Hiroki Kitaura

Somatic Mutations in the MTOR gene cause focal cortical dysplasia type IIb

Focal cortical dysplasia (FCD) type IIb is a cortical malformation characterized by cortical architectural abnormalities, dysmorphic neurons, and balloon cells. It has been suggested that FCDs are caused by somatic mutations in cells in the developing brain. Here, we explore the possible involvement of somatic mutations in FCD type IIb.

Enduring critical period plasticity visualized by transcranial flavoprotein imaging in mouse primary visual cortex

Experience-dependent plasticity in the visual cortex was investigated using transcranial flavoprotein fluorescence imaging in mice anesthetized with urethane. On- and off-responses in the primary visual cortex were elicited by visual stimuli. Fluorescence responses and field potentials elicited by grating patterns decreased similarly as contrasts of visual stimuli were reduced. Fluorescence responses also decreased as spatial frequency of grating stimuli increased. Compared with intrinsic signal imaging in the same mice, fluorescence imaging showed faster responses with ∼10 times larger signal changes. Retinotopic maps in the primary visual cortex and area LM were constructed using fluorescence imaging. After monocular deprivation (MD) of 4 d starting from postnatal day 28 (P28), deprived eye responses were suppressed compared with nondeprived eye responses in the binocular zone but not in the monocular zone. Imaging faithfully recapitulated a critical period for plasticity with maximal effects of MD observed around P28 and not in adulthood even under urethane anesthesia. Visual responses were compared before and after MD in the same mice, in which the skull was covered with clear acrylic dental resin. Deprived eye responses decreased after MD, whereas nondeprived eye responses increased. Effects of MD during a critical period were tested 2 weeks after reopening of the deprived eye. Significant ocular dominance plasticity was observed in responses elicited by moving grating patterns, but no long-lasting effect was found in visual responses elicited by light-emitting diode light stimuli. The present results indicate that transcranial flavoprotein fluorescence imaging is a powerful tool for investigating experience-dependent plasticity in the mouse visual cortex.

Roles of nitric oxide as a vasodilator in neurovascular coupling of mouse somatosensory cortex

Neural activities trigger regional vasodilation in the brain. Diffusible messengers such as nitric oxide (NO) and prostanoids are considered to work as vasodilators in neurovascular coupling. However, their roles are still controversial. In the present study, cortical images of neural activities and vasodilation were recorded through the intact skull of C57BL/6 mice anesthetized with urethane. Flavoprotein fluorescence responses elicited by vibratory hindpaw stimulation were followed by darkening of arteriole images reflecting vasodilation in the somatosensory cortex. Vasodilation was also observed in light reflection images at the wavelength of 570 nm in the same mice. We perfused the surface of the cortex under the skull with 100 microM N(G)-nitro-l-arginine (l-NA), an inhibitor of NO synthase (NOS), and 10 microM indomethacin, an inhibitor of cyclooxygenase (COX). These drugs suppressed vasodilation without changing flavoprotein fluorescence responses. A mixture of l-NA and indomethacin almost completely eliminated vasodilation. In mice lacking neuronal NOS (nNOS), activity-dependent vasodilation was significantly suppressed compared with that in littermate control mice, while that in mice lacking cytosolic phospholipase A2 alpha (cPLA2alpha) was unchanged. These results indicate that NO works as a vasodilator in neurovascular coupling of the mouse somatosensory cortex.

Activity-dependent glial swelling is impaired in aquaporin-4 knockout mice.

We investigated the role of aquaporin-4 (AQP4), a water channel expressed in glial cells, in neural activity mediated morphological changes observed in brain slice preparation. Changes in flavoprotein fluorescence (FF) and infrared light scattering (LS) signals were measured before and after repetitive stimulation of layer VI in rostral somatosensory cortical slices taken from AQP4 knockout (KO) and wild-type (WT) mice. Changes in FF, which reflect neural aerobic activities, were comparable for the two groups in all cortical layers. However, changes in LS signals, which are indicative of cell swelling, were significantly decreased in layer I of AQP4 KO mice compared to that of WT mice. We conclude that AQP4 likely plays a significant role in neural activity-dependent glial swelling.

