Mapping and modulating neuronal circuits
We map neuronal circuits using activity reporter Targeted Recombination in Active Populations (TRAP) mice, combined with local field potential recordings, immunohistochemistry, tissue clearing confocal, two photon and light sheet microscopy. We use high resolution & expansion microscopy to localize synapses. Chemogenetic and closed loop optogenetic approaches help us modulate neuronal circuits.
Seizures activate the indirect basal ganglia pathway neurons. (A) C57BL/6 mice were implanted with cobalt and a bilateral cannula. Sumanirole or aCSF was injected via a bilateral cannula into the striatum at 18 h after cobalt (red arrow). The number of seizures was counted by a blinded experimenter before the injection (15–18 h after cobalt), following (18–20.5 h after cobalt), and after the injection (20.5–23.5 h after cobalt). (B) A representative EEG 20 min after bilateral sumanirole injection into the striatum. (C) The effect of either sumanirole (red) or aCSF (green) injection (red arrow) on the number of seizures before the injection (15–18 h after cobalt) (sum per mouse, n = 5 mice): D2 agonist: 2.2 ± 0.58, aCSF: 2.4 ± 0.87 (Kolmogorov-Smirnov test), following the injection (18–20.5 h after cobalt): D2 agonist: 0.0 ± 0.0, aCSF: 1.4 ± 0.68 (P = 0.0476, Kolmogorov-Smirnov test), and after the injection (20.5–25.5 h after cobalt): D2 agonist: 2.2 ± 0.37, aCSF: 2.0 ± 0.45 (unpaired t-test). (D) Light microscope image of the traces left from the bilateral cannula targeting dorsal anterior striatum (red arrows). (E) DRD2 (green) and tdTomato (red) expression in the striatum. (F) DRD1 (green) and tdTomato expression in the striatum. (G) ENK (green) and tdTomato expression in the striatum. Boxed inset: tdTomato-positive neurons co-localized with ENK. (H) DYN (green) and tdTomato expression in the striatum. Boxed inset: The majority of tdTomato-positive neurons did not co-localize with DYN. (I) % Co-localization of tdTomato-positive cells with either DRD2 or DRD1 in the striatum. Co-localization of tdTomato with: DRD2 in the ipsilateral striatum (red): 79.71 ± 1.857%; DRD2 in the contralateral striatum (blue): 78.40 ± 2.542%; DRD1 in the ipsilateral striatum (green): 10.93 ± 1.085%; DRD1 in the contralateral striatum (black): 11.55 ± 1.736%, (n = 4–5 mice). (J) Point line graph of the data in (I) across bregma from the anterior (1.6 mm) to posterior (−0.8 mm) striatum. (K) % Co-localization of tdTomato-positive cells with either ENK or DYN in the striatum. Co-localization of tdTomato with: ENK in the ipsilateral striatum (red): 79.46 ± 1.158%; ENK in the contralateral striatum (blue): 75.09 ± 2.092%; DYN in the ipsilateral striatum (green): 15.43 ± 1.170%; DYN in the contralateral striatum (black): 21.12 ± 0.3507%, (n = 3 mice). Data are mean ± SEM, Kolmogorov-Smirnov test, n.s. = non-significant. *P < 0.05.
Chemogenetic suppression of the ipsilateral VL does not change the onset latency in the contralateral VL or contralateral cortex. (A) KORD/CamKII.Cre was injected in the right VL. (B) Patch‐clamp recordings were done on KORD‐expressing neurons in the right VL. (C) Resting membrane potential (mV) of transduced cells before and after SALB application, where the left graph is repeated recordings from a single cell, and the right graph is the average for 5 mice. (D) Mean number of seizures in C57Bl/6 mice after cobalt insertion in C57Bl/6 mice that were injected with SALB (green) or saline (red) at 20 hours after Co (black arrow) at the peak of seizures. (E) One hundred percent of all mice developed seizures by 20 hours after Co. (F) LFPs of a seizure recorded before and after SALB injection. Power was suppressed in the right VL (red box). (G) A schematic illustrates anatomical projections to the anterior and posterior VL. (H) Mean seizure duration (seconds) remained the same before (16–20 hours after Co) and after (20–24 hours after Co) SALB or saline injection. (I) Seizure onset was delayed only in the right VL after SALB injection but not in the left cortex or left VL. (J) Right cortex was suppressed after right VL suppression (in 6 out of 11 mice). (K, L) Seizure duration decreased after posterior VL suppression. Co = cobalt; VL = ventrolateral; SALB = salvinorin B.
