Accepted Manuscript Title: Models of cortical malformation - chemical and physical Author: Heiko J. Luhmann PII: DOI: Reference:
S0165-0270(15)00135-1 http://dx.doi.org/doi:10.1016/j.jneumeth.2015.03.034 NSM 7201
To appear in:
Journal of Neuroscience Methods
Received date: Revised date: Accepted date:
12-3-2015 27-3-2015 30-3-2015
Please cite this article as: Luhmann HJ, Models of cortical malformation - chemical and physical, Journal of Neuroscience Methods (2015), http://dx.doi.org/10.1016/j.jneumeth.2015.03.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights Chemical and physical manipulations during early development induce cortical malformations.
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These malformations resemble cortical pathologies in humans with epilepsy. Animal models show cortical hyperexcitability due to excitatory-inhibitory imbalance.
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Animal models may uncover mechanisms of epileptic disorders.
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JNM Special Issue Series on “Models and Methods for Neurological and Psychiatric Diseases”
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Models of cortical malformation - chemical and physical
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Heiko J. Luhmann
Heiko J. Luhmann Institute of Physiology University Medical Center Mainz Duesbergweg 6 D-55128 Mainz Germany
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Number of tables: 1
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Number of pages: 31
+49 6131 39 26070 +49 6131 39 26071 [email protected]
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phone: fax: e-mail:
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Corresponding author:
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Institute of Physiology, University Medical Center of the Johannes Gutenberg University, Duesbergweg 6, D-55128 Mainz, Germany
Keywords: Neocortex, hippocampus, developmental disorders, neuronal migration, epilepsy, model, rat, mouse
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Abstract Pharmaco-resistant epilepsies, and also some neuropsychiatric disorders, are often associated with malformations in hippocampal and neocortical structures. The mechanisms leading to these cortical malformations causing an imbalance between the excitatory and inhibitory system are largely unknown. Animal models using chemical or physical
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manipulations reproduce different human pathologies by interfering with cell generation and neuronal migration. The model of in utero injection of methylazoxymethanol (MAM) acetate
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mimics periventricular nodular heterotopia. The freeze lesion model reproduces
(poly)microgyria, focal heterotopia and schizencephaly. The in utero irradiation model causes
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microgyria and heterotopia. Intraperitoneal injections of carmustine 1-3-bis-chloroethylnitrosurea (BCNU) to pregnant rats produces laminar disorganization, heterotopias and cytomegalic neurons. The ibotenic acid model induces focal cortical malformations, which
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resemble human microgyria and ulegyria. Cortical dysplasia can be also observed following prenatal exposure to ethanol, cocaine or antiepileptic drugs.
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All these models of cortical malformations are characterized by a pronounced hyperexcitability, few of them also produce spontaneous epileptic seizures. This dysfunction results from an impairment in GABAergic inhibition and/or an increase in glutamatergic
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synaptic transmission. The cortical region initiating or contributing to this hyperexcitability may not necessarily correspond to the site of the focal malformation. In some models widespread molecular and functional changes can be observed in remote regions of the brain,
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where they cause pathophysiological activities.
This paper gives a overview on different animal models of cortical malformations, which are
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mostly used in rodents and which mimic the pathology and to some extent the pathophysiology of neuronal migration disorders associated with epilepsy in humans.
Introduction
Cortical malformations are frequently the pathological substrate of epilepsy and can be often found in patients with pharmaco-resistant epilepsy. Models of cortical malformations, which are experimentally induced by chemical or physical manipulations of the developing cortex, resemble in many aspects the pathology and pathophysiology of epilepsy associated with malformations in human cortex. These chemical and physical manipulations interfere with different developmental processes occurring during prenatal and (depending on the species) early postnatal stages: (i) The generation of neurons (neurogenesis) and/or glial cells (gliogenesis) may be impaired. (ii) The insult may induce cell death or the process of programmed cell death (apoptosis) may be modified, e.g. cells destined to die may survive. 3 Page 3 of 32
(iii) Spontaneous activity patterns, which are important for the maturation of cortical networks may be altered. (iv) Most importantly, the tangential migration of inhibitory interneurons and the radial migration of pyramidal cells is often altered following these chemical or physical insults. The resulting malformations can be focal or diffuse extending over large cortical regions, resembling the spectrum of cortical pathologies described in tissue resected from patients with pharmaco-resistant epilepsy. The extent and type of the experimentally induced
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cortical malformation depends more on the time point of the insult in development and not so much on the cause. Therefore, in all animal models the timing of the experimental
manipulation is most critical. The same protocol of chemical or physical insult will cause
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different pathologies when induced at different developmental stages.
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It is mostly unclear how these structural lesions lead to seizure activity and animal models are required to understand the mechanisms of pathogenesis, epileptogenesis, and
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epileptogenicity of cortical malformations. An important and clinically unresolved issue relates to the question, whether the lesion itself is the main epileptogenic zone, or whether subtle molecular, structural and functional disturbances in the region surrounding the obvious
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lesion are the trigger for epileptic discharges (Luhmann et al., 2014).
This review will focus on different animal models, largely in rodents, of acquired cortical
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malformations (Table 1). For a review of genetic models of cortical malformations the reader is referred to the paper by S. Roper and R. Spreafico in this issue. This review will cover
neocortex.
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malformations in the hippocampus, an allocortical structure, and in the cerebral cortex, the
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Human pathology and pathophysiology of cortical malformations One of the first reports on the pathology of cortical malformations associated with epilepsy goes back to Rudolf Virchow, who in 1867 reported in the cerebral cortex of a 44 years old male patient local cell clusters and bulges ("Haufen und Wülste", p. 140) as an excellent example of heterotopia ("ausgezeichnetes Beispiel von Heterotopie") (Virchow, 1867). A more recent overview on the pathology of cortical malformations in humans and some models has been published by Najm et al. (Najm et al., 2007) and Takano (Takano, 2011). Cortical malformations, especially focal cortical dysplasia, represent the most common pathological finding in pediatric epilepsy surgery patients, and range from mild to severe malformations including hippocampal sclerosis (Krsek et al., 2008). A number of molecular and cell physiological data have been obtained in cortical structures from human epileptic patients affected by periventricular nodular heterotopia, subcortical band beterotopia, or focal cortical dysplasia. Immunoblotting and immunoprecipitation analyses in human cortex 4 Page 4 of 32
resected from EEG-verified epileptic and distal nonepileptic areas demonstrated a higher expression of NMDA (NMDA) receptor subunits 1 and 2A/B in epileptic dysplastic cortex compared with the nonepileptic cortex, indicating that an increased NR1-NR2A/B coassembly may contribute to the epileptogenicity of the dysplastic cortex (Mikuni et al., 1999). These observations are supported by coimmunoprecipitation and immunoblotting studies in resected cortical tissues from patients with pharma-coresistant epilepsy associated
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with neocortical dysplasia, which showed an increased coassembly of NR1 and NR2B with PSD-95 when compared with non-epileptic tissue (Ying et al., 2004). A more distinct pattern of NMDA receptor modification has been reported by Finardi et al. (Finardi et al., 2006), who
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observed a selective increase in the NR2B subunit in all cortical dysplasia, but a reduced expression level of NR2A and NR2B subunits in all patients with heterotopia. These data
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suggest that different developmental malformations are associated with distinct alterations in NMDA receptor density and function. Differences in NMDA receptor function have been also
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reported at the cellular level with electrophysiological methods in pediatric cortical dysplasia. Cytomegalic neurons from human cortical dysplasia tissue showed NMDA currents with decreased Mg2+ sensitivity as compared to neurons from non-dysplastic tissue.
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Immunofluorescence analyses revealed a decrease in NR2B subunit expression in cytomegalic neurons and in a number of normal appearing pyramidal neurons from
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dysplastic tissue (André et al., 2004).
Beside these molecular and electrophysiological changes in the glutamatergic system, prominent alterations have been also documented in the structure and function of the
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GABAergic system in human cortical malformations. Using quantitative immunohistochemistry Thom et al. (Thom et al., 2004) reported in cases of grey matter
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heterotopia in postmortem tissue from patients with epilepsy an overall normal density and distribution of GABA-containing interneurons, but morphologically these cells were less organized and more randomly orientated compared to control cortex. In contrast, a "scattering" of GABAergic interneurons has been demonstrated by Calcagnotto et al. in human dysplastic cortex (Calcagnotto et al., 2005). The same authors also showed with patch-clamp recordings from identified neurons a significant decrease in the frequency of spontaneous inhibitory synaptic currents (IPSCs), an increase in the decay-time constant of evoked and spontaneous IPSCs and a decrease in transporter-mediated GABA reuptake function. In slices from cortical tissue resected for the treatment of pharmaco-resistant epilepsy in children (0.2-14 years), Cepeda et al. observed spontaneous GABA-mediated membrane depolarizations, which frequently elicited action potentials, indicating an excitatory role of GABA in pediatric cortical dysplasia (Cepeda et al., 2007). This assumption is supported by a report on changes in the expression of the chloride transporters KCC2 and 5 Page 5 of 32
NKCC1 in neocortical tissue resected in children with intractable focal epilepsy using quantitative western blot analyses (Jansen et al., 2010). A significant decrease in the mRNA and protein levels of the chloride outward transporter KCC2 has been demonstrated in human dysplastic tissue (Shimizu-Okabe et al., 2011), indicating that the chloride reversal potential is more depolarized, as reported by Cepeda et al. (Cepeda et al., 2007). Finally, beside deficits in glutamatergic and GABAergic synaptic function contributing to the
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epileptogenicity of cortical malformations, abnormal intrinsic membrane properties have been also reported dysplastic cortex (Cepeda et al., 2003).
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In the following paragraphs seven animal models of acquired cortical malformations
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associated with hyperexcitability or epileptic seizures will be reviewed. The MAM model
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The model of in utero injection of methylazoxymethanol (MAM) acetate into pregnant rats has been introduced by Spatz and Laqueur in 1968 (Spatz and Laqueur, 1968). Singh (Singh, 1977) documented in more detail the resulting hippocampal malformations, which are
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similar to periventricular nodular heterotopia in human patients with drug-resistant focal epilepsy. MAM is a potent cytotoxic agent, which induces a time-specific aberrant DNA methylation and alkylation leading to abnormal patterns of cell formation and migration. MAM
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seems to selectively affect developing brain structures and not other embryonic organs undergoing cell proliferation at the time of its administration. Neuronal precursors that are undergoing their final mitosis at the time of MAM exposure are specifically ablated, glial cells
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are only indirectly affected. Noctor et al. (Noctor et al., 1999) reported in the ferret neocortex, that injection of MAM during embryogenesis produces distorted radial glial cells and
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concluded that this interference with early cortical development causes premature differentiation of radial glial cells into astrocytes. Beside its antimitotic effects, MAM also seems to directly influence neuronal migration. The migration speed and the exploratory behavior of migrating neurons is significantly reduced after MAM treatment (Abbah and Juliano, 2014). MAM also modifies the migration pattern of interneurons, but this deficit is not intrinsic to the migrating neurons, but rather mediated by cues, most likely GABA, dictating proper orientation of interneurons migrating into the cortex (Poluch et al., 2008). For a recent overview on the role of GABA in controlling neuronal migration see review by Luhmann et al. (Luhmann et al., 2015). The site and type of malformations depends critically on the time of MAM administration. Concentrations between 10 and 30 mg/kg maternal body weight have been used. When a single (25 mg/kg) or a double (15 mg/kg each at 12 h interval) injection of MAM is given on embryonic day (E) 15, malformations are mostly present in the hippocampal CA1 and CA2 region, to a lesser extent also in the striatum, thalamus, 6 Page 6 of 32
hypothalamus and cerebral cortex. Battaglia et al. (Battaglia et al., 2003) demonstrated that the MAM-induced ablation of early generated neurons is sufficient to change the migration and differentiation of subsequently generated neurons, which then form the different heterotopia. For a comprehensive overview on the cellular actions of MAM and its timedependent effects on different brain regions the reader is referred to a review by Cattabeni and Di Luca (Cattabeni and Di Luca, 1997). Experimental details on the MAM models can be
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found in the review by Battaglia and Bassanini (Battaglia and Bassanini, 2006).