Functional local connections with differential activity-dependence and critical periods surrounding the primary auditory cortex in rat cerebral slices

Sensory information is processed in neural networks connecting the primary sensory cortices with surrounding higher areas. Here, we investigated the properties of local connections between the primary auditory cortex (area 41) and surrounding areas (areas 20, 36, 18a and 39) in rat cerebral slices. Neural activities elicited by repetitive electrical stimulation were visualized using the activity-dependent changes in endogenous fluorescence derived from mitochondrial flavoproteins, which mostly reflect activities produced by polysynaptic glutamatergic transmission. Polysynaptic feedforward propagation was dominant compared with the corresponding polysynaptic feedback propagation between the primary (area 41) and secondary (areas 20 and 36) auditory cortices, while such a tendency was less clear in other pathways. Long inter-areal (>1 mm) propagation with the same dominancy was observed after layer V stimulation between areas 41 and 20, and was not affected by cutting the underlying white matter. Activity-dependent changes in neural activities induced by low-frequency stimulation in the presence of 1 microM bicuculline were investigated using Ca2+ imaging. Significant potentiation of the polysynaptic Ca2+ activities was only observed in polysynaptic feedforward pathways from the primary to secondary auditory cortices. Experience-dependence of the connections between areas 41 and 20 was investigated using flavoprotein fluorescence imaging. The activities from areas 41 to 20 were reduced by cochlear lesions produced at P12 but not at P28, while the activities from areas 20 to 41 were reduced by the lesions at P28, suggesting the critical period for the polysynaptic feedforward connection was before P28, while for the polysynaptic feedback connection was after P28.

Periventricular nodular heterotopia functionally couples with the overlying hippocampus

Patients with periventricular nodular heterotopia (PVNH) often have severe epilepsy. However, it is unclear how the heterotopia contributes to epileptogenesis. Recently, electrophysiologic studies using intraoperative depth electrodes have indicated that interaction between the heterotopia and overlying cortex is crucial for seizure onset. We performed an in vitro physiologic study using slices of resected brain from a 22‐year‐old man with PVNH, who manifested medically refractory mesial temporal lobe epilepsy. Preoperative evaluation indicated that the right mesial temporal structure and PVNH were the epileptogenic focus. The resected tissue was immediately immersed in cold artificial cerebrospinal fluid, and then slices of the brain tissue including the heterotopic nodules and overlying hippocampus were prepared. We electrically stimulated the incubated slices, and the elicited neural activities were analyzed as changes in the flavoprotein fluorescence signals. When we stimulated either the heterotopic nodule or the overlying hippocampus, clear functional coupling of neural activities between these structures was observed. The coupling response evoked by stimulation of the subiculum and developing within the heterotopic nodule was enhanced by application of bicuculline. Therefore, activities of the hippocampus and the nodule are closely correlated.

Spatiotemporal dynamics of epileptiform propagations: Imaging of human brain slices

Seizure activities often originate from a localized region of the cerebral cortex and spread across large areas of the brain. The properties of these spreading abnormal discharges may account for clinical phenotypes in epilepsy patients, although the manner of their propagation and the underlying mechanisms are not well understood. In the present study we performed flavoprotein fluorescence imaging of cortical brain slices surgically resected from patients with partial epilepsy caused by various symptomatic lesions. Elicited neural activities in the epileptogenic tissue spread horizontally over the cortex momentarily, but those in control tissue taken from patients with brain tumors who had no history of epilepsy demonstrated only localized responses. Characteristically, the epileptiform propagation comprised early and late phases. When the stimulus intensity was changed gradually, the early phase showed an all-or-none behavior, whereas the late phase showed a gradual increase in the response. Moreover, the two phases were propagated through different cortical layers, suggesting that they are derived from distinct neural circuits. Morphological investigation revealed the presence of hypertrophic neurons and loss of dendritic spines, which might participate in the aberrant activities observed by flavoprotein fluorescence imaging. These findings indicate that synchronized activities of the early phase may play a key role in spreading abnormal discharges in human cortical epilepsies.

