Aopwiki

SNAPSHOT

Created at: 2018-08-09 12:38

AOP ID and Title:


AOP 10: Binding to the picrotoxin site of ionotropic GABA receptors leading to epileptic seizures in adult brain
Short Title: Blocking iGABA receptor ion channel leading to seizures

Graphical Representation


Authors


Ping Gong, Edward J. Perkins, US Army Engineer Research and Development Center

Email: ping.gong@usace.army.mil or edward.j.perkins@usace.army.mil

Point of contact for this AOP entry: Dr. Ping Gong


Status

Author status OECD status OECD project SAAOP status
Open for comment. Do not cite EAGMST Under Review 1.15 Included in OECD Work Plan

Abstract


This AOP begins with a molecular initiating event (MIE) where a chemicla binds to the picrotoxin binding site at or near the central pore of the ionotropic GABA receptor complex causing blockage of the ion channel. As a result, the first key event (KE) is a decrease in inward chloride conductance through the ligand-gated ion channel. This leads to the second KE, 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), i.e., the third KE. The large EPSP is reflected as a spike (rise) of intracellular Ca2+ 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 sequence of events represents the fourth KE 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 are 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 adult brain.


Background


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, iGABARs are widely distributed in both vertebrates and invertebrates (McGonigle and Lummis 2010; Garcia-Reyero et al. 2011). In vertebrates, the iGABAR includes two subclasses of fast-responding ion channels, GABAA receptor (GABAA-R) and GABAC receptor (GABAC-R). Invertebrate iGABARs do not readily fit the vertebrate GABAA/GABAC receptor categories (Sieghart 1995). The majority of insect iGABARs are distinguished from vertebrate GABAA receptors by their insensitivity to bicuculline and differ from GABAC-Rs in that they are subject to allosteric modulation, albeit weakly, by benzodiazepines and barbiturates (Hosie et al. 1997).

Chemical interactions with iGABARs can cause a variety of pharmacological and neurotoxicological effects depending on the location of the active or allosteric site affected. Three distinct types of interactions at binding sites on iGABARs can antagonize the postsynaptic inhibitory functions of GABA and lead to epileptic seizures and death. These three types of interactions correspond to three AOPs (Gong et al. 2015). One of the three types of interaction is non-competitive channel blocking at the picrotoxin convulsant site located inside of the iGABAR pore that spans neuronal cell membranes (this MIE). The other two types of interactions are negative modulation at allosteric sites and competitive binding at the active orthosteric sites (MIEs to be developed in the future).


Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Sequence Type Event ID Title Short name
1 MIE 667 Binding at picrotoxin site, iGABAR chloride channel Binding at picrotoxin site, iGABAR chloride channel
2 KE 64 Reduction, Ionotropic GABA receptor chloride channel conductance Reduction, Ionotropic GABA receptor chloride channel conductance
3 KE 669 Reduction, Neuronal synaptic inhibition Reduction, Neuronal synaptic inhibition
4 KE 682 Generation, Amplified excitatory postsynaptic potential (EPSP) Generation, Amplified excitatory postsynaptic potential (EPSP)
5 KE 616 Occurrence, A paroxysmal depolarizing shift Occurrence, A paroxysmal depolarizing shift
6 AO 613 Occurrence, Epileptic seizure Occurrence, Epileptic seizure

Key Event Relationships

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
Binding at picrotoxin site, iGABAR chloride channel adjacent Reduction, Ionotropic GABA receptor chloride channel conductance High High
Reduction, Ionotropic GABA receptor chloride channel conductance adjacent Reduction, Neuronal synaptic inhibition High High
Reduction, Neuronal synaptic inhibition adjacent Generation, Amplified excitatory postsynaptic potential (EPSP) High Moderate
Generation, Amplified excitatory postsynaptic potential (EPSP) adjacent Occurrence, A paroxysmal depolarizing shift Moderate Moderate
Occurrence, A paroxysmal depolarizing shift adjacent Occurrence, Epileptic seizure High Moderate

Stressors


Name Evidence
Picrotoxin High
Lindane High
Dieldrin High
Heptachlor High
Endosulfan High
RDX High
Fipronil High

Picrotoxin

Picrotoxin seizures are well defined mechanistically. They arise from GABAA receptor chloride channel blockage. (See Page 131 in "Models of Seizures and Epilepsy", edited by A. Pitkanen, P.A. Schwartzkroin, S.L. Moshe. Elsevier Academic Press. 2006)

As picrotoxin effectively inhibits chloride influx in GABAA and other ionotropic receptors, it represents a universal "reference" channel blocker with whom other ligands may be compared. (A.V. Kalueff. 2007. Mapping convulsants' binding to the GABA-A receptor chloride ionophore: a proposed model for channel binding sites. Neurochem Int. 50(1): 61-8.)

Lindane

Neurotoxic pesticides, such as lindane, alpha-endosulfan and dieldrin, share structural similarities (and compete for the binding site) with picrotoxin, inhibit TBPS binding, induce seizures and block Cl-currents through the ionophore. (A.V. Kalueff. 2007. Mapping convulsants' binding to the GABA-A receptor chloride ionophore: a proposed model for channel binding sites. Neurochem Int. 50(1): 61-8.)

Dieldrin

See evidence text for picrotoxin and lindane.

Heptachlor

Organochlorine insecticides, including DDT, dieldrin, heptachlor, lindane, and β-hexachlorocyclohexane (β-HCH), have varied targets and mechanisms of action within the central nervous system that affect the function of sodium and calcium channels and transporters. They also interfer with γ-aminobutyric acid (GABA) neurotransmission by blocking specific GABA receptors, contributing to their neurotoxic effects (Narahashi et al., 1995). 