The MAM induced heterotopia are generally localized along the border of the lateral
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ventricles and share structural features with the periventricular nodules in human
periventricular or subcortical nodular heterotopia. Cells in hippocampal heterotopia reveal
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neuronal morphology and do not stain with immunohistochemical markers for glia (Baraban et al., 2000). It has been suggested that neurons in hippocampal heterotopia are originally
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destined to the cerebral cortex (Chevassus et al., 1998b;Chevassus et al., 1998a;Castro et al., 2001), but this hypothesis needs to be proven by immunohistochemical markers specific for neocortical layers. Intracellular and whole-cell patch-clamp recordings in hippocampal
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slices were performed to study the electrophysiological properties of dysplastic neurons in MAM-treated animals. Baraban and Schwartzkroin (Baraban and Schwartzkroin, 1995) could not find any significant difference in resting membrane potential, time constant, input
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resistance, action potential amplitude and duration of CA1 neurons recorded in MAM-treated animals when compared to controls. However, a higher percentage of neurons (62% vs. 10%) fired an intrinsic burst of action potentials in response to suprathreshold current
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injection. Intrinsic burst firing of neurons located in the heterotopia has been also reported by Sancini et al. (Sancini et al., 1998). Abnormal intrinsic firing properties with sustained
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repetitive bursts of action potentials have been further observed in subcortical heterotopic nodules (Colacitti et al., 1999). In situ hybridization and immunohistochemical studies demonstrated in heterotopic cell regions of the hippocampus of MAM-exposed rats a pronounced reduction in the expression of the Kv4.2 subunit of the A-type voltage-dependent potassium channel (Castro et al., 2001). Castro et al. also reported that heterotopic neurons lack functional A-type Kv4.2 potassium channels and show hyperexcitable firing, indicating that heterotopic neuronal clusters may trigger epileptiform activity. The experimental data so far suggest that this intrinsic hyperexcitability is mostly mediated by alterations in voltagedependent potassium channels. Voltage-dependent calcium currents of the L-, T-, N-, P/Q and P-type in hippocampal heterotopic neurons show similar activation and inactivation kinetics and pharmacological sensitivity to nifedipine, amiloride, omega-conotoxin GVIA, omega-agatoxin KT, and sFTX-3.3 as control neurons (Calcagnotto and Baraban, 2003).
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The important question, whether neurons in heterotopic clusters are connected with neurons located in other brain regions, has been addressed by a number of groups with anatomical and electrophysiological techniques. Using anterograde and retrograde tract tracing, Colacitti et al. (Colacitti et al., 1998) demonstrated the existence of short- and long-range reciprocal connections between neocortical heterotopia and ipsilateral as well as contralateral cortical areas. Furthermore abnormal cortico-hippocampal and cortico-cortical connections were
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observed in these neocortical heterotopia. Similar abnormal connections have been reported in hippocampal CA3 pyramidal neurons of MAM-treated rats, which showed an exuberant
mossy fibres innervation with ectopic mossy boutons on their basal dendrites (Chevassus-
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Au-Louis et al., 1999). The same authors verified with staining of the activity marker c-fos the functional connectivity between hippocampal and neocortical regions (Chevassus et al.,
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1998b). Further support for the existence of functional connections between the limbic system and the cerebral cortex in MAM-treated animals comes from patch-clamp recordings
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from intrahippocampal heterotopic as well as from neocortical and hippocampal neurons (Chevassus et al., 1998a). Heterotopic neurons are functionally integrated in both neocortical and hippocampal networks and receive mono- and polysynaptic pathophysiological inputs
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from these structures. This observation has been confirmed with extracellular field potential recordings in hippocampal slices from MAM-exposed animals, which demonstrated in the bicuculline and 4-aminopyridine model that heterotopic neurons can generate epileptiform
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activity, which is transmitted to the neocortex and hippocampus (Baraban et al., 2000). Spontaneous epileptiform discharges were also observed in the majority of slices from MAMtreated animals when the excitability was only slightly increased by elevation of the
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extracellular potassium concentration from 3 to 6 mM (Baraban and Schwartzkroin, 1995). A study by Tschuluun et al. (Tschuluun et al., 2005) confirmed with neuroanatomical and single
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cell electrophysiological methods the bidirectional connectivity between the heterotopia and the hippocampus and cerebral cortex, but failed to identify the heterotopia as trigger for epileptiform activity and to demonstrate a propagation of this activity to the cerebral cortex. Using c-fos activation in organotypic hippocampal slice cultures from MAM treated rats Doisy et al. (Doisy et al., 2015) recently demonstrated that nodular heterotopia are not responsible for hyperexcitability and do not trigger epileptiform activity in this in vitro culture model.
Beside abnormal connectivity and intrinsic pathophysiology, neurons in heterotopia are also characterized by modifications in their synaptic properties. Using whole-cell patch-clamp recordings Calcagnotto and Baraban (Calcagnotto and Baraban, 2005) demonstrated in hippocampal heterotopic neurons a significant increase in the amplitude and slower decaytime constant of the N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic current (EPSC) component as compared to normotopic pyramidal cells. In 8 Page 8 of 32
contrast, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor mediated EPSC was not different in both groups. The authors propose changes in the composition and function of the NMDA receptor subunits in MAM-treated animals. Changes in the distribution and expression of glutamate receptors have been reported by in situ hybridization and immunohistochemistry, but these demonstrated in the heterotopiae a 26% reduction in the expression of the NMDA NR1 subunit and a 40% increase in the expression
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of the AMPA GluR2 flip subunit (Rafiki et al., 1998). A selective impairment of both the targeting and the alphaCaMKII-dependent phosphorylation of the NR2A/B subunits in the
postsynaptic membranes of heterotopic pyramidal cells have been demonstrated by Gordoni
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et al. (Gardoni et al., 2003).
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Inhibitory synaptic function is also changed in hippocampal slices from rats exposed to MAM in utero. The decay time constants of spontaneous and evoked inhibitroy postsynaptic
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currents (IPSCs) were increased by about 200%, whereas IPSC amplitude or rise time were unchanged (Calcagnotto et al., 2002). Based on immunohistochemical staining for the GABA transporters GAT-1 and GAT-3 and pharmacological experiments with the GABA transport
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inhibitors tiagabine and NO-711 the authors conclude that GABA reuptake may be reduced in heterotopic neurons leading to an enhanced GABAergic function. However, it remains to be studied whether this increase in GABAergic function in the MAM model is associated with
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an increase in inhibitory function or due to possible alterations in chloride transporter function leading to a depolarizing or excitatory action of GABA.
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Spontaneous epileptic seizures have so far not been reported in MAM-treated animals. However, as in many other models, this question has yet not been studied in detail in the
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MAM model. Long-term, preferentially multi-site telemetric EEG recordings or depth electrode recordings combined with continuous video monitoring over several days and in different age groups are required to unequivocally answer the question, whether MAMtreated animals show spontaneous epileptic seizures. Two months old rats exposed to MAM in utero showed a significantly lower threshold for flurothyl-induced seizures (Baraban and Schwartzkroin, 1996). Furthermore, shorter seizure latencies correlated with an increased number of heterotopic neurons. A lower threshold for the induction of epileptic seizures in MAM-treated animals have been also reported in the kainic acid and bicuculline model (Defeo et al., 1995;Germano and Sperber, 1997) and in hyperthermia (Germano et al., 1996). Using an experimental 'double-hit' model of prenatal MAM exposure and postnatal pilocarpine-induced status epilepticus Colciaghi et al. (Colciaghi et al., 2011) could show that MAM-treated rats develop more severe epilepsy than non-MAM animals. This enhanced seizure activity in the MAM group was associated with cortical malformations, abnormally 9 Page 9 of 32
large cortical pyramidal neurons with neurofilament overexpression and recruitment of NMDA receptor subunits to the postsynaptic membrane.
In summary, the large amount of molecular, anatomical and physiological data clearly demonstrate pathological alterations in hippocampal and neocortical regions of MAM-treated animals. These malformations mimic the pathology of periventricular nodular heterotopia
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described in humans suffering from severe forms of epilepsy. It remains to be studied in more detail whether spontaneous epileptic seizures are present in this model and, if yes,
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whether anticonvulsants may have an effect.
The freeze (cryogenic) lesion model
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The first report of the cortical freeze lesion model goes back to 1883, when Openchowski (Openchowski, 1883) demonstrated that local cooling of the neocortical surface of a dog
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produced focal lesions and cortical dysfunction. However, only adult animals were used in the study of Openchowski as well as in a small number of subsequent studies performed over the following 70 years (see (Balthasar, 1957). The freeze lesion model in the cerebral
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cortex of newborn animals has been introduced by Dvorak and Feit (Dvorak and Feit, 1977) and Dvorak et al. (Dvorak et al., 1978), who studied the consequences of a focal cortical freeze lesion in rats shortly after birth. This model can be easily established and reproduces
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the pathology of certain types of human neuronal migration disorders associated with epileptic seizures, e.g. polymicrogyria, focal heterotopia, cortical dysplasia or a cortical cleft
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(schizencephaly). In most experimental studies the focal neocortical malformation consisted of a four-layered microgyrus, as described in human pathology (McBride and Kemper, 1982).
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For a clinical classification system of focal cortical dysplasias proposed by an International League Against Epilepsy task force consortium see Blümcke et al. (Blümcke et al., 2011). The neocortical freeze lesion model has been widely used in rats, but comparable results can be obtained in mice (Supèr et al., 1997;Rosen et al., 1995). As in the MAM model, the animal´s age of the experimental induction of the freeze lesion is very critical. At different ages the identical lesion protocol produces very different cortical pathologies. In adult rodents, the freeze lesion model has been used as a model for traumatic brain injury or brain edema (Stoffel et al., 2001;Murakami et al., 1999;Chan et al., 1987). For the induction of the typical focal cortical malformations, best reproducible results can be obtained when the lesion is induced during the first 24 hours after birth. Recently prenatal freeze lesioning has been performed at E18 in utero (Kamada et al., 2013;Takase et al., 2008). A detailed experimental protocol has been given previously by the author of this review (Luhmann, 2006). In brief, a stainless or copper probe with a tip diameter of 1 mm is cooled with liquid nitrogen and applied for 8-10 s on the exposed calvarium above the neocortical area of 10 Page 10 of 32
interest, mostly the parietal cortex. Smaller (0.5 mm) and larger (2 mm) probes can be also used, but depending on the application time will induce other malformations (Rosen and Galaburda, 2000;Ferrer et al., 1993). The severity and type of the cortical malformation is directly related to the number and duration of freeze lesions, indicating that a single patho(physio)logical event of variable severity at a distinct developmental stage can induce different malformations (Rosen and Galaburda, 2000). Larger freezing probes and freezing
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times (20-30 s) result in the formation of a neocortical cleft resembling the pathology of schizencephaly described in humans. The cooled probe may be positioned in an identical
manner at other locations, producing for example a 4-6 mm long microsulcus in rostro-caudal
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direction. Sham-operated animals are treated the same way as freeze-lesioned animals with the exception that the copper cylinder is not cooled. The mortality of this model is below 5%
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of treated animals.
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The most consistent result of a focal cortical freeze in the newborn rodent is the formation of a four-layered microgyrus (Dvorak and Feit, 1977;Dvorak et al., 1978), which occurs in two stages. In the first stage neurons and glial cells are destroyed by the freezing procedure,
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followed by a regrowth of damaged radial glial fibers and repair of the damaged cortical region by migrating neurons. At the day of birth marginal zone/layer 1 and layers 4 to 6 are already present and the freezing procedure induces here a focal necrotic lesion, which is
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invaded within 24 hours by reactive astrocytes and macrophages (Rosen et al., 1992). A pronounced increase in GFAP immunoreactivity and clusters of proliferative BrdU-positive astrocytes have been demonstrated at the site of the lesion (Bordey et al., 2001). Whole-cell
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patch-clamp recordings from BrdU-positive astrocytes in slices from freeze-lesionded animals demonstrated a lack of KIR channel expression and an increase in delayed rectifier K+ (KDR)
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channels. Furthermore these proliferative astrocytes showed almost no dye coupling, suggesting a dysfunction in K+ buffering (Bordey et al., 2001). In the second stage migrating neurons destined to form layer 2/3 invade the necrotic core. In freeze-lesioned cortex upper layer Cajal-Retzius neurons survive for longer developmental periods (Supèr et al., 1997) and radial glial cells are preserved in adult cortex (Rosen et al., 1994). Furthermore, neurogenesis in response to the lesion may take place postnatally (Rosen et al., 1996). The damaged cortex begins to assume its adult-like microgyric appearance from P5 to P10. The microgyrus and the paramicrogyral zone is characterized by an abnormal organization of afferent and efferent connections. Thalamocortical as well as corticothalamic projections are markedly reduced and disorganized (Jacobs et al., 1999b;Rosen et al., 2000). Furthermore, callosally projecting pyramidal neurons located in the paramicrogyral zone reveal a different laminar distribution compared to control animals, indicating disturbances in interhemispheric interactions (Giannetti et al., 1999). Autoradiographic measurements demonstrated a 11 Page 11 of 32
significant reduction of basic cortical [14C]deoxyglucose metabolism up to 1 mm lateral to the lesion (Kraemer et al., 2001). This decrease in basic metabolism is accompanied by a reduction in Na+/K+-ATPase in the paramicrogyral cortex as demonstrated by immunohistochemistry and in situ hybridization expression of the alpha3 isoform of Na+/K+ATPase (Chu et al., 2009). In adult freeze-lesioned rats functional magnetic resonance imaging showed a significantly reduced cortical activation in the lesioned hemisphere
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following sensory stimulation of the sensory periphery (Schwindt et al., 2004), further
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supporting the reduction and disorganization of thalamocortical afferents described above.
