Cingulate seizure-like activity reveals neuronal avalanche regulated by network excitability and thalamic inputs

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Thalamic modulation of cingulate seizure activity via the regulation of gap junctions in mice thalamocingulate slice.

The thalamus is an important target for deep brain stimulation in the treatment of seizures. However, whether the modulatory effect of thalamic inputs on cortical seizures occurs through the modulation of gap junctions has not been previously studied. Therefore, we tested the effects of different gap junction blockers and couplers in a drug-resistant seizure model and studied the role of gap junctions in the thalamic modulation on cortical seizures. Multielectrode array and calcium imaging were used to record the cortical seizures induced by 4-aminopyridine (250 µM) and bicuculline (5-50 µM) in a novel thalamocingulate slice preparation. Seizure-like activity was significantly attenuated by the pan-gap junction blockers carbenoxolone and octanol and specific neuronal gap junction blocker mefloquine. The gap junction coupler trimethylamine significantly enhanced seizure-like activity. Gap junction blockers did not influence the initial phase of seizure-like activity, but they significantly decreased the amplitude and duration of the maintenance phase. The development of seizures is regulated by extracellular potassium concentration. Carbenoxolone partially restored the amplitude and duration after removing the thalamic inputs. A two-dimensional current source density analysis showed that the sink and source signals shifted to deeper layers after removing the thalamic inputs during the clonic phase. These results indicate that the regulatory mechanism of deep brain stimulation in the thalamus occurs partially though gap junctions

Gap junction blocker modulates the spatiotemporal propagation pattern of seizure-like activity.

<p>(A) Typical seizure-like activity before and after the application of CBX. The black and red lines indicate the segments are enlarged below. (B) The ictal-tonic phase of seizure-like activity (black line in A) was magnified. The spatiotemporal change in 2D-CSD was plotted according to different time-points. Notice that sink signals (blue) were more concentrated in layer V during the ictal-tonic phase after the application of 50 µM CBX. Sink signals were enhanced in the superficial layer. During the clonic phase, sink signals were enhanced in layer V and decreased in layer II/III after the application of CBX. (C) A typical seizure-like event was divided into ictal-tonic and clonic phases. The 2D-CSD pseudocolor map shows the spatiotemporal evolution of seizure-like activity without thalamic inputs under the influence of CBX. (D) The patterns of sink-source distributions from five different slices were superimposed. (E) The statistical results showed that sink signals significantly increased in layer II/III and decreased in layer V after the application of CBX during the ictal-tonic phase. Source signals significantly increased in layer V and decreased in layer VI. During the clonic phase, sink signals significantly increased in layer V and decreased in layer II/III, whereas source signals significantly increased in layer II/III after CBX application. (F) The statistical results showed that the sink signals were located in layer V when the thalamic inputs were intact, whereas source signals were also located in layer V during the ictal-tonic phase. Removing the thalamic inputs did not significantly change the sink-source distribution during the ictal-tonic phase. In the clonic phase, removing the thalamic inputs significantly increased sink signals in layer V and decreased them in layer VI, whereas source signals were enhanced in layers V and VI.</p

Gap junction opener TriMA enhances seizure-like activity.

<p>(A) Typical example of ensemble seizure-like activity. The channel surrounded by the blue square is magnified in B. (B) Typical seizure-like activity showed that the application of 5 mM TriMA enhanced the clonic phase of seizure-like activity (red lines). The effects of TriMA were reversible. Brain slice washes with 4-AP and bicuculline restored seizure-like activity to original levels. (C) The statistical results showed that the amplitude of seizure-like activity was not changed by TriMA in either the ictal-tonic or clonic phase. The duration of the ictal-tonic phase was significantly enhanced after 5 mM TriMA application. The clonic phase was enhanced by 3 mM TriMA application. Cx, cortex; Str, striatum; Th, thalamus.</p

Gap junctions are involved in the maintenance of seizure-like activity.

