Article Figures & Data
Figures
- Figure 1.
Putative L2/3 PNs from human epileptic foci have diverse morphology and spiking properties. Ai–Ei, Depolarizing current steps at rheobase (pA; dark trace) and rheobase + 100 pA (2× rheobase, light trace) elicit action potentials (APs) of various shapes and frequencies. Each group was determined based on firing property and AHP shape. Aii–Eii, Representative trace of each AP from neuron subtype (scale bars: 10 mV, 10 ms). Aiii–Eiii, Corresponding 3D reconstruction of neuron morphology (x-axis scale bar: 200 µm). Each neuron's location in the representative cortical layers was determined by the respective distance of their soma from pial surface (dendrites: subtype color scheme, axon branches: blue).
- Figure 2.
Putative L2/3 PN subtypes show subtle differences in AP kinetics and firing properties. A, Neuron subtype percentage (%) of the entire population of recorded neurons (ACC, accommodating neurons; RS, regular spiking neurons; Not, notch neurons; ST, stuttering neurons; ES, early spiking neurons). B, Representative traces of L2/3 PN subtype AP (scale bars: 10 mV, 10 ms). C, Overlay of average AP of each neuron subtype. D, Overlay of average phase plot (dV/dT vs voltage) indicating differences in subtype AP kinetics. E, Notch neurons showed the shortest AP half-width with a significant difference compared with accommodating neurons. F, Stutter neurons showed a significant reduction in AP spike amplitude. G, Notch neurons have the shortest AHP latency with a significantly shorter AHP latency compared with RS and ACC neurons. H, FR accommodation ratio of each neuron subtype taken at 2× rheobase indicated ES neurons had a significantly lower chance of accommodating compared with stutter neurons. I, Initial instantaneous frequency ± SEM versus injected current (pA). J, Initial instantaneous frequency was taken at the rheobase + 100 pA current step and indicated significant differences between subtype initial instantaneous firing frequency. K, Final instantaneous frequency ± SEM versus injected current (pA). Scatterplots include mean values ± SD.
- Figure 3.
Epileptic L2/3 PNs with fAHP or mAHP do not differ in their firing rates or firing rate accommodation. A, Representative traces of L2/3 PN AP shapes showing neurons with a (Ai) fAHP followed by mAHP, (Aii) fAHP followed by ADP and mAHP, and (Aiii) mAHP only. B, Overlay of average AP of neuron split by AHP shape. C, Overlay of average phase plots (dV/dT vs voltage) of neuron split by AHP shape. D, There is no significant differences in the maximum depolarization slope of the AP (dV/dT) between subtypes. E, mAHP PNs have a significantly slower repolarization slope (dV/dT) when compared with fAHP PNs. F, AHP latency is shorter for fAHP and ADP PNs as expected compared with mAHP neurons. G, fAHP and ADP PNs show a more positive ΔAHP. H, mAHP PNs have more AP amplitude adaptation compared with ADP PNs. I, mAHP PNs have higher overall voltage sag (%) compared with fAHP PNs. We observe no differences in J, initial instantaneous frequency ± SEM versus injected current (pA); K, mean instantaneous frequency ± SEM versus injected current (pA); L, final instantaneous frequency ± SEM versus injected current (pA); or M, FR accommodation ratio ± SEM versus injected current (pA). N, Percentage (%) of AHP PN based on cell type characterized in Figure 2. f, fAHP; ADP, fAHP-ADP; m mAHP. Scatterplots include mean values ± SD.
- Figure 4.
