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Ping Gong, Edward J. Perkins, US Army Engineer Research and Development Center
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OECD Project 1.15: The Adverse Outcome Pathways for Neurotoxicity induced by ionotropic GABA receptor inhibition.
This AOP was last modified on 5/5/2014.
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Ionotropic GABA receptors (iGABARs) are ligand-gated ion channels which play important functional roles in the nervous system. As the major player in inhibitory neurotransmission of vertebrates, iGABARs have also been described in many different phyla of invertebrates such as social amoeba (Dictyostelium discodeum), cnidarians, mollusks, annelids, arthropods, nematodes, and chordates. Chemical interactions with iGABARs can cause a variety of pharmacological and neurotoxicological effects depending on the location of the active or allosteric site affected. Interactions at three different types of binding sites on iGABARs can antagonize the postsynaptic inhibitory functions of GABA and lead to epileptic seizures and death. One of the three types of binding sites is the picrotoxin convulsant site located inside of the iGABAR pore that spans neuronal cell membranes. This AOP begins with a molecular initiating event (MIE) where chloride conductance through the ion channel is blocked due to chemical binding at or near the central pore of the receptor complex (e.g., the picrotoxin site). As a result, the first key event is a decrease in inward chloride conductance through the ligand-gated ion channel. This leads to the second key event, a reduction in postsynaptic inhibition, reflected as reduced frequency and amplitude of spontaneous inhibitory postsynaptic current (sIPSC) or abolishment of GABA-induced firing action in GABAergic neuronal membranes. Consequently, the resistance of excitatory neurons to fire is decreased, resulting in the generation of a large excitatory postsynaptic potential (EPSP). Although the underlying mechanism for the large EPSP is not well understood, a spike (rise) of intracellular Ca2+ is observed in the affected region, where a large group of excitatory neurons begin firing in an abnormal, excessive, and synchronized manner. Such a giant Ca2+-mediated excitatory firing (depolarization) causes voltage gated Na+ to open, which results in action potentials. The depolarization is followed by a period of hyper-polarization mediated by Ca2+-dependent K+ channels or GABA-activated Cl− influx. During seizure development, the post-depolarlization hyperpolarization becomes smaller, gradually disappears, and is replaced by a depolarization. This characteristic depolarization-shrinking hyperpolarization sequent of events represents the fourth key event known as “paroxysmal depolarizing shift” (PDS), which forms a “seizure focus”. A PDS is, essentially, an indication of epilepsy at the cellular level, which serves as the foci to initiate the adverse outcome at the organismal level of epileptic seizure. The severity of symptoms is often dose- and duration- dependent, while the toxicological symptoms are associated with the type and location of affected iGABARs. Mortality can occur if the individual sustains a prolonged or pronounced convulsion or seizure. Neurotoxicity, of which seizures is an end point, is a regulated outcome for chemicals. This AOP allows for screening chemicals for the potential to cause neurotoxicity through the use of in vitro assays that demonstrate binding to the picrotoxin site, electrophysiological assays demonstrating depolarization of neuronal membranes, or electroencephalography that records electrical activity of the brain.