<p>(A) In the 4-AP and bicuculline control group, seizure-like activity began to appear after 10 min and reached a maximum after 80 min (upper panel). The application of 100 µM CBX 30 min prior to the application of 4-AP and bicuculline did not delay the onset of seizure-like activity. However, in the CBX group, seizure-like activity was not potentiated (lower panel). (B) Statistical results of the changes in the duration and amplitude of seizure-like activity over time. The duration and amplitude in the 4-AP and bicuculline control group were significantly greater than in the CBX group after 50 min of seizure induction. (C) The statistical results showed that CBX did not significantly influence the initial phase, but CBX could significantly decreased the duration and amplitude of seizure-like activity in the maintenance phase. (D) Time-lapse recording of potassium concentration. (E) Development of seizure-like activity. The potassium concentration increased before seizure-like activity turned into a longer duration. (F) Statistical results of time-lapse recording of potassium concentration. The application of 4-AP and bicuculline significantly increased potassium concentration, whereas the application of CBX significantly decreased it. (G) The duration of seizure-like activity was positively correlated with the concentration of extracellular potassium ions.</p

Effect of different gap junction uncouplers on seizure-like activity in thalamocingulate slice.

<p>(A) Representative traces of 4-AP+bicuculline-induced seizure-like activity. The application of 100 µM CBX significantly decreased the amplitude and duration of seizure-like activity in both the tonic and clonic phases. The application of 3 µM SPL did not change the effect of CBX. Brain slice washes with 4-AP and bicuculline partially restored seizure-like activity. (B) The statistical results showed that the duration of the clonic phase was significantly shortened after the application of 50 µM CBX. The duration and amplitude of both the tonic and clonic phases of seizure-like activity were significantly decreased after 100 µM CBX application. (C) Another gap junction uncoupler, octanol, also effectively decreased seizure-like activity. (D) The statistical results showed that 0.1 mM octanol was sufficient to significantly shorten the duration of the clonic phase of seizure-like activity. The duration and amplitude of the tonic phase was shortened by the application of 0.2 mM octanol. The amplitude of the clonic phase was also shortened by 0.2 mM octanol application. (E) The neuronal-specific gap junction blocker MFQ was tested and also modulated seizure-like activity. (F) The statistical results showed that 10 µM MFQ did not change the duration of the tonic phase of seizure-like activity. However, 10 µM MFQ significantly decreased the amplitude of the tonic phase. The clonic phase was sensitive to MFQ application. MFQ (10 µM) significantly decreased the amplitude and duration of the clonic phase. CBX was significantly more effective in suppressing seizure-like activity compared with the MFQ treatment groups.</p

Influence of gap junctions on cingulate seizures.

<p>(A) Position of the MEA against a MT-ACC slice. Ensemble seizure-like activity induced by 4-AP and bicuculline is shown. The channel surrounded by the blue square is magnified in C. (B) Simultaneous extracellular (e) and intracellular (i) recording showed that the tonic phase corresponded to a depolarization phase in the intracellular recording, whereas the clonic phase was correlated with bursting activity. After the application of octanol, the depolarization phase and bursting activity were shortened. (C) Typical example of changes in seizure-like activity after the application of octanol. 4-AP- and bicuculline-induced seizure-like activity is composed of ictal onset (arrow), a tonic phase (green line), and a clonic phase (red line). The application of 0.2 mM octanol shortened the tonic and clonic phases. (D) Western blot showed that both glial gap junction Cx43 and neuronal-specific gap junction Cx36 are expressed in the cingulate cortex.</p

Gap junctions decrease theta range oscillation.