A number of electrophysiological studies on neocortical slices from freeze-lesioned rats
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demonstrated a pronounced hyperexcitability in the cortical region surrounding the microgyrus (Jacobs et al., 1996;Luhmann and Raabe, 1996;Hablitz and DeFazio, 1998). These data indicate that the neocortical region adjacent to the microgyrus and not the
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microgyrus itself is the generator for epileptiform activity. This paramicrogyral zone shows a number of molecular and functional modifications, which may contribute to this hyperexcitability. Layer 5 pyramidal neurons located 1-2 mm from the microgyrus showed a
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hyperpolarized resting membrane potential, increased input resistances and a smaller inwardly rectifying hyperpolarization-activated h-current (Albertson et al., 2011). The
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reduction in h-current contributes to increased single cell and network excitability in cortical dysplasia. Local electrical stimulation of the paramicrogyral zone elicited epileptiform responses that propagated over >4 mm in horizontal direction (Luhmann and Raabe, 1996),
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indicating widespread structural and/or functional modifications in the lesioned cortical hemisphere. Application of a NMDA antagonist blocked the late recurrent component of the
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propagating activity in field potential recordings (Luhmann et al., 1998b) and intracellular recordings from supragranular neurons (Luhmann et al., 1998a). An overabundance of excitatory inputs to the paramicrogyral zone has been postulated by Jacobs et al. (Jacobs et al., 1999a) and subsequently demonstrated with various techniques. Spontaneous and miniature EPSCs recorded from pyramidal neurons adjacent to a microgyrus were significantly increased in frequency, suggesting that these neurons receive more excitatory synaptic inputs compared to neurons in control cortex (Jacobs and Prince, 2005). An increased EPSC frequency was also documented by combining laser scanning photostimulation with glutamate uncaging (Brill and Huguenard, 2010). This hyperinnervation of pyramidal neurons by excitatory afferents occurs at an age before onset of cortical epileptiform activity (Zsombok and Jacobs, 2007).
Using quantitative in vitro receptor autoradiography of glutamatergic receptors demonstrated an up-regulation of AMPA receptors over the whole lesioned hemisphere and an increase in 12 Page 12 of 32
NMDA and kainate receptors in the paramicrogyral zone (Zilles et al., 1998). However, neither receptor autoradiography nor immunohistochemical stains for NMDA receptor subunits (Hagemann et al., 2003) revealed any widespread alterations in remote cortical regions, indicating that NMDA receptors are mainly increased in density and/or affinity in the microgyrus itself and in the immediate surrounding tissue. These molecular data are supported by in vitro electrophysiological and neuropharmacological experiments, which
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demonstrated that NR2B-containing NMDA receptors are functionally enhanced in freezelesioned cortex (Defazio and Hablitz, 2000) and, as shown by voltage-sensitive dye imaging, contribute to the local spread of paroxysmal activity in freeze lesioned cortex
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(Bandyopadhyay and Hablitz, 2006). Pathophysiological increases in NMDA receptor
function are further mediated by changes of glutamate transporters, specifically the glia
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glutamate transporter (GLT-1), which in dysplastic cortex causes larger tonic NMDA currents
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(Campbell and Hablitz, 2008).
Beside molecular, structural and functional changes in the glutamatergic system, prominent alterations in the GABAergic system also contribute to the hyperexcitability in freeze lesioned
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cortex. A reduction in the expression of parvalbumin immunoreactivity has been documented in layers 2/3 within the microgyrus and up to 2 mm adjacent to it, but this disinhibition was only transient and disappeared by P21 (Rosen et al., 1998). In adult cortex, the widespread
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functional changes described above are not related to a general loss of inhibitory interneurons (Schwarz et al., 2000). A significant decrease in the number of immunoreactive neurons for parvalbumin, calretinin and calbindin has been reported only for the region within
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the microsulcus, but not for the paramicrogyral zone or remote cortical regions (Hablitz and DeFazio, 1998). A subpopulation of GABAergic interneurons that co-express the GluR1
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subunit and the calcium-binding protein calbindin is also reduced within the microgyrus (Kharazia et al., 2003). In contrast to these more local changes, which are restricted to the lesion itself, quantitative in vitro receptor autoradiography revealed a widespread downregulation of GABA-A receptors (Zilles et al., 1998). Immunohistochemical studies for the GABA-A receptor subunits alpha1, alpha2, alpha3, alpha5 and gamma2 confirmed this widespread down-regulation in GABAergic function (Redecker et al., 2000). Furthermore, the downregulation of GABA-A receptor subunits even involved the ipsilateral hippocampal formation and restricted contralateral neocortical areas (Redecker et al., 2000). A decrease in the alpha subunit composition of the GABA-A receptor has been also demonstrated by recordings of miniature IPSCs and by a reduced affinity for zolpidem in layer 2/3 pyramidal cells located 1-2 mm lateral to the microgyrus (Defazio and Hablitz, 1999). These modifications would result in an elimination of the type 1 benzodiazepine receptor, which
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would be of relevance for pharmacological treatment of epileptic seizures associated with cortical dysplasia.
Whereas intracellular recordings from layer 2/3 cells in the paramicrogyral zone suggested a decrease of glutamatergic inputs onto inhibitory interneurons (Luhmann et al., 1998a), an increase of functional excitatory synapses on both interneurons and pyramidal cells has been
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also demonstrated (Jacobs and Prince, 2005). Recordings from pyramidal neurons located 1-2 mm lateral of the microgyrus showed for layer 5 an increased excitatory input in freeze lesioned cortices, while no significant differences were seen in layer 2/3 cells (Brill and
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Huguenard, 2010), indicating layer-specific modifications in connectivity.
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An upregulation in the mRNA expression of the chloride inward transporter NKCC1 and a simultaneous downregulation of the chloride outward transporter KCC2 has been
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demonstrated by in situ hybridization histochemistry in the upper layers of the microgyrus, indicating disturbances in intracellular chloride homeostasis (Shimizu-Okabe et al., 2007). More recently a downregulation of KCC2 was documented for the microgyrus forming
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GABAergic and E17.5 born glutamatergic neurons at P4, which resulted in GABA-A receptormediated calcium oscillations in microgyrus forming cells (Wang et al., 2014). The imbalance in excitatory and inhibitory synaptic transmission in freeze-lesioned cortex also results in
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interesting alterations of synaptic plasticity. Field potential recordings in layer 2/3 demonstrated an increased long-term potentiation (LTP) following theta-burst stimulation in layer 6, but a lack of LTP in layer 4 (Peters et al., 2004). These layer-specific modifications in
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synaptic plasticity may result from the reduction in GABA-A receptor gamma2 subunit
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expression (Peters et al., 2004).
As in the MAM model, spontaneous seizures cannot be observed without a provoking event in adult rodents freeze-lesioned in the first postnatal days (for review (Luhmann, 2009;Luhmann, 2006). However, the threshold temperature to elicit hyperthermia-induced seizures is significantly reduced in freeze-lesioned immature rats as compared to shamtreated controls (Scantlebury et al., 2004). A novel rat model with multiple focal cortical dysplasia has been created by Takase et al. (Takase et al., 2008) by freeze lesioning at embryonic day (E) 18 through the uterus wall. At adult stages these animals revealed in the EEG spontaneous epileptic spikes and a faster development of hippocampal kindling following electrical stimulation. Furthermore, these animals showed an upreglation of the NMDA receptor subunits NR1 and NR2B and a rise in the expression of glutamate/aspartate transporters (Takase et al., 2008). A subsequent study of the same lab using the identical model of E18 freeze lesioning confirmed the expression of spontaneous seizures in a high 14 Page 14 of 32
percentage (69%) of treated animals and the increase in the expression levels of NMDA receptor subunits NR1, NR2A, NR2B, glutamate/aspartate transporter and glial glutamate transporter 1 (GLT1) (Kamada et al., 2013).
A recent study by Andresen et al. (Andresen et al., 2014) reported that a one-week treatment with the anticonvulsant and analgesic gabapentin immediately after freeze lesioning
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prevented the development of hyperexcitability in vitro and in vivo. This anti-epileptogenic effect of gabapentin is mediated by its inhibitory action on thrombospondin and alpha 2 delta1 signaling, the latter representing an auxiliary calcium channel subunit, which are both
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upregulated during the formation of the microgyrus (Andresen et al., 2014).
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In summary, the freeze lesion model produces a pathology which resembles cortical dysplasia reported in humans. A large number of experimental studies have demonstrated
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wide-spread molecular and functional changes in paramicrogyral regions remote from the lesion itself. This pathology may resemble the human condition, since clinical studies have demonstrated that the epileptogenic zone is usually more extensive than the structural lesion
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(Palmini et al., 1994;Palmini et al., 1991) (for review (Luhmann et al., 2014). The novel in utero freeze lesion model produces spontaneous epileptic seizures in vivo and this model requires further investigations by other groups. It will be an experimental challenge to study
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in this model the process of epileptogenesis and early therapeutical interventions. Of further interest is the differential role of GABA-A mediated synaptic phasic inhibition versus extrasynaptic tonic inhibition, which are mediated by gamma-subunit and delta-subunit
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containing receptors, respectively (for review (Kilb et al., 2013). The experimental studies obtained in animal models suggest region and layer specific changes in the expression of
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these subunits in dysplastic cortex. The in utero irradiation model
This model has been initially used in rats to study the consequences of radiation exposure on early brain development (Riggs et al., 1956) and more specifically on neuronal migration (Altman et al., 1968). Mostly rodents have been used as experimental animals, but Algan and Rakic (Algan and Rakic, 1997) applied ionizing irradiation at selected prenatal stages in macaque monkeys to delete specific neuronal cell populations in the visual cortex and to study the consequences on the development of cortical cytoarchitecture. A detailed description of the methods in rats is given in the review by Lin and Roper (Lin and Roper, 2006). Usually pregnant rats on E17 are exposed to 145 to 225 cGy of gamma-irradiation and offspring are used for experiments. Lower doses (e.g. 100 cGy) induce minor pathological changes (Kellinghaus et al., 2004), higher doses (e.g. 300 cGy) elicit severe 15 Page 15 of 32
malformations such as absence of corpus callosum (Lent and Schmidt, 1986). Different cortical malformations can be produced in rats with irradiation given at different embryonic stages (Ferrer et al., 1984). Irradiation with a single dose of 200 cGy on E14 causes large cortical ectopic cell clusters (Ferrer, 1993). A four-layered microgyrus can be produced by irradiation on E16 and segmentation of the cerebral cortex can be observed after irradiation on E15, E17 or E19 (Ferrer, 1993). Beside these focal cortical malformations, which depend
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on the dose and on the time point of irradiation, gross cytoarchitectural abnormalities have been described in the somatosensory cortex of adult rats subjected to this in utero treatment. The cortical representation of the whiskers in primary somatosensory cortex, the so-called
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barrel cortex (for review (Feldmeyer et al., 2013), is severely disturbed and structural barrels
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fail to develop, despite a normal functional thalamocortical input (Ito, 1995).
Whereas the overall neuronal density in adult dysplastic cortex is not significantly different
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compared to controls, the density of parvalbumin and calbindin immunoreactive inhibitory interneurons is significantly reduced in irradiated cortex (Roper et al., 1999). Stereological measurements in the cerebral cortex on E21 and P6 showed that the total number of
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neurons and of GABAergic neurons is reduced by about 50% of controls at both time points (Deukmedjian et al., 2004). Whereas the total number of neurons doubled during this developmental period in both control and irradiated animals, the number of GABAergic cells
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did not increase in treated rats. Since GABAergic neurons in the controls increased by the factor of 10 between E21 and P6, these data suggest that the GABAergic system is irreversibly damaged by in utero radiation (Deukmedjian et al., 2004). Using antibodies to
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vesicular glutamate transporter 1 (VGLUT1), vesicular glutamate transporter 2 (VGLUT2), vesicular GABA transporter (VGAT), and parvalbumin to quantify glutamatergic and
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GABAergic presynaptic terminals, Zhou and Roper (Zhou and Roper, 2010) demonstrated in dysplastic cortex an overall increase in excitatory synaptic connectivity and decrease in inhibitory synaptic connectivity.
As other models of focal cortical malformations, the in utero irradiation model is also characterized by a pronounced hyperexcitability when studied with in vitro electrophysiological methods. Neocortical slices from adult rats exposed to embryonic irradiation show more robust epileptiform activity compared to controls when treated with bicuculline (Roper et al., 1997;Roper, 1998). Whole-cell patch-clamp recordings from pyramidal neurons demonstrated in dysplastic cortex a reduction in the frequency of spontaneous and miniature IPSCs and in the amplitude of spontaneous IPSCs (Zhu and Roper, 2000). Monosynaptic evoked IPSCs were also impaired. Fast-spiking inhibitory interneurons recorded in layer 4 dysplastic cortex show a reduction in the frequency of 16 Page 16 of 32
miniature and spontaneous IPSCs (Zhou et al., 2009). Recordings from identified GABAergic interneurons immunoreactive for calretinin, somatostatin and parvalbumin revealed a reduction in excitatory drive for the two latter cell types in dysplastic cortex (Zhou and Roper, 2011). These data indicate a prominent impairment in the function of intracortical GABAergic inhibition (Zhu and Roper, 2000). The mechanisms underlying this impairment were examined in more details by Zhou and Roper (Zhou and Roper, 2014) by paired recordings
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from various types of identified neuronal types. This study showed a reduced synaptic efficiency of GABAergic inhibition originating from fast-spiking, parvalbumin-positive
interneurons to their postsynaptic target cell. Interestingly, electrical, gap junctions mediated
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connections within this cell type were also impaired (Zhou and Roper, 2014).
