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Relationship: 871


The title of the KER should clearly define the two KEs being considered and the sequential relationship between them (i.e., which is upstream and which is downstream). Consequently all KER titles take the form “upstream KE leads to downstream KE”.  More help

GABAergic interneurons, Decreased leads to Synaptogenesis, Decreased

Upstream event
Upstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help
Downstream event
Downstream event in the Key Event Relationship. On the KER page, clicking on the Event name under Upstream Relationship will bring the user to that individual KE page. More help

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes. Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

This table is automatically generated upon addition of a KER to an AOP. All of the AOPs that are linked to this KER will automatically be listed in this subsection. Clicking on the name of the AOP in the table will bring you to the individual page for that AOP. More help
AOP Name Adjacency Weight of Evidence Quantitative Understanding Point of Contact Author Status OECD Status
Inhibition of Na+/I- symporter (NIS) leads to learning and memory impairment adjacent Moderate Low Anna Price (send email) Open for citation & comment TFHA/WNT Endorsed

Taxonomic Applicability

Select one or more structured terms that help to define the biological applicability domain of the KER. In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. Authors can indicate the relevant taxa for this KER in this subsection. The process is similar to what is described for KEs (see pages 30-31 and 37-38 of User Handbook) More help
Term Scientific Term Evidence Link
human Homo sapiens High NCBI
rat Rattus norvegicus High NCBI
mouse Mus musculus High NCBI
Xenopus laevis Xenopus laevis Moderate NCBI

Sex Applicability

Authors can indicate the relevant sex for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of the User Handbook). More help
Sex Evidence
Mixed High

Life Stage Applicability

Authors can indicate the relevant life stage for this KER in this subsection. The process is similar to what is described for KEs (see pages 31-32 of User Handbook). More help
Term Evidence
During brain development High

Key Event Relationship Description

Provide a brief, descriptive summation of the KER. While the title itself is fairly descriptive, this section can provide details that aren’t inherent in the description of the KEs themselves (see page 39 of the User Handbook). This description section can be viewed as providing the increased specificity in the nature of upstream perturbation (KEupstream) that leads to a particular downstream perturbation (KEdownstream), while allowing the KE descriptions to remain generalised so they can be linked to different AOPs. The description is also intended to provide a concise overview for readers who may want a brief summation, without needing to read through the detailed support for the relationship (covered below). Careful attention should be taken to avoid reference to other KEs that are not part of this KER, other KERs or other AOPs. This will ensure that the KER is modular and can be used by other AOPs. More help

Early in cortical development, the GABAergic interneurons have been found to contribute to key aspects of the brain development. A precise balance between excitatory and inhibitory synapses in cortical neurons is crucial for the formation and maturation of the neuronal connections and eventually the proper neural circuitry function. In the cerebral cortex, the young neurons first receive GABAergic depolarizing inputs before forming any synapses (Owens et al., 1999; Tyzio et al., 1999; Hennou et al., 2002), and thus the GABAergic system is believed to be the initial regulator of synaptogenesis.  Indeed, initial depolarizing GABAergic transmission is required for the formation of the glutamatergic synapses and is therefore responsible for the regulation of the balance between excitation and inhibition in the developing cortex (Wang and Kriegstein, 2009; Owens et al., 1999; Tyzio et al., 1999; Hennou et al., 2002; Ben-Ari, 2006). Nascent GABAergic synapses contain both presynaptic and postsynaptic elements, and produce synaptic transmission (Ahmari and Smith, 2002). GABAA receptors form clusters before presynaptic terminals emerge (Scotti and Reuter, 2001), and this clustering occur in the absence of scaffolding proteins and GABA release (Scotti and Reuter, 2001; Christie et al., 2002). Also, during maturation, GABAA receptors become selectively clustered across from terminals that release the neurotransmitter GABA (Craig et al., 1994; Swanwick et al., 2006).

Evidence Supporting this KER

Assembly and description of the scientific evidence supporting KERs in an AOP is an important step in the AOP development process that sets the stage for overall assessment of the AOP (see pages 49-56 of the User Handbook). To do this, biological plausibility, empirical support, and the current quantitative understanding of the KER are evaluated with regard to the predictive relationships/associations between defined pairs of KEs as a basis for considering WoE (page 55 of User Handbook). In addition, uncertainties and inconsistencies are considered. More help
Biological Plausibility
Define, in free text, the biological rationale for a connection between KEupstream and KEdownstream. What are the structural or functional relationships between the KEs? For example, there is a functional relationship between an enzyme’s activity and the product of a reaction it catalyses. Supporting references should be included. However, it is recognised that there may be cases where the biological relationship between two KEs is very well established, to the extent that it is widely accepted and consistently supported by so much literature that it is unnecessary and impractical to cite the relevant primary literature. Citation of review articles or other secondary sources, like text books, may be reasonable in such cases. The primary intent is to provide scientifically credible support for the structural and/or functional relationship between the pair of KEs if one is known. The description of biological plausibility can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured (see page 40 of the User Handbook for further information).   More help