Neuronal circuits are three dimensional best visualized in videos.
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Patch clamp electrophysiology
We use patch-clamp electrophysiology to study plasticity of GABAergic and glutamatergic synapses, and neuronal excitability.
The enhancement of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor–mediated synaptic transmission during status epilepticus (SE) was blocked, but the diminution in γ-aminobutyric acid receptor–mediated synaptic inhibition during SE occurred in GluA1 knockout (KO) animals. (A) Averaged spontaneous excitatory postsynaptic current (sEPSC) traces recorded from CA1 pyramidal neurons of a representative control GluA1 wild-type (WT) animal (black) and a WT animal in SE (red). (B) Averaged sEPSC traces from a control GluA1-KO animal and in a KO animal in SE. (C) The mean of the median amplitude and the average frequency of sEPSCs recorded from control and SE WT animals. The amplitude in WT controls was 16.4 ± 1.0pA (n = 8 cells, n = 4 animals), and that in WT SE animals was 19.7 ± 0.9pA (n = 8 cells, 5 animals; *p = 0.028, unpaired t test). The frequency in control WT animals was 0.54 ± 0.9Hz, and that in WT SE animals was 0.54 ± 0.07Hz (n is the same as for amplitude measurements; p = 0.98, unpaired t test). (D) The mean of the median amplitude and the average frequency of sEPSCs recorded from control and SE KO animals. The amplitude in KO control and KO SE animals was 16.2 ± 1.0pA (5 cells from 3 animals) and 14.6 ± 0.43pA (8 cells from 5 animals), respectively (p = 0.132, unpaired t test). The frequency in control and SE animals was 0.57 ± 0.05Hz and 0.46 ± 0.09Hz, respectively (n is the same as for amplitude measurements; p = 0.4, unpaired t test). (E) Averaged spontaneous inhibitory postsynaptic current (sIPSC) recorded from the CA1 pyramidal neurons of a control GluA1-WT animal (black) and a WT animal in SE (blue). (F) Averaged sIPSCs recorded from CA1 pyramidal neurons of control and SE GluA1-KO animals. (F) Mean of the median sIPSC amplitude in WT control (49.5 ± 3.3pA, 8 cells from 5 animals) and WT SE animals (36.6 ± 3.3pA, 7 cells from 5 animals; p = 0.017, unpaired t test) and the mean frequency of events in the control and SE animals (1.5 ± 0.25Hz and 1.8 0.33Hz, respectively; p = 0.49, unpaired t test). (G) The mean of the median amplitude and the mean frequency of sIPSCs recorded from GluA1-KO control and SE animals. (H) The mean sIPSC amplitude in the control and SE animals was 48.9 ± 4.3pA (8 cells from 5 animals) and 26.9 ± 2.7pA (6 cells from 5 animals), respectively (**p = 0.0018, unpaired t test). Mean sIPSC frequency in control and SE animals was 1.1 ± 0.12Hz and 0.86 ± 0.19Hz, respectively (p = 0.27, unpaired t test).
