View Full Text | Abstract |
Article as PDF | Print this Article |
Pubmed | PMC |
PubReader | Export to Citation |
Email Alerts | Open Access |
Exp Neurobiol 2022; 31(2): 116-130
Published online April 30, 2022
https://doi.org/10.5607/en22007
© The Korean Society for Brain and Neural Sciences
Bomi Chang1,2,3, Junweon Byun1, Ko Keun Kim1, Seung Eun Lee2, Boyoung Lee1, Key-Sun Kim2, Hoon Ryu2, Hee-Sup Shin1* and Eunji Cheong3*
1Center for Cognition and Sociality, Institute for Basic Science, Daejeon 34126, 2Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792, 3Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Korea
Correspondence to: *To whom correspondence should be addressed.
Eunji Cheong, TEL: 82-2-2123-5885, FAX: 82-2-362-7265
e-mail: eunjicheong@yonsei.ac.kr
Hee-Sup Shin, TEL: 82-42-878-9155, FAX: 82-42-878-9151
e-mail: shin@ibs.re.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Absence seizures are caused by abnormal synchronized oscillations in the thalamocortical (TC) circuit, which result in widespread spike-and-wave discharges (SWDs) on electroencephalography (EEG) as well as impairment of consciousness. Thalamic reticular nucleus (TRN) and TC neurons are known to interact dynamically to generate TC circuitry oscillations during SWDs. Clinical studies have suggested the association of
Keywords: Thalamocortical neuronal system, Absence seizure, Spike and wave discharges, Thalamic reticular nucleus,
Absence seizures refer to nonconvulsive status epilepticus (NCSE) caused by abnormal synchronized oscillations in the thalamocortical (TC) circuit, which result in widespread spike-and-wave discharges (SWDs) on electroencephalography (EEG) along with impairment of consciousness [1]. Symptomatically, there are two essential diagnostic components of absence seizures, behavioral arrest during the clinical ictal state, with severe impairment of consciousness, and signs of SWDs on EEG that spread throughout the brain [2]. The frequency of hypersynchrony ranges from 2.5 to 4 Hz, gradually decreasing over time, and the seizures usually last for up to 15~20 s [3]. The age of onset ranges from 4 and 7 years [4], and cognitive impairment can develop if a diagnosis is not made in a timely manner [5, 6]. In most cases, absence seizures disappear during adolescence. However, they sometimes progress to longer or more intense seizures, which can be prevented by earlier diagnosis and treatment [7, 8]. Therefore, understanding the cellular mechanisms and identifying genetic risk factors underlying the pathogenesis of absence epilepsies are necessary for the development of appropriate diagnostic and therapeutic tools [9].
The thalamic reticular nucleus (TRN) plays a significant role in controlling TC circuits and SWD generations. The TRN is a shell-like structure comprising GABAergic neurons that cover most of the rostral, lateral, and ventral parts of the thalamus [10] and provide major inhibitory synaptic input to the TC neurons [11, 12]. TRN neurons generate two distinctive patterns of action potential firings, tonic and burst. Tonic modulation of the TRN has been implicated in the genesis of absence seizures [13, 14]. Moreover, rhythmic burst firing mediated by T-type channels in TRN neurons is critical for the generation of TC oscillations during SWDs [2, 15, 16]. More importantly, a recent study suggested that altered intrinsic excitability of TRN neurons and disruption of TRN–TRN synaptic inhibition are sufficient to regulate thalamic and cortical network synchrony and generate absence seizures [14]. Therefore, TRN neurons can serve as a central modulator of TC network states and of absence seizure generation.