Transcranial imaging of somatotopic map plasticity after tail cut in mice.

Peripheral afferent denervation induces reorganization of somatotopic maps in the primary somatosensory cortex (S1). In the present study, we investigated somatotopic map plasticity after tail cut. Neonatal mice at postnatal days (P) 2-3 and adult mice at eight weeks of age were anesthetized with ether, and approximately two thirds of the tail was cut from the tip. Both groups of mice were anesthetized with urethane (1.7g/kg, i.p.) at 10weeks of age, and transcranial flavoprotein fluorescence imaging was performed in the S1. Neural activities in the S1 were elicited by vibratory stimulation applied to the contralateral hindpaw or the tail in control mice. The cortical areas activated by hindpaw, tail base, and tail tip stimuli were placed in this order according to the medial and posterior direction. In mice with tail cut, the tail base area moved to the more medial and posterior area corresponding to the tail tip in control mice. The shift of the tail base area was observed in both neonatal and adult tail cut mice, indicating the absence of a critical period before eight weeks. Medial and posterior shift of the tail base area with regard to the bregma was confirmed in tail cut mice. These data suggest that transcranial flavoprotein fluorescence imaging is a useful technique for investigating somatosensory map plasticity in mice.

Hypertrophy of hippocampal end folium neurons in patients with mesial temporal lobe epilepsy

Hypertrophic and dysmorphic neurons have been identified in the hippocampal end folium of patients with mesial temporal lobe epilepsy (mTLE). No data are available regarding the correlation between these cellular alterations and the severity of hippocampal sclerosis (HS), and the significance of this phenomenon has been unclear. We evaluated both the perikaryon and nuclear areas of residual neurons in the hippocampal end folium of 47 patients with mTLE, seven with lesional neocortical temporal lobe epilepsy (LTLE), and 10 controls without seizure episodes. According to the severity of neuron loss in the end folium, we defined mTLE cases showing slight (<10%) or no, moderate (10–50%) and severe (>50%) loss as groups A, B and C, respectively. We also performed immunohistochemistry with antibodies against heat shock protein 70 and the phosphorylated epitope of neurofilament. In both mTLE and LTLE cases, the perikaryon and nuclear areas of the end folium neurons were significantly greater than those in the controls (P < 0.0001), and those in mTLE were significantly greater than those in LTLE. There were no differences in areas between groups A and B, but the areas in group C were significantly greater than those of both groups A and B. Neurons with large, bizarre morphology were labeled with both antibodies. Neuronal hypertrophy is evident in patients with epilepsy, and appears to advance gradually as the hippocampal sclerosis becomes more severe. This alteration may be a consequence of cellular stress incurred by neurons.

Ca2+-permeable AMPA receptors associated with epileptogenesis of hypothalamic hamartoma

Hypothalamic hamartoma (HH), composed of neurons and glia without apparent cytologic abnormalities, is a rare developmental malformation in humans. Patients with HH often have characteristic medically refractory gelastic seizures, and intrinsic epileptogenesis within the lesions has been speculated. Herein we provide evidence to suggest that in HH neurons, Ca2+ permeability through α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors is aberrantly elevated. In needle biopsy specimens of HH tissue, field potential recordings demonstrated spontaneous epileptiform activities similar to those observed in other etiologically distinct epileptogenic tissues. In HH, however, these activities were clearly abolished by application of Joro Spider Toxin (JSTX), a specific inhibitor of the Ca2+‐permeable AMPA receptor. Consistent with these physiologic findings, the neuronal nuclei showed disappearance of adenosine deaminase acting on RNA 2 (ADAR2) immunoreactivity. Furthermore, examination of glutamate receptor 2 (GluA2) messenger RNA (mRNA) revealed that editing efficiency at the glutamine/arginine site was significantly low. These results suggest that neurons in HH may bear Ca2+‐permeable AMPA receptors due to dislocation of ADAR2.