66 PLATFORM SESSION

whole brains were removed rapidly and frozen. Au- toradiographic techniques was used for the localiza- tion of readily diffusible radioligands together with radiosensitive sheets called imaging plates, which allowed detailed mapping of the BZD receptors by an imaging analyzer. Radioactivity was standardized by the caudate putamen.

Two hours after the KA injection in the left amyg- dala, limbic seizure status was observed, and the receptor binding of Iz5I-IMZ was increased in all regions. Four weeks after the KA injection, the re- ceptor binding of '251-IMZ was decreased in the hip- pocampus, especially in ipsilateral CA3. Histologic studies revealed neuronal cell loss in CA3 of the pyramidal cell layer on the side of the injected KA. This cell loss appeared to coincide with the decrease in density of the GABA/BZD receptor complex.

In the acute phase, the receptor binding of Iz5I- IMZ was increased in the bilateral hippocampus.

In contrast, the receptor binding of Iz5I-IMZ was decreased in the hippocampus, especially in ipsilat- era1 CA3, in the chronic phase. Quantitative mor- phometric analysis indicated a remarkable increase of BZD receptor binding in the granular cell layer of the dentate gyrus.

In chronic rats, a decrease of BZD-receptor bind- ing in CA3 may play an important role in the mecha- nism of seizure induction and is well correlated to continuous epileptic excitation of the hippocampus. '251-IMZ may be a promising BZD-receptor ligand for clarifying the role of the BZD receptor in epilep- tic inhibitory phenomena, not only in animal models but also in patients with epilepsy, by using single- photon emission-computed tomography (SPECT) or positron emission tomography (PET) studies. Further investigation is needed to understand the relation between intractable complex partial sei- zures and BZD receptors.

Immunohistochemistry of Neurotransmitters and Their Receptors in Hemimegalencephaly . * t Hiros hi Arai, "Toshisaburo Nagai, tMasahiro Nakayama, $Hiroshi Kimura, and "Shintaro Okada (*Department of Pediatrics, Osaka University Medical School, tDepartment of Pathology, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka; and $Center of Molecular Neurobiology , Shiga Otsu, Japan).

Hemimegalencephaly is often associated with early-onset intractable epilepsy. Clinical EEG and single-photon emission-computed tomography (SPECT) studies revealed neuronal hyperactivity of the megalencephalic hemisphere (MH). To investi- gate the pathologic background for epileptogenicity , we performed an immunohistochemical study of two major neurotransmitters, y-aminobutyric acid (GABA) and glutamate, and their receptors on the MH.

Surgically resected parietal cortex of the MH of a 4-year-old boy was immediately fixed in a mixture of 4% paraformaldehyde and 0.3% glutaraldehyde in 4°C phosphate-buffered solution and processed for immunostaining for GABA and glutamate. Pari- etal cortex from the MH of the autopsies of two patients (1-year-old girl and 17-year-old boy) were routinely fixed in 10% neutral formaldehyde solution and embedded in paraffin blocks for immunostaining of the a and p subunits of the GABA, receptor (GABA,R a, p) and of GluR1, one of the AMPA-

University of Medical Science,

receptor subunits. Part of cortex of the former pa- tient was frozen and used for Western blotting. Staining results were compared with those of the opposite hemisphere (OH).

Immunoreactivity to GABA and glutamate in the surgical specimens was similar to that of the control tissues, although GABA- or glutamate-positive fi- bers had coarse structures with large varicosities and were morphologically abnormal. Electronmi- croscopic observation revealed poor synapse forma- tion of GABA terminals. Western blotting and im- munostaining of postmortum specimens showed im- munoreactivity to both GABAAR a and p of the MH weaker than that of OH. Only margins of several giant neurons and quite a few fibers were immuno- stained for GABAAR. On the contrary, immunoreac- tivity to GluRl of the MH was much stronger than that of the OH, especially in the deep cortical areas. Most pyramidal cells were strongly immunostained for GluR1, and dense GluR1-positive fibers were ob- served.