Endosulfan

See "Toxicological Profile for Endosulfan" (Agency for Toxic Substances and Disease Registry, 2000). Dose- and time-dependent correlations between picrotoxin site binding by chlorinated pesticides and convulsions were observed in mice (Cole and Casida 1986), whereas poisoning with the organochlorine insecticide endosulfan caused seizure, status epilepticus, or refractory status epilepticus in humans (Durukan et al. 2009; Moon et al. 2017; Moses and Peter 2010; Parbhu et al. 2009; Roberts et al. 2004), and eventually led to the death of a farmer (Roberts et al. 2004) and a toddler (Parbhu et al. 2009).

RDX

See Williams et al. (2011).

Fipronil

See Chen et al. (2006).

Overall Assessment of the AOP

Biological plausibility

The 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; Casida and Durkin 2015). 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; Casida and Durkin 2015). 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 such GABA antagonists as 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 electrophysirological esponse 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). Moreover, 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 the 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 the 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. 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). For instance, dose- and time-dependent correlations between picrotoxin site binding by chlorinated pesticides and convulsions were observed in mice (Cole and Casida 1986), whereas poisoning with the organochlorine insecticide endosulfan caused seizure, status epilepticus, or refractory status epilepticus in humans (Durukan et al. 2009; Moon et al. 2017; Moses and Peter 2010; Parbhu et al. 2009; Roberts et al. 2004), and eventually led to the death of a farmer (Roberts et al. 2004) and a toddler (Parbhu et al. 2009).

Uncertainties, inconsistencies, and data gaps

No inconsistencies have been reported so far, though some uncertainties and data gaps do exist. For instance, 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 are less well understood (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 also not yet fully understood (Dichter and Ayala 1987). The remarkable plasticity of GABAergic neurons and iGABARs (e.g., iGABAR regulation by phosphorylation, and expression of potassium chloride cotransporters (KCCs) or sodium dependent anion exchangers (NDAE)) in response to insults and injury constitutes additional complexity and creates another layer of uncertainties in the emergence of epileptic seizures (Ben-Ari 2006; Galanopoulou 2008; Scharfman and Brooks-Kayal 2014).


Domain of Applicability

Life Stage Applicability
Life Stage Evidence
Adults High
Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens High NCBI
mouse Mus musculus High NCBI
rat Rattus norvegicus High NCBI
bobwhite quail Colinus virginianus High NCBI
zebrafish Danio rerio Moderate NCBI
Sex Applicability
Sex Evidence
Male High
Female High

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 during their embryonic and early developmental stages when GABA acts as an excitatory neurotransmitter due to increased intracellular Clˉ concentration in immature or developing neurons (Taketo and Yoshioka 2000; Galanopoulou 2008; Ben-Ari 2006). A key feature of the immature type function of GABAA 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 GABAA 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).

Essentiality of the Key Events

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., GABAA 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).

Weight of Evidence Summary

A novel subject matter expertise driven approach was developed for weight of evidence (WoE) assessment (Collier et al. 2016). This approach, tailored toward the needs of AOPs and in compliance with the AOP Users' Handbook (OECD 2017), was based on criteria and metrics related to data quality and causality (i.e., the strength of causal linkage between key events). The methodology consists of three main steps: (1) assembling evidence (preparing the AOP), (2) weighting evidence (criteria weighting and scoring), and (3) weighting the body of evidence (aggregating lines of evidence). We adopted the General Assessment Factors (GAF) established by the US EPA as the criteria for data quality evaluation, and a set of five criteria known as Bradford Hill criteria to measure the strength of causal linkages (see Table below). The numerical scoring scale corresponds to the descriptive scoring scale recommended in the Users' Handbook as follows: 4 or 5 is equivalent to High, 3 is equivalent to Moderate, and 1 or 2 is equivalent to Low. Detailed description on, supporting evidence for and quantitative understanding of each KE/KER can be found by clicking the link on each KE/KER above. 

The authors of Collier et al. (2016), who served as the developers for several AOPs (including this one), represented subject matter experts, and they applied their expertise and best professional judgment to assign weights to the criteria and scores to each line of evidence. Final criteria scoring represented the consensus scores agreed upon after debates among the authors. For example, the MIE has been intensively reviewed where numerous documented studies provided supporting evidence. Hence, the MIE received high scores for all five GAF criteria. However, the Bradford Hill criteria connecting KE2-->KE3 and KE3-->KE4 received relatively lower scores because there still exist knowledge gaps in the spread of epileptic activity throughout the normal CNS and the mechanism underlying the generalized epilepsies. The following table shows the results of our WoE assessment (note that scores may be inexact due to rounding).

ScoreTable.jpg

Quantitative Consideration

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.

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 acting through 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).

References


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.

Ben-Ari Y. (2006) Seizures beget seizures: the quest for GABA as a key player. Crit Rev Neurobiol. 18(1-2):135-44.

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/.

Casida JE, Durkin KA. (2015) Novel GABA receptor pesticide targets. Pesticide Biochem Physiol. 121:22-30.

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.

Cole LM, Casida JE. (1986) Polychlorocycloalkane insecticide-induced convulsions in mice in relation to disruption of the GABA-regulated chloride ionophore. Life Sci. 39 (20):1855-62.

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. Regul Toxicol Pharmacol. 75:46-57.

Dichter MA, Ayala GF. (1987) Cellular mechanisms of epilepsy: A status report. Science 237: 157-164.

