Hiroatsu Murakami

Dynamic imaging of somatosensory cortical activity in the rat visualized by flavoprotein autofluorescence

We used autofluorescence of mitochondrial flavoproteins to image cortical neural activity in the rat. Green autofluorescence in blue light was examined in slices obtained from rat cerebral cortex. About half of the basal autofluorescence was modulated by the presence or absence of O2 or glucose in the medium. Repetitive electrical stimulation at 20 Hz for 1 s produced a localized fluorescence increase in the slices. The amplitude of the increase was 27 ± 2 % (mean ±s.d., n= 35). Tetrodotoxin or diphenyleneiodonium, an inhibitor of flavoproteins, blocked the autofluorescence responses. The autofluorescence responses were not observed in slices perfused with calcium‐, glucose‐ or O2‐free medium. In the primary somatosensory cortex of rats anaesthetized with urethane (1.5 g kg−1, i.p.), an activity‐dependent increase in autofluorescence of 20 ± 4 % (n= 6) was observed after electrical cortical stimulation at 100 Hz for 1 s, and an increase of 2.6 ± 0.5 % (n= 33) after vibratory skin stimulation at 50 Hz for 1 s applied to the plantar hindpaw. These responses were large enough to allow visualization of the neural activity without having to average a number of trials. The distribution of the fluorescence responses after electrical or vibratory skin stimulation was comparable to that of the cortical field potentials in the same rats. The fluorescence responses were followed by an increase in arterial blood flow. The former were resistant to an inhibitor of nitric oxide synthase, while the latter was inhibited. Thus, activity‐dependent changes in the autofluorescence of flavoproteins are useful for functional brain imaging in vivo.

Minimally invasive magnetic resonance imaging-guided stereotactic radiofrequency thermocoagulation for epileptogenic hypothalamic hamartomas.

OBJECTIVETo validate the safety and efficacy of magnetic resonance imaging (MRI)-guided stereotactic radiofrequency thermocoagulation (SRT) for epileptogenic hypothalamic hamartoma (HH), we evaluated surgical outcomes and revised the MRI classification. METHODSWe retrospectively reviewed 25 consecutive patients with HH (age range, 2–36 years; mean age, 14.8 years) with gelastic seizures. Other seizure types were exhibited in 22 patients (88.0%), precocious puberty in 8 (32.0%), behavioral disorder in 10 (40.0%), and mental retardation in 14 (56.0%). We classified HH into 3 subtypes according to coronal MRI: intrahypothalamic, parahypothalamic, and mixed hypothalamic type. Maximum diameter ranged from 8 to 30 mm (mean, 15.3 mm). All patients underwent SRT (74°C, 60 seconds) for HH. RESULTSHH subtype and size were correlated with precocious puberty, mental retardation, and behavioral disorder. Thirty-one SRT procedures were performed, requiring 1 to 8 tracks (mean, 3.8 tracks) and involving 1 to 18 lesions (mean, 7.2 lesions). There were no adaptive limitations, regardless of size or subtype. Mixed-type HHs needed more tracks and more lesions. No permanent complications persisted after SRT, and gelastic seizures disappeared in all but 2 patients. Complete seizure freedom was achieved in 19 patients (76.0%). These patients had not only disappearance of all seizure types and behavioral disorder but also intellectual improvement. CONCLUSIONThe present SRT procedure has favorable efficacy and invasiveness and has no adaptive limitations. SRT should therefore be considered before adulthood. The new HH classification is useful to understand clinical symptoms and to determine surgical strategies.

Short-term plasticity visualized with flavoprotein autofluorescence in the somatosensory cortex of anaesthetized rats