<p>(A) The frequency analysis showed that the frequency of seizure-like activity predominantly occurred in the delta (1.5–4 Hz) and theta (4–8) ranges. The application of CBX attenuated seizure-like activity in the theta range. The statistical results showed that CBX significantly lowered θ range oscillation. (B) Average color plot of theta distribution during seizure-like activity and after the application of CBX. The application of CBX significantly decreased the distribution area of theta oscillations. (C) The coherence coefficient map of theta oscillation showed that the correlation between each channel was strong, even after CBX application, during the ictal and tonic phase. In the clonic phase, the correlation between channels in the superficial layer deceased after the application of CBX. (D) The statistical results showed that the coherence coefficient between layers was not significantly different between the control and CBX groups during the ictal-tonic phase. After the application of CBX, the theta correlation was significantly decreased in the superficial layer. ACC, anterior cingulate cortex; cc, corpus callosum; Bg, basal ganglia; Th, thalamus.</p

Gap junction blocker attenuates cross-correlation and calcium transient of seizure-like activity.

<p>(A) Average cross-correlation coefficient pseudocolor map between control, CBX, and TriMA. (B) The statistical results showed that the cross-correlation coefficient of seizure-like activity was significantly decreased after the application of CBX and increased after the application of 3 mM TriMA in both the ictal-tonic and clonic phases. (C) Calcium imaging focused on the most frequent seizure-initiation onset (black square). Calcium transients were induced by 4-AP and bicuculline. (D) Individual and average traces of calcium transients. Notice that the amplitude of the calcium transient was gradually attenuated after the application of CBX. (E) Changes in fluorescence were compared. The amplitude of the calcium transient was significantly larger before applying CBX. (F) The statistical results showed that the calcium transients were significantly decreased after the application of 50 µM CBX. (G) The synchronization index of the calcium transients between each cell was decreased after the application of CBX. (H) The statistical results showed that CBX significantly decreased the synchronization index of the calcium transients. cc, corpus callosum; Cx, cortex; Str, striatum; Th, thalamus.</p

Spontaneous inflammatory pain model from a mouse line with <it>N</it>-ethyl-<it>N</it>-nitrosourea mutagenesis

<p>Abstract</p> <p>Background</p> <p><it>N</it>-ethyl-<it>N</it>-nitrosourea mutagenesis was used to induce a point mutation in C57BL/6 J mice. Pain-related phenotype screening was performed in 915 G3 mice. We report the detection of a heritable recessive mutant in meiotic recombinant N1F1 mice that caused an abnormal pain sensitivity phenotype with spontaneous skin inflammation in the paws and ears.</p> <p>Methods</p> <p>We investigated abnormal sensory processing, neuronal peptides, and behavioral responses after the induction of autoinflammatory disease. Single-nucleotide polymorphism (SNP) markers and polymerase chain reaction product sequencing were used to identify the mutation site.</p> <p>Results</p> <p>All affected mice developed paw inflammation at 4–8 weeks. Histological examinations revealed hyperplasia of the epidermis in the inflamed paws and increased macrophage expression in the spleen and paw tissues. Mechanical and thermal nociceptive response thresholds were reduced in the affected mice. Locomotor activity was decreased in affected mice with inflamed hindpaws, and this reduction was attributable to the avoidance of contact of the affected paw with the floor. Motor strength and daily activity in the home cage in the affected mice did not show any significant changes. Although Fos immunoreactivity was normal in the dorsal horn of affected mice, calcitonin gene-related peptide immunoreactivity significantly increased in the deep layer of the dorsal horn. The number of microglia increased in the spinal cord, hippocampus, and cerebral cortex in affected mice, and the proliferation of microglia was maintained for a couple of months. Two hundred eighty-five SNP markers were used to reveal the affected gene locus, which was found on the distal part of chromosome 18. A point mutation was detected at A to G in exon 8 of the <it>pstpip2</it> gene, resulting in a conserved tyrosine residue at amino acid 180 replaced by cysteine (Y180 C).</p> <p>Conclusions</p> <p>The data provide definitive evidence that a mutation in <it>pstpip2</it> causes autoinflammatory disease in an <it>N</it>-ethyl-<it>N</it>-nitrosourea mutagenesis mouse model. Thus, our <it>pstpip2</it> mutant mice provide a new model for investigating the potential mechanisms of inflammatory pain.</p