Durukan P, Ozdemir C, Coskun R, Ikizceli I, Esmaoglu A, Kurtoglu S, Guven M. (2009) Experiences with endosulfan mass poisoning in rural areas. Eur J Emerg Med. 16(1):53-6.

Galanopoulou AS. (2008) GABAA Receptors in Normal Development and Seizures: Friends or Foes? Curr Neuropharmacol. 6(1): 1–20.

Garcia-Reyero N, Habib T, Pirooznia M, Gust KA, Gong P, Warner C, Wilbanks M, Perkins E. (2011) Conserved toxic responses across divergent phylogenetic lineages: a meta-analysis of the neurotoxic effects of RDX among multiple species using toxicogenomics. Ecotoxicology. 20(3):580-94.

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.

Hosie AM, Aronstein K, Sattelle DB, ffrench-Constant RH. (1997) Molecular biology of insect neuronal GABA receptors. Trends Neurosci. 20(12): 578-583.

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.

McGonigle I, Lummis SC. (2010) Molecular characterization of agonists that bind to an insect GABA receptor. Biochemistry. 49(13):2897-902.

Moon JM, Chun BJ, Lee SD. (2017) In-hospital outcomes and delayed neurologic sequelae of seizure-related endosulfan poisoning. Seizure. 51:43-49.

Moses V, Peter JV. (2010) Acute intentional toxicity: endosulfan and other organochlorines. Clin Toxicol (Phila). 48(6):539-44.

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.

OECD. (2017) Users' Handbook Supplement to the Guidance Document for Developing and Assessing AOPs. OECD Environment Directorate, Environment, Health and Safety Division, Series on Testing & Assessment No. 233, Series on Adverse Outcome Pathways No. 1, ENV/JM/MONO(2016)12, Paris, France.

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.

Parbhu B, Rodgers G, Sullivan JE. (2009) Death in a toddler following endosulfan ingestion. Clin Toxicol (Phila). 47(9):899-901.

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.

Roberts DM, Dissanayake W, Sheriff MHR, Eddleston M. (2004) Refractory status epilepticus following self-poisoning with the organochlorine pesticide endosulfan. J Clinical Neurosci. 11(7): 760-2.

Scharfman HE, Brooks-Kayal AR. (2014) Is Plasticity of GABAergic Mechanisms Relevant to Epileptogenesis? In: Scharfman HE and Buckmaster PS (eds.), Issues in Clinical Epileptology: A View from the Bench, Advances in Experimental Medicine and Biology 813, pp.133-150.

Sieghart W.(1995) Structure and pharmacology of gamma-aminobutyric acid A receptor subtypes. Pharmacol.Rev. 47(2):181-234

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.

 


Appendix 1

List of MIEs in this AOP

Event: 667: Binding at picrotoxin site, iGABAR chloride channel

Short Name: Binding at picrotoxin site, iGABAR chloride channel

Key Event Component

Process Object Action
GABA receptor binding gamma-aminobutyric acid receptor subunit alpha-1 increased
GABA receptor binding gamma-aminobutyric acid receptor subunit alpha-5 increased
GABA receptor binding gamma-aminobutyric acid receptor subunit alpha-6 increased

AOPs Including This Key Event

Stressors

Name
Picrotoxin
Lindane
Fipronil
RDX
Alpha-endosulfan
Penicillin

Biological Context

Level of Biological Organization
Molecular

Cell term

Cell term
neuron

Organ term

Organ term
brain

Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

Chemicals non-competitively bind at or near the central pore of the receptor complex (e.g., the picrotoxin site) and directly block chloride conductance through the ion channel (Kalueff 2007). It has been postulated that they fit a single "big picrotoxin binding pocket" in the chloride channel lumen lined by five TM2 segments (Kalueff 2007; Olsen 2006). This hypothesis was examined with the β3 homopentamer by mutagenesis, pore structure studies, ligand binding, and molecular modeling (Chen et al. 2006). Results suggest that they fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and hydrophobic interactions with A2', T6', and L9' alkyl substituents, thereby blocking the channel. More computational evidence can be found in Sander et al. (2011), Carpenter et al. (2013) and Zheng et al. (2014).



Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
fruit fly Drosophila melanogaster High NCBI
mouse Mus musculus High NCBI
dogs Canis lupus familiaris High NCBI
Life Stage Applicability
Life Stage Evidence
Adult High
Sex Applicability
Sex Evidence
Unspecific High

Theoretically, this MIE is applicable to any organisms that possess ionotropic GABA receptors (iGABARs) in their central and/or peripheral nervous systems. Many reviews (e.g., Hoisie et al. 1997; Buckingham et al. 2005; Michels and Moss 2007; Olsen and Sieghart 2009) have summarized evidence of ubiquitous existence of iGABARs (GABAA-R in vertebrates including the humans) in species spanning from invertebrates to human. For instance, an ionotropic GABA receptor gene (GABA-receptor subunit-encoding Rdl gene) was isolated from a naturally occurring dieldrin-resistant strain of D. melanogaster (Ffrench-Constant et al., 1991,1993; Ffrench-Constant and Rocheleau, 1993). Nineteen GABAA receptor genes have been identified in the human genome (Simon et al. 2004). Direct evidence is mostly derived from in silico molecular modeling that docks ligands to the binding pockets of iGABARs in human (Carpenter et al. 2013; Chen et al. 2006; Sander et al. 2011), fruitfly and zebrafish (Zheng et al. 2014).

It should be noted that this MIE is limited to adult brains because of the age- or developmental stage-dependent nature of iGABAR where GABA acts as an excitatory (instead of inhibitory) neurotransmitter due to a higher intracellular chloride concentration in immature or developing neurons (Taketo and Yoshioka 2000; Galanopoulou 2008; Ben-Ari 2006).