Early in the development of the neocortex, GABAergic interneurons play a role in the formation of spontaneous synchronized activity, which has a fundamental role in the activation of glutamatergic synapses, the synchronization of synaptogenesis and the establishment of long–range cortico-cortical connections (Voigt et al., 2001; 2005). Increasing evidence suggests that GABAergic signaling is the main regulator of this early neuronal activity, as it is established before the glutamatergic one in the neocortex (Wang and Kriegstein, 2009; Owens et al., 1999; Tyzio et al., 1999; Hennou et al., 2002; Ben-Ari, 2006). Despite the fact that GABA is the main inhibitory neurotransmitter in the adult CNS, it exerts depolarizing actions in the immature brain (Ben-Ari et al., 2007), caused by the low levels of Cl- concentration in the post-synaptic cells (Rivera et al., 1999; Ehrlich et al., 1999). K-Cl co-transporter 2 (KCC2) is the main Cl- efflux mechanism with a developmentally-regulated expression profile in the brain and it is therefore thought to be the regulator of GABA signalling during early neuronal development. The effects of KCC2 on the levels of [Cl-]I in immature neurons and the subsequent effects on the shift of the GABA signaling has been extensively studied during the last decades:

• Existing data indicate that KCC2 expressed by GABA neurons is sufficient to shift from the depolarizing and excitatory period of GABA during cortical neuron development (Lee et al., 2005; Chudotvorova et al., 2005) and to effectively decrease the [Cl-]I in immature rat neurons (Chudotvorova et al., 2005).

• Transcriptional repression of KCC2 in rat cortical neurons delayed the GABA switch corresponding to significant changes of [Cl-]I in the same neurons (Yeo et al., 2009).

Several studies focused on the effects of GABA signaling on synaptogenesis and they all had convergent results leading to a strong biological plausibility of this KER.

• Too early shift of GABA-induced excitation-to-inhibition not only affects synaptic integration, but it also results in deficient circuitry development (Wang and Kriegstein, 2008). This has been demonstrated in rodents and mammals cortical neurons in culture.

• Premature GABA switch has also morphological effects in cortical neurons, as it has been shown to drive in fewer and shorter dendrites with defective effects in synaptic formation (Cancedda et al., 2007).

• In the dentate gyrus of the adult hippocampus, newborn granule cells are tonically activated by ambient GABA before being sequentially innervated by GABA- and glutamate-mediated synaptic inputs. GABA initially exerts an excitatory action on newborn neurons owing to their high cytoplasmic Cl- ion content (Ge et al., 2006).

• An early hyperpolarizing shift in Cl reversal potential, by premature expression of KCC2, has been shown to increase the ratio of inhibitory-to-excitatory inputs both in Xenopus tectal neurons and rat cortical neurons in vitro (Chudotvorova et al., 2005; Akerman and Cline, 2006).

The mechanistic details of this relationship are not entirely known, but the most possible mechanism entails a functional relationship between GABA and NMDA receptor activation (Wang and Kriegstein, 2008; Cserép et al., 2012). Cortical neurons begin to express functional NMDA receptors when they migrate to the cortical plate, but these initial glutamatergic synapses are “silent” because of the Mg2+ block of NMDA receptors at the resting membrane potential (LoTurco et al., 1991; Akerman and Cline, 2006). GABAergic depolarization can facilitate relief of this voltage-dependent Mg2+ block and allow Ca2+ entry to initiate intracellular signalling cascades (Leinekugel et al., 1997). This mechanism suggests that the initial depolarizing GABAergic transmission is required for the formation of the glutamatergic synapses and is therefore responsible for the regulation of the balance between excitation and inhibition in the developing cortex (Wang and Kriegstein, 2009). 

