Intact Functional Inhibition in the Surround of Experimentally Induced Focal Cortical Dysplasias in Rats

G. Hagemann, C. Redecker, and O. W. Witte

Department of Neurology, Heinrich-Heine-University, D-40225 Duesseldorf, Germany


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Hagemann, G., C. Redecker, and O. W. Witte. Intact Functional Inhibition in the Surround of Experimentally Induced Focal Cortical Dysplasias in Rats. J. Neurophysiol. 84: 600-603, 2000. Early postnatal injections of ibotenate into the rat neopallium induce cortical dysplasias mimicking human polymicrogyria which often goes along with seizure disorders. Under in vitro conditions these experimentally induced dysplasias cause widespread hyperexcitability. The underlying mechanisms are as yet not fully understood. Electrophysiologically there is clear evidence of widespread alterations of the excitatory system. Intracellular recordings also showed some changes of the inhibitory system but have concentrated on recordings from focal areas close to the microgyrus. We investigated the integrity of functional inhibition using a paired-pulse paradigm to map the whole ipsilateral hemisphere. In rat cortical slices double-pulses were applied in layer VI/white matter and field potentials recorded in layer II/III. The ratio of the field potential amplitude did not show significant alterations in the dysplasias or their surround as compared with control and sham-injected animals. This result was obtained with two different locations of the dysplasias, excluding a mere areal specific effect. Our results show that despite prominent hyperexcitability in the surround of ibotenate-induced cortical dysplasias the inhibitory network appears to be functionally intact.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

With the advent of modern imaging techniques malformations of cortical development are increasingly recognized in association with minor neuropsychological disorders, mental delay or retardation, and epilepsy (Guerrini et al. 1999; Raymond et al. 1995). The mechanisms responsible for functional impairment are less clear (Jacobs et al. 1999; Redecker et al. 1998a).

Electrophysiologically, dysplastic human cortex as well as in vitro studies of rodent models of cortical dysgenesis revealed intrinsic epileptogenecity of such lesions, which is present in the cortical malformation itself as well as in its vicinity (Jacobs et al. 1996; Luhmann et al. 1998a; Mattia and Avoli 1995; Palmini et al. 1995; Redecker et al. 1998a). Such a hyperexcitability can be explained by altered intrinsic membrane properties or a change in network characteristics with an imbalance of inhibition and excitation. Accordingly, in animal models of cortical dysgenesis, GABAA-receptor binding is downregulated and glutamate-receptor binding is upregulated (Zilles et al. 1998) in the surround of the malformation. Furthermore, the number of parvalbumin-reactive interneurons is locally diminished and also, to a lesser extent and only temporarily, reduced in more widespread areas (Jacobs et al. 1996; Rosen et al. 1998). However, intracellular recordings revealed intact monosynaptic inhibition but a regionally differential change of inhibitory network efficacy: in the microgyrus polysynaptic inhibitory postsynaptic currents (IPSCs) were diminished whereas the paramicrogyral area showed increased IPSC amplitude (Jacobs and Prince 1999; Luhmann et al. 1998b; Prince et al. 1997). Although these intracellular studies have been limited to the microgyrus or paramicrogyral area there are also indirect pharmacological findings suggesting that the inhibitory system is functionally intact in more widespread areas. This was concluded from the occurrence of spontaneous epileptiform activity after pharmacological blockade of potassium currents with 4-aminopyridine which requires inhibitory networks to be present (Hablitz and DeFazio 1998).

Here we investigated the effectiveness of functional inhibition in a previously described model of cortical malformation (Redecker et al. 1998a,b). We used the paired-pulse paradigm, which proved to be helpful in detecting widespread impairment of functional inhibition in models of chronic epilepsy and of focal cortical lesions in adult rats (Buchkremer-Ratzmann et al. 1996; Hagemann et al. 1999). Our results give evidence for an unimpaired functional inhibition in the surround of ibotenate-induced cortical dysplasias.


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Ibotenate injections

All experiments were in strict adherence with local government requirements and performed according to approved guidelines. In several litters of newborn Wistar rats ibotenate (Sigma) diluted in 0.1 M phosphate buffered saline (PBS, pH 7.4) was injected in the neopallium at day P0. The injections were performed under diethylether inhalation as previously described (Redecker et al. 1998a,b). Two different injection sites were chosen: one group was injected in anterior (fronto-parietal group) whereas a second group was injected in posterior brain areas (parieto-occipital group). This was necessary to exclude a mere areal specific effect as electrophysiological properties can vary in different cytarchitectonic areas (Castro Alamancos et al. 1995; Hagemann et al. 1999; Neumann-Haefelin et al. 1996). A 27-gauge needle was inserted 2.5 mm beneath the skin surface between the coronal and lambdoidal sutures, approximately 2 mm from the midline. A dose of 10 µg ibotenate per rat pup was applied by successive injections of two 1 µl boluses with an interval of 45 s. The needle was then left in place for an additional 45 s. Sham-injected animals received 2 µl PBS. After the injection procedure the pups were allowed to recover from anesthesia and returned to their dam.