This MIE is not applicable to GABAB receptors because, as metabotropic G-protein-coupled receptors, they mediate slow and sustained inhibitory responses and are involved in absence epilepsy (Han et al. 2012).


Key Event Description

Figure1.png

Figure 1. Structure of ionotropic GABA receptors based on the consensus in multiple literature reviews (Source: Gong et al. 2015). Shown is a common subtype α1β2γ2 of GABAA receptors found in the mammalian CNS. (A) Five subunits from three subunit subfamilies assemble to form a heteropentameric chloride permeable channel. (B) Stoichiometry and subunit arrangement of the GABAA receptor. Also shown are the binding sites for GABA and BZ. (C) Receptor subunits consist of four hydrophobic transmembrane domains (TM1-4), where TM2 is believed to line the pore of the channel. The large extracellular N-terminus is the site for ligand binding as well as the site of action of various drugs (e.g., picrotoxin and dieldrin). Each receptor subunit also contains a large intracellular domain between TM3 and TM4, which is the site for various protein–protein interactions as well as the site for post-translational modifications that modulate receptor activity. BZ: Benzodiazepines; CNS: Central nervous system; TM: Transmembrane.

-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
As shown in Figure 1, binding of GABA opens up the chloride channel in the iGABAR complex, leading to neuronal inhibition (Olsen et al. 2004). Non-competitive channel blockers (e.g., fipronil, lindane, picrotoxin, penicillin and alpha-endosulfan) indirectly modulate the iGABAR activity (i.e., alter the response of the receptor to agonist) by noncompetitively binding at or near the central pore of the receptor complex (e.g., the picrotoxin site), an allosteric site distinct from that of the orthosteric agonist binding site, and inducing a conformational change within the receptor (Ernst et al. 2005; Johnston 2005).

Mounting evidence indicates that iGABAR chloride channel blockers bind to overlapping but not identical sites (Kalueff 2007). Based on the mapping of convulsants' binding domains to the iGABAR chloride ionophore (Olsen 2006), Kalueff (2007) proposed a "big picrotoxin binding pocket" model for the ligand-binding area of ionophore. For instance, bicyclophosphates, butyrolactones, pentylenetetrazole and penicillin act on the N-terminus of TM2 (‘‘main’’ picrotoxin binding site), whereas picrotoxin may act on the "main" as well as a hypothetical second ‘‘allosteric’’ picrotoxin site located on the C-terminus of TM2 (Kalueff 2007; Olsen 2006). In spite of minor differences, these blockers share similarity in channel blockage characteristics (e.g., voltage-dependency, binding to closed channel, reversible, and non-competitive) and effects of channel state (open/closed frequency and duration) (Kalueff 2007). Nevertheless, the 3D structure of iGABAR ionophore is unavailable. More homology modeling work (similar to the electron microscopic structure of nicotinic receptor channel (Unwin 2005)) may allow us to gain in-depth understanding of the picrotoxin site's accessibility for and interactions with channel blockers (i.e., convulsants), clarify the biological functions of the binding sites, and verify the impact of mutations in TM2 on ligand binding activity (Kalueff 2007).


How it is Measured or Detected

Binding to a specific site on iGABAR can be determined using a variety of methods including mutagenesis, pore structure studies, ligand binding, and molecular modeling (more details on methods can be found in Chen et al. 2006). One should choose a method in accordance with specific goal and also on the basis of available laboratory facilities. For example, Atack et al. (2007) chose the radioligand [35S]t-butylbicyclophosphorothionate (TBPS) binding assay to determine the binding properties (i.e., inhibition by TBPS, picrotoxin, loreclezole and pentobarbital and modulation by GABA) at the convulsant binding site.


References

Atack JR, Ohashi Y, McKernan RM. 2007. Characterization of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in postmortem human brain. Br J Pharmacol. 150(8):1066-74.

Buckingham SD, Biggin PC, Sattelle BM, Brown LA, Sattelle DB. 2005. Insect GABA receptors: splicing, editing, and targeting by antiparasitics and insecticides. Mol Pharmacol 68(4):942-951.

Ben-Ari Y. 2006. Seizures beget seizures: the quest for GABA as a key player. Crit Rev Neurobiol 18(1-2): 135-44.

Carpenter TS, Lau EY, Lightstone FC. 2013. Identification of a possible secondary picrotoxin-binding site on the GABAA receptor. Chem Res Toxicol. 26(10):1444-54.

Chen L, Durkin KA, Casida JE. 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.

Ernst M, Bruckner S, Boresch S, Sieghart W. 2005. Comparative models of GABAA receptor extracellular and transmembrane domains: important insights in pharmacology and function. Mol Pharmacol 68(5):1291-1300.

Ffrench-Constant RH, Mortlock DP, Shaffer CD, MacIntyre RJ, Roush RT. 1991. Molecular cloning and transformation of cyclodiene resistance in Drosophila: an invertebrate gamma-aminobutyric acid subtype A receptor locus. Proc Natl Acad Sci USA 88:7209–7213.

Ffrench-Constant RH and Rocheleau TA. 1993 Drosophila gamma-aminobutyric acid receptor gene Rdl shows extensive alternative splicing. J Neurochem 60:2323–2326.

Ffrench-Constant RH, Steichen JC, Rocheleau TA, Aronstein K, and Roush RT. 1993. A single-amino acid substitution in a gamma-aminobutyric acid subtype A receptor locus is associated with cyclodiene insecticide resistance in Drosophila populations. Proc Natl Acad Sci USA 90:1957–1961.

