Yasco Aracava’s research while affiliated with University of Maryland, Baltimore and other places

Preclinical studies have reported that, compared to the muscarinic receptor (mAChR) antagonist atropine, (R,S)-trihexyphenidyl (THP) more effectively counters the cholinergic crisis, seizures, and neuropathology triggered by organophosphorus (OP)-induced acetylcholinesterase (AChE) inhibition. The greater effectiveness of THP was attributed to its ability to block mAChRs and N-methyl-d-aspartate-type glutamatergic receptors (NMDARs) in the brain. However, THP also inhibits α7 nicotinic receptors (nAChRs). The present study examined whether THP-induced inhibition of mAChRs, α7 nAChRs, and NMDARs is required to suppress glutamatergic synaptic transmission, whose overstimulation sustains OP-induced seizures. In primary hippocampal cultures, THP (1-30 μM) suppressed the frequency of excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) recorded from neurons in nominally Mg2+-free solution. A single sigmoidal function adequately fit the overlapping concentration-response relationships for THP-induced suppression of IPSC and EPSC frequencies yielding an IC50 of 6.3 ± 1.3 μM. Atropine (1 μM), the NMDAR antagonist d,l-2-amino-5-phosphonopentanoic acid (D,L-AP5, 50 μM), and the α7 nAChR antagonist methyllycaconitine (MLA, 10 nM) did not prevent THP-induced inhibition of synaptic transmission. THP (10 μM) did not affect the probability of transmitter release because it had no effect on the frequency of miniature IPSCs and EPSCs recorded in the presence of tetrodotoxin. Additionally, THP had no effect on the amplitudes and decay-time constants of miniature IPSCs and EPSCs; therefore, it did not affect the activity of postsynaptic GABAA and glutamate receptors. This study provides the first demonstration that THP can suppress action potential-dependent synaptic transmission via a mechanism independent of NMDAR, mAChR, and α7 nAChR inhibition.

Ketamine can rapidly alleviate symptoms of treatment-resistant major depression within hours of a single administration. While this finding offers a new and promising trajectory for novel therapeutic development, identifying rapid antidepressant mechanisms of action have proven especially difficult. The goal of the current study was to investigate the cellular actions of (2R,6R)-hydroxynorketamine (HNK) – a metabolite of ketamine that has been shown preclinically to exert rapid antidepressant-like effects without dissociative-like properties and abuse potential. The Wistar Kyoto rat model of treatment-resistant major depression was used to examine the mechanisms by which (2R,6R)-HNK promotes hippocampal-dependent plasticity and behavior. Pharmacological and electrophysiological approaches were used to identify the signaling mechanisms that are required for (2R,6R)-HNK to potentiate hippocampal activity in vitro. Fiber photometry was used during an object displacement task to establish whether (2R,6R)-HNK promotes hippocampal function in vivo. (2R,6R)-HNK promotes Schaffer collateral plasticity through a rapid synapse-selective calcium-mediated increase in evoked glutamate release. Acutely, this presynaptic potentiation involves the rapid induction of cyclic adenosine monophosphate-dependent protein kinase independent of neurotrophic signaling. (2R,6R)-HNK restores Schaffer collateral-dependent plasticity and function in vivo, sustained effects that are mediated by activity-dependent neurotrophic signaling that occlude further potentiation by high frequency stimulation. By applying an in vitro-à-in vivo approach, we establish a cellular mechanism by which (2R,6R)-HNK rapidly potentiates hippocampal activity, which may give rise to the sustained neurotrophic-dependent adaptations in synaptic transmission and behavior. These findings provide a novel mechanistic framework for the development of improved therapeutic options for depression.