Hippocampal CA1 neurons tagged by a seizure have larger dendritic spines and are more excitable.a) A fluorescent/DIC image showing hippocampal CA1 region in an acute hippocampal slice obtained from a c-fos/EGFP mouse after a single seizure. Tagged neurons are shown as bright ones in the image. The scale bar is 50 μm. Total 11 tagged neurons and 11 untagged surrounding neurons were tested for their membrane properties. b) A tagged CA1 neuron reconstructed through tracing using Imaris program (red). A non-tagged CA1 neuron (black) is shown for comparison. The scale bar represents to 100 μm. c) and d) Soma diameter and total dendritic length analyzed for tagged (GFP+ve) and non-tagged (GFP-ve) CA1 neurons (5 cells from 5 animals for each group, mean ± SD, P = 0.8526 for the dendritic length, P = 0.8472 for the soma diameter). e) Examples of dendritic sections obtained from a tagged and a non-tagged neurons, which were 200 to 250 μm away from the somas (s. radiatum area). Spine dimensional analysis was performed for tagged neurons and untagged neurons (5 neurons from 5 animals for each group, see F, G, H, I and J). f) Spine-head diameter for spines 200 to 250 mm away from the somas. Mean ± SD for 432 spines from 5 untagged neurons (black), and 444 spines from 5 tagged neurons (red). ***P < 0.0001. g) Spine-neck length for spines analyzed in c). P = 0.276. h) Spine number in 10 μm dendritic sections (density) for those spines analyzed in c). P = 0.931. i) A schematic illustrating classification of spines based on their morphology. j) Percentage of spines of each type (illustrated in panel i) for tagged and untagged neurons. * P = 0.0204 for mushroom shaped spines (red) and * P= 0.0431 for thin spines (blue). Long-thin spine (yellow), P = 0.4172; filopodia, black, P = 0.5861; Stubby, white, P = 0.8839. k) Action-potential (AP) threshold for tagged (red) and untagged (black) neurons (−48.23 ± 3.264 mV vs. −44.89 ± 2.891 mV, paired t-test, ** P = 0.0017). AP was evoked by current injection. AP threshold was measured from the first AP evoked during current injections. Initial point of AP upstroke phase is the threshold. l) Membrane resistance vs. rheobass for tagged (red circles) and untagged (black circles) neurons (** P = 0.007 for rheobass, P = 0.213 for Rm. Dashed lines show the mean values). m) Traces illustrate AP’s evoked at different current injections for a tagged and an untagged neurons. Current injections was performed from −100 pA to 300 pA with 20 pA increasing step, and lasted for 500 ms. To compare firing patterns between neurons, membrane potential was adjusted to −65 mV by using a small amount of current before the injections. n) Frequency-current (F-I) plot illustrating the frequency of AP’s evoked by each current injection, values are mean ± SD (n=11 per group. P < 0.0001, Two-way ANOVA). o) Instantaneous frequency vs. action potential sequence based on analysis of APs evoked by current injection, examples shown in m. The values represent mean ± SD, n=11 in each group. The lines represent two-phase exponential fit of the data (fast decay tau is 0.356, slow decay tau is 6.380).
Imaging neuronal activity and synaptic transmission
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Modern biosensors allow us to visualize neuronal excitability and chemical synaptic transmission. Fluorescence is detected via microscopy and fiber fluorometry.
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Video monitoring combined with electrophysiology
We monitor animal behavior with video recording while simultaneously recording EEG, or local field potentials.
Electroencephalography (EEG) patterns marking early focal intermittent seizures, continuous generalized convulsive seizures, and coma phase during status epilepticus (SE). EEG and associated behavior patterns during cobalt (Co)-homocysteine-induced SE. A, The total power of EEG recorded from the ipsilateral frontal (Fi) electrode was plotted against time. The power is in the range of 0-40 Hz. The scale bar represents power in µV2. Time 0 corresponds homocysteine injection. B-E, EEG traces recorded from four cortical electrodes—ipsilateral frontal (Fi), contralateral frontal (Fc), ipsilateral parietal (Pi), and contralateral parietal (Pc)—illustrating activity associated with behaviors corresponding to the early focal intermittent seizure phase. (B) Unilateral forepaw clonus (stage1) and (C) focal dystonia with clonus (stage 2), continuous generalized convulsive seizure phase, that is, (D) tonic stiffening (stage 3) and bilateral clonus (stage 4), and (E) generalized tonic-clonic seizure with loss of posture (stage 5). Please note the changes in EEG recorded from frontal electrode (F-I) EEG patterns observed during the coma and burst suppression phase. F, periodic epileptiform discharges, G, periodic epileptiform discharges with burst patterns (H) burst suppression patterns, and (I) complete suppression of electrographic activity, which marked the end of SE (n = 7). J, Kaplan-Meier curve illustrating the duration of SE. Percentage of animals in SE and 95% confidence intervals plotted against time (n = 7). The total duration of the SE was divided into 10-min slots. The event (end of SE) was plotted slot-wise in a Kaplan-Meier curve. K, The pie chart shows the percentage of time spent by the animals in each stage of behavior observed during SE. The numbers in the wedges represent the average percentage of time spent in the respective phase (n = 5). L, Kaplan-Meier curve illustrating the survival of animals after SE. Percentage of animals survived and 95% confidence intervals were plotted against time (n = 7). The total duration after SE was divided into a 1 h slot. The event (death of the animal) was plotted slot-wise in the Kaplan-Meier curve.