Galanopoulou AS. 2008. GABAA 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.

Han HA, Cortez MA, Snead OC III. 2012. GABAB Receptor and Absence Epilepsy. In: Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies [Internet]. 4th edition. Bethesda (MD): National Center for Biotechnology Information (US); 2012. Available from: https://www.ncbi.nlm.nih.gov/books/NBK98192/.

Hosie AM, Aronstein K, Sattelle DB, Ffrench-Constant RH. 1997. Molecular biology of insect neuronal GABA receptors. Trends Neurosci 20(12):578-583.

Johnston GA. 2005. GABA(A) receptor channel pharmacology. Curr Pharm Des 11(15):1867-1885.

Kalueff AV. 2007. Mapping convulsants' binding to the GABA-A receptor chloride ionophore: a proposed model for channel binding sites. Neurochem Int 50(1): 61-68.

Michels G, Moss SJ. 2007. GABAA receptors: properties and trafficking. Crit Rev Biochem Mol Biol 42(1):3-14.

Olsen RW. 2006. Picrotoxin-like channel blockers of GABAA receptors. Proc Natl Acad Sci USA. 103:6081-82.

Olsen RW, Chang C-SS, Li G, Hanchar HJ, Wallner M. 2004. Fishing for allosteric sites on GABAa receptors. Biochem Pharmacol. 68:1675-84.

Olsen RW, Sieghart W. 2009. GABAA receptors: subtypes provide diversity of function and pharmacology. Neuropharmacology. 56(1):141-8.

Sander T, Frolund B, Bruun AT, Ivanov I, McCammon JA, Balle T. 2011. New insights into the GABAA receptor structure and orthosteric ligand binding: receptor modeling guided by experimental data. Proteins. 79(5):1458-77.

Simon J, Wakimoto H, Fujita N, Lalande M, Barnard EA. 2004. Analysis of the set of GABA(A) receptor genes in the human genome. J. Biol. Chem. 279(40), 41422–41435.

Taketo M , Yoshioka T. 2000. Developmental change of GABAA receptor-mediated current in rat hippocampus. Neuroscience 96(3):507-514.

Unwin N. 2005. Refined structure of the nicotinic acetylcholine receptor at 4 AËš resolution. J Mol Biol. 346:967-89.

Zheng N, Cheng J, Zhang W, Li W, Shao X, Xu Z, Xu X, Li Z. 2014. Binding difference of fipronil with GABAARs in fruitfly and zebrafish: insights from homology modeling, docking, and molecular dynamics simulation studies. J Agric Food Chem 62(44):10646-53.

 


List of Key Events in the AOP

Event: 64: Reduction, Ionotropic GABA receptor chloride channel conductance

Short Name: Reduction, Ionotropic GABA receptor chloride channel conductance

Key Event Component

Process Object Action
GABA-gated chloride ion channel activity chloride decreased

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
neuron

Organ term

Organ term
brain

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens High NCBI
rats Rattus norvegicus High NCBI
mouse Mus musculus High NCBI
Drosophila melanogaster Drosophila melanogaster High NCBI

Banerjee et al. (1999) reported functional modulation of GABAA receptors by Zn2+, pentobarbital, neuroactive steroid alphaxalone, and flunitrazepam in the cerebral cortex and cerebellum of rats undergoing status epilepticus induced by pilocarpine.

Babot et al. (2007) measured the reduction in mouse GABAA receptor function by 3 μM dieldrin using the GABA-induced 36Cl- uptake method.

Bromfield et al. (2006) reviewed evidence for GABAA receptors in human and mammalian brains, whereas Narahashi (1996) and Costa (2015) reviewed organochlorine and some pyrethroid compounds as insecticides with the target site of chloride channel.

Grolleau and Sattelle (2000) reported a complete blocking of inward current by 100 μM picrotoxin in the wild-type RDL (iGABAR) of Drosophila melanogaster.


Key Event Description

This key event occurs at the cellular level and is characterized by a dose-dependent post-synaptic inhibition of membrane currents in iGABAR-containing cells, especially neuronal cells (Dichter and Ayala 1987; Bromfield et al. 2006), leading to the reduction of iGABAR chloride channel conductance.


How it is Measured or Detected

The change in membrane conductance can be measured by determining the alteration (i.e., inhibition) in muscimol-stimulated (Banerjee et al. 1999) or GABA-induced uptake (Babot et al. 2007) of 36Cl- in cortical and cerebellar membranes or primary cerebellar granule cell cultures, prior to and after exposure to a GABA antagonist. Inglefield and Schwartz-Bloom (1998) reported a Cl--sensitive fluorescent dye-based method where to measure real-time changes in intracellular chloride concentration with UV laser scanning confocal microscopy.


References

Babot Z, Vilaro MT, 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.

Banerjee PK, Olsen RW, Snead OC, III. (1999) Zinc inhibition of gamma-aminobutyric acid(A) receptor function is decreased in the cerebral cortex during pilocarpine-induced status epilepticus. J Pharmacol Exp Ther 1999; 291(1):361-366.

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

Costa LG. (2015) The neurotoxicity of organochlorine and pyrethroid pesticides. Handb Clin Neurol. 131:135-48.

Dichter MA, Ayala GF. (1987) Cellular mechanisms of epilepsy: a status report. Science 237(4811), 157-164.

Gong P. Hong HH, Perkins EJ. (2015) Ionotropic GABA receptor antagonism-induced adverse outcome pathways for potential neurotoxicity biomarkers. Biomark. Med. 9(11):1225-39.