Uncertainties and Inconsistencies
In addition to outlining the evidence supporting a particular linkage, it is also important to identify inconsistencies or uncertainties in the relationship. Additionally, while there are expected patterns of concordance that support a causal linkage between the KEs in the pair, it is also helpful to identify experimental details that may explain apparent deviations from the expected patterns of concordance. Identification of uncertainties and inconsistencies contribute to evaluation of the overall WoE supporting the AOPs that contain a given KER and to the identification of research gaps that warrant investigation (seep pages 41-42 of the User Handbook).Given that AOPs are intended to support regulatory applications, AOP developers should focus on those inconsistencies or gaps that would have a direct bearing or impact on the confidence in the KER and its use as a basis for inference or extrapolation in a regulatory setting. Uncertainties that may be of academic interest but would have little impact on regulatory application don’t need to be described. In general, this section details evidence that may raise questions regarding the overall validity and predictive utility of the KER (including consideration of both biological plausibility and empirical support). It also contributes along with several other elements to the overall evaluation of the WoE for the KER (see Section 4 of the User Handbook).  More help

In vivo evidence for the role of GABA in synaptogenesis is controversial. Ji et al., 1999 have shown that in GAD65/67-deficient mice, in which the production of GABA was reduced to less than 5%, the development of brain morphology until birth was normal. These mice die at birth and therefore synaptogenesis and circuit development could not be controlled, however no morphological defects were detected in the neocortex, cerebellum and hippocampus of these animals by the time of their death. The authors of this study suggested that GABA may not be crucial for development. However, functional changes were not assessed in this study. One hypothesis is that glutamate, glycine and taurine could compensate for the lack of GABA (LoTurco et al., 1995; Flint et al., 1998).

In KCC2 knock out mice, apart from lung atelectasis, no other obvious histological changes in the brain were observed in neonatal mice (Hubner et al., 2001). Moreover, these mice died at birth, before the GABA switch takes place, and neuronal electrical activity or synaptogenesis were not evaluated.

Additionally, after premature expression of KCC2 transporter an increase of the excitatory synapses was observed, but the glutamatergic synapses were not affected (Chudotvorova et al., 2005), as in the case of NKCC1 knock out mice (Wang and Kriegstein, 2008). These contradictory results reveal the complexity of the developmental brain and suggest that many different mechanisms are involved in the regulation of the temporal profile of the two main neuronal co-transporters, namely the KNCC1 and KCC2. However, in all cases the importance of Cl- homeostasis in the developmental cortex and its correlation with the proper synapse formation is demonstrated.

Response-response Relationship
This subsection should be used to define sources of data that define the response-response relationships between the KEs. In particular, information regarding the general form of the relationship (e.g., linear, exponential, sigmoidal, threshold, etc.) should be captured if possible. If there are specific mathematical functions or computational models relevant to the KER in question that have been defined, those should also be cited and/or described where possible, along with information concerning the approximate range of certainty with which the state of the KEdownstream can be predicted based on the measured state of the KEupstream (i.e., can it be predicted within a factor of two, or within three orders of magnitude?). For example, a regression equation may reasonably describe the response-response relationship between the two KERs, but that relationship may have only been validated/tested in a single species under steady state exposure conditions. Those types of details would be useful to capture.  More help
This sub-section should be used to provide information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). This can be useful information both in terms of modelling the KER, as well as for analyzing the critical or dominant paths through an AOP network (e.g., identification of an AO that could kill an organism in a matter of hours will generally be of higher priority than other potential AOs that take weeks or months to develop). Identification of time-scale can also aid the assessment of temporal concordance. For example, for a KER that operates on a time-scale of days, measurement of both KEs after just hours of exposure in a short-term experiment could lead to incorrect conclusions regarding dose-response or temporal concordance if the time-scale of the upstream to downstream transition was not considered. More help
Known modulating factors
This sub-section presents information regarding modulating factors/variables known to alter the shape of the response-response function that describes the quantitative relationship between the two KEs (for example, an iodine deficient diet causes a significant increase in the slope of the relationship; a particular genotype doubles the sensitivity of KEdownstream to changes in KEupstream). Information on these known modulating factors should be listed in this subsection, along with relevant information regarding the manner in which the modulating factor can be expected to alter the relationship (if known). Note, this section should focus on those modulating factors for which solid evidence supported by relevant data and literature is available. It should NOT list all possible/plausible modulating factors. In this regard, it is useful to bear in mind that many risk assessments conducted through conventional apical guideline testing-based approaches generally consider few if any modulating factors. More help
Known Feedforward/Feedback loops influencing this KER
This subsection should define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits? In some cases where feedback processes are measurable and causally linked to the outcome, they should be represented as KEs. However, in most cases these features are expected to predominantly influence the shape of the response-response, time-course, behaviours between selected KEs. For example, if a feedback loop acts as compensatory mechanism that aims to restore homeostasis following initial perturbation of a KE, the feedback loop will directly shape the response-response relationship between the KERs. Given interest in formally identifying these positive or negative feedback, it is recommended that a graphical annotation (page 44) indicating a positive or negative feedback loop is involved in a particular upstream to downstream KE transition (KER) be added to the graphical representation, and that details be provided in this subsection of the KER description (see pages 44-45 of the User Handbook).  More help

Domain of Applicability

As for the KEs, there is also a free-text section of the KER description that the developer can use to explain his/her rationale for the structured terms selected with regard to taxonomic, life stage, or sex applicability, or provide a more generalizable or nuanced description of the applicability domain than may be feasible using standardized terms. More help

Most of the available studies have been performed in rodent models and human cortical neurons, referenced in the "Biological plausibility" section.