The
In this study, we found that
The mice were housed at room temperature (22℃), fed
Skull surface screw electrodes for EEG recording were implanted at the following coordinates in mice under 16 weeks of age that were anesthetized with ketamine using a stereotaxic device (David Kopf Instruments): anteroposterior (AP), -1.5 mm; mediolateral (ML), +1.5 mm; AP, -1.3 mm; and ML, +1.3 mm. The ground electrode was implanted in the skull above the occipital region of the brain. For EMG signal recording, a Teflon-coated tungsten electrode was inserted into the neck muscle to record postural tone. The animals were allowed to fully recover for 7 days before the experiments. We recorded EEG and EMG signals in all the groups using a monopolar setting in real time (sampling frequency, 500 Hz). EEG activity was recorded for 60 min using a pClamp10. All EEGs were acquired using an analog amplifier (8~16℃, Grass Technologies) and digitized by an analog-digital converter (Axon Digidata 1320A, Molecular Devices). Each signal was band-pass filtered using a digital Butterworth filter with a cutoff frequency of 0.5 and 30 Hz.
We classified SWDs based on two criteria: (1) the amplitude of SWDs was required to be greater than twice the amplitude of EEG waveforms observed during the awake state, and (2) the duration of SWDs was required to be at least 0.5 s. pClamp10 and MATLAB were utilized to detect SWDs based on the amplitudes, peak-to-peak periods, and shapes of EEG signals. We applied a resolution of 250 ms and a frequency range of 2~7 Hz for detection. Waveforms with a low total power and non-SWD harmonic and high-frequency content were filtered automatically. Temporal spectrograms were obtained by filtering the signals with a second-order Butterworth infinite impulse response (IIR) via a high-pass filter with a cutoff frequency before fast Fourier transformation. The power spectrum was normalized frequency-wise from the mean baseline power density estimated between the first and tenth minute of the baseline recording. In this study, we mainly focused on the role of PLCβ1 in absence seizures, so we attempted to exclude EEG recordings during convulsive seizures to analyze and carefully examine SWDs.
To design the AAV-sh
In in vivo tests for selective
Immunohistochemistry analysis was performed as described previously [13] using the following primary antibodies: anti-PLCβ1 (mouse, 1:300; Santa Cruz), anti-PV (mouse, 1:3,000~5,000; Swant, 235) and anti-cholera toxin-B subunit (goat, 1:20,000~30,000). The secondary antibodies included Alexa 488, Cy3, and Cy5 (1:500, Jackson ImmunoResearch), and images were captured using a confocal laser scanning system (Nikon).
Slice preparation was performed as described previously [13]. For patch-clamp recordings, 20- to 28-day-old mouse brains were quickly removed and placed in carbogen-equilibrated ice-cold slicing solution containing 2.5 mM KCl, 10 mM MgSO4, 1.25 mM NaH2PO4, 24 mM NaHCO3, 0.5 mM CaCl2-2H2O, 11 mM glucose, and 234 mM sucrose. From dorsal to ventral, 270-μm-thick brain slices containing the TRN region were selectively collected and incubated in an ACSF solution containing 125 mM NaCl, 2.5 mM KCl, 5 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, 3 mM CaCl2-2H2O, and 25 mM glucose for 30 min before recording.
In K+-based whole-cell current clamp mode, the intrinsic firing properties were measured in recording solutions comprising 125 mM NaCl, 2.5 mM KCl, 1.3 mM MgCl2, 1.25 mM NaH2PO4, 26 mM NaHCO3, 2 mM CaCl2-2H2O, and 25 mM glucose [310~320 mOsm/L] bubbled with 95% (vol/vol) O2/5% (vol/vol) CO2 at 36℃. The solution heater was used to maintain the temperature at 36℃ during recording solution perfusion. Recording electrodes were pulled from fabricated standard-wall borosilicate glass capillary tubes (G150F-4: outer diameter (OD), 1.50 mm; inner diameter (ID), 0.86 mm; Warner Instruments) and had a 4~7 MΩ tip resistance when filled with an intracellular solution containing 140 mM K-gluconate, 0.1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 0.02 mM EGTA, and 4 mM Mg-ATP [(pH 7.2) 300~310 mOsm/L]. Recordings of Ca2+ currents in intact TRN neurons in slices were performed as described previously. Brain slices were superfused with a bathing solution containing 120 mM NaCl, 20 mM tetraethylammonium (TEA)-Cl, 5 mM CsCl, 3 mM KCl, 2 mM MgCl2, 10 mM HEPES, 2.5 mM CaCl2-2H2O, 1 mM 4-amino pyridine (4-AP), and 0.001 mM tetrodotoxin (TTX) bubbled with 95% O2/5% CO2 at 36℃. The electrode solution contained 117 mM Cs-gluconate, 13 mM KCl, 10 mM TEA-Cl, 1 mM MgCl2, 0.07 mM CaCl2-2H2O, 10 mM HEPES, 10 mM 1,2-bis(o-amino phenoxy)ethane-N,N’,N’-tetraacetic acid, 4 mM Mg-ATP, and 0.3 Na-GTP; the pH was adjusted to 7.35 with CsOH, and the osmolarity was adjusted to 290 mosmol/L.