Grolleau F, Sattelle DB. (2000) Single channel analysis of the blocking actions of BIDN and fipronil on a Drosophila melanogaster GABA receptor (RDL) stably expressed in a Drosophila cell line. Br J Pharmacol. 130(8):1833-42.

Inglefield JR, Schwartz-Bloom RD. (1998) Optical imaging of hippocampal neurons with a chloride-sensitive dye: early effects of in vitro ischemia. J Neurochem. 70(6):2500-9.

Narahashi T. (1996). Neuronal ion channels as the target sites of insecticides. Pharmacol Toxicol. 79(1):1-14.


Event: 669: Reduction, Neuronal synaptic inhibition

Short Name: Reduction, Neuronal synaptic inhibition

Key Event Component

Process Object Action
chemical synaptic transmission decreased

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
neuron

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
rat Rattus norvegicus High NCBI
guinea pig Cavia porcellus High NCBI
human Homo sapiens High NCBI
Japanese quail Coturnix japonica High NCBI
Life Stage Applicability
Life Stage Evidence
Adult High
Sex Applicability
Sex Evidence
Unspecific High

See Juarez et al. (2013) for supporting evidence for Guinea pig. For rat, whole-cell in vitro recordings in the rat basolateral amygdala (BLA) showed that RDX reduces the frequency and amplitude of GABAA receptor mediated sIPSCs and the amplitude of GABA-evoked postsynaptic currents, whereas in extracellular field recordings from the BLA, RDX induced prolonged, seizure-like neuronal discharges (Williams et al, 2011).


Key Event Description

A reduction in GABA-mediated inhibition of neuronal synaptic signaling is reflected as decreased frequency and amplitude of iGABAR-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) or abolishment of GABA-induced firing action (Newland and Cull-Candy 1992).


How it is Measured or Detected

Juarez et al. (2013) used primary cultured neurons obtained from the guinea-pig small intestine to detect picrotoxin concentration-dependent (and reversible) inhibition of GABA-induced membrane currents. Williams et al. (2011) used whole-cell in vitro recordings in the rat basolateral amygdala (BLA) to detect the reduced frequency and amplitude of GABAA receptor mediated spontaneous inhibitory postsynaptic currents (sIPSCs) and the amplitude of GABA-evoked postsynaptic currents, both of which were induced by RDX.


References

Newland C F, Cull-Candy S G. On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat. J Physiol 1992; 447: 191–213.

Juarez E H, Ochoa-Cortes F, Miranda-Morales M, Espinosa-Luna R, Montano L M, Barajas-Lopez C. Selectivity of antagonists for the Cys-loop native receptors for ACh, 5-HT and GABA in guinea-pig myenteric neurons. Auton Autacoid Pharmacol 2013; 34(1-2):1-8.

Williams L R, Aroniadou-Anderjaska V, Qashu F, Finne H, Pidoplichko V, Bannon D I et al. 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 2011; 119(3):357-363.

 


Event: 682: Generation, Amplified excitatory postsynaptic potential (EPSP)

Short Name: Generation, Amplified excitatory postsynaptic potential (EPSP)

Key Event Component

Process Object Action
excitatory postsynaptic potential occurrence

Biological Context

Level of Biological Organization
Cellular

Organ term

Organ term
brain

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
mouse Mus musculus High NCBI
rat Rattus norvegicus High NCBI
guinea pig Cavia porcellus High NCBI
Life Stage Applicability
Life Stage Evidence
Adult High
Sex Applicability
Sex Evidence
Unspecific High

Miura et al. (1997) reported supporting evidence from guinea pigs whereas Dichter and Ayala (1987) and Bromfield et al. (2006) summarized relevant studies on humans. Acker et al. (2016) perform simultaneous two-photon voltage-sensitive dye recording with two-photon glutamate uncaging in order to measure the characteristics (amplitude and duration) of uncaging-evoked EPSPs in acute mouse brain slices.


Key Event Description

In neuroscience, an excitatory postsynaptic potential (EPSP) is defined as a neurotransmitter-induced postsynaptic potential change that depolarizes the cell, and hence increases the likelihood of initiating a postsynaptic action potential (Purves et al. 2001). On the contrary, an inhibitory postsynaptic potential (IPSP) decreases this likelihood. Whether a postsynaptic response is an EPSP or an IPSP depends on the type of channel that is coupled to the receptor, and on the concentration of permeant ions inside and outside the cell. In fact, the only factor that distinguishes postsynaptic excitation from inhibition is the reversal potential of the postsynaptic potential (PSP) in relation to the threshold voltage for generating action potentials in the postsynaptic cell. When an active presynaptic cell releases neurotransmitters into the synapse, some of them bind to receptors on the postsynaptic cell. Many of these receptors contain an ion channel capable of passing positively charged ions (e.g., Na+ or K+) or negatively charged ions (e.g., Cl-) either into or out of the cell. In epileptogenesis, discharges reduced GABAA receptor-mediated hyperpolarizing IPSPs by shifting their reversal potentials in a positive direction. At the same time, the amplitudes of Schaffer collateral-evoked RS-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated EPSPs and action potential-independent miniature EPSPs were enhanced, whereas N-methyl-d-aspartate receptor-mediated EPSPs remained unchanged. Together, these changes in synaptic transmission produce a sustained increase in hippocampal excitability (Lopantsev et al. 2009).


How it is Measured or Detected

EPSPs are usually recorded by measuring electrical responses and changes in intracellular calcium concentration using intracellular electrodes (Miura et al. 1997) or recording extracellular electrical activity or potential using >20 electroencephalogram (EEG) electrodes (often in clinical settings) (Bromfield et al. 2006). Recently, voltage-sensitive dyes have been successfully used for measuring voltage responses from large neuronal populations in acute brain slice preparations (Popovic et al. 2015; Acker et al. 2016). 


