavioural reactivity, emotional state and fear/anxiety-driven behaviour were also examined using open field (OF), acoustic startle reflex (ASR) and elevated plus-maze (EPM) tests. A single daily injection of DZ (1.5mg/kg, s.c.), over gestational days (GD) 14-20, induced, in an age-independent manner, a severe deficit in learning performance, a decrease in locomotor and explorative activity and an increase in peak amplitude in the ASR. Furthermore, anxiety-driven behaviour in EPM was disrupted. Daily maternal separation for 15 min over postnatal days 2-21 exerted opposite effects in all the paradigms examined. Prenatally DZ-exposed maternal separated rats, in contrast to respective non-separated rats, showed an improvement in learning performance, a decrease in emotionality and a normalization of the exploratory behaviour in EPM. These results suggest that a greater maternal care, induced by separation, can serve as a source for the developing brain to enhance neuronal plasticity and to prevent the behavioural abnormalities induced by prenatal DZ exposure.