The relationship between KCC2 function and GABA signalling has been also demonstrated in the retinotectal circuit of Xenopus (Akerman and Cline, 2006).


List of the literature that was cited for this KER description using the appropriate format. Ideally, the list of references should conform, to the extent possible, with the OECD Style Guide (OECD, 2015). More help

Ahmari SE, Smith SJ. (2002). Knowing a nascent synapse when you see it. Neuron. 34:333–336.

Akerman CJ, Cline HT. (2006). Depolarizing GABAergic conductances regulate the balance of excitation to inhibition in the developing retinotectal circuit in vivo. J Neurosci 26: 5117–5130.

Ben-Ari Y. (2006). Basic developmental rules and their implications for epilepsy in the immature brain. Epileptic Disord. Jun; 8(2):91-102.

Ben Ari Y, Gaiarsa JL, Tyzio R, Khazipov R. (2007). GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiol Rev 87:1215–84.

Ben-Ari Y, Khalilov I, Kahle KT, Cherubini E. (2012). The GABA excitatory/inhibitory shift in brain maturation and neurological disorders. Neuroscientist. 18(5):467-486.

Cancedda L, Fiumelli H, Chen K, Poo MM. (2007). Excitatory GABA action is essential for morphological maturation of cortical neurons in vivo. J Neurosci 27: 5224–5235.

Chudotvorova I, Ivanov A, Rama S, Hubner CA, Pellegrino C, Ben-Ari Y, Medina I. (2005). Early expression of KCC2 in rat hippocampal cultures augments expression of functional GABA synapses. J Physiol 566: 671–679.

Christie SB, Miralles CP, De Blas AL. GABAergic innervation organizes synaptic and extrasynaptic GABAA receptor clustering in cultured hippocampal neurons. J Neurosci. 2002;22:684–697.

Craig AM, Blackstone CD, Huganir RL, Banker G. Selective clustering of glutamate and gamma-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters. Proc Natl Acad Sci U S A. 1994;91:12373–12377.

Cserép C, Szabadits E, Szőnyi A, Watanabe M, Freund TF, Nyiri G. (2012). NMDA receptors in GABAergic synapses during postnatal development. PLoS One. 7(5):e37753.

Ehrlich I, Lohrke S, Friauf E. (1999). Shift from depolarizing to hyperpolarizing glycine action in rat auditory neurones is due to age-dependent Cl- regulation. J Physiol. 1:121-137.

Fisher JW, Li S, Crofton K, Zoeller RT, McLanahan ED, Lumen A, Gilbert ME. (2013). Evaluation of iodide deficiency in the lactating rat and pup using a biologically based dose-response model. Toxicol Sci. 132(1):75-86.

Flint AC, Liu X, Kriegstein AR. (1998). Nonsynaptic glycine receptor activation during early neocortical development. Neuron 20: 43–53.

Friauf E, Wenz M, Oberhofer M, Nothwang HG, Balakrishnan V, Knipper M, Löhrke S. (2008). Hypothyroidism impairs chloride homeostasis and onset οφ inhibitory neurotransmission in developing auditory brainstem and hippocampal neurons. Eur J Neurosci 28:2371-2380.

Ge S, Goh EL, Sailor KA, Kitabatake Y, Ming GL, Song H. (2006). GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature 43: 589–593.

Gilbert ME, Hedge JM, Valentin-Blasini L, Blount BC, Kannan K, Tietge J, Zoeller RT, Crofton KM, Jarrett JM, Fisher JW (2013) An animal model of marginal iodine deficiency during development: the thyroid axis and neurodevelopmental outcome. Toxicol Sci 132:177-195.

Hadjab-Lallemend S, Wallis K, van Hogerlinden M, Dudazy S, Nordström K, Vennström B, Fisahn A. (2010). A mutant thyroid hormone receptor alpha1 alters hippocampal circuitry and reduces seizure susceptibility in mice. Neuropharmacol 58(7):1130-9.

Hennou S, Khalilov I, Diabira D, Ben-Ari Y, Gozlan H. (2002). Early sequential formation of functional GABAA and glutamatergic synapses on CA1 interneurons of the rat foetal hippocampus. Eur J Neurosci 16: 197–208.