References

Acker CD, Hoyos E, Loew LM. (2016) EPSPs Measured in Proximal Dendritic Spines of Cortical Pyramidal Neurons. eNeuro. 3(2) ENEURO.0050-15.2016.

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/.

Dichter MA, Ayala GF. (1987) Cellular mechanisms of epilepsy: A status report. Science 237:157-64.

Lopantsev V, Both M, Draguhn A. 2009. Rapid Plasticity at Inhibitory and Excitatory Synapses in the Hippocampus Induced by Ictal Epileptiform Discharges. Eur J Neurosci 29(6):1153–64.

Miura M, Yoshioka M, Miyakawa H, Kato H, Ito KI. (1997) Properties of calcium spikes revealed during GABAA receptor antagonism in hippocampal CA1 neurons from guinea pigs. J Neurophysiol. 78(5):2269-79.

Popovic MA, Carnevale N, Rozsa B, Zecevic D. (2015)  Electrical behaviour of dendritic spines as revealed by voltage imaging. Nature Communications. 6:8436.

Purves D, Augustine GJ, Fitzpatrick D, Katz LC, LaMantia A-S, McNamara JO, Williams SM (Eds). 2001. Neuroscience. 2nd edition. Chapter 7. Neurotransmitter Receptors and Their Effects. Sunderland (MA): Sinauer Associates. Available from: http://www.ncbi.nlm.nih.gov/books/NBK10799/.


Event: 616: Occurrence, A paroxysmal depolarizing shift

Short Name: Occurrence, A paroxysmal depolarizing shift

Key Event Component

Process Object Action
membrane depolarization occurrence

Biological Context

Level of Biological Organization
Tissue

Organ term

Organ term
brain

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
Life Stage Applicability
Life Stage Evidence
Adult High
Sex Applicability
Sex Evidence
Unspecific High

Most of the supporting evidence comes from studies on human and rodents. See the reviews of Bromfield (2006) and Lomen-Hoerth and Messing (2010) for examples.


Key Event Description

A paroxysmal depolarizing shift (PDS) or depolarizing shift is a cellular manifestation of epilepsy. As summarized by Lomen-Hoerth and Messing (2010), brain electrical activity is nonsynchronous under normal conditions. In epileptic seizures, a large group of neurons begin firing in an abnormal, excessive, and synchronized manner, which results in a wave of depolarization known as a paroxysmal depolarizing shift (Somjen, 2004). Normally after an excitatory neuron fires it becomes more resistant to firing for a period of time, owing in part to the effect of inhibitory neurons, electrical changes within the excitatory neuron, and the negative effects of adenosine. However, in epilepsy the resistance of excitatory neurons to fire during this period is decreased, likely due to changes in ion channels or inhibitory neurons not functioning properly. This then results in a specific area from which seizures may develop, known as a "seizure focus".

Increased, abnormal neuron firing causes a wave of depolarization throughout the brain/neuronal tissue. 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 also called paroxysmal depolarizing shift (PDS). The bursting activity resulting from the relatively prolonged depolarization of the neuronal membrane is due to influx of extracellular Ca2+, which leads to the opening of voltage-dependent Na+ channels, influx of Na+, and generation of repetitive action potentials. The subsequent hyperpolarizing afterpotential is mediated by iGABA receptors and Cl- influx, or by K+ efflux, depending on the cell type (Bromfield et al. 2006).


How it is Measured or Detected

Paroxysmal depolarizing shifts can be measured in vitro using patch-clamp recording technique (Kapur 2009) or micro-electrode arrays (Novellino et al. 2011) to determin effects of chemicals on action potential patterns of neurons.

PDS can be detected in vivo using electroencephalography techniques (Niedermeyer and da Silva 2005).


References

Bromfield EB, Cavazos JE, Sirven JI. 2006. An Introduction to Epilepsy [Internet]. West Hartford (CT): American Epilepsy Society; Chapter 1, Basic Mechanisms Underlying Seizures and Epilepsy. Available from: http://www.ncbi.nlm.nih.gov/books/NBK2510/.

Kapur J. 2009. GABA | Pathophysiology of Status Epilepticus. Encyclopedia of Basic Epilepsy Research, pp 304-8.

Lomen-Hoerth C, Messing RO. 2010. Chapter 7: Nervous system disorders. Edited by Stephen J. McPhee, and Gary D. Hammer, Pathophysiology of disease: an introduction to clinical medicine (6th Edition). New York: McGraw-Hill Medical. ISBN 9780071621670.

Novellino A, Scelfo B, Palosaari T, Price A, Sobanski T, Shafer TJ, Johnstone AF, Gross GW, Gramowski A, Schroeder O, Jügelt K, Chiappalone M, Benfenati F, Martinoia S, Tedesco MT, Defranchi E, D'Angelo P, Whelan M. 2011. Development of micro-electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility with neuroactive chemicals. Front Neuroeng. 4:4.

Niedermeyer E, da Silva FL. 2005. Electroencephalography: basic principles, clinical applications, and related fields. Lippincott Williams & Wilkins.

Somjen GG. 2004. Ions in the Brain Normal Function, Seizures, and Stroke. New York: Oxford University Press. p. 167.