Summary of the AOP
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Molecular Initiating Event
|Molecular Initiating Event||Support for Essentiality|
|iGABAR chloride channel, Binding at picrotoxin site||Strong|
|Epileptic seizure, Occurrence|
Relationships Among Key Events and the Adverse Outcome
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Life Stage Applicability
|bobwhite quail||Colinus virginianus||Strong||NCBI|
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Overall Assessment of the AOP
Biological mechanisms underlying epilepsy (defined as a disorder of the central nervous system characterized by recurrent seizures unprovoked by an acute systemic or neurologic insult) have been investigated for more than six decades and are well understood except for a few intermediate details (Bromfield et al. 2006; Lomen-Hoerth and Messing 2010). As one of the cellular mechanisms of action, blocking postsynaptic GABA-mediated inhibition can lead to epileptic seizure (Dichter and Ayala 1987; Gong et al. 2015). It has been extensively documented that non-competitive ion channel blockers such as picrotoxin, lindane, α-endosulfan and fipronil act through binding to iGABARs (Chen et al. 2006). Despite large structural diversity, it has been postulated that these blockers fit a single binding site in the chloride channel lumen lined by five TM2 (transmembrane domain 2) segments, which was supported in the β3 homopentamer by mutagenesis, pore structure studies, ligand binding, and molecular modeling (Chen et al. 2006). The downstream cascading key events of this AOP have also been reviewed in multiple publications (e.g., Dichter and Ayala 1987; Bromfield et al. 2006; Lomen-Hoerth and Messing 2010). Based on the extensive evidence supporting the MIE, KEs and the AO, there is a high likelihood and certainty that GABA antagonists including non-competitive channel blockers produce seizures in both invertebrates and vertebrates that possess GABAergic inhibitory neurotransmission in central nervous systems (Treiman 2001; Raymond-Delpech et al. 2005).
Concordance of dose-response relationships
Numerous pharmacological studies have reported quantitative dose-response relationships between the dose of non-competitive antagonists and the recorded electrophysiresponse of epileptic seizures. See examples for picrotoxin (Newland and Cull-Candy 1992; Ikeda 1998; Stilwell et al. 2006), RDX (Williams et al. 2011) and dieldrin (Babot et al. 2007; Ikeda 1998).
Temporal concordance among the key events and the adverse outcome
Given that the basic mechanism of neuronal excitability is the action potential, a hyperexcitable state can result from many causes including decreased inhibitory neurotransmission (KE2). Action potentials occur due to depolarization of the neuronal membrane, with membrane depolarization propagating down the axon to induce neurotransmitter release at the axon terminal. The action potential occurs in an all-or-none fashion as a result of local changes in membrane potential brought about by net positive inward ion fluxes. Membrane potential thus varies with activation of ligand- gated channels, whose conductance is affected by binding to neurotransmitters. For instance, the conductance is decreased (KE1) due to the binding at allosteric sites in the chloride channel of iGABAR by non-competitive blockers (MIE).
Seizure initiation: The hypersynchronous discharges that occur during a seizure may begin in a very discrete region of cortex and then spread to neighboring regions. Seizure initiation is characterized by two concurrent events: 1) high-frequency bursts of action potentials, and 2) hypersynchronization of a neuronal population. The synchronized bursts from a sufficient number of neurons result in a so-called spike discharge on the EEG (electroencephalogram), i.e., amplified excitatory postsynaptic potential (KE3). At the level of single neurons, epileptiform activity consists of sustained neuronal depolarization resulting in a burst of action potentials, a plateau-like depolarization associated with completion of the action potential burst, and then a rapid repolarization followed by hyperpolarization. This sequence is called the paroxysmal depolarizing shift (KE4).
Seizure propagation (AO), the process by which a partial seizure spreads within the brain, occurs when there is sufficient activation to recruit surrounding neurons. This leads to a loss of surround inhibition and spread of seizure activity into contiguous areas via local cortical connections, and to more distant areas via long association pathways such as the corpus callosum. The propagation of bursting activity is normally prevented by intact hyperpolarization and a region of surrounding inhibition created by inhibitory neurons. With sufficient activation there is a recruitment of surrounding neurons via a number of mechanisms. Repetitive discharges lead to: 1) an increase in extracellular K+, which blunts the extent of hyperpolarizing outward K+ currents, tending to depolarize neighboring neurons; 2) accumulation of Ca++ in presynaptic terminals, leading to enhanced neurotransmitter release; and 3) depolarization-induced activation of the NMDA subtype of the excitatory amino acid receptor, which causes more Ca++ influx and neuronal activation. The above description is excerpted and summarized from Bromfield et al. (2006).