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Whereas the intracortical GABAergic system is functionally reduced in adult animals treated by irradiation during embryonic stages, the glutamatergic excitatory system seems to be
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enhanced. Spontaneous EPSCs recorded in pyramidal neurons are increased in amplitude and frequency in dysplastic cortex (Zhu and Roper, 2000). Presynaptic release probability of excitatory synapses is significantly enhanced, most likely due to a reduced tonic activity of
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presynaptic metabotropic glutamate receptors of the type GluR2/3 (Chen et al., 2007). However, these changes appear to be specific for glutamatergic synaptic connections between excitatory neurons, since miniature and spontaneous EPSCs recorded in
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GABAergic interneurons are significantly reduced in frequency (Xiang et al., 2006;Zhou et al., 2009), suggesting that inhibitory interneurons receive less excitatory drive in dysplastic
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cortex.
Using epidural electrodes Roper et al. (Roper et al., 1995) demonstrated in irradiated rats an
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increased propensity for seizures in the presence of the anaesthetic agents acepromazine and xylazine, or a combination of both drugs. Prolonged continuous EEG recordings with bifrontal epidural and hippocampal depth electrodes uncovered spontaneous seizures arising independently from the hippocampus or the frontal neocortex (Kondo et al., 2001). Using the same long-term EEG recordings in combination with video monitoring revealed interictal spikes in rats treated at E17 with low- or medium-dose radiation causing mild or moderate malformations, but not in severe radiation-treated animals (Kellinghaus et al., 2004). Spontaneous epileptiform spikes and seizures on EEG have been also demonstrated during the chronic phase in a second hit model combining irradiation at E17 and a low dose of pentylenetetrazole at adult stages (Oghlakian et al., 2009).
In summary, the utero irradiation model dose-dependently mimics different types of cortical malformations. In rats, medium-dose radiation at E17 produces focal cortical malformations, 17 Page 17 of 32
which can generate spontaneous seizures. The mechanisms underlying this hyperexcitability appear to be largely mediated by an impairment in intracortical GABAergic inhibition. It remains to be studied in more detail whether the dysplasia itself or the region surrounding the malformation represent the trigger zone for hyperexcitability and epileptic seizures. The BCNU model
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Intraperitoneal injections of carmustine 1-3-bis-chloroethyl-nitrosurea (BCNU) to pregnant rats on E15 produce cortical dysplasia in the offspring consisting of laminar disorganization, cytomegalic neurons, heterotopias and clusters of Cajal-Retzius cells (Benardete and
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Kriegstein, 2002). Furthermore radial glial fibers are disrupted (Moroni et al., 2011), as also reported in the freeze lesion model. Thus the BCNU model mimics the pathology of cortical
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dysplasia in human patients, such as altered cortical layering, presence of heterotopia and dysmorphic neurons. In cortical slices exposed to bicuculline methiodide, BCNU treated
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tissue showed a larger number of spontaneous and evoked epileptiform network discharges as compared to controls. At the single cell level, pyramidal neurons demonstrated a decreased sensitivity to GABA (Benardete and Kriegstein, 2002). By the use of transcription
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factors, which are specific for cortical layer 6 (Nurr1), 5 (Er81), 4 (Ror-beta) and supragranular layers (Cux2 ) Moroni et al. (Moroni et al., 2009) could demonstrate by in situ hybridization and immunohistochemistry that the cortical thinning in the BCNU model is
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mostly restricted to the supragranular layers and that heterotopia show a rudimentary pattern of laminar organization with layer 2/3 neurons in the core and deeper layer neurons in the periphery (Moroni et al., 2009;Moroni et al., 2011). The disturbance of tangential fibres in this
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model leads to modifications in the distribution of GABAergic cells (Moroni et al., 2011). BCNU-treated rats reveal normal motor activity, anxiety-related behavior, and intact long-
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term aversive memory, but impaired short-term working memory (Inverardi et al., 2013). Hippocampal slices from these animals show a decrease in excitatory synaptic transmission, impaired paired pulse facilitation, and enhanced LTP associated with hyperexcitability (Inverardi et al., 2013).
The BCNU model needs to be characterized in more detail and it is currently unknown, whether spontaneous epileptic seizures can be observed in this model under in vivo conditions. The ibotenic acid model Innocenti and Berbel (Innocenti and Berbel, 1991) induced in cat visual cortex focal malformations, which resemble human microgyria and ulegyria by intracortical injections of the glutamatergic agonist ibotenate at P2 or P3. The ibotenate model has been used to some 18 Page 18 of 32
extent in the golden hamster, which offer the advantage that they are born more immature than rats and mice and early developmental processes in the cortex can be manipulated in postnatal animals. An injection of ibotenate in newborn hamsters induces an arrest of migrating neurons destined for layers 2, 3 and 4 and malformations including microgyria, periventricular nodular heterotopias and subcortical band heterotopias (Marret et al., 1995b;Takano et al., 2004). Radial glial cells and the extracellular matrix are not damaged
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indicating that these guiding structures are intact. Coinjection of an NMDA receptor antagonist, but not a metabotropic glutamate receptor antagonist, prevented these
malformations (Marret et al., 1996), indicating that an excessive calcium influx through
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NMDA channels induced these migration disorders. In addition, ibotenate also interferes with the termination of the migration process (Takano et al., 2004). Interestingly a systemic bolus
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injection of magnesium, a non-competitive NMDA channel blocker, did not prevent microgyia,
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but did prevent ulegyrias and porencephalic cyst formation (Marret et al., 1995a).
Cortical malformations consisting of microgyrus and heterotopias can be also obtained in rats when injected with ibotenate at the day of birth (Redecker et al., 1998a). Extracellular
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recordings in neocortical slices from ibotenate injected rats at 2 months of age demonstrated a pronounced hyperexcitability in remote regions from the microgyrus (Redecker et al., 1998b), very similar as observed in the freeze lesion model. Stimulus-evoked epileptiform
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activity was blocked by an NMDA receptor antagonist, indicating wide-spread changes in the function of NMDA receptors. In contrast, the GABAergic inhibitory system seems to be
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largely intact in this model of focal cortical malformation (Hagemann et al., 2000).
Spontaneous epileptic seizures have so far not been described in this model, but as in other
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models of focal cortical malformations detailed and long-term EEG recordings have yet not been performed in the ibotenate model. Antiepileptic drugs
Antiepileptic drugs have not been used as a model for cortical malformation, but rather gained recent attention for their adverse side effects on neuronal migration. When pregnant rats receive from E14 to E19 daily intraperitoneal injections of vigabatrin and valproate at clinically relevant concentrations, the offspring show focal neuronal migration disorders in the hippocampus and cerebral cortex (Manent et al., 2007). Vigabatrin and valproate, which both modulate GABAergic function, induced hippocampal and cortical dysplasias. In contrast, prenatal exposure to carbamazepine caused no dysplasias (Manent et al., 2007). Hippocampal and cortical malformations were also observed in animals exposed prenatally to clinically relevant concentrations of lamotrigine, but not in animals treated with topiramate, 19 Page 19 of 32
levetiracetam or phenobarbital (Manent et al., 2008). This result is interesting since phenobarbital is an anti-epileptic drug, which also acts on the GABAergic system.
The consequences of antiepileptic drug use during pregnancy is of central clinical relevance. Alone in the USA, each year approximately 30,000 children are born to epileptic mothers and it is well-known that antiepileptic drugs may have teratogenic effects (for review (Hill et al.,
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2010;Tomson and Battino, 2012). Although the most common malformations of in utero exposure to antiepileptic drugs are cardiac abnormalities, neuronal malformations and
neurodevelopmental deficits have been also reported. Children exposed to valproate (mono-
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and polytherapy) have a significantly higher risk of motor, cognitive (e.g. lower IQ score) and behavioural problems than those children exposed to other antiepileptics, such as
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carbamazepine, lamotrigine, phenobarbital, topiramate or vigabatrin (Tomson and Battino, 2012). The poorer performance of children exposed in utero to valproate is a consisting
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finding. The long-term effects of other antiepileptic drugs are often less clear, because the number of exposed cases are too low to allow statistical analyses. Therefore animal studies require further attention to understand the molecular mechanisms of cortical malformations
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and dysfunction induced by antiepileptic drugs. Ethanol and cocaine
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Neuropathological data from human neonates who were exposed to large amounts of ethanol during gestation show cortical malformation resulting from neuronal migration disorders and glial dysfunction (Clarren et al., 1978). Experimental animal studies provide
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evidence that ethanol does not only inhibit neuronal migration in the cerebral cortex, but also prolongs the cell cycle leading to a reduction in overall neuron number (Hirai et al., 1999).
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The same authors also reported an abnormally dense staining pattern for N-CAM on the surface of migrating neurons in ethanol treated animals, indicating that abnormal expression of N-CAM may lead to neuronal migration disorders (Hirai et al., 1999). A relatively low level of ethanol in utero (maternal and fetal blood alcohol level of 25 mg/dl) promotes premature tangential migration of cortical GABAergic interneurons (Cuzon et al., 2008), most likely because ethanol exposure in utero increases ambient extracellular GABA level and the sensitivity of migrating interneurons to GABA. Exposure of organotypic slice cultures prepared from E17 rat fetuses to different concentrations of ethanol (0, 200, 400 or 800 mg/dl) and for periods of 2 to 32 hours showed a concentration- and time-dependent expression of cortical malformations. Ethanol induced the focal formation of warts and heterotopias. Furthermore ethanol caused morphological alterations in radial glia cells leading to infiltration of migrating neurons into the marginal zone (Mooney et al., 2004). Using organotypic slice cultures Cuzon et al. (Cuzon et al., 2008) also provide evidence that 20 Page 20 of 32
ethanol consumption during pregnancy modifies the migration process of cortical GABAergic interneurons in the embryonic brain.
Another drug, which affects developmental processes in the embryonic brain and causes cortical malformations is cocaine. When injected into pregnant mice from E8 to birth the offspring show disturbances in neocortical architecture, such as loss of horizontal and
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vertical lamination and abnormal organization of axonal-dendritic bundles (Gressens et al., 1992). Recurrent exposure of mouse embryos to cocaine from E8 to E15 induces selective impairment of tangential migration of cortical GABAergic interneurons, whereas GABAergic
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neurons of the olfactory bulb are not affected (Crandall et al., 2004). Furthermore Lee et al. (Lee et al., 2011) reported that prenatal cocaine exposure does not only disturb tangential
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migration of GABAergic neurons, but also interrupts radial migration of both glutamatergic
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and GABAergic neurons within the neocortex.
Hippocampal slices from 2-3 months old rats exposed to prenatal cocaine reveal a reduced threshold for both electrical stimulation- and potassium-induced epileptiform discharges,
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indicating that cocaine exposure during gestation is associated with at high risk for the development of seizure activity (Baraban et al., 1997). This hyperexcitability cannot be explained by cocaine induced modifications in passive or action potential properties, but
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rather by reduction of spike frequency adaptation, post-spiking afterhyperpolarizations and an impairment in GABA-A receptor mediated synaptic inhibition (Baraban and Schwartzkroin, 1997). Using oligo microarrays followed by real-time RT-PCR Novikova et al. (Novikova et
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al., 2005) found in the cerebral wall of E18 mice treated with cocaine from E8 to E18 an upregulation of beta-catenin, the key functional component of both Wnt and cadherin
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systems. These data indicate that at least some cortical malformations observed in animals and humans exposed to prenatal cocaine may be related to alterations in the Wnt/cadherin molecular network.
Awake, freely behaving rats exposed to cocaine in utero also show a significant reduction in thresholds for both flurothyl- and kainic acid-induced seizures providing further support for the suggestion that cortical malformations induced by cocaine exposure during gestation represent a high risk for the development of seizure activity (Baraban et al., 1997).
Acknowledgements The author is most thankful to his coworkers who over the years contributed to the research on the cortical freeze lesion model. This work was supported by the DFG. 21 Page 21 of 32
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ip t Time point of insult
Reproducibility
depends on species and brain region of interest, mostly E15
Mouse, rat, ferret
easy
Freeze lesion
Mouse, rat
very easy, in utero difficult
In utero irridiation
Mouse, rat, monkey
easy
BCNU
Rat
easy
ibotenic acid
hamster, rat, cat
Pathophysiology
inexpensive
periventricular nodular heterotopia
Hyperexcitable, but no spontaneous seizures. Reduced seizure threshold. Hyperexcitable. Reduced seizure threshold. Spontaneous seizures when lesioned at E18. Hyperexcitable. Reduced seizure threshold and spontaneous seizures.
very reproducible, (multi-)focal
inexpensive
microgyrus, heterotopia, schizencephaly
E12-E17, for cortical dysplasia E17
Reproducible, but location of focal malformation(s) vary (diffuse) Reproducible, but location of focal malformation(s) vary (diffuse) Reproducible, but location of focal malformation(s) vary (diffuse)
inexpensive, if access to gamma radiation source
microgyrus, heterotopia
E15
Ac c moderately easy
Reproducible, but location of focal malformation(s) vary (diffuse)
Neuropathology
Costs
P0-P1, E18 possible
ep te
MAM
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Experimental induction
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Species
d
Model
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Table 1: Summary of animal models of cortical malformations
species-dependent rat, hamster: P0 cat: P2/3
inexpensive
inexpensive
laminar disorganization, heterotopias, cytomegalic neurons microgyria, ulegyria, periventricular nodular heterotopias, schizencephaly
Hyperexcitable. Impaired short-term working memory.