Earlier reports suggested that galantamine, a drug approved to treat mild-to-moderate Alzheimer's disease (AD), and other centrally acting reversible acetylcholinesterase (AChE) inhibitors can serve as adjunct pretreatments against poisoning by organophosphorus (OP) compounds, including the nerve agent soman. The present study was designed to determine whether pretreatment with a clinically relevant oral dose of galantamine HBr mitigates the acute toxicity of 4.0xLD50 soman (15.08 µg/kg) in Macaca fascicularis posttreated intramuscularly with the conventional antidotes atropine (0.4 mg/kg), 2-PAM (30 mg/kg), and midazolam (0.32 mg/kg). The pharmacokinetic profile and maximal degree of blood AChE inhibition (~ 25-40%) revealed that the oral doses of 1.5 and 3.0 mg/kg galantamine HBr in these non-human primates (NHPs) translate to human equivalent doses that are within the range used for AD treatment. Subsequent experiments demonstrated that 100% of NHPs pretreated with either dose of galantamine, challenged with soman, and posttreated with conventional antidotes survived 24 h. By contrast, given the same posttreatments, 0% and 40% of the NHPs pretreated, respectively, with vehicle and pyridostigmine bromide (1.2 mg/kg, oral), a peripherally acting reversible AChE inhibitor approved as pretreatment for military personnel at risk of exposure to soman, survived 24 h after the challenge. In addition, soman caused extensive neurodegeneration in the hippocampi of saline- or pyridostigmine-pretreated NHPs, but not in the hippocampi of galantamine-pretreated animals. To our knowledge, this is the first study to demonstrate the effectiveness of clinically relevant oral doses of galantamine to prevent the acute toxicity of supralethal doses of soman in NHPs. SIGNIFICANCE STATEMENT: This is the first study to demonstrate that a clinically relevant oral dose of galantamine effectively prevents lethality and neuropathology induced by a supralethal dose of the nerve agent soman in Cynomolgus monkeys posttreated with conventional antidotes. These findings are of major significance for the continued development of galantamine as an adjunct pretreatment against nerve agent poisoning.

For over three-quarters of a century, organophosphorus (OP) insecticides have been ubiquitously used in agricultural, residential, and commercial settings and in public health programs to mitigate insect borne diseases. Their broad-spectrum insecticidal effectiveness is accounted for by the irreversible inhibition of acetylcholinesterase (AChE), the enzyme that catalyzes acetylcholine (ACh) hydrolysis, in the nervous system of insects. However, because AChE is evolutionarily conserved, OP insecticides are also toxic to mammals, including humans, and acute OP intoxication remains a major public health concern in countries where OP insecticide usage is poorly regulated. Environmental exposures to OP levels that are generally too low to cause marked inhibition of AChE and to trigger acute signs of intoxication, on the other hand, represent an insidious public health issue worldwide. Gestational exposures to OP insecticides are particularly concerning because of the exquisite sensitivity of the developing brain to these insecticides. The present article overviews and discusses: (i) the health effects and therapeutic management of acute OP poisoning during pregnancy, (ii) epidemiological studies examining associations between environmental OP exposures during gestation and health outcomes of offspring, (iii) preclinical evidence that OP insecticides are developmental neurotoxicants, and (iv) potential mechanisms underlying the developmental neurotoxicity of OP insecticides. Understanding how gestational exposures to different levels of OP insecticides affect pregnancy and childhood development is critical to guiding implementation of preventive measures and direct research aimed at identifying effective therapeutic interventions that can limit the negative impact of these exposures on public health.

