SEARCH

SEARCH BY CITATION

Keywords:

  • Human;
  • Optical imaging;
  • Flavoprotein fluorescence;
  • Periventricular nodular heterotopia;
  • NMDA receptor

Summary

  1. Top of page
  2. Summary
  3. Patient and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

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.

Periventricular nodular heterotopia (PVNH) is a neuronal migration disorder characterized by subependymal gray matter nodules (Battaglia et al., 2006). Within these nodules, glial cells and well-differentiated pyramidal neurons with no laminar organization can be identified (Kakita et al., 2002). A large proportion of patients with PVNH have seizures, and electroclinical studies have indicated that both the heterotopic nodules and overlying cortical tissues likely contribute to the epileptogenesis. However, the precise mechanisms underlying this syndrome remain largely unclear (Tassi et al., 2005; Valton et al., 2008).

In the present study, using in vitro flavoprotein fluorescence imaging, we investigated the functional interaction between heterotopic nodules and overlying hippocampal tissue that had been surgically removed from a male patient with bilateral PVNH. Neural activities are strongly coupled with changes in endogenous flavoprotein fluorescence derived from metabolic changes in mitochondria, and monitoring of flavoprotein fluorescence signals has been used for functional brain imaging both in vivo and in vitro in several animal studies (Shibuki et al., 2003; Theyel et al., 2010). As we demonstrated recently, this method can be applied successfully to surgically resected human brain tissues, yielding significant information about the spatiotemporal dynamics of epileptogenic tissues (Kitaura et al., 2011).

Patient and Methods

  1. Top of page
  2. Summary
  3. Patient and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

The patient provided written informed consent, and the study was performed with the approval of the ethics committee of the University of Niigata.

The patient, a 22-year-old Japanese man, had medically refractory complex partial seizures (CPS) for 4 years. Semiologically the seizures were characterized by a strange sensation followed by motionless staring, oral automatism, and gestural automatism. A brain magnetic resonance imaging (MRI) study revealed some heterotopic nodules predominantly abutting along the bilateral inferior horns of the lateral ventricle (Fig. 1A), bearing some resemblance to bilateral PVNH (Battaglia et al., 2006). Invasive video–electroencephalography (EEG) monitoring using grid and depth electrodes demonstrated high-voltage and fast activities, developing simultaneously in the hippocampus and heterotopic nodules on the right side (Fig. 1B). The patient underwent a right temporal lobectomy including the mesial temporal structure and some of the heterotopic nodules. He has since remained seizure free for 1 year.

image

Figure 1.  Clinical and pathologic features of the heterotopic nodules. (A) Preoperative (upper) and postoperative (lower) T2-reversed MRI images. Arrows indicate bilateral multiple heterotopic nodules, and asterisks indicate irregularly formed mesial temporal structures. (B) Recording from depth electrodes and electrocorticogram (ECoG). The positions of depth electrodes (left) and ictal ECoG (right) are shown. The seizure pattern largely arising from channels 2–4 of D1 and 2–3 of D2 indicates simultaneous involvement of the hippocampus and heterotopic nodules at seizure onset. BT, basal temporal; D1, depth 1; D2, depth 2; LT, lateral temporal; ECG, electrocardiogram. (C) Photograph of resected brain tissues containing heterotopic nodules. The surface used for preparing experimental slices (left) and the opposite surface (right) of the brain block are shown. Arrows indicate subiculum of the hippocampal formation and arrowheads show heterotopic nodules. (D) Low-magnification views of histopathologic specimens of hippocampal formations (left upper) and a heterotopic nodule (left lower) stained with Klüver-Barrera. Hippocampal sclerosis is not evident. Higher-magnification views of the subiculum (right upper) and heterotopic nodule (right lower) of the specimen. Scale bars: left panels: 1 mm, right panels: 100 μm.

Download figure to PowerPoint

Procedures of flavoprotein fluorescence imaging using human brain slices were described previously in detail (Kitaura et al., 2011), and the summary was provided in Data S1. In brief, the resected brain tissues were immediately immersed in ice-cold artificial cerebrospinal fluid (ACSF) bubbled with 95% O2 and 5% CO2 in the operating room. Then slices, 500-μm thick, were prepared and stimulated either on a heterotopic nodule or the overlying subiculum of the hippocampus at 10 Hz for 1 s. Because of tissue damage caused by the surgical procedure, the hippocampal CA subfields were not suitable for the imaging study. Endogenous green fluorescence images (λ = 510–550 nm) in blue light (470–490 nm) were recorded as flavoprotein fluorescence signals.

In Data S1, procedures of histologic examination and protein analysis (Takei et al., 2009) were provided.