Hyperexcitable.
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S0165-0270(15)00135-1 http://dx.doi.org/doi:10.1016/j.jneumeth.2015.03.034 NSM 7201
To appear in:
Journal of Neuroscience Methods
Received date: Revised date: Accepted date:
12-3-2015 27-3-2015 30-3-2015
Please cite this article as: Luhmann HJ, Models of cortical malformation - chemical and physical, Journal of Neuroscience Methods (2015), http://dx.doi.org/10.1016/j.jneumeth.2015.03.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights Chemical and physical manipulations during early development induce cortical malformations.
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These malformations resemble cortical pathologies in humans with epilepsy. Animal models show cortical hyperexcitability due to excitatory-inhibitory imbalance.
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Animal models may uncover mechanisms of epileptic disorders.
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JNM Special Issue Series on “Models and Methods for Neurological and Psychiatric Diseases”
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Models of cortical malformation - chemical and physical
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Heiko J. Luhmann
Heiko J. Luhmann Institute of Physiology University Medical Center Mainz Duesbergweg 6 D-55128 Mainz Germany
Number of figures: 0
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Number of tables: 1
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Number of pages: 31
+49 6131 39 26070 +49 6131 39 26071 [email protected]
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phone: fax: e-mail:
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Corresponding author:
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Institute of Physiology, University Medical Center of the Johannes Gutenberg University, Duesbergweg 6, D-55128 Mainz, Germany
Keywords: Neocortex, hippocampus, developmental disorders, neuronal migration, epilepsy, model, rat, mouse
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Abstract Pharmaco-resistant epilepsies, and also some neuropsychiatric disorders, are often associated with malformations in hippocampal and neocortical structures. The mechanisms leading to these cortical malformations causing an imbalance between the excitatory and inhibitory system are largely unknown. Animal models using chemical or physical
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manipulations reproduce different human pathologies by interfering with cell generation and neuronal migration. The model of in utero injection of methylazoxymethanol (MAM) acetate
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mimics periventricular nodular heterotopia. The freeze lesion model reproduces
(poly)microgyria, focal heterotopia and schizencephaly. The in utero irradiation model causes
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microgyria and heterotopia. Intraperitoneal injections of carmustine 1-3-bis-chloroethylnitrosurea (BCNU) to pregnant rats produces laminar disorganization, heterotopias and cytomegalic neurons. The ibotenic acid model induces focal cortical malformations, which
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resemble human microgyria and ulegyria. Cortical dysplasia can be also observed following prenatal exposure to ethanol, cocaine or antiepileptic drugs.
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All these models of cortical malformations are characterized by a pronounced hyperexcitability, few of them also produce spontaneous epileptic seizures. This dysfunction results from an impairment in GABAergic inhibition and/or an increase in glutamatergic
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synaptic transmission. The cortical region initiating or contributing to this hyperexcitability may not necessarily correspond to the site of the focal malformation. In some models widespread molecular and functional changes can be observed in remote regions of the brain,
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where they cause pathophysiological activities.
This paper gives a overview on different animal models of cortical malformations, which are
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mostly used in rodents and which mimic the pathology and to some extent the pathophysiology of neuronal migration disorders associated with epilepsy in humans.
Introduction
Cortical malformations are frequently the pathological substrate of epilepsy and can be often found in patients with pharmaco-resistant epilepsy. Models of cortical malformations, which are experimentally induced by chemical or physical manipulations of the developing cortex, resemble in many aspects the pathology and pathophysiology of epilepsy associated with malformations in human cortex. These chemical and physical manipulations interfere with different developmental processes occurring during prenatal and (depending on the species) early postnatal stages: (i) The generation of neurons (neurogenesis) and/or glial cells (gliogenesis) may be impaired. (ii) The insult may induce cell death or the process of programmed cell death (apoptosis) may be modified, e.g. cells destined to die may survive. 3 Page 3 of 32
(iii) Spontaneous activity patterns, which are important for the maturation of cortical networks may be altered. (iv) Most importantly, the tangential migration of inhibitory interneurons and the radial migration of pyramidal cells is often altered following these chemical or physical insults. The resulting malformations can be focal or diffuse extending over large cortical regions, resembling the spectrum of cortical pathologies described in tissue resected from patients with pharmaco-resistant epilepsy. The extent and type of the experimentally induced
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cortical malformation depends more on the time point of the insult in development and not so much on the cause. Therefore, in all animal models the timing of the experimental
manipulation is most critical. The same protocol of chemical or physical insult will cause
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different pathologies when induced at different developmental stages.
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It is mostly unclear how these structural lesions lead to seizure activity and animal models are required to understand the mechanisms of pathogenesis, epileptogenesis, and
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epileptogenicity of cortical malformations. An important and clinically unresolved issue relates to the question, whether the lesion itself is the main epileptogenic zone, or whether subtle molecular, structural and functional disturbances in the region surrounding the obvious
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lesion are the trigger for epileptic discharges (Luhmann et al., 2014).
This review will focus on different animal models, largely in rodents, of acquired cortical
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malformations (Table 1). For a review of genetic models of cortical malformations the reader is referred to the paper by S. Roper and R. Spreafico in this issue. This review will cover
neocortex.
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malformations in the hippocampus, an allocortical structure, and in the cerebral cortex, the
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Human pathology and pathophysiology of cortical malformations One of the first reports on the pathology of cortical malformations associated with epilepsy goes back to Rudolf Virchow, who in 1867 reported in the cerebral cortex of a 44 years old male patient local cell clusters and bulges ("Haufen und Wülste", p. 140) as an excellent example of heterotopia ("ausgezeichnetes Beispiel von Heterotopie") (Virchow, 1867). A more recent overview on the pathology of cortical malformations in humans and some models has been published by Najm et al. (Najm et al., 2007) and Takano (Takano, 2011). Cortical malformations, especially focal cortical dysplasia, represent the most common pathological finding in pediatric epilepsy surgery patients, and range from mild to severe malformations including hippocampal sclerosis (Krsek et al., 2008). A number of molecular and cell physiological data have been obtained in cortical structures from human epileptic patients affected by periventricular nodular heterotopia, subcortical band beterotopia, or focal cortical dysplasia. Immunoblotting and immunoprecipitation analyses in human cortex 4 Page 4 of 32
resected from EEG-verified epileptic and distal nonepileptic areas demonstrated a higher expression of NMDA (NMDA) receptor subunits 1 and 2A/B in epileptic dysplastic cortex compared with the nonepileptic cortex, indicating that an increased NR1-NR2A/B coassembly may contribute to the epileptogenicity of the dysplastic cortex (Mikuni et al., 1999). These observations are supported by coimmunoprecipitation and immunoblotting studies in resected cortical tissues from patients with pharma-coresistant epilepsy associated
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with neocortical dysplasia, which showed an increased coassembly of NR1 and NR2B with PSD-95 when compared with non-epileptic tissue (Ying et al., 2004). A more distinct pattern of NMDA receptor modification has been reported by Finardi et al. (Finardi et al., 2006), who
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observed a selective increase in the NR2B subunit in all cortical dysplasia, but a reduced expression level of NR2A and NR2B subunits in all patients with heterotopia. These data
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suggest that different developmental malformations are associated with distinct alterations in NMDA receptor density and function. Differences in NMDA receptor function have been also
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reported at the cellular level with electrophysiological methods in pediatric cortical dysplasia. Cytomegalic neurons from human cortical dysplasia tissue showed NMDA currents with decreased Mg2+ sensitivity as compared to neurons from non-dysplastic tissue.
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Immunofluorescence analyses revealed a decrease in NR2B subunit expression in cytomegalic neurons and in a number of normal appearing pyramidal neurons from
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dysplastic tissue (André et al., 2004).
Beside these molecular and electrophysiological changes in the glutamatergic system, prominent alterations have been also documented in the structure and function of the
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GABAergic system in human cortical malformations. Using quantitative immunohistochemistry Thom et al. (Thom et al., 2004) reported in cases of grey matter
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heterotopia in postmortem tissue from patients with epilepsy an overall normal density and distribution of GABA-containing interneurons, but morphologically these cells were less organized and more randomly orientated compared to control cortex. In contrast, a "scattering" of GABAergic interneurons has been demonstrated by Calcagnotto et al. in human dysplastic cortex (Calcagnotto et al., 2005). The same authors also showed with patch-clamp recordings from identified neurons a significant decrease in the frequency of spontaneous inhibitory synaptic currents (IPSCs), an increase in the decay-time constant of evoked and spontaneous IPSCs and a decrease in transporter-mediated GABA reuptake function. In slices from cortical tissue resected for the treatment of pharmaco-resistant epilepsy in children (0.2-14 years), Cepeda et al. observed spontaneous GABA-mediated membrane depolarizations, which frequently elicited action potentials, indicating an excitatory role of GABA in pediatric cortical dysplasia (Cepeda et al., 2007). This assumption is supported by a report on changes in the expression of the chloride transporters KCC2 and 5 Page 5 of 32
NKCC1 in neocortical tissue resected in children with intractable focal epilepsy using quantitative western blot analyses (Jansen et al., 2010). A significant decrease in the mRNA and protein levels of the chloride outward transporter KCC2 has been demonstrated in human dysplastic tissue (Shimizu-Okabe et al., 2011), indicating that the chloride reversal potential is more depolarized, as reported by Cepeda et al. (Cepeda et al., 2007). Finally, beside deficits in glutamatergic and GABAergic synaptic function contributing to the
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epileptogenicity of cortical malformations, abnormal intrinsic membrane properties have been also reported dysplastic cortex (Cepeda et al., 2003).
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In the following paragraphs seven animal models of acquired cortical malformations
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associated with hyperexcitability or epileptic seizures will be reviewed. The MAM model
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The model of in utero injection of methylazoxymethanol (MAM) acetate into pregnant rats has been introduced by Spatz and Laqueur in 1968 (Spatz and Laqueur, 1968). Singh (Singh, 1977) documented in more detail the resulting hippocampal malformations, which are
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similar to periventricular nodular heterotopia in human patients with drug-resistant focal epilepsy. MAM is a potent cytotoxic agent, which induces a time-specific aberrant DNA methylation and alkylation leading to abnormal patterns of cell formation and migration. MAM
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seems to selectively affect developing brain structures and not other embryonic organs undergoing cell proliferation at the time of its administration. Neuronal precursors that are undergoing their final mitosis at the time of MAM exposure are specifically ablated, glial cells
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are only indirectly affected. Noctor et al. (Noctor et al., 1999) reported in the ferret neocortex, that injection of MAM during embryogenesis produces distorted radial glial cells and
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concluded that this interference with early cortical development causes premature differentiation of radial glial cells into astrocytes. Beside its antimitotic effects, MAM also seems to directly influence neuronal migration. The migration speed and the exploratory behavior of migrating neurons is significantly reduced after MAM treatment (Abbah and Juliano, 2014). MAM also modifies the migration pattern of interneurons, but this deficit is not intrinsic to the migrating neurons, but rather mediated by cues, most likely GABA, dictating proper orientation of interneurons migrating into the cortex (Poluch et al., 2008). For a recent overview on the role of GABA in controlling neuronal migration see review by Luhmann et al. (Luhmann et al., 2015). The site and type of malformations depends critically on the time of MAM administration. Concentrations between 10 and 30 mg/kg maternal body weight have been used. When a single (25 mg/kg) or a double (15 mg/kg each at 12 h interval) injection of MAM is given on embryonic day (E) 15, malformations are mostly present in the hippocampal CA1 and CA2 region, to a lesser extent also in the striatum, thalamus, 6 Page 6 of 32
hypothalamus and cerebral cortex. Battaglia et al. (Battaglia et al., 2003) demonstrated that the MAM-induced ablation of early generated neurons is sufficient to change the migration and differentiation of subsequently generated neurons, which then form the different heterotopia. For a comprehensive overview on the cellular actions of MAM and its timedependent effects on different brain regions the reader is referred to a review by Cattabeni and Di Luca (Cattabeni and Di Luca, 1997). Experimental details on the MAM models can be
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found in the review by Battaglia and Bassanini (Battaglia and Bassanini, 2006).
The MAM induced heterotopia are generally localized along the border of the lateral
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ventricles and share structural features with the periventricular nodules in human
periventricular or subcortical nodular heterotopia. Cells in hippocampal heterotopia reveal
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neuronal morphology and do not stain with immunohistochemical markers for glia (Baraban et al., 2000). It has been suggested that neurons in hippocampal heterotopia are originally
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destined to the cerebral cortex (Chevassus et al., 1998b;Chevassus et al., 1998a;Castro et al., 2001), but this hypothesis needs to be proven by immunohistochemical markers specific for neocortical layers. Intracellular and whole-cell patch-clamp recordings in hippocampal
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slices were performed to study the electrophysiological properties of dysplastic neurons in MAM-treated animals. Baraban and Schwartzkroin (Baraban and Schwartzkroin, 1995) could not find any significant difference in resting membrane potential, time constant, input
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resistance, action potential amplitude and duration of CA1 neurons recorded in MAM-treated animals when compared to controls. However, a higher percentage of neurons (62% vs. 10%) fired an intrinsic burst of action potentials in response to suprathreshold current
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injection. Intrinsic burst firing of neurons located in the heterotopia has been also reported by Sancini et al. (Sancini et al., 1998). Abnormal intrinsic firing properties with sustained
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repetitive bursts of action potentials have been further observed in subcortical heterotopic nodules (Colacitti et al., 1999). In situ hybridization and immunohistochemical studies demonstrated in heterotopic cell regions of the hippocampus of MAM-exposed rats a pronounced reduction in the expression of the Kv4.2 subunit of the A-type voltage-dependent potassium channel (Castro et al., 2001). Castro et al. also reported that heterotopic neurons lack functional A-type Kv4.2 potassium channels and show hyperexcitable firing, indicating that heterotopic neuronal clusters may trigger epileptiform activity. The experimental data so far suggest that this intrinsic hyperexcitability is mostly mediated by alterations in voltagedependent potassium channels. Voltage-dependent calcium currents of the L-, T-, N-, P/Q and P-type in hippocampal heterotopic neurons show similar activation and inactivation kinetics and pharmacological sensitivity to nifedipine, amiloride, omega-conotoxin GVIA, omega-agatoxin KT, and sFTX-3.3 as control neurons (Calcagnotto and Baraban, 2003).