The present study was designed to evaluate the effectiveness of galantamine administered orally as a pre-treatment to mitigate the acute toxicity of 4.0xLD50 soman in Cynomolgus monkeys post-treated with atropine, 2-PAM, and midazolam. Pharmacokinetic experiments revealed that the oral doses of 1.5 and 3.0 mg/kg galantamine HBr were quickly absorbed and produced plasma concentrations of galantamine that generated approximately 20% to 40% reversible inhibition of blood acetylcholinesterase (AChE) activity. This degree of reversible AChE inhibition has been shown to be safe and sufficient to protect AChE from the irreversible inhibition by nerve agents, and, thereby, suppress the acute toxicity of these agents. Thus, in subsequent experiments, adult male Cynomolgus monkeys were pretreated orally with 1.5 or 3.0 mg/kg galantamine, challenged intramuscularly with 4.0xLD50, and post-treated with intramuscular injections of 0.4 mg/kg atropine, 30 mg/kg 2-PAM, and 0.32 mg/kg midazolam. All animals subjected to these treatments survived the soman. By contrast, none of the animals that were pretreated with saline and only 40% of the animals that were pretreated with pyridostigmine survived the soman challenge when post-treated with the same conventional antidotal therapy as that delivered to the galantamine-pretreated, soman-challenged monkeys. In addition, large numbers of degenerating neurons were visualized in the hippocampi of soman-challenged monkeys that had been pretreated with pyridostigmine or saline, but not in the hippocampi of animals that had been pretreated with galantamine. To our knowledge, this is the first study to demonstrate the effectiveness of clinically relevant oral doses of galantamine to prevent the acute toxicity of supra-lethal doses of soman in non-human primates.

Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) retains the rapid and sustained antidepressant-like actions of ketamine, but is spared its dissociative-like properties and abuse potential. While (2R,6R)-HNK is thought to exert its antidepressant-like effects by potentiating α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission, it is unknown how it exerts this effect. The acute synaptic effects of (2R,6R)-HNK were examined by recording field excitatory postsynaptic potentials (fEPSPs) and miniature excitatory postsynaptic currents (mEPSCs) in rat hippocampal slices. (2R,6R)-HNK bath application caused a rapid and persistent potentiation of AMPAR-mediated Schaffer collateral (SC)-CA1 fEPSPs in slices derived from male and female rats. The (2R,6R)-HNK-induced potentiation occurred independent of N-methyl-D-aspartate receptor (NMDAR) activity, was accompanied by a concentration-dependent decrease in paired pulse ratios, and was occluded by raising glutamate release probability. In additon, in the presence of tetrodotoxin, (2R,6R)-HNK increased the frequency, but not amplitude, of mEPSC events, confirming a presynaptic site of action that is independent of glutamatergic network disinhibition. A dual extracellular recording configuration revealed that the presynaptic effects of (2R,6R)-HNK were synapse-selective, occurring in CA1-projecting SC terminals, but not in CA1-projecting temporoammonic terminals. Overall, we found that (2R,6R)-HNK enhances excitatory synaptic transmission in the hippocampus through a concentration-dependent, NMDAR-independent, and synapse-selective increase in glutamate release probability with no direct actions on AMPAR function. These findings provide novel insight regarding (2R,6R)-HNK’s acute mechanism of action, and may inform novel antidepressant drug mechanisms that could yield superior efficacy, safety, and tolerability.

Significance Standard antidepressant treatments require weeks to show effectiveness. A single subanesthetic dose of ketamine rapidly attenuates many clinical signs and symptoms of depression; however, ketamine treatment also has many adverse effects, including dissociation and potential for abuse, which are mediated by NMDA glutamate receptor (NMDAR) inhibition. Previous work has revealed that the ketamine metabolite (2 R ,6 R )-hydroxynorketamine (HNK) induces antidepressant-like responses in rodents while minimizing the adverse effects observed with ketamine. The results of this study, using a multitude of experimental approaches, confirm that antidepressant-relevant concentrations of (2 R ,6 R )-HNK are not sufficient to block NMDARs. This provides a basis for work directed at alternative molecular targets and toward novel drugs that exert rapid antidepressant effects independent of NMDAR inhibition and NMDAR-mediated adverse effects.