Epileptic L2/3 PNs show minor differences between etiology, but crucial differences compared with control neurons. A, Representative traces from current (pA) ramp protocol (1,000 pA, 1 s) of L2/3 PNs from control (blue), MCD epileptic (purple), and other epileptic (OE) tissue (green). B, Representative traces from L2/3 PNs from control and epileptic subtype showing the corresponding −250 pA hyperpolarizing step (darker negative trace), rheobase step (darker positive trace), and 2× rheobase (lighter positive trace) depolarizing current step (600 ms steps). C, Percentage (%) of PNs with certain AHP cell type based on control and epilepsy subtype. D, Overlay of average AP of control and epileptic subtype L2/3 PNs. E, Overlay of average phase plots (dV/dT vs voltage) of control and epileptic subtype L2/3 PNs. F, MCD L2/3 PNs have a depolarized AP threshold compared with control. G, OE L2/3 PNs have significantly longer AP half-widths compared with MCD with trends toward longer half-widths in both epileptic subtype PNs compared with control PNs. H, Depolarization slopes (dV/dT) were significantly slower in both epileptic subtypes compared with control. I, AHP magnitude (mV) is significantly larger in both epileptic subtypes compared with control. J, Epileptic L2/3 PNs show a wide variability in input resistance. MCD L2/3 PNs on average have significantly higher input resistances compared with control and OE L2/3 PNs. K, Voltage sag is significantly smaller in both epileptic subtypes compared with control. L, Left, Mean FR ± SEM versus injected current (pA). L, Right, We observed lower max firing rate for both epileptic subtypes compared with control. M, Left, FR accommodation ratio ± SEM versus injected current (pA). M, Right, FR accommodation ratios indicate a lack of FR accommodation by OE L2/3 PNs compared with MCD L2/3 PNs with no differences compared with control. Although, on average, control PNs accommodate their frequency the most compared with the epileptic subtype PNs. Scatterplots include mean values ± SD.
- Figure 5.
4-AP increases L2/3 PN AP half-width and AHP latency and decreases AHP magnitude leading to sustained firing. A, Overlay of a representative AP before and after 4-AP wash on (scale bars: 10 mV, 10 ms). Bi, Overlay of average AP before and after 4-AP wash on. Bii, Overlay of phase plots. C, Input resistance (MΩ) decreased after 4-AP wash on. D, AP half-width increased after 4-AP wash on. E, AP threshold became hyperpolarized after 4-AP wash on. F, AHP magnitude was reduced after 4-AP wash on G, AHP latency got slower after 4-AP wash on. H, AP amplitude was not different after 4-AP wash on. I, Left, Mean FR ± SEM versus injected current (pA). I, Right, No significant difference was found for mean Max FR after 4-AP wash on. J, FR accommodation ratio ± SEM versus injected current (pA) before and after 4-AP wash on at 400, 500, 600, 750, 800, and 850 pA depolarizing current steps indicates a significant absence of accommodation after 4-AP wash on. Scatterplots include mean values ± SD.
Tables
Final pathological diagnosis Experimental group Region of tissue resection Patient age at surgery (years) Patient sex Anaplastic
Pleomorphic Xanthoastrocytoma
Control Parietal lobe 21 M Germinoma Control Parietal lobe 11 M Embryoma Control Temporal lobe 9 M Atypical meningioma Control Parietal lobe 8 F Glioma Control Temporal lobe 1 M FCD 1B MCD Temporal tip 9 F FCD IA MCD Lateral temporal lobe 17 F FCD IC MCD Frontal lobe, temporal tip 7 M FCD IIA MCD Frontal lobe 2 F Mild MCD MCD Frontal lobe 3 F Tuberous Sclerosis MCD Frontal lobe 10 M FCD IIA MCD Occipital lobe 19 M FCD IIB MCD Frontal lobe 2 F FCD IIID MCD Lateral temporal lobe 6 M FCD IIID MCD Temporal lobe, frontal lobe 18 M Tuberous Sclerosis MCD Temporal lobe 15 M FCD IIA MCD Frontal lobe 2 F FCD IIB MCD Frontal lobe 7 F Chaslin's subpial gliosis Other epilepsy Lateral temporal lobe 18 M Chaslin's subpial gliosis Other epilepsy Lateral temporal lobe 20 M Encephalomalacia Other epilepsy Frontal lobe 18 M Chaslin's subpial gliosis Other epilepsy Lateral temporal lobe 5 F Ganglioglioma Other epilepsy Temporal tip 16 F Chaslin's subpial gliosis Other epilepsy Frontal lobe 5 M Chaslin's subpial gliosis Other epilepsy Lateral temporal lobe 10 M Gliosis and Meningioangiomatosis Other epilepsy Parietal lobe 16 F Gliosis Other epilepsy Frontal lobe 6 M Gliosis Other epilepsy Temporal lobe 5 M Low-grade glial neoplasm Other epilepsy Temporal lobe 1 F DNET Other epilepsy Temporal lobe 10 F Low-grade glioma, WHO grade I Other epilepsy Temporal lobe 8 M Chaslin's subpial gliosis Other epilepsy Temporal lobe, frontal lobe 16 M DNET Other epilepsy Frontal lobe 6 F Demographic information, diagnosis, region of tissue resection and experimental group.
- Table 2.