Results

  1. Top of page
  2. Summary
  3. Patient and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

In the resected brain block, multiple heterotopic nodules (HNs) of gray matter were observed within the white matter of the parahippocampal gyrus and periventricular area (Fig. 1C). Microscopically, thin bundles of myelinated nerve fibers separated one nodule from another, the nodules from the subiculum, and the nodules from the ventricular wall. All of the nodules were composed of pyramidal and nonpyramidal neurons and glial cells. The histologic characteristics of the neurons were similar to those of mature neocortical neurons, but no cortical lamination was evident. Myelinated nerve fibers were also found within the nodules (Fig. 1D). In addition, single heterotopic neurons were scattered within the white matter adjacent to the nodules. The presence of myelinated nerve fibers between the nodules and the ependymal lining suggested that neurons generated within the ventricular zone had migrated in some extent. Hippocampal sclerosis was not evident. All CA subfields were well organized without misplaced neurons. On the other hand, in the subiculum, the pyramidal neurons showed uneven directions of the apical dendrites, suggesting mild disorganization (Fig. 1D).

In the optical imaging study when an HN was stimulated, a fluorescence response was observed within the stimulated HN and a weak response was observed simultaneously within the subiculum, a nonstimulated structure. We adopted the term “coupling response” for the response observed within such a nonstimulated structure. Conversely, when we stimulated the subiculum, the coupling response in the HNs was not apparent, but subsequent application of 5-μm bicuculline methiodide yielded a clear coupling response (Fig. 2Ab middle). For quantitative analysis, a circular window (diameter 0.6 mm) was placed in the HN or subiculum in order to maximize the fluorescence responses. In the presence of bicuculline, the initial rising slopes of the flavoprotein fluorescence responses of the subiculum and HN evoked by stimulation of the subiculum were very similar (Fig. 2B left). On the other hand, the coupling response in the subiculum evoked by stimulation of the HN was not enhanced by bicuculline (Fig. 2B right). In addition, application of 50-μm D-2-amino-5-phosphonovalerate (APV), an N-methyl-d-aspartate (NMDA) receptor antagonist, markedly suppressed the coupling response in the HN (Fig. 2Ab right). To evaluate the effects of these drugs, we calculated the coupling ratio as the maximal ΔF/F0 amplitude in the coupling area divided by that in the stimulated area. The coupling ratio was 91.6% in the HN during application of bicuculline, and was suppressed to 37.9% during application of APV. On the other hand, the coupling ratios in the subiculum were largely unchanged during application of these drugs (Fig. 2C).

image

Figure 2.  Functional coupling from the overlying subiculum (Sub) to the heterotopic nodule (HN) is suppressed by feedforward inhibition. (A) Macroscopic appearance of the brain block (left) and a translucent image of the prepared slice (right). The examined area is indicated by a blue square. (b) Flavoprotein fluorescence images after stimulation of the heterotopic nodule (HN; upper panels) or subiculum (Sub; lower panels). Responses before any drug application, after application of bicuculline 5 μm, and after application of both APV 50 μm and bicuculline 5 μm are compared. Note clear coupling response in HN during application of bicuculline 5 μm, and its abrogation by APV 50 μm (arrowheads). All images were obtained at 1.2 s after stimulation onset. (B) Time courses of flavoprotein fluorescence signals in HN and Sub stimulated by Sub (left) or HN (right) in the presence of bicuculline. These traces were analyzed from the circular windows (Ab) shown in the middle panel. (C) Coupling ratio of HN and Sub during drug application. (left: after HN stimulation; right: after subicular stimulation). (D) Western blot analysis of NR1 and NR2B expression. β-Actin and NSE are shown as controls. (E) Schematic illustration of connectivity between the NH and subiculum. Int, inhibitory interneuron. Modified from a schema that appeared in Tschuluun et al. (2011).

Download figure to PowerPoint

We then analyzed the protein levels of the NMDA receptors. Western blot analysis demonstrated an immunoreactive band for NR1 and NR2B (NMDA receptor subunits) in both the HNs and subiculum, but the signals in the HN were weaker than those in the subiculum (61% for NR1 and 67% for NR2B) (Fig. 2D). The levels of expression of both β-actin and neuron-specific enolase (NSE) in both the HNs and subiculum appeared to be similar, suggesting that the populations of neurons were roughly even.

Discussion

  1. Top of page
  2. Summary
  3. Patient and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

By applying the flavoprotein fluorescence imaging technique to human epileptogenic tissue, we clarified that the evoked neural activity in the HN was functionally coupled to that in the overlying hippocampus. This was consistent with the features of the electrocorticogram recorded using depth electrodes, in which the seizures began simultaneously from the HNs and the overlying cortex (Fig. 1B). Similar phenomena have been reported previously. For example, ictal spikes detected by invasive EEG recording have shown synchronicity between the HNs and overlying cortex, whereas interictal spikes rarely do so (Tassi et al., 2005; Valton et al., 2008). Moreover, the synchronous interictal spiking activity has been shown in both heterotopic nodules and neocortex immediately after ceasing the seizure (Battaglia et al., 2006). Therefore, these findings indicate bidirectional excitatory tight connections between the HNs and overlying cortex. Consistent with this, a carbocyanine dye (DiI) tracing study using autopsied tissue from humans with PVNH has demonstrated that the HNs and overlying cortex appeared to be anatomically connected by axon fibers (Kakita et al., 2002).