Epikpsia. Vol. 37, Suppl. 3, 1996

PLATFORM SESSION 67

Morphologic (ultrastructural) abnormality of GABA terminals and poor expression of GABAAR suggested impaired GABA transmission in MH, which could explain why seizures of hemimega- lencephaly are rarely controlled by antiepileptic drugs (AEDs) that modulate the GABAergic system. Whether the abnormality was primary or secondary to chronic epileptic activity remained unclear. Ex- cessive GluR1 expression compared with that of GABAAR may be one of the main histochemical

background factors of epilepsy in hemimegalenceph- aly. Our results could be applied to certain types of focal cortical dysgenesis, because advanced imaging technique and epilepsy surgery disclosed that histo- logic features of the dysgenesis sometimes resem- bles those of hemimegalencephaly .

Impaired GABA transmission with poor GABAAR and excessive GluR1 in MH may cause neuronal hyperactivity, which leads to severe epi- lepsy.

Perirhinal Cortical Kindling: A Behavioral and Immunohistochemical Study. Toshiki Sato, Norihito Yamada, "Yoshihiro Kitamura, Kenro Otsuki, Akihisa Orita, $Shyuji Uemura, "fiyoshi Morimoto, and Sigetoshi Kuroda (Department of Neuropsychiatry; Okayama Uni- versity Medical School, Okayama; *Department of Integrative Physi- ology, National Institute for Physiological Sciences, Okazaki; TDe- partment of Neuropsychiatry, Kagawa University Medical School, Kagawa; and $Institute of Molecular Neurobiology, Shiga University of Medical Science, Otsu, Japan)

We previously reported that perirhinal cortex (PRC) has an important role in the generalization of kindled seizures. In our study, we kindled rat PRC and made a behavioral comparison with amygdala (AM) kindling. We also stained for Fos protein in both PRC- and AM-kindled brains by using an immu- nohistochemistry technique.

Male Sprague-Dawley rats were implanted with a chronic electrode into the left PRC or left AM and received daily kindling stimulation (biphasic square pulses for 2 s at 50 Hz once daily),. After a l-week seizure-free period after the first stimulation trial or after the last fully kindled seizure, the animals were given a final stimulation. The animals were killed under deep anesthesia 1 h after the last stimulation trial, and brain sections were obtained for immuno- histochemical staining for Fos protein.

There were no significant differences between AM- and PRC-kindled groups in afterdischarge (AD) thresholds and the number of stimulations required to reach the first stage 4 or 5 seizures. AD duration of AD and the latency to forelimb clonus in the fully PRC-kindled group (21.3 k 7.2 s, 1.3 t 1.4 s, respectively) were significantly shorter than those in the AM-kindled group (68.5 ? 36.3 s, 5.3 k 1.9 s, respectively). Typically, PRC-kindled rats dem- onstrated movement arrest or searching behavior for - 10 days, followed by the sudden appearance

of stage 4 or stage 5 seizures or running fits. In- termediate partial seizures, such as stage 2 or 3 seizures, were not normally observed. After the first stimulation, PRC-stimulated brains showed positive Fos-protein staining in the neocortex, whereas AM-kindled brains did not. Fully PRC- kindled seizures produced massive induction of Fos protein in bilateral PRC, piriform cortex, entorhinal cortex, AM, and neocortex, although there was no induction of Fos protein in the hippo- campus.

The pattern of Fos protein-staining after a single PRC AD indicates that the epileptic activity could be propagated directly from the PRC to the neocortex. This result might account for the extraordinarily short latency to generalized motor seizures in fully PRC-kindled seizures. The pattern of development of PRC kindling could also support this hypothesis, in that PRC-kindled epileptogenesis appears to be propagated from the PRC to the neocortex responsi- ble for generalized convulsions, with little involve- ment of other limbic systems such as the hippocam- pus. The lack of induction of Fos protein in the hippocampus in fully PRC-kindled animals is possi- bly related to the lack of intermediate partial seizures or the short AD durations (Shin et al., 1990; Clark et al., 1991; Chiasson et al., 1995) of PRC-kindled seizures.