Intrinsic properties of putative L2/3 PN subtypes based on firing properties and AP shape
Intrinsic property Accommodating (n = 7, 4) Regular Spiker (n = 44, 21) Notch (n = 18, 10) Stutter (n = 6, 4) Early Spiker (n = 7, 6) KW Test (p value, H) Resting membrane potential (mV) −68.47 ± 7.50 −66.53 ± 7.46 −68.33 ± 2.95 −72.86 ± 5.25 −65.77 ± 5.72 p = 0.1943, H(4) = 6.07 Input resistance (MΩ) 137.60 ± 68.50 136.8 ± 74.64 88.42 ± 33.15 136.10 ± 69.14 95.73 ± 44.51 p = 0.1202, H(4) = 7.31 Voltage sag (%) 5.69 ± 3.27 5.27 ± 2.59 5.22 ± 2.73 4.74 ± 2.79 4.10 ± 1.14 p = 0.8742, H(4) = 1.22 Membrane decay (ms) 35.34 ± 6.99 26.49 ± 7.45 24.14 ± 5.97 24.68 ± 6.95 27.03 ± 5.94 p = 0.0340, H(4) = 10.41 AP threshold (mV) −37.57 ± 3.76 −36.31 ± 5.47 −36.84 ± 3.59 −36.87 ± 10.16 −41.50 ± 5.47 p = 0.2593, H(4) = 5.28 AP amplitude (mV) 86.50 ± 7.92 79.47 ± 8.54 82.98 ± 6.30 68.67 ± 10.09 85.49 ± 6.76 p = 0.0016, H(4) = 17.41 AP half-width (ms) 1.69 ± 0.43 1.30 ± 0.30 1.12 ± 0.22 1.22 ± 0.40 1.5 ± 0.34 p = 0.0062, H(4) = 14.38 AHP magnitude (mV) −15.90 ± 2.71 −14.75 ± 3.67 −15.27 ± 2.55 −14.54 ± 3.14 −16.48 ± 2.61 p = 0.7774, H(4) = 1.77 AHP latency (ms) 11.56 ± 8.36 14.96 ± 12.72 3.57 ± 1.12 5.05 ± 2.14 9.10 ± 5.45 p < 0.0001, H(4) = 25.45 ΔAHP (mV) −5.12 ± 3.95 −3.72 ± 3.07 −1.89 ± 1.53 −5.53 ± 2.86 −4.46 ± 2.68 p = 0.0396, H(4) = 10.05 AP broadening ratio 1.30 ± 0 0.08 1.27 ± 0.11 1.24 ± 0.10 1.32 ± 0.07 1.22 ± 0.07 p = 0.1265, H(4) = 7.18 AP amplitude adaptation ratio 0.91 ± 0.06 0.93 ± 0.06 0.96 ± 0.04 0.91 ± 0.05 0.91 ± 0.05 p = 0.0794, H(4) = 8.355 Initial instantaneous frequency (Hz) 19.88 ± 3.67 34.55 ± 14.20 34.05 ± 21.42 45.48 ± 13.20 17.27 ± 4.10 p = 0.0002, H(4) = 22.11 Maximum firing rate (Hz) 24.35 ± 4.24 34.96 ± 11.76 32.94 ± 13.32 35.06 ± 8.37 23.69 ± 5.75 p = 0.0141, H(4) = 12.49 Final instantaneous frequency (Hz) 8.10 ± 2.34 11.21 ± 3.37 9.59 ± 1.89 9.43 ± 7.67 8.48 ± 1.56 p = 0.0152, H(4) = 12.31 Accommodation ratio 0.39 ± 0.10 0.37 ± 0.16 0.36 ± 0.12 0.20 ± 0.14 0.49 ± 0.11 p = 0.0202, H(4) = 11.64 Intrinsic properties of accommodating, regular spiking, notch, stutter, and early spiking neurons.
- Table 3.