Galantamine, a drug currently approved for treatment of Alzheimer's disease, has recently emerged as an effective pretreatment against the acute toxicity and delayed cognitive deficits induced by organophosphorus (OP) nerve agents, including soman. Since cognitive deficits can result from impaired glutamatergic transmission in the hippocampus, the present study was designed to test the hypothesis that hippocampal glutamatergic transmission declines following an acute exposure to soman and that this effect can be prevented by galantamine. To test this hypothesis, spontaneous excitatory postsynaptic currents (EPSCs) were recorded from CA1 pyramidal neurons in hippocampal slices obtained at 1h, 24 h, or 6-9 days after guinea pigs were injected with: (i) 1xLD50 soman (26.3μg/kg, s.c.); (ii) galantamine (8mg/kg, i.m.) followed 30min later by 1xLD50 soman, (iii) galantamine (8mg/kg, i.m.), or (iv) saline (0.5ml/kg, i.m.). In soman-injected guinea pigs that were not pretreated with galantamine, the frequency of EPSCs was significantly lower than that recorded from saline-injected animals. There was no correlation between the severity of soman-induced acute toxicity and the magnitude of soman-induced reduction of EPSC frequency. Pretreatment with galantamine prevented the reduction of EPSC frequency observed at 6-9 days after the soman challenge. Prevention of soman-induced long-lasting reduction of hippocampal glutamatergic synaptic transmission may be an important determinant of the ability of galantamine to counter cognitive deficits that develop long after an acute exposure to the nerve agent.

The translational capacity of data generated in preclinical toxicological studies is contingent upon several factors, including the appropriateness of the animal model. The primary objectives of this article are: (i) to analyze the natural history of acute and delayed signs and symptoms that develop following an acute exposure of humans to organophosphorus (OP) compounds, with an emphasis on nerve agents, (ii) to identify animal models of the clinical manifestations of human exposure to OPs, and (iii) to review the mechanisms that contribute to the immediate and delayed OP neurotoxicity. As discussed here, clinical manifestations of an acute exposure of humans to OP compounds can be faithfully reproduced in rodents and non-human primates. These manifestations include an acute cholinergic crisis in addition to signs of neurotoxicity that develop long after the OP exposure, particularly chronic neurological deficits consisting of anxiety-related behavior and cognitive deficits, structural brain damage, and increased slow electroencephalographic frequencies. Because guinea pigs and non-human primates, like humans, have low levels of circulating carboxylesterases-- the enzymes that metabolize and inactivate OP compounds-- they stand out as appropriate animal models for studies of OP intoxication. These are critical points for the development of safe and effective therapeutic interventions against OP poisoning because approval of such therapies by the Food and Drug Administration is likely to rely on the Animal Efficacy Rule, which allows exclusive use of animal data as evidence of the effectiveness of a drug against pathological conditions that cannot be ethically or feasibly tested in humans.

Galantamine has emerged as a potential antidote to prevent the acute toxicity of organophosphorus (OP) compounds. Changes in inhibitory GABAergic activity in different brain regions can contribute to both induction and maintenance of seizures in subjects exposed to the OP nerve agent soman. Here, we tested the hypothesis that galantamine can prevent immediate and delayed effects of soman on hippocampal inhibitory synaptic transmission. Spontaneous inhibitory postsynaptic currents (IPSCs) were recorded from CA1 pyramidal neurons in hippocampal slices obtained at 1 h, 24 h, or 6 to 9 days after the injection of guinea pigs with saline (0.5 ml/kg i.m.), 1xLD(50) soman (26.3 microg/kg s.c.), galantamine (8 mg/kg i.m.), or galantamine at 30 min before soman. Soman-challenged animals that were not pretreated showed mild, moderate, or severe signs of acute intoxication. At 1 h after the soman injection, the mean IPSC amplitude recorded from slices of mildly intoxicated animals and the mean IPSC frequency recorded from slices of severely intoxicated animals were larger and lower, respectively, than those recorded from slices of control animals. Regardless of the severity of the acute toxicity, at 24 h after the soman challenge the mean IPSC frequency was lower than that recorded from slices of control animals. At 6 to 9 days after the challenge, the IPSC frequency had returned to control levels, whereas the mean IPSC amplitude became larger than control. Pretreatment with galantamine prevented soman-induced changes in IPSCs. Counteracting the effects of soman on inhibitory transmission can be an important determinant of the antidotal effectiveness of galantamine.