Electrophysiological recordings

Electrophysiological recordings were obtained in adult animals (>P60) weighing 280-310 g. The rats were anesthetized with diethylether and decapitated. The brains were rapidly removed and chilled to 4-10°C with artificial cerebrospinal fluid (ACSF in mM: 124 NaCl, 26 NaHCO3, 5 KCl, 2 CaCl2, 2 MgSO4, 1.25 Na H2 PO4, and 10 glucose; equilibrated with 95% O2-5% CO2 to pH 7.4). Serial slices (400 µm) were taken from the area of cortical dysplasia or from corresponding brain sections of control or sham-injected animals. The slices were kept in an interface recording chamber at 33°C and allowed at least 1 h to recover. Extracellular field potentials were recorded by a glass electrode placed in cortical layer II/III; the bipolar stimulation electrode was situated in layer VI/white matter beneath the recording electrode (on column). Both electrodes were moved in parallel across slices and recordings were obtained in 1.0 mm steps. A double pulse stimulation protocol was applied to investigate functional inhibition (pulses of 50 ms/5-50 V with 20 ms intervals). The ratio of the amplitudes of the field potential (fp) elicited by the second versus the first stimulus was calculated (Q = fp2/fp1). All data were digitally stored to disk and analyzed off-line using Signal Averager (Science Products, Frankfurt, Germany). After electrophysiological recordings the slices were histologically processed and the relative position of each slice to bregma was estimated by comparison with the atlas of Paxinos and Watson (Paxinos and Watson 1986). For each position relative to midline the mean and standard deviation (SD) were computed. All statistical analysis was performed using the software package SPSS for Windows (SPSS, Chicago, Illinois; version 8.0). Homogeneity of variance was assessed using Levine's test. For group comparisons analysis of variance (ANOVA) was used with post hoc testing for multiple comparisons (Bonferroni correction). Significant differences were assumed with P < 0.05.


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All adult ibotenate-injected animals (n = 20) displayed typical focal cortical dysplasias at the site of injection similar to those previously reported (Redecker et al. 1998a). These lesions are characterized by neuronal depopulation of deep cortical layers, ectopic neurons in superficial layers, and sulcus formation. The core of the lesion was easily identified in the recording chamber and was situated between 3.5 and 5 mm from midline. None of the animals developed overt seizures or showed obvious behavioral changes. Only slices with the dysplasia and directly adjacent slices were investigated. In the first group, evoked field potentials were analyzed in 22 slices of 10 animals with fronto-parietal dysplasias and compared with 12 slices of 5 control animals (CTRL) as well as with 12 slices of 4 sham-operated animals. Slice positions ranged from +0.5 to -2.2 relative to bregma [mainly forelimb, hindlimb, and parietal cortical areas (FL, Par1, and HL according to Paxinos and Watson 1986)]. These results were compared with data obtained in a second group incorporating 18 slices of 10 animals with parieto-occipital dysplasias and 9 slices of 4 control animals. Slices of this group covered an area of -2.6 to -5.9 relative to bregma [mainly hindlimb, parietal, and occipital cortical areas (HL, Par1, and Oc1)]. Data were pooled within groups for each position relative to midline. Under in vitro conditions, no spontaneous epileptiform discharges were observed. Typical field responses from control or ibotenate-injected animals are depicted in Fig. 1A. They were discarded if smaller than 1 mV in amplitude. In the fronto-parietal area there was no statistical difference in the ratio fp2/fp1 between ibotenate- and sham-injected, as well as control animals (Fig. 1). To avoid overlooking areal specific effects of cortical dysplasias the same protocol was used to assess functional inhibition in more posterior cortical areas. However, changing the location to parietal-occipital areas did not alter the result and gave no significant differences in field potential ratio (Fig. 1B).



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Fig. 1. Results of experimentally induced cortical dysplasias (arrow) in frontal (A) and in parieto-occipital (B) cortical areas. A1 and B1: cresyl-violet stained slices used for electrophysiological recording with schematic illustration of electrode positions. A2 and B2: ratio of field potential amplitudes (amplitude of second field potential/amplitude of first field potential) elicited by paired-pulse stimulation in slices of control and ibotenate-injected rats. Ratios are plotted against the distance from the interhemispheric fissure. Inset: examples of field potentials recorded at the same position in a slice of a control animal (down-triangle), or in a slice of an ibotenate-injected animal (). For simplification just one pair of recordings is depicted; field potential configuration did not vary between groups. Stimulus artifact was upgoing and is truncated.