 


List of Adverse Outcomes in this AOP

Event: 613: Occurrence, Epileptic seizure

Short Name: Occurrence, Epileptic seizure

Key Event Component

Process Object Action
seizures occurrence

Biological Context

Level of Biological Organization
Individual

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI
honeybee Apis mellifera High NCBI
eisenia fetida eisenia fetida High NCBI
Life Stage Applicability
Life Stage Evidence
Adult High
Sex Applicability
Sex Evidence
Unspecific High

Substance-induced epileptic seizures have been documented in a wide range of species including invertebrates and vertebrates (see Tingle et al. (2003) and Gunasekara et al. (2007) for reviews on the list of aquatic and terrestrial species affected by fipronil). For instance, fipronil can induce seizures in fruit flies (Stilwell et al. 2006) and house flies (Gao et al. 2007).


Key Event Description

Blockage of the GABA-gated chloride channel reduces neuronal inhibition and induces focal seizure. This may further lead to generalized seizure, convulsions and death (Bloomquist 2003; De Deyn et al. 1990; Werner and Covenas 2011). For instance, exposure to fipronil produces hyperexcitation at low doses and convulsion or tonic-clonic seizure and seizure-related death at high doses (Gunasekara et al. 2007; Tingle et al. 2003; Jackson et al. 2009).

As described in Bromfield et al. (2006), sizure propagation, 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. Of equal interest, but less well understood, is 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).


How it is Measured or Detected

Electrophysiological measurements and physical (visual) observation (for mortality) are the methods often used to detect epileptic seizure-related effects (Ulate-Campos et al. 2016). One may also visit http://www.mayoclinic.org/diseases-conditions/epilepsy/diagnosis-treatment/diagnosis/dxc-20117234 for more information on how medical doctors diagnose epilepsy in patients.

Recently, a new technique called micro-electrode array (MEA) recording has been developed and tested both in vitro (Novellino et al. 2011) and ex vivo (Dossi et al. 2014). MEAs, which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide a unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue (Dossi et al. 2014). Thus, MEAs recordings constitute an excellent tool for studying the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events, the mechanisms underlying seizure onset and propagation, and electrophysiological activity of the neurons in response to chemical exposures (Novellino et al. 2011; Dossi et al. 2014).


Regulatory Significance of the AO

As a neurotoxicity endpoint, information with regard to the seizure or epilepsy is often used by regulators such as EPA, FDA and DHS for human and environmental health assessment and regulation of chemicals, drugs and other materials. For instance, the Office of Pesticide Programs (OPP) in US EPA, regulates, monitors and investigates the use of all pesticides in accordance with the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (https://www.epa.gov/laws-regulations/summary-federal-insecticide-fungicide-and-rodenticide-act). Many pesticides like fipronil target the iGABAR causing seizure and mortality. Another example is the regulatory actions of US FDA to ensure drug safety (see https://www.fda.gov/Drugs/DrugSafety/ucm436494.htm).


References

Bloomquist JR. 2003. Chloride channels as tools for developing selective insecticides. Arch. Insect Biochem. Physiol 54(4), 145-156.

Bromfield EB, Cavazos JE, Sirven JI, editors. 2006. An Introduction to Epilepsy [Internet]. West Hartford (CT): American Epilepsy Society. Chapter 1 Basic Mechanisms Underlying Seizures and Epilepsy. Available from: http://www.ncbi.nlm.nih.gov/books/NBK2510/

De Deyn PP, Marescau B, Macdonald RL. 1990. Epilepsy and the GABA-hypothesis a brief review and some examples. Acta Neurol. Belg. 90(2), 65-81.

Dossi E, Blauwblomme T, Nabbout R, Huberfeld G, Rouach N. 2014. Multi-electrode array recordings of human epileptic postoperative cortical tissue.J Vis Exp. (92):e51870.

Gao JR, Kozaki T, Leichter CA, Rinkevich FD, Shono T, Scott JG. 2007. The A302S mutation in Rdl that confers resistance to cyclodienes and limited crossresistance to fipronil is undetectable in field populations of house flies from the USA. Pestic. Biochem. Physiol. 88, 66−70.

Gunasekara AS, Truong T, Goh KS, Spurlock F, Tjeerdema RS. 2007. Environmental fate and toxicology of fipronil. J. Pestic. Sci. 32(3), 189-199.

Jackson D, Cornell CB, Luukinen B, Buhl K, Stone D. 2009. Fipronil Technical Fact Sheet. National Pesticide Information Center, Oregon State University Extension Services,

Novellino A, Scelfo B, Palosaari T, Price A, Sobanski T, Shafer TJ, Johnstone AF, Gross GW, Gramowski A, Schroeder O, Jügelt K, Chiappalone M, Benfenati F, Martinoia S, Tedesco MT, Defranchi E, D'Angelo P, Whelan M. 2011. Development of micro-electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility with neuroactive chemicals.Front Neuroeng. 4:4.

Stilwell GE, Saraswati S, J. Troy Littleton JT, Chouinard SW. 2006. Development of a Drosophila seizure model for in vivo high-throughput drug screening. European J Neurosci. 24, 2211-2222.

Tingle CC, Rother JA, Dewhurst CF, Lauer S, King WJ. 2003. Fipronil: environmental fate, ecotoxicology, and human health concerns. Rev. Environ. Contam Toxicol. 176, 1-66.

Ulate-Campos A, Coughlin F, Gaínza-Lein M, Fernández IS, Pearl PL, Loddenkemper T. 2016. Automated seizure detection systems and their effectiveness for each type of seizure. Seizure. 40:88-101.

Werner FM, Covenas R. 2011. Classical neurotransmitters and neuropeptides involved in generalized epilepsy: a focus on antiepileptic drugs. Curr. Med. Chem. 18(32), 4933-4948.

 


Appendix 2

List of Key Event Relationships in the AOP