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The important question, whether neurons in heterotopic clusters are connected with neurons located in other brain regions, has been addressed by a number of groups with anatomical and electrophysiological techniques. Using anterograde and retrograde tract tracing, Colacitti et al. (Colacitti et al., 1998) demonstrated the existence of short- and long-range reciprocal connections between neocortical heterotopia and ipsilateral as well as contralateral cortical areas. Furthermore abnormal cortico-hippocampal and cortico-cortical connections were
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observed in these neocortical heterotopia. Similar abnormal connections have been reported in hippocampal CA3 pyramidal neurons of MAM-treated rats, which showed an exuberant
mossy fibres innervation with ectopic mossy boutons on their basal dendrites (Chevassus-
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Au-Louis et al., 1999). The same authors verified with staining of the activity marker c-fos the functional connectivity between hippocampal and neocortical regions (Chevassus et al.,
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1998b). Further support for the existence of functional connections between the limbic system and the cerebral cortex in MAM-treated animals comes from patch-clamp recordings
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from intrahippocampal heterotopic as well as from neocortical and hippocampal neurons (Chevassus et al., 1998a). Heterotopic neurons are functionally integrated in both neocortical and hippocampal networks and receive mono- and polysynaptic pathophysiological inputs
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from these structures. This observation has been confirmed with extracellular field potential recordings in hippocampal slices from MAM-exposed animals, which demonstrated in the bicuculline and 4-aminopyridine model that heterotopic neurons can generate epileptiform
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activity, which is transmitted to the neocortex and hippocampus (Baraban et al., 2000). Spontaneous epileptiform discharges were also observed in the majority of slices from MAMtreated animals when the excitability was only slightly increased by elevation of the
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extracellular potassium concentration from 3 to 6 mM (Baraban and Schwartzkroin, 1995). A study by Tschuluun et al. (Tschuluun et al., 2005) confirmed with neuroanatomical and single
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cell electrophysiological methods the bidirectional connectivity between the heterotopia and the hippocampus and cerebral cortex, but failed to identify the heterotopia as trigger for epileptiform activity and to demonstrate a propagation of this activity to the cerebral cortex. Using c-fos activation in organotypic hippocampal slice cultures from MAM treated rats Doisy et al. (Doisy et al., 2015) recently demonstrated that nodular heterotopia are not responsible for hyperexcitability and do not trigger epileptiform activity in this in vitro culture model.
Beside abnormal connectivity and intrinsic pathophysiology, neurons in heterotopia are also characterized by modifications in their synaptic properties. Using whole-cell patch-clamp recordings Calcagnotto and Baraban (Calcagnotto and Baraban, 2005) demonstrated in hippocampal heterotopic neurons a significant increase in the amplitude and slower decaytime constant of the N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic current (EPSC) component as compared to normotopic pyramidal cells. In 8 Page 8 of 32
contrast, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor mediated EPSC was not different in both groups. The authors propose changes in the composition and function of the NMDA receptor subunits in MAM-treated animals. Changes in the distribution and expression of glutamate receptors have been reported by in situ hybridization and immunohistochemistry, but these demonstrated in the heterotopiae a 26% reduction in the expression of the NMDA NR1 subunit and a 40% increase in the expression
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of the AMPA GluR2 flip subunit (Rafiki et al., 1998). A selective impairment of both the targeting and the alphaCaMKII-dependent phosphorylation of the NR2A/B subunits in the
postsynaptic membranes of heterotopic pyramidal cells have been demonstrated by Gordoni
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et al. (Gardoni et al., 2003).
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Inhibitory synaptic function is also changed in hippocampal slices from rats exposed to MAM in utero. The decay time constants of spontaneous and evoked inhibitroy postsynaptic
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currents (IPSCs) were increased by about 200%, whereas IPSC amplitude or rise time were unchanged (Calcagnotto et al., 2002). Based on immunohistochemical staining for the GABA transporters GAT-1 and GAT-3 and pharmacological experiments with the GABA transport
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inhibitors tiagabine and NO-711 the authors conclude that GABA reuptake may be reduced in heterotopic neurons leading to an enhanced GABAergic function. However, it remains to be studied whether this increase in GABAergic function in the MAM model is associated with
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an increase in inhibitory function or due to possible alterations in chloride transporter function leading to a depolarizing or excitatory action of GABA.
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Spontaneous epileptic seizures have so far not been reported in MAM-treated animals. However, as in many other models, this question has yet not been studied in detail in the
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MAM model. Long-term, preferentially multi-site telemetric EEG recordings or depth electrode recordings combined with continuous video monitoring over several days and in different age groups are required to unequivocally answer the question, whether MAMtreated animals show spontaneous epileptic seizures. Two months old rats exposed to MAM in utero showed a significantly lower threshold for flurothyl-induced seizures (Baraban and Schwartzkroin, 1996). Furthermore, shorter seizure latencies correlated with an increased number of heterotopic neurons. A lower threshold for the induction of epileptic seizures in MAM-treated animals have been also reported in the kainic acid and bicuculline model (Defeo et al., 1995;Germano and Sperber, 1997) and in hyperthermia (Germano et al., 1996). Using an experimental 'double-hit' model of prenatal MAM exposure and postnatal pilocarpine-induced status epilepticus Colciaghi et al. (Colciaghi et al., 2011) could show that MAM-treated rats develop more severe epilepsy than non-MAM animals. This enhanced seizure activity in the MAM group was associated with cortical malformations, abnormally 9 Page 9 of 32
large cortical pyramidal neurons with neurofilament overexpression and recruitment of NMDA receptor subunits to the postsynaptic membrane.
In summary, the large amount of molecular, anatomical and physiological data clearly demonstrate pathological alterations in hippocampal and neocortical regions of MAM-treated animals. These malformations mimic the pathology of periventricular nodular heterotopia
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described in humans suffering from severe forms of epilepsy. It remains to be studied in more detail whether spontaneous epileptic seizures are present in this model and, if yes,
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whether anticonvulsants may have an effect.
The freeze (cryogenic) lesion model
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The first report of the cortical freeze lesion model goes back to 1883, when Openchowski (Openchowski, 1883) demonstrated that local cooling of the neocortical surface of a dog
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produced focal lesions and cortical dysfunction. However, only adult animals were used in the study of Openchowski as well as in a small number of subsequent studies performed over the following 70 years (see (Balthasar, 1957). The freeze lesion model in the cerebral
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cortex of newborn animals has been introduced by Dvorak and Feit (Dvorak and Feit, 1977) and Dvorak et al. (Dvorak et al., 1978), who studied the consequences of a focal cortical freeze lesion in rats shortly after birth. This model can be easily established and reproduces
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the pathology of certain types of human neuronal migration disorders associated with epileptic seizures, e.g. polymicrogyria, focal heterotopia, cortical dysplasia or a cortical cleft
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(schizencephaly). In most experimental studies the focal neocortical malformation consisted of a four-layered microgyrus, as described in human pathology (McBride and Kemper, 1982).
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For a clinical classification system of focal cortical dysplasias proposed by an International League Against Epilepsy task force consortium see Blümcke et al. (Blümcke et al., 2011). The neocortical freeze lesion model has been widely used in rats, but comparable results can be obtained in mice (Supèr et al., 1997;Rosen et al., 1995). As in the MAM model, the animal´s age of the experimental induction of the freeze lesion is very critical. At different ages the identical lesion protocol produces very different cortical pathologies. In adult rodents, the freeze lesion model has been used as a model for traumatic brain injury or brain edema (Stoffel et al., 2001;Murakami et al., 1999;Chan et al., 1987). For the induction of the typical focal cortical malformations, best reproducible results can be obtained when the lesion is induced during the first 24 hours after birth. Recently prenatal freeze lesioning has been performed at E18 in utero (Kamada et al., 2013;Takase et al., 2008). A detailed experimental protocol has been given previously by the author of this review (Luhmann, 2006). In brief, a stainless or copper probe with a tip diameter of 1 mm is cooled with liquid nitrogen and applied for 8-10 s on the exposed calvarium above the neocortical area of 10 Page 10 of 32
interest, mostly the parietal cortex. Smaller (0.5 mm) and larger (2 mm) probes can be also used, but depending on the application time will induce other malformations (Rosen and Galaburda, 2000;Ferrer et al., 1993). The severity and type of the cortical malformation is directly related to the number and duration of freeze lesions, indicating that a single patho(physio)logical event of variable severity at a distinct developmental stage can induce different malformations (Rosen and Galaburda, 2000). Larger freezing probes and freezing
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times (20-30 s) result in the formation of a neocortical cleft resembling the pathology of schizencephaly described in humans. The cooled probe may be positioned in an identical
manner at other locations, producing for example a 4-6 mm long microsulcus in rostro-caudal
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direction. Sham-operated animals are treated the same way as freeze-lesioned animals with the exception that the copper cylinder is not cooled. The mortality of this model is below 5%
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of treated animals.
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The most consistent result of a focal cortical freeze in the newborn rodent is the formation of a four-layered microgyrus (Dvorak and Feit, 1977;Dvorak et al., 1978), which occurs in two stages. In the first stage neurons and glial cells are destroyed by the freezing procedure,
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followed by a regrowth of damaged radial glial fibers and repair of the damaged cortical region by migrating neurons. At the day of birth marginal zone/layer 1 and layers 4 to 6 are already present and the freezing procedure induces here a focal necrotic lesion, which is
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invaded within 24 hours by reactive astrocytes and macrophages (Rosen et al., 1992). A pronounced increase in GFAP immunoreactivity and clusters of proliferative BrdU-positive astrocytes have been demonstrated at the site of the lesion (Bordey et al., 2001). Whole-cell
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patch-clamp recordings from BrdU-positive astrocytes in slices from freeze-lesionded animals demonstrated a lack of KIR channel expression and an increase in delayed rectifier K+ (KDR)
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channels. Furthermore these proliferative astrocytes showed almost no dye coupling, suggesting a dysfunction in K+ buffering (Bordey et al., 2001). In the second stage migrating neurons destined to form layer 2/3 invade the necrotic core. In freeze-lesioned cortex upper layer Cajal-Retzius neurons survive for longer developmental periods (Supèr et al., 1997) and radial glial cells are preserved in adult cortex (Rosen et al., 1994). Furthermore, neurogenesis in response to the lesion may take place postnatally (Rosen et al., 1996). The damaged cortex begins to assume its adult-like microgyric appearance from P5 to P10. The microgyrus and the paramicrogyral zone is characterized by an abnormal organization of afferent and efferent connections. Thalamocortical as well as corticothalamic projections are markedly reduced and disorganized (Jacobs et al., 1999b;Rosen et al., 2000). Furthermore, callosally projecting pyramidal neurons located in the paramicrogyral zone reveal a different laminar distribution compared to control animals, indicating disturbances in interhemispheric interactions (Giannetti et al., 1999). Autoradiographic measurements demonstrated a 11 Page 11 of 32
significant reduction of basic cortical [14C]deoxyglucose metabolism up to 1 mm lateral to the lesion (Kraemer et al., 2001). This decrease in basic metabolism is accompanied by a reduction in Na+/K+-ATPase in the paramicrogyral cortex as demonstrated by immunohistochemistry and in situ hybridization expression of the alpha3 isoform of Na+/K+ATPase (Chu et al., 2009). In adult freeze-lesioned rats functional magnetic resonance imaging showed a significantly reduced cortical activation in the lesioned hemisphere
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following sensory stimulation of the sensory periphery (Schwindt et al., 2004), further
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supporting the reduction and disorganization of thalamocortical afferents described above.