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This study demonstrates that functional inhibition is intact in the surround of experimentally induced cortical dysplasias. As areal specific consequences of cortical lesioning as well as specific electrophysiological properties of different cortical areas have been reported in recent years we investigated two different lesion locations thus excluding such an effect (Castro Alamancos et al. 1995; Hagemann et al. 1999; Neumann-Haefelin et al. 1996).

The role of inhibition for induction and spread of epileptic activity has been discussed extensively but remains elusive (Olsen and Avoli 1997; Prince et al. 1997). Although there might be a need for disinhibition to elicit epileptic activity, functional inhibition could also play a role in restricting the spread of epileptic activity thereby representing the "inhibitory surround" (Bruehl and Witte 1995; Chagnac-Amitai and Connors 1989; Golomb and Amitai 1997; Prince and Wilder 1967). In animal models mimicking human polymicrogyria intracellular electrophysiological evidence for alterations of the inhibitory system is restricted to the microgyrus or paramicrogyral area thus probably reflecting such a "surround inhibition." However, there are no intracellular studies focusing on more widespread areas. Extracellularly long-lasting epileptiform activity could be evoked in widespread ipsilateral areas, persisting even after transcortically resecting the microgyrus from the investigated slice preparation (Prince et al. 1997). This might be indirect evidence for alterations of the inhibitory system as well, as in intact tissue a pharmacological blockade of inhibition is necessary for eliciting spread of epileptiform activity (Golomb and Amitai 1997; Olsen and Avoli 1997).

Evidence for vastly extending changes also comes from receptor autoradiographic studies revealing a widespread reduction of GABAA-receptor-binding and an upregulation of excitatory neurotransmitter binding ipsilaterally (Zilles et al. 1998). Furthermore, Jacobs and Rosen could show a reduced density of parvalbumin-immunoreactive interneurons in the dysplastic cortex but also, to a lesser extent and only temporarily, in more widespread areas (Jacobs et al. 1996; Rosen et al. 1998).

In light of these data, it is surprising that we could not detect any changes of the field-potential ratio in this model as we could show earlier that this paradigm may pick up alterations in functional inhibition ipsilateral and even contralateral to small photothrombotic cortical lesions (Buchkremer-Ratzmann et al. 1996). Due to the interstimulus interval of 20 ms the paired-pulse paradigm predominantly reflects GABAA-dependent inhibition and has been shown to correlate well with GABAA-receptor autoradiography (Qu et al. 1998). Furthermore, the magnitude of the changes in GABA-receptor binding found in the photothrombosis model was the same as in experimentally induced cortical dysplasias (Qu et al. 1998; Zilles et al. 1998). This stresses the point that consequences of cortical lesioning acquired in youth or adulthood are fundamentally different.

The mechanisms underlying these complex changes are as yet unclear. Unchanged or even enlarged inhibition on the cellular level has been interpreted with sprouting of axon collaterals and thereby strengthening the excitatory drive onto interneurons (Luhmann et al. 1998b; Prince et al. 1997). Furthermore, recent data revealing an increased amplitude of miniature IPSCs can best be explained by a change of composition of postsynaptic GABA receptors which could also be demonstrated lately (Jacobs and Prince 1999; Redecker et al. 1999). Again, these immunohistochemical studies reveal that there are widespread changes of GABAA-receptor subunit distribution. Keeping in mind the complexity of field-potential generation our finding of intact functional inhibition could therefore be explained by a balanced change of the excitatory as well as inhibitory system, probably achieved by a change of GABA receptor distribution, thereby increasing inhibitory efficacy. Whether this reflects adaptive changes or is a coincidental proportional alteration of the interplay of excitation and inhibition remains to be elucidated.


    ACKNOWLEDGMENTS

This work was supported in part by a grant of the Heinrich-Heine-University to G. Hagemann and by grants from the Deutsche Forschungsgemeinschaft (Re 1315/1-1 to C. Redecker and SFB 194 B2).


    FOOTNOTES

Address for reprint requests: G. Hagemann, Dept. of Neurology, Heinrich-Heine-University, Moorenstrasse 5, D-40225 Duesseldorf, Germany (E-mail: Georg.Hagemann{at}uni-duesseldorf.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 December 1999; accepted in final form 5 April 2000.


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