Strength, consistency, and specificity of association of adverse effect and initiating event
Drug- or chemical-induced focal or generalized seizures are not limited to any specific group of chemical structures, neuroreceptors or taxonomy. This AOP addresses a specific group of chemicals that are capable of binding to the picrotoxin convulsant site of iGABARs, leading to epileptic seizures. Literature evidence strongly and consistently supports such a forward association, i.e., binding to the picrotoxin site leads to epileptic seizures (see reviews Gong et al. 2015; Bromfield et al. 2006; Raymond-Delpech et al. 2005; Treiman 2001; Dichter and Ayala 1987).
Uncertainties, inconsistencies, and data gaps
No inconsistencies have been reported so far, though some uncertainties and data gaps do exist. For instance, it is less well understood about the process by which seizures typically end, usually after seconds or minutes, and what underlies the failure of this spontaneous seizure termination in the life-threatening condition known as status epilepticus (Bromfield et al. 2006). The spread of epileptic activity throughout the brain, the development of primary generalized epilepsy, the existence of “gating: mechanisms in specific anatomic locations, and the extrapolation of hypotheses derived from simple models of focal epilepsy to explain more complex forms of epilepsies observed in human and other animals, all are not yet fully understood (Dichter and Ayala 1987).
Weight of Evidence Summary
See Collier et al. (2016) for details.
Essentiality of the Key Events
Molecular Initiating Event Summary,
Key Event Summary
The MIE, four key events and resulted adverse outcome listed for this AOP are all essential based on current knowledge and understanding of the structure, pharmacology, localization, classification of ionotropic GABA receptors (e.g., GABA-A receptors) (Olsen 2015; Olsen and Sieghart 2009), the basic neurophysiology, neurochemistry and cellular mechanisms underlying epilepsies (Dichter and Ayala 1987; Bromfield et al. 2006), and the pathophysiology of seizures (Lomen-Hoerth and Messing 2010).
Many studies have reported quantitative relationships between chemicals such as drugs and pesticides and electrophysiological response. For instance, long-term exposure of primary cerebellar granule cell cultures to 3 µM dieldrin reduced the GABAA receptor function to 55% of control, as measured by the GABA-induced 36Cl- uptake (Babot et al. 2007). Juarez et al. (2013) observed that picrotoxin exerted concentration-dependent and reversible inhibition of GABA-induced membrane currents in primary cultured neurons obtained from the guinea-pig small intestine. The stepwise qualitative relationships between consecutive events (MIE, KEs and AO) are well established but quantitative ones are rarely documented.
Applicability of the AOP
Life Stage Applicability,
This AOP is applicable to all vertebrates and invertebrates possessing iGABARs, without restrictions pertaining to sex and taxonomy. This AOP may not be applicable to young animals because GABA acts as an excitatory neurotransmitter due to increased intracellular Clˉ concentration during development of the nervous system (Taketo and Yoshioka 2000). A key feature of the immature type function of GABA(A) receptors is the depolarizing signaling, attributed to the inability of young neurons to maintain low intracellular chloride. The regulation of GABAergic switch is different in neurons with depolarizing vs hyperpolarizing GABAergic signaling. In mature neurons, recurrent and prolonged seizures may trigger a pathological reemergence of immature features of GABA(A) receptors, which compromises the efficacy of GABA-mediated inhibition. In immature neurons with depolarizing GABAergic signaling, the physiological and pathological regulation of this system is completely different, possibly contributing to the different outcomes of early life seizures (Galanopoulou 2008).
Considerations for Potential Applications of the AOP (optional)
This AOP can be used to establish the mode of neurotoxicological actions for chemicals capable of binding to the picrotoxin convulsant site of iGABARs. It can also be applied to risk assessment where AOP can assist in predictive modeling of chemical toxicity. Chemicals possessing this AOP can be distinguished from neurotoxicants acting on other types of iGABAR sites (e.g., orthosteric or allosteric binding sites) or other types of neuroreceptors (e.g., ardrenergic, dopaminergic, glutaminergic, cholinergic and serotonergic receptors). More information relevant to this topic can be found in Gong et al. (2015).