A number of electrophysiological studies on neocortical slices from freeze-lesioned rats
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demonstrated a pronounced hyperexcitability in the cortical region surrounding the microgyrus (Jacobs et al., 1996;Luhmann and Raabe, 1996;Hablitz and DeFazio, 1998). These data indicate that the neocortical region adjacent to the microgyrus and not the
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microgyrus itself is the generator for epileptiform activity. This paramicrogyral zone shows a number of molecular and functional modifications, which may contribute to this hyperexcitability. Layer 5 pyramidal neurons located 1-2 mm from the microgyrus showed a
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hyperpolarized resting membrane potential, increased input resistances and a smaller inwardly rectifying hyperpolarization-activated h-current (Albertson et al., 2011). The
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reduction in h-current contributes to increased single cell and network excitability in cortical dysplasia. Local electrical stimulation of the paramicrogyral zone elicited epileptiform responses that propagated over >4 mm in horizontal direction (Luhmann and Raabe, 1996),
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indicating widespread structural and/or functional modifications in the lesioned cortical hemisphere. Application of a NMDA antagonist blocked the late recurrent component of the
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propagating activity in field potential recordings (Luhmann et al., 1998b) and intracellular recordings from supragranular neurons (Luhmann et al., 1998a). An overabundance of excitatory inputs to the paramicrogyral zone has been postulated by Jacobs et al. (Jacobs et al., 1999a) and subsequently demonstrated with various techniques. Spontaneous and miniature EPSCs recorded from pyramidal neurons adjacent to a microgyrus were significantly increased in frequency, suggesting that these neurons receive more excitatory synaptic inputs compared to neurons in control cortex (Jacobs and Prince, 2005). An increased EPSC frequency was also documented by combining laser scanning photostimulation with glutamate uncaging (Brill and Huguenard, 2010). This hyperinnervation of pyramidal neurons by excitatory afferents occurs at an age before onset of cortical epileptiform activity (Zsombok and Jacobs, 2007).
Using quantitative in vitro receptor autoradiography of glutamatergic receptors demonstrated an up-regulation of AMPA receptors over the whole lesioned hemisphere and an increase in 12 Page 12 of 32
NMDA and kainate receptors in the paramicrogyral zone (Zilles et al., 1998). However, neither receptor autoradiography nor immunohistochemical stains for NMDA receptor subunits (Hagemann et al., 2003) revealed any widespread alterations in remote cortical regions, indicating that NMDA receptors are mainly increased in density and/or affinity in the microgyrus itself and in the immediate surrounding tissue. These molecular data are supported by in vitro electrophysiological and neuropharmacological experiments, which
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demonstrated that NR2B-containing NMDA receptors are functionally enhanced in freezelesioned cortex (Defazio and Hablitz, 2000) and, as shown by voltage-sensitive dye imaging, contribute to the local spread of paroxysmal activity in freeze lesioned cortex
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(Bandyopadhyay and Hablitz, 2006). Pathophysiological increases in NMDA receptor
function are further mediated by changes of glutamate transporters, specifically the glia
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glutamate transporter (GLT-1), which in dysplastic cortex causes larger tonic NMDA currents
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(Campbell and Hablitz, 2008).
Beside molecular, structural and functional changes in the glutamatergic system, prominent alterations in the GABAergic system also contribute to the hyperexcitability in freeze lesioned
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cortex. A reduction in the expression of parvalbumin immunoreactivity has been documented in layers 2/3 within the microgyrus and up to 2 mm adjacent to it, but this disinhibition was only transient and disappeared by P21 (Rosen et al., 1998). In adult cortex, the widespread
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functional changes described above are not related to a general loss of inhibitory interneurons (Schwarz et al., 2000). A significant decrease in the number of immunoreactive neurons for parvalbumin, calretinin and calbindin has been reported only for the region within
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the microsulcus, but not for the paramicrogyral zone or remote cortical regions (Hablitz and DeFazio, 1998). A subpopulation of GABAergic interneurons that co-express the GluR1
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subunit and the calcium-binding protein calbindin is also reduced within the microgyrus (Kharazia et al., 2003). In contrast to these more local changes, which are restricted to the lesion itself, quantitative in vitro receptor autoradiography revealed a widespread downregulation of GABA-A receptors (Zilles et al., 1998). Immunohistochemical studies for the GABA-A receptor subunits alpha1, alpha2, alpha3, alpha5 and gamma2 confirmed this widespread down-regulation in GABAergic function (Redecker et al., 2000). Furthermore, the downregulation of GABA-A receptor subunits even involved the ipsilateral hippocampal formation and restricted contralateral neocortical areas (Redecker et al., 2000). A decrease in the alpha subunit composition of the GABA-A receptor has been also demonstrated by recordings of miniature IPSCs and by a reduced affinity for zolpidem in layer 2/3 pyramidal cells located 1-2 mm lateral to the microgyrus (Defazio and Hablitz, 1999). These modifications would result in an elimination of the type 1 benzodiazepine receptor, which
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would be of relevance for pharmacological treatment of epileptic seizures associated with cortical dysplasia.
Whereas intracellular recordings from layer 2/3 cells in the paramicrogyral zone suggested a decrease of glutamatergic inputs onto inhibitory interneurons (Luhmann et al., 1998a), an increase of functional excitatory synapses on both interneurons and pyramidal cells has been
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also demonstrated (Jacobs and Prince, 2005). Recordings from pyramidal neurons located 1-2 mm lateral of the microgyrus showed for layer 5 an increased excitatory input in freeze lesioned cortices, while no significant differences were seen in layer 2/3 cells (Brill and
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Huguenard, 2010), indicating layer-specific modifications in connectivity.
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An upregulation in the mRNA expression of the chloride inward transporter NKCC1 and a simultaneous downregulation of the chloride outward transporter KCC2 has been
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demonstrated by in situ hybridization histochemistry in the upper layers of the microgyrus, indicating disturbances in intracellular chloride homeostasis (Shimizu-Okabe et al., 2007). More recently a downregulation of KCC2 was documented for the microgyrus forming
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GABAergic and E17.5 born glutamatergic neurons at P4, which resulted in GABA-A receptormediated calcium oscillations in microgyrus forming cells (Wang et al., 2014). The imbalance in excitatory and inhibitory synaptic transmission in freeze-lesioned cortex also results in
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interesting alterations of synaptic plasticity. Field potential recordings in layer 2/3 demonstrated an increased long-term potentiation (LTP) following theta-burst stimulation in layer 6, but a lack of LTP in layer 4 (Peters et al., 2004). These layer-specific modifications in
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synaptic plasticity may result from the reduction in GABA-A receptor gamma2 subunit
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expression (Peters et al., 2004).
As in the MAM model, spontaneous seizures cannot be observed without a provoking event in adult rodents freeze-lesioned in the first postnatal days (for review (Luhmann, 2009;Luhmann, 2006). However, the threshold temperature to elicit hyperthermia-induced seizures is significantly reduced in freeze-lesioned immature rats as compared to shamtreated controls (Scantlebury et al., 2004). A novel rat model with multiple focal cortical dysplasia has been created by Takase et al. (Takase et al., 2008) by freeze lesioning at embryonic day (E) 18 through the uterus wall. At adult stages these animals revealed in the EEG spontaneous epileptic spikes and a faster development of hippocampal kindling following electrical stimulation. Furthermore, these animals showed an upreglation of the NMDA receptor subunits NR1 and NR2B and a rise in the expression of glutamate/aspartate transporters (Takase et al., 2008). A subsequent study of the same lab using the identical model of E18 freeze lesioning confirmed the expression of spontaneous seizures in a high 14 Page 14 of 32
percentage (69%) of treated animals and the increase in the expression levels of NMDA receptor subunits NR1, NR2A, NR2B, glutamate/aspartate transporter and glial glutamate transporter 1 (GLT1) (Kamada et al., 2013).
A recent study by Andresen et al. (Andresen et al., 2014) reported that a one-week treatment with the anticonvulsant and analgesic gabapentin immediately after freeze lesioning
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prevented the development of hyperexcitability in vitro and in vivo. This anti-epileptogenic effect of gabapentin is mediated by its inhibitory action on thrombospondin and alpha 2 delta1 signaling, the latter representing an auxiliary calcium channel subunit, which are both
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upregulated during the formation of the microgyrus (Andresen et al., 2014).
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In summary, the freeze lesion model produces a pathology which resembles cortical dysplasia reported in humans. A large number of experimental studies have demonstrated
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wide-spread molecular and functional changes in paramicrogyral regions remote from the lesion itself. This pathology may resemble the human condition, since clinical studies have demonstrated that the epileptogenic zone is usually more extensive than the structural lesion
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(Palmini et al., 1994;Palmini et al., 1991) (for review (Luhmann et al., 2014). The novel in utero freeze lesion model produces spontaneous epileptic seizures in vivo and this model requires further investigations by other groups. It will be an experimental challenge to study
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in this model the process of epileptogenesis and early therapeutical interventions. Of further interest is the differential role of GABA-A mediated synaptic phasic inhibition versus extrasynaptic tonic inhibition, which are mediated by gamma-subunit and delta-subunit
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containing receptors, respectively (for review (Kilb et al., 2013). The experimental studies obtained in animal models suggest region and layer specific changes in the expression of
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these subunits in dysplastic cortex. The in utero irradiation model
This model has been initially used in rats to study the consequences of radiation exposure on early brain development (Riggs et al., 1956) and more specifically on neuronal migration (Altman et al., 1968). Mostly rodents have been used as experimental animals, but Algan and Rakic (Algan and Rakic, 1997) applied ionizing irradiation at selected prenatal stages in macaque monkeys to delete specific neuronal cell populations in the visual cortex and to study the consequences on the development of cortical cytoarchitecture. A detailed description of the methods in rats is given in the review by Lin and Roper (Lin and Roper, 2006). Usually pregnant rats on E17 are exposed to 145 to 225 cGy of gamma-irradiation and offspring are used for experiments. Lower doses (e.g. 100 cGy) induce minor pathological changes (Kellinghaus et al., 2004), higher doses (e.g. 300 cGy) elicit severe 15 Page 15 of 32
malformations such as absence of corpus callosum (Lent and Schmidt, 1986). Different cortical malformations can be produced in rats with irradiation given at different embryonic stages (Ferrer et al., 1984). Irradiation with a single dose of 200 cGy on E14 causes large cortical ectopic cell clusters (Ferrer, 1993). A four-layered microgyrus can be produced by irradiation on E16 and segmentation of the cerebral cortex can be observed after irradiation on E15, E17 or E19 (Ferrer, 1993). Beside these focal cortical malformations, which depend
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on the dose and on the time point of irradiation, gross cytoarchitectural abnormalities have been described in the somatosensory cortex of adult rats subjected to this in utero treatment. The cortical representation of the whiskers in primary somatosensory cortex, the so-called
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barrel cortex (for review (Feldmeyer et al., 2013), is severely disturbed and structural barrels
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fail to develop, despite a normal functional thalamocortical input (Ito, 1995).
Whereas the overall neuronal density in adult dysplastic cortex is not significantly different
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compared to controls, the density of parvalbumin and calbindin immunoreactive inhibitory interneurons is significantly reduced in irradiated cortex (Roper et al., 1999). Stereological measurements in the cerebral cortex on E21 and P6 showed that the total number of
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neurons and of GABAergic neurons is reduced by about 50% of controls at both time points (Deukmedjian et al., 2004). Whereas the total number of neurons doubled during this developmental period in both control and irradiated animals, the number of GABAergic cells
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did not increase in treated rats. Since GABAergic neurons in the controls increased by the factor of 10 between E21 and P6, these data suggest that the GABAergic system is irreversibly damaged by in utero radiation (Deukmedjian et al., 2004). Using antibodies to
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vesicular glutamate transporter 1 (VGLUT1), vesicular glutamate transporter 2 (VGLUT2), vesicular GABA transporter (VGAT), and parvalbumin to quantify glutamatergic and
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GABAergic presynaptic terminals, Zhou and Roper (Zhou and Roper, 2010) demonstrated in dysplastic cortex an overall increase in excitatory synaptic connectivity and decrease in inhibitory synaptic connectivity.
As other models of focal cortical malformations, the in utero irradiation model is also characterized by a pronounced hyperexcitability when studied with in vitro electrophysiological methods. Neocortical slices from adult rats exposed to embryonic irradiation show more robust epileptiform activity compared to controls when treated with bicuculline (Roper et al., 1997;Roper, 1998). Whole-cell patch-clamp recordings from pyramidal neurons demonstrated in dysplastic cortex a reduction in the frequency of spontaneous and miniature IPSCs and in the amplitude of spontaneous IPSCs (Zhu and Roper, 2000). Monosynaptic evoked IPSCs were also impaired. Fast-spiking inhibitory interneurons recorded in layer 4 dysplastic cortex show a reduction in the frequency of 16 Page 16 of 32
miniature and spontaneous IPSCs (Zhou et al., 2009). Recordings from identified GABAergic interneurons immunoreactive for calretinin, somatostatin and parvalbumin revealed a reduction in excitatory drive for the two latter cell types in dysplastic cortex (Zhou and Roper, 2011). These data indicate a prominent impairment in the function of intracortical GABAergic inhibition (Zhu and Roper, 2000). The mechanisms underlying this impairment were examined in more details by Zhou and Roper (Zhou and Roper, 2014) by paired recordings
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from various types of identified neuronal types. This study showed a reduced synaptic efficiency of GABAergic inhibition originating from fast-spiking, parvalbumin-positive
interneurons to their postsynaptic target cell. Interestingly, electrical, gap junctions mediated
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connections within this cell type were also impaired (Zhou and Roper, 2014).