In the present study, short‐term plasticity of somatosensory neural responses was investigated using flavoprotein autofluorescence imaging in rats anaesthetized with urethane (1.5 g/kg, i.p.) Somatosensory neural activity was elicited by vibratory skin stimulation (50 Hz for 1 s) applied on the surface of the left plantar hindpaw. Changes in green autofluorescence (λ = 500–550 nm) in blue light (λ = 450–490 nm) were elicited in the right somatosensory cortex. The normalised maximal fluorescence responses (ΔF/F) was 2.0 ± 0.1% (n = 40). After tetanic cortical stimulation (TS), applied at a depth of 1.5–2.0 mm from the cortical surface, the responses elicited by peripheral stimulation were significantly potentiated in both peak amplitude and size of the responsive area (both P < 0.02; Wilcoxon signed rank test). This potentiation was clearly observed in the recording session started 5 min after the cessation of TS, and returned to the control level within 30 min. However, depression of the responses was observed after TS applied at a depth of 0.5 mm. TS‐induced changes in supragranular field potentials in cortical slices showed a similar dependence on the depth of the stimulated sites. When TS was applied on the ipsilateral somatosensory cortex, marked potentiation of the ipsilateral responses and slight potentiation of the contralateral responses to peripheral stimulation were observed after TS, suggesting the involvement of commissural fibers in the changes in the somatosensory brain maps. The present study clearly demonstrates that functional brain imaging using flavoprotein autofluorescence is a useful technique for investigating neural plasticity in vivo.

Correlating magnetoencephalography to stereo-electroencephalography in patients undergoing epilepsy surgery

Magnetoencephalography and stereo-electroencephalography are often necessary in the course of the non-invasive and invasive presurgical evaluation of challenging patients with medically intractable focal epilepsies. In this study, we aim to examine the significance of magnetoencephalography dipole clusters and their relationship to stereo-electroencephalography findings, area of surgical resection, and seizure outcome. We also aim to define the positive and negative predictors based on magnetoencephalography dipole cluster characteristics pertaining to seizure-freedom. Included in this retrospective study were a consecutive series of 50 patients who underwent magnetoencephalography and stereo-electroencephalography at the Cleveland Clinic Epilepsy Center. Interictal magnetoencephalography localization was performed using a single equivalent current dipole model. Magnetoencephalography dipole clusters were classified based on tightness and orientation criteria. Magnetoencephalography dipole clusters, stereo-electroencephalography findings and area of resection were reconstructed and examined in the same space using the patient’s own magnetic resonance imaging scan. Seizure outcomes at 1 year post-operative were dichotomized into seizure-free or not seizure-free. We found that patients in whom the magnetoencephalography clusters were completely resected had a much higher chance of seizure-freedom compared to the partial and no resection groups ( P = 0.007). Furthermore, patients had a significantly higher chance of being seizure-free when stereo-electroencephalography completely sampled the area identified by magnetoencephalography as compared to those with incomplete or no sampling of magnetoencephalography results ( P = 0.012). Partial concordance between magnetoencephalography and interictal or ictal stereo-electroencephalography was associated with a much lower chance of seizure freedom as compared to the concordant group ( P = 0.0075). Patients with one single tight cluster on magnetoencephalography were more likely to become seizure-free compared to patients with a tight cluster plus scatter ( P = 0.0049) or patients with loose clusters ( P = 0.018). Patients whose magnetoencephalography clusters had a stable orientation perpendicular to the nearest major sulcus had a better chance of seizure-freedom as compared to other orientations ( P = 0.042). Our data demonstrate that stereo-electroencephalography exploration and subsequent resection are more likely to succeed, when guided by positive magnetoencephalography findings. As a corollary, magnetoencephalography clusters should not be ignored when planning the stereo-electroencephalography strategy. Magnetoencephalography tight cluster and stable orientation are positive predictors for a good seizure outcome after resective surgery, whereas the presence of scattered sources diminishes the probability of favourable outcomes. The concordance pattern between magnetoencephalography and stereo-electroencephalography is a strong argument in favour of incorporating localization with non-invasive tools into the process of presurgical evaluation before actual placement of electrodes. * Abbreviations : SECD : single equivalent current dipole SEEG : stereo-electroencephalography

Neuromagnetic activation of primary and secondary somatosensory cortex following tactile-on and tactile-off stimulation