The series resistance compensation function (>60%) was used routinely, with a final access resistance of ~30 MΩ. The currents were corrected for capacitive currents and leak currents using a P/4 leak subtraction protocol. Twenty to thirty minutes were required to achieve acceptable perforation, with final series resistances ranging from 15 to 30 MΩ. The membrane holding potential was -60 mV unless otherwise specified.
Data acquisition and analysis were performed using pClamp10, MATLAB (MathWorks) and GraphPad Prism (GraphPad Software, San Diego, USA) in combination. We performed one-way repeated ANOVA and Bonferroni post hoc analysis of the electrophysiological experimental results using comparisons between groups and within-subject repeated conditions, including the temporal evolution of SWD density (i.e., main factors of group and time and Group×Time interaction). Large (>10 mice) and small sample size comparisons were performed using the two-tailed Student’s t test and Wilcoxon rank-sum test, respectively. All data are presented as the mean±SEM unless stated otherwise.
To understand the role of
To separate the role of
To specifically delete
With another strategy to confirm the selective role of
To determine whether the intrinsic properties of TRN neurons were altered upon deletion of the
For the burst firing pattern, in acute brain slices, the current injection hyperpolarized the membrane potential to -100 mV and followed by subsequent burst firing patterns (Fig. 5A). In
To understand the cellular mechanism underlying the decrease in TRN excitability in
Next, to assess LVA calcium currents, the TRN neurons were held at -60 mV as the resting membrane potential and deactivated by a hyperpolarizing prepulse step at -100 mV, followed by activation steps ranging from -90 to -40 mV. These steps activated LVA Ca2+ channels and induced the fast-inactivating current of T-type Ca2+ channels (Fig. 6C) [36]. The normalized I~V curves were unchanged by the peak density of a fast-inactivating current evoked at test potentials ranging from -90 to -40 mV in the TRN neurons of
The patients with
Previous studies have demonstrated that patients with the
Previously, we reported Plcβ4-deficient mice as a potential animal model for absence seizures [30, 46]. In terms of absence seizures, either Plcβ1 or Plcβ4 deficiency leads to absence seizures. Therefore, PLCβ1 and PLCβ4 are important for inducing absence seizures. However, there are clear differences between the two models in terms of brain targets and underlying mechanisms. In the thalamocortical circuit, PLCβ1 is mainly expressed in TRN neurons, whereas PLCβ4 is highly expressed in TC neurons [30]. Another important difference is that PLCβ4 modulates TC firing modes through simultaneous regulation of T- and L-type Ca2+ currents in TC neurons [34]. However, in the current study, we found no changes in T- and L-type Ca2+ currents in TRN neurons of
We observed reduced quantities of tonic firing in the TRN neurons of
Voltage-gated Ca2+ channels (VGCCs) and calcium-activated potassium channels play critical roles in the excitability of TRN neurons [13, 48]. Furthermore, LVA T-type calcium channels in particular are considered a key mechanism underlying the generation of burst firing [49]. In this study, we unexpectedly found that the properties of VGCCs and calcium-activated potassium channels were not altered in
Overall, while
This research was supported by the Institute for Basic Science (IBS), Center for Cognition and Sociality (IBS-R001-D2).