Babot Z, Vilaro M T, Sunol C. (2007) Long-term exposure to dieldrin reduces gamma-aminobutyric acid type A and N-methyl-D-aspartate receptor function in primary cultures of mouse cerebellar granule cells. J Neurosci Res, 85(16):3687-3695.
Bromfield EB, Cavazos JE, Sirven JI. (2006) Chapter 1, Basic Mechanisms Underlying Seizures and Epilepsy. In: An Introduction to Epilepsy [Internet]. West Hartford (CT): American Epilepsy Society; Available from: http://www.ncbi.nlm.nih.gov/books/NBK2510/.
Chen L, Durkin KA, Casida J. (2006) Structural model for gamma-aminobutyric acid receptor noncompetitive antagonist binding: widely diverse structures fit the same site. Proc Natl Acad Sci USA, 103(13):5185-5190.
Collier ZA, Gust KA, Gonzalez-Morales B, Gong P, Wilbanks MS, Linkov I, Perkins EJ. (2016) A weight of evidence assessment approach for adverse outcome pathways. Regulatory Toxicology and Pharmacology 75:46-57.
Dichter MA, Ayala GF. (1987) Cellular mechanisms of epilepsy: A status report. Science 237: 157-164.
Galanopoulou AS. (2008) GABA(A) Receptors in Normal Development and Seizures: Friends or Foes? Curr Neuropharmacol. 6(1): 1–20.
Gong P, Hong H, Perkins EJ. (2015) Ionotropic GABA receptor antagonism-induced adverse outcome pathways for potential neurotoxicity biomarkers. Biomarkers in Medicine 9(11):1225-39.
Ikeda T, Nagata K, Shono T, Narahashi T. (1998) Dieldrin and picrotoxinin modulation of GABA(A) receptor single channels. Neuroreport 9(14):3189-3195.
Juarez EH, Ochoa-Cortes F, Miranda-Morales M, Espinosa-Luna R, Montano L M, Barajas-Lopez C. (2013) Selectivity of antagonists for the Cys-loop native receptors for ACh, 5-HT and GABA in guinea-pig myenteric neurons. Auton Autacoid Pharmacol, 34(1-2):1-8.
Lomen-Hoerth C, Messing RO. (2010) Chapter 7: Nervous system disorders. In: Stephen J. McPhee, and Gary D. Hammer (Eds), Pathophysiology of disease: an introduction to clinical medicine (6th Edition). New York: McGraw-Hill Medical. ISBN 9780071621670.
Newland CF, Cull-Candy SG. (1992) On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat. J Physiol, 447: 191–213.
Olsen RW. (2015) Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes. Adv Pharmacol. 73:167-202.
Olsen RW, Sieghart W. (2009) GABA A receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology. 56(1):141-8.
Raymond-Delpech V, Matsuda K, Sattelle BM, Rauh JJ, Sattelle DB. (2005) Ion channels: molecular targets of neuroactive insecticides. Invert Neurosci, 5(3-4):119-133.
Stilwell GE, Saraswati S, Littleton JT, Chouinard SW. (2006) Development of a Drosophila seizure model for in vivo high-throughput drug screening. Eur J Neurosci, 24(8):2211-22.
Taketo M , Yoshioka T (2000) Developmental change of GABA(A) receptor-mediated current in rat hippocampus. Neuroscience 96(3):507-514.
Treiman DM. (2001) GABAergic mechanisms in epilepsy. Epilepsia, 42(Suppl. 3):8–12.
Williams LR, Aroniadou-Anderjaska V, Qashu F, Finne H, Pidoplichko V, Bannon D I et al. (2011) RDX binds to the GABA(A) receptor-convulsant site and blocks GABA(A) receptor-mediated currents in the amygdala: a mechanism for RDX-induced seizures. Environ Health Perspect, 119(3):357-363.