Intrinsic properties of epileptic L2/3 PNs separated based on AHP shape and latency
Intrinsic property fAHP neurons (n = 16, 12) fAHP + ADP neurons (n = 35, 18) mAHP neurons (n = 28, 13) Statistical comparisons (p value) Resting membrane potential (mV) −70.29 ± 5.23 −68.09 ± 7.25 −65.73 ± 6.49 KW test
p = 0.0577
Input resistance (MΩ) 135.10 ± 53.61 104.40 ± 45.38 137.90 ± 82.44 KW test
p = 0.2531
Voltage sag (%) 4.19 ± 2.97 5.11 ± 2.61 6.51 ± 2.63 KW test
p = 0.0194
Membrane decay (ms) 26.89 ± 6.02 26.16 ± 7.25 27.73 ± 7.29 ANOVA
p = 0.7114
AP threshold (mV) −35.17 ± 6.58 −37.46 ± 4.62 −37.76 ± 5.68 KW test
p = 0.6493
AP amplitude (mV) 78.92 ± 10.74 85.02 ± 7.40 84.98 ± 8.19 KW test
p = 0.2345
AP half-width (ms) 1.26 ± 0.35 1.26 ± 0.36 1.40 ± 0.31 KW test
p = 0.1375
AHP magnitude (mV) −16.42 ± 2.74 −15.25 ± 3.56 −15.14 ± 4.01 KW test
p = 0.6069
AHP latency (ms) 4.35 ± 1.31 4.11 ± 1.36 20.84 ± 12.36 KW test
p < 0.0001
ΔAHP (mV) −2.61 ± 3.06 −2.49 ± 2.47 −5.21 ± 2.91 KW test
p = 0.0003
AP broadening ratio 1.26 ± 0.09 1.26 ± 0 0.11 1.27 ± 0.10 KW test
p = 0.8880
AP amplitude adaptation ratio 0.93 ± 0.06 0.95 ± 0.03 0.90 ± 0.05 KW test
p = 0.0026
Initial instantaneous frequency (Hz) 32.49 ± 15.72 33.53 ± 18.93 32.30 ± 13.25 KW test
p = 0.9414
Maximum firing rate (Hz) 37.21 ± 13.94 35.09 ± 13.10 33.67 ± 12.03 KW test
p = 0.6588
Final instantaneous frequency (Hz) 11.43 ± 4.71 9.78 ± 2.019 11.13 ± 4.36 KW test
p = 0.4298
Accommodation ratio 0.38 ± 0.15 0.38 ± 0.15 0.39 ± 0.13 ANOVA
p = 0.9230
Max depolarization slope (dV/dT) 207.80 ± 56.22 228.80 ± 39.37 206.70 ± 49.12 ANOVA
p = 0.1265
Max repolarization slope (dV/dT) −50.44 ± 17.06 −54.64 ± 10.75 −44.74 ± 10.48 ANOVA
p = 0.0072
Intrinsic properties of fAHP, fAHP-ADP, and mAHP epileptic L2/3 PNs.
Intrinsic property Control (n = 26, 5) MCD (n = 37, 13) Other epilepsies (n = 45, 15) Statistical comparison (p value) Resting membrane potential (mV) −69.98 ± 1.19 −68.12 ± 7.13 −67.76 ± 5.20 ANOVA
p = 0.3464
Input resistance (MΩ) 95.21 ± 36.54 137.20 ± 63.21 111.80 ± 67.72 KW test
p = 0.0068
Voltage sag (%) 8.42 ± 4.95 5.20 ± 2.34 5.26 ± 2.88 KW test
p = 0.0137
Membrane decay (ms) 26.23 ± 7.95 26.7 ± 7.37 26.77 ± 7.43 KW test
p = 0.9238
AP threshold (mV) −45.26 ± 5.61 −35.62 ± 6.09 −38.16 ± 4.75 KW test
p < 0.0001
AP amplitude (mV) 84.07 ± 8.73 75.60 ± 8.37 84.64 ± 7.08 KW test
p < 0.0001
AP half-width (ms) 1.33 ± 0.26 1.21 ± 0.33 1.38 ± 0.33 KW test
p = 0.0064
AHP magnitude (mV) −12.87 ± 3.19 −15.29 ± 3.19 −14.94 ± 3.36 ANOVA
p = 0.0102
AHP latency (ms) 10.58 ± 5.12 8.92 ± 6.32 8.65 ± 5.56 KW test
p = 0.0843
ΔAHP (mV) −3.44 ± 3.10 −3.86 ± 3.28 −3.42 ± 2.73 KW test
p = 0.9027
AP broadening ratio 1.23 ± 0.09 1.28 ± 0.10 1.25 ± 0.10 KW test
p = 0.1324
AP amplitude adaptation ratio 0.95 ± 0.05 0.91 ± 0.06 0.94 ± 0.04 Welch's
p = 0.0421
Maximum firing rate (Hz) 32.80 ± 10.29 26.68 ± 10.33 24.34 ± 8.10 ANOVA
p = 0.0022
Accommodation ratio 0.46 ± 0.21 0.38 ± 0.16 0.48 ± 0.18 Welch's
p = 0.0359
Maximum depolarization slope (dV/dT) 250.20 ± 44.25 211.80 ± 50.45 218.50 ± 48.12 ANOVA
p = 0.0062
Maximum repolarization slope (dV/dT) −53.85 ± 14.93 −52.61 ± 12.97 −48.26 ± 11.89 KW test
p = 0.0917
Intrinsic properties of L2/3 PNs from control, MCD and OE ex vivo brain tissue.