OBJECTIVE Magnetoencephalography (MEG) recordings were performed to investigate the cortical activation following tactile-on and tactile-off stimulation. METHODS We used a 306-ch whole-head MEG system and a tactile stimulator driven by a piezoelectric actuator. Tactile stimuli were applied to the tip of right index finger. The interstimulus interval was set at 2000 ms, which included a constant stimulus of 1000 ms duration. RESULTS Prominent somatosensory evoked magnetic fields were recorded from the contralateral hemisphere at 57.5 ms and 133.0 ms after the onset of tactile-on stimulation and at 58.2 ms and 138.5 ms after the onset of tactile-off stimulation. All corresponding equivalent current dipoles (ECDs) were located in the primary somatosensory cortex (SI). Moreover, long-latency responses (168.7 ms after tactile-on stimulation, 169.8 ms after tactile-off stimulation) were detected from the ipsilateral hemisphere. The ECDs of these signals were identified in the secondary somatosensory cortex (SII). CONCLUSIONS The somatosensory evoked magnetic fields waveforms elicited by the two tactile stimuli (tactile-on and tactile-off stimuli) with a mechanical stimulator were strikingly similar. These mechanical stimuli elicited both contralateral SI and ipsilateral SII activities. SIGNIFICANCE Tactile stimulation with a mechanical stimulator provides new possibilities for experimental designs in studies of the human mechanoreceptor system.

Ictogenesis and symptomatogenesis of gelastic seizures in hypothalamic hamartomas: An ictal SPECT study

Purpose:  To topographically localize the ictogenic zone within hypothalamic hamartomas (HHs) and the symptomatogenic zone for gelastic seizure (GS), we analyzed data from both interictal and ictal single photon emission computed tomography (SPECT).

Muscle-afferent projection to the sensorimotor cortex after voluntary movement and motor-point stimulation: An MEG study

OBJECTIVE To investigate the projection of muscle afferents to the sensorimotor cortex after voluntary finger movement by using magnetoencephalography (MEG). METHODS The movement-evoked magnetic fields (MEFs) after voluntary index-finger extension were recorded by a 204-channel whole-head MEG system. Somatosensory-evoked magnetic fields (SEFs) were recorded after motor-point stimulation was applied to the right extensor indicis muscle by using a pair of wire electrodes. RESULTS The MEF waveforms were observed at 35.8±9.7 ms after movement onset (MEF1). The most concentrated SEFs were identified at 78.7±5.6 ms (M70), and the onset latency of M70 was 39.0±5.5 ms after motor-point stimulation. The mean locations of the equivalent current dipoles (ECDs) of MEF1 and M70 were significantly medial and superior to that of N20m elicited by median-nerve stimulation. The ECD locations and directions of both MEF1 and M70 were concordant in the axial, coronal and sagittal planes. CONCLUSIONS MEF1 and M70 might be elicited by muscle-afferent feedback following muscle contraction. In addition, these ECDs may be located in area 4. SIGNIFICANCE Motor-point stimulation is a useful tool for confirming the projection of muscle-afferent feedback to the sensorimotor cortex after voluntary movement.

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.

Suppressed Expression of Autophagosomal Protein LC3 in Cortical Tubers of Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is characterized by benign tumors and hamartomas, including cortical tubers. Hamartin and tuberin, encoded by the TSC 1 and 2 genes, respectively, constitute a functional complex that negatively regulates the mammalian target of rapamycin (mTOR) signaling pathway, eventually promoting the induction of autophagy. In the present study, we assessed the induction of autophagy in cortical tubers surgically removed from seven patients with TSC in comparison with five controls of cortical tissue taken from non‐TSC patients with epilepsy. Immunoblotting demonstrated a marked reduction of LC3B‐I and LC3B‐II in tubers relative to the controls. In tubers, strong, diffuse and dot‐like immunoreactivity (IR) for LC3B was observed in dysmorphic neurons and balloon cells, but LC3B‐IR in other neurons with normal morphology was significantly weaker than that in neurons in the controls. Immunoelectron microscopy revealed diffuse distribution of LC3B‐IR within the cytoplasm of balloon cells. The dot‐like pattern may correspond to abnormal aggregation bodies involving LC3. In an autopsy patient with TSC, we observed that LC3B‐IR in neurons located outside of the tubers was preserved. Thus, autophagy is suppressed in tubers presumably through the mTOR pathway, and possibly a pathological autophagy reaction occurs in the dysmorphic neurons and balloon cells.

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.