Hübner CA, Stein V, Hermans-Borgmeyer I, Meyer T, Ballanyi K, Jentsch TJ. (2001). Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition. Neuron. 30:515-524.

Ji F, Kanbara N, Obata K. (1999). GABA and histogenesis in fetal and neonatal mouse brain lacking both the isoforms of glutamic acid decarboxylase. Neurosci Res 33: 187–194.

Lee H, Chen CX, Liu YJ, Aizenman E, Kandler K. (2005). KCC2 expression in immature rat cortical neurons is sufficient to switch the polarity of GABA responses. Eur J Neurosci. 21: 2593-2599.

Leinekugel X, Medina I, Khalilov I, Ben-Ari Y, Khazipov R. (1997). Ca2+ oscillations mediated by the synergistic excitatory actions of GABAA and NMDA receptors in the neonatal hippocampus. Neuron 18: 243–255.

López-Espíndola D, Morales-Bastos C, Grijota-Martínez C, Liao XH, Lev D, Sugo E, Verge CF, Refetoff S, Bernal J, Guadaño-Ferraz A. (2014). Mutations of the thyroid hormone transporter MCT8 cause prenatal brain damage and persistent hypomyelination. J Clin Endocrinol Metab. Dec;99(12):E2799-804.

LoTurco JJ, Blanton MG. Kriegstein AR (1991). Initial expression and endogenous activation of NMDA channels in early neocortical development. J Neurosci 11: 792–799.

LoTurco JJ, Owens DF, Heath MJ, Davis MB, Kriegstein AR. (1995). GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron 15: 1287–1298.

Owens DF, Liu X, Kriegstein AR. (1999). Changing properties of GABAA receptor-mediated signalling during early neocortical development. J Neurophysiol 82: 570–583.

Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kaila K. (1999). The K/Cl co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397: 251–255.

Scotti AL, Reuter H. Synaptic and extrasynaptic gamma -aminobutyric acid type A receptor clusters in rat hippocampal cultures during development. Proc Natl Acad Sci U S A. 2001;98:3489–3494.

Swanwick CC, Murthy NR, Mtchedlishvili Z, Sieghart W, Kapur J. Development of gamma-aminobutyric acidergic synapses in cultured hippocampal neurons. J Comp Neurol. 2006 Apr 10;495(5):497-510

Tyzio R, Represa A, Jorquera I, Ben-Ari Y, Gozlan H, Aniksztejn L. (1999). The establishment of GABAergic and glutamatergic synapses on CA1 pyramidal neurons is sequential and correlates with the development of the apical dendrite. J Neurosci 19: 10372–10382.

Voigt T, Opitz T, De Lima AD. (2001). Synchronous oscillatory activity in immature cortical network is driven by GABAergic preplate neurons. J Neurosci 21: 8895–8905.

Voigt T,Opitz T, deLima AD. (2005). Activation of early silent synapses by spontaneous synchronous network activity limits the range of neocortical connections. J. Neurosci. 25: 4605–4615.

Wang DD, Kriegstein AR. (2008). GABA regulates excitatory synapse formation in the neocortex via NMDA receptor activation. J Neurosci 28: 5547–5558.

Wang DD, Kriegstein AR. (2009). Defining the role of GABA in cortical development. J Physiol. 587:1873-1879.

Westerholz S, de Lima AD, Voigt T. (2010). Regulation of early spontaneous network activity and GABAergic neurons development by thyroid hormone. Neuroscience 168: 573–589.

Westerholz S, de Lima AD, Voigt T. (2013). Thyroid hormone-dependent development of early cortical networks: temporal specificity and the contribution of trkB and mTOR pathways. Front Cell Neurosci 7:121.

Wu Y, Beland FA1, Fang JL. (2016). Effect of triclosan, triclocarban, 2,2',4,4'-tetrabromodiphenyl ether, and bisphenol A on the iodide uptake, thyroid peroxidase activity, and expression of genes involved in thyroid hormone synthesis. Toxicol In Vitro. Apr;32:310-9.

Yeo M, Berglund K, Augustine G, Liedtke W. (2009). Novel repression of Kcc2 transcription by REST-RE-1 controls developmental switch in neuronal chloride. J Neurosci 29:14652–14662.

Yeo M, Berglund K, Hanna M, Guo JU, Kittur J, Torres MD, Abramowitz J, Busciglio J, Gao Y, Birnbaumer L, Liedtke WB. (2013). Bisphenol A delays the perinatal chloride shift in cortical neurons by epigenetic effects on the Kcc2 promoter. Proc Natl Acad Sci U S A. 110(11):4315-20.