Intrinsic property No 4-AP (n = 17, 8) +4-AP (100 μM) (n = 17, 8) Statistical comparisons (p value) Input resistance (MΩ) 146.00 ± 67.25 111.40 ± 53.23 Paired t test
p = 0.0002
Voltage sag (%) 6.43 ± 3.58 6.68 ± 3.48 Wilcoxon test
p = 0.3060
Membrane decay (ms) 28.15 ± 7.90 33.38 ± 17.54 Wilcoxon test
p = 0.3303
AP threshold (mV) −38.14 ± 4.28 −40.59 ± 4.93 Paired t test
p = 0.0433
AP amplitude (mV) 86.80 ± 7.62 87.40 ± 4.62 Paired t test
p = 0.7729
AP half-width (ms) 1.28 ± 0.32 1.61 ± 0.39 Paired test
p = 0.0091
AHP magnitude (mV) −16.85 ± 2.76 −14.13 ± 3.26 Wilcoxon test
p = 0.0021
AHP latency (ms) 8.59 ± 3.79 15.53 ± 5.19 Paired t test
p = 0.0002
ΔAHP (mV) −5.45 ± 3.22 −5.87 ± 4.50 Paired t test
p = 0.6689
AP broadening ratio 1.26 ± 0.10 1.34 ± 0.25 Wilcoxon test
p = 0.2078
AP amplitude adaptation ratio 0.91 ± 0.05 0.92 ± 0.07 Paired t test
p = 0.2812
Maximum firing rate (Hz) 26.08 ± 8.70 25.29 ± 7.17 Paired t test
p = 0.3543
Accommodation ratio (400 pA step) 0.29 ± 0.10 0.44 ± 0.29 Wilcoxon test
p = 0.0273
Accommodation ratio (500 pA step) 0.27 ± 0.08 0.35 ± 0.16 Wilcoxon test
p = 0.0342
Accommodation ratio (600 pA step) 0.23 ± 0.09 0.34 ± 0.13 Paired t test
p = 0.0143
Accommodation ratio (750 pA step) 0.23 ± 0.07 0.35 ± 0.19 Wilcoxon test
p = 0.0105
Accommodation ratio (800 pA step) 0.22 ± 0.07 0.30 ± 0.14 Wilcoxon test
p = 0.0034
Accommodation ratio (850 pA step) 0.22 ± 0.09 0.45 ± 0.50 Wilcoxon test
p = 0.0068
Intrinsic properties of L2/3 epileptic PNs before and after 4-AP (100 µM) wash on.
Figure 2-1
Other intrinsic properties of putative L2/3 PN subtypes. Other analyzed intrinsic properties were graphed to show spread and variability as follows: A) resting membrane potential (RMP, mV), B) input resistance (MΩ), C) voltage sag (%), D) membrane decay, E) AHP amplitude, F) Mean FR ± SEM vs injected current (pA), G) Max FR, H) final instantaneous frequency, and I) FR accommodation ratio ± SEM vs injected current (pA). Scatter plots include mean values ± SD. Download Figure 2-1, TIF file.
Figure 2-2
PCA and K-means clustering of epileptic putative L2/3 PNs. A) Scree Plot used to determine the number of clusters for K-means clustering. B) Silhouette Value for each epileptic L2/3 PN based on cluster assignment. C) PCA 3D plot with neurons clustered based on 6 intrinsic properties with cluster centroids marked by an ‘X’. Download Figure 2-2, TIF file.
Table 2-1
Post hoc test p-values for statistically significant properties based on putative L2/3 PN subtype. The correlated post-hoc test for comparing intrinsic properties from Table 2. Download Table 2-1, DOCX file.
Table 2-2
Definitions of electrophysiologic parameters. How each intrinsic property was calculated based on the square 600ms current steps. Download Table 2-2, DOC file.
Figure 3-1
Other intrinsic properties of L2/3 PN subtypes based on AHP shape. Other analyzed intrinsic properties were graphed to show spread and variability as follows: A) AP