Of interest, functional coupling activity evoked in the HNs appeared in the presence of bicuculline (Fig. 2C right). On the other hand, the coupling response in the subiculum was detected before application of bicuculline, albeit weakly, and the response was largely unaffected by bath application of bicuculline. Moreover, application of 50-μm APV clearly suppressed the coupling response in HNs, but not in the subiculum. We also detected the expression of both NR1 and NR2B in the HNs, although at a lower level than that in the subiculum (Fig. 2D). Consistent with this finding, a previous study also demonstrated lower expression of both NR1 and NR2B in HNs than in the overlying cortex (Battaglia et al., 2002). These results suggest that the excitatory connections between HNs and the subiculum involve different mechanisms. The coupling response in the HNs was detectable in the presence of bicuculline. This evidence suggests possible feedforward inhibition from the subiculum to the HN (Fig. 2E). If this connection is weakened, the excitatory loop through the HN and subiculum might be amplified, resulting in epileptiform activities. This speculation seems reasonable when considering a previous study using brain slices from model rats with PVNH. In that study, spontaneous discharges induced by bicuculline were time-locked between the HN and the hippocampus, and the discharges were always initiated in the hippocampus, and not in the HN (Tschuluun et al., 2011).

In conclusion, our in vitro study shows highly integrated activities between the HN and the overlying cortex exist in a patient with PVNH, and is consistent with the hypothesis that interaction between the heterotopia and overlying cortex is crucial for seizure onset.

Acknowledgments

  1. Top of page
  2. Summary
  3. Patient and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

This work was supported by Grants-in-Aid (21300134, 22700376) for Scientific Research from MEXT, Japan; a Grant (21B-5) for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare, Japan; and a Project Research Promotion Grant from the University of Niigata.

Disclosure

  1. Top of page
  2. Summary
  3. Patient and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

References

  1. Top of page
  2. Summary
  3. Patient and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information
  • Battaglia G, Pagliardini S, Ferrario A, Gardoni F, Tassi L, Setola V, Garbelli R, LoRusso G, Spreafico R, Di Luca M, Avanzini G. (2002) AlphaCaMKII and NMDA-receptor subunit expression in epileptogenic cortex from human periventricular nodular heterotopia. Epilepsia 43:209216.
  • Battaglia G, Chiapparini L, Franceschetti S, Freri E, Tassi L, Bassanini S, Villani F, Spreafico R, D’Incerti L, Granata T. (2006) Periventricular nodular heterotopia: classification, epileptic history, and genesis of epileptic discharges. Epilepsia 47:8697.
  • Kakita A, Hayashi S, Moro F, Guerrini R, Ozawa T, Ono K, Kameyama S, Walsh CA, Takahashi H. (2002) Bilateral periventricular nodular heterotopia due to filamin 1 gene mutation: widespread glomeruloid microvascular anomaly and dysplastic cytoarchitecture in the cerebral cortex. Acta Neuropathol 104:649657.
  • Kitaura H, Hiraishi T, Murakami H, Masuda H, Fukuda M, Oishi M, Ryufuku M, Fu YJ, Takahashi H, Kameyama S, Fujii Y, Shibuki K, Kakita A. (2011) Spatiotemporal dynamics of epileptiform propagations: imaging of human brain slices. NeuroImage 58:5059.
  • Shibuki K, Hishida R, Murakami H, Kudoh M, Kawaguchi T, Watanabe M, Watanabe S, Kouuchi T, Tanaka R. (2003) Dynamic imaging of somatosensory cortical activity in the rat visualized by flavoprotein autofluorescence. J Physiol 549:919927.
  • Takei N, Kawamura M, Ishizuka Y, Kakiya N, Inamura N, Namba H, Nawa H. (2009) Brain-derived neurotrophic factor enhances the basal protein synthesis by increasing active eukaryotic elongation factor 2 levels and promoting translation elongation in cortical neurons. J Biol Chem 284:2634026348.
  • Tassi L, Colombo N, Cossu M, Mai R, Francione S, Lo Russo G, Galli C, Bramerio M, Battaglia G, Garbelli R, Meroni A, Spreafico R. (2005) Electroclinical, MRI and neuropathological study of 10 patients with nodular heterotopia, with surgical outcomes. Brain 128:321337.
  • Theyel BB, Llano DA, Sherman SM. (2010) The corticothalamocortical circuit drives higher-order cortex in the mouse. Nat Neurosci 13:8488.
  • Tschuluun N, Wenzel HJ, Doisy ET, Schwartzkroin PA. (2011) Initiation of epileptiform activity in a rat model of periventricular nodular heterotopia. Epilepsia 52:23042314.
  • Valton L, Guye M, McGonigal A, Marquis P, Wendling F, Regis J, Chauvel P, Bartolomei F. (2008) Functional interactions in brain networks underlying epileptic seizures in bilateral diffuse periventricular heterotopia. Clin Neurophysiol 119:212223.

Supporting Information

  1. Top of page
  2. Summary
  3. Patient and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Data S1. Supplementary methods.

FilenameFormatSizeDescription
EPI_3509_sm_Supplementary-Methods.doc26KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.