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Whereas the intracortical GABAergic system is functionally reduced in adult animals treated by irradiation during embryonic stages, the glutamatergic excitatory system seems to be
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enhanced. Spontaneous EPSCs recorded in pyramidal neurons are increased in amplitude and frequency in dysplastic cortex (Zhu and Roper, 2000). Presynaptic release probability of excitatory synapses is significantly enhanced, most likely due to a reduced tonic activity of
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presynaptic metabotropic glutamate receptors of the type GluR2/3 (Chen et al., 2007). However, these changes appear to be specific for glutamatergic synaptic connections between excitatory neurons, since miniature and spontaneous EPSCs recorded in
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GABAergic interneurons are significantly reduced in frequency (Xiang et al., 2006;Zhou et al., 2009), suggesting that inhibitory interneurons receive less excitatory drive in dysplastic
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cortex.
Using epidural electrodes Roper et al. (Roper et al., 1995) demonstrated in irradiated rats an
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increased propensity for seizures in the presence of the anaesthetic agents acepromazine and xylazine, or a combination of both drugs. Prolonged continuous EEG recordings with bifrontal epidural and hippocampal depth electrodes uncovered spontaneous seizures arising independently from the hippocampus or the frontal neocortex (Kondo et al., 2001). Using the same long-term EEG recordings in combination with video monitoring revealed interictal spikes in rats treated at E17 with low- or medium-dose radiation causing mild or moderate malformations, but not in severe radiation-treated animals (Kellinghaus et al., 2004). Spontaneous epileptiform spikes and seizures on EEG have been also demonstrated during the chronic phase in a second hit model combining irradiation at E17 and a low dose of pentylenetetrazole at adult stages (Oghlakian et al., 2009).
In summary, the utero irradiation model dose-dependently mimics different types of cortical malformations. In rats, medium-dose radiation at E17 produces focal cortical malformations, 17 Page 17 of 32
which can generate spontaneous seizures. The mechanisms underlying this hyperexcitability appear to be largely mediated by an impairment in intracortical GABAergic inhibition. It remains to be studied in more detail whether the dysplasia itself or the region surrounding the malformation represent the trigger zone for hyperexcitability and epileptic seizures. The BCNU model
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Intraperitoneal injections of carmustine 1-3-bis-chloroethyl-nitrosurea (BCNU) to pregnant rats on E15 produce cortical dysplasia in the offspring consisting of laminar disorganization, cytomegalic neurons, heterotopias and clusters of Cajal-Retzius cells (Benardete and
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Kriegstein, 2002). Furthermore radial glial fibers are disrupted (Moroni et al., 2011), as also reported in the freeze lesion model. Thus the BCNU model mimics the pathology of cortical
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dysplasia in human patients, such as altered cortical layering, presence of heterotopia and dysmorphic neurons. In cortical slices exposed to bicuculline methiodide, BCNU treated
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tissue showed a larger number of spontaneous and evoked epileptiform network discharges as compared to controls. At the single cell level, pyramidal neurons demonstrated a decreased sensitivity to GABA (Benardete and Kriegstein, 2002). By the use of transcription
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factors, which are specific for cortical layer 6 (Nurr1), 5 (Er81), 4 (Ror-beta) and supragranular layers (Cux2 ) Moroni et al. (Moroni et al., 2009) could demonstrate by in situ hybridization and immunohistochemistry that the cortical thinning in the BCNU model is
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mostly restricted to the supragranular layers and that heterotopia show a rudimentary pattern of laminar organization with layer 2/3 neurons in the core and deeper layer neurons in the periphery (Moroni et al., 2009;Moroni et al., 2011). The disturbance of tangential fibres in this
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model leads to modifications in the distribution of GABAergic cells (Moroni et al., 2011). BCNU-treated rats reveal normal motor activity, anxiety-related behavior, and intact long-
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term aversive memory, but impaired short-term working memory (Inverardi et al., 2013). Hippocampal slices from these animals show a decrease in excitatory synaptic transmission, impaired paired pulse facilitation, and enhanced LTP associated with hyperexcitability (Inverardi et al., 2013).
The BCNU model needs to be characterized in more detail and it is currently unknown, whether spontaneous epileptic seizures can be observed in this model under in vivo conditions. The ibotenic acid model Innocenti and Berbel (Innocenti and Berbel, 1991) induced in cat visual cortex focal malformations, which resemble human microgyria and ulegyria by intracortical injections of the glutamatergic agonist ibotenate at P2 or P3. The ibotenate model has been used to some 18 Page 18 of 32
extent in the golden hamster, which offer the advantage that they are born more immature than rats and mice and early developmental processes in the cortex can be manipulated in postnatal animals. An injection of ibotenate in newborn hamsters induces an arrest of migrating neurons destined for layers 2, 3 and 4 and malformations including microgyria, periventricular nodular heterotopias and subcortical band heterotopias (Marret et al., 1995b;Takano et al., 2004). Radial glial cells and the extracellular matrix are not damaged
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indicating that these guiding structures are intact. Coinjection of an NMDA receptor antagonist, but not a metabotropic glutamate receptor antagonist, prevented these
malformations (Marret et al., 1996), indicating that an excessive calcium influx through
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NMDA channels induced these migration disorders. In addition, ibotenate also interferes with the termination of the migration process (Takano et al., 2004). Interestingly a systemic bolus
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injection of magnesium, a non-competitive NMDA channel blocker, did not prevent microgyia,
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but did prevent ulegyrias and porencephalic cyst formation (Marret et al., 1995a).
Cortical malformations consisting of microgyrus and heterotopias can be also obtained in rats when injected with ibotenate at the day of birth (Redecker et al., 1998a). Extracellular
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recordings in neocortical slices from ibotenate injected rats at 2 months of age demonstrated a pronounced hyperexcitability in remote regions from the microgyrus (Redecker et al., 1998b), very similar as observed in the freeze lesion model. Stimulus-evoked epileptiform
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activity was blocked by an NMDA receptor antagonist, indicating wide-spread changes in the function of NMDA receptors. In contrast, the GABAergic inhibitory system seems to be
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largely intact in this model of focal cortical malformation (Hagemann et al., 2000).
Spontaneous epileptic seizures have so far not been described in this model, but as in other
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models of focal cortical malformations detailed and long-term EEG recordings have yet not been performed in the ibotenate model. Antiepileptic drugs
Antiepileptic drugs have not been used as a model for cortical malformation, but rather gained recent attention for their adverse side effects on neuronal migration. When pregnant rats receive from E14 to E19 daily intraperitoneal injections of vigabatrin and valproate at clinically relevant concentrations, the offspring show focal neuronal migration disorders in the hippocampus and cerebral cortex (Manent et al., 2007). Vigabatrin and valproate, which both modulate GABAergic function, induced hippocampal and cortical dysplasias. In contrast, prenatal exposure to carbamazepine caused no dysplasias (Manent et al., 2007). Hippocampal and cortical malformations were also observed in animals exposed prenatally to clinically relevant concentrations of lamotrigine, but not in animals treated with topiramate, 19 Page 19 of 32
levetiracetam or phenobarbital (Manent et al., 2008). This result is interesting since phenobarbital is an anti-epileptic drug, which also acts on the GABAergic system.
The consequences of antiepileptic drug use during pregnancy is of central clinical relevance. Alone in the USA, each year approximately 30,000 children are born to epileptic mothers and it is well-known that antiepileptic drugs may have teratogenic effects (for review (Hill et al.,
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2010;Tomson and Battino, 2012). Although the most common malformations of in utero exposure to antiepileptic drugs are cardiac abnormalities, neuronal malformations and
neurodevelopmental deficits have been also reported. Children exposed to valproate (mono-
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and polytherapy) have a significantly higher risk of motor, cognitive (e.g. lower IQ score) and behavioural problems than those children exposed to other antiepileptics, such as
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carbamazepine, lamotrigine, phenobarbital, topiramate or vigabatrin (Tomson and Battino, 2012). The poorer performance of children exposed in utero to valproate is a consisting
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finding. The long-term effects of other antiepileptic drugs are often less clear, because the number of exposed cases are too low to allow statistical analyses. Therefore animal studies require further attention to understand the molecular mechanisms of cortical malformations
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and dysfunction induced by antiepileptic drugs. Ethanol and cocaine
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Neuropathological data from human neonates who were exposed to large amounts of ethanol during gestation show cortical malformation resulting from neuronal migration disorders and glial dysfunction (Clarren et al., 1978). Experimental animal studies provide
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evidence that ethanol does not only inhibit neuronal migration in the cerebral cortex, but also prolongs the cell cycle leading to a reduction in overall neuron number (Hirai et al., 1999).
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The same authors also reported an abnormally dense staining pattern for N-CAM on the surface of migrating neurons in ethanol treated animals, indicating that abnormal expression of N-CAM may lead to neuronal migration disorders (Hirai et al., 1999). A relatively low level of ethanol in utero (maternal and fetal blood alcohol level of 25 mg/dl) promotes premature tangential migration of cortical GABAergic interneurons (Cuzon et al., 2008), most likely because ethanol exposure in utero increases ambient extracellular GABA level and the sensitivity of migrating interneurons to GABA. Exposure of organotypic slice cultures prepared from E17 rat fetuses to different concentrations of ethanol (0, 200, 400 or 800 mg/dl) and for periods of 2 to 32 hours showed a concentration- and time-dependent expression of cortical malformations. Ethanol induced the focal formation of warts and heterotopias. Furthermore ethanol caused morphological alterations in radial glia cells leading to infiltration of migrating neurons into the marginal zone (Mooney et al., 2004). Using organotypic slice cultures Cuzon et al. (Cuzon et al., 2008) also provide evidence that 20 Page 20 of 32
ethanol consumption during pregnancy modifies the migration process of cortical GABAergic interneurons in the embryonic brain.
Another drug, which affects developmental processes in the embryonic brain and causes cortical malformations is cocaine. When injected into pregnant mice from E8 to birth the offspring show disturbances in neocortical architecture, such as loss of horizontal and
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vertical lamination and abnormal organization of axonal-dendritic bundles (Gressens et al., 1992). Recurrent exposure of mouse embryos to cocaine from E8 to E15 induces selective impairment of tangential migration of cortical GABAergic interneurons, whereas GABAergic
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neurons of the olfactory bulb are not affected (Crandall et al., 2004). Furthermore Lee et al. (Lee et al., 2011) reported that prenatal cocaine exposure does not only disturb tangential
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migration of GABAergic neurons, but also interrupts radial migration of both glutamatergic
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and GABAergic neurons within the neocortex.
Hippocampal slices from 2-3 months old rats exposed to prenatal cocaine reveal a reduced threshold for both electrical stimulation- and potassium-induced epileptiform discharges,
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indicating that cocaine exposure during gestation is associated with at high risk for the development of seizure activity (Baraban et al., 1997). This hyperexcitability cannot be explained by cocaine induced modifications in passive or action potential properties, but
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rather by reduction of spike frequency adaptation, post-spiking afterhyperpolarizations and an impairment in GABA-A receptor mediated synaptic inhibition (Baraban and Schwartzkroin, 1997). Using oligo microarrays followed by real-time RT-PCR Novikova et al. (Novikova et
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al., 2005) found in the cerebral wall of E18 mice treated with cocaine from E8 to E18 an upregulation of beta-catenin, the key functional component of both Wnt and cadherin
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systems. These data indicate that at least some cortical malformations observed in animals and humans exposed to prenatal cocaine may be related to alterations in the Wnt/cadherin molecular network.
Awake, freely behaving rats exposed to cocaine in utero also show a significant reduction in thresholds for both flurothyl- and kainic acid-induced seizures providing further support for the suggestion that cortical malformations induced by cocaine exposure during gestation represent a high risk for the development of seizure activity (Baraban et al., 1997).
Acknowledgements The author is most thankful to his coworkers who over the years contributed to the research on the cortical freeze lesion model. This work was supported by the DFG. 21 Page 21 of 32
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cr
ip t Time point of insult
Reproducibility
depends on species and brain region of interest, mostly E15
Mouse, rat, ferret
easy
Freeze lesion
Mouse, rat
very easy, in utero difficult
In utero irridiation
Mouse, rat, monkey
easy
BCNU
Rat
easy
ibotenic acid
hamster, rat, cat
Pathophysiology
inexpensive
periventricular nodular heterotopia
Hyperexcitable, but no spontaneous seizures. Reduced seizure threshold. Hyperexcitable. Reduced seizure threshold. Spontaneous seizures when lesioned at E18. Hyperexcitable. Reduced seizure threshold and spontaneous seizures.
very reproducible, (multi-)focal
inexpensive
microgyrus, heterotopia, schizencephaly
E12-E17, for cortical dysplasia E17
Reproducible, but location of focal malformation(s) vary (diffuse) Reproducible, but location of focal malformation(s) vary (diffuse) Reproducible, but location of focal malformation(s) vary (diffuse)
inexpensive, if access to gamma radiation source
microgyrus, heterotopia
E15
Ac c moderately easy
Reproducible, but location of focal malformation(s) vary (diffuse)
Neuropathology
Costs
P0-P1, E18 possible
ep te
MAM
an
Experimental induction
M
Species
d
Model
us
Table 1: Summary of animal models of cortical malformations
species-dependent rat, hamster: P0 cat: P2/3
inexpensive
inexpensive
laminar disorganization, heterotopias, cytomegalic neurons microgyria, ulegyria, periventricular nodular heterotopias, schizencephaly
Hyperexcitable. Impaired short-term working memory.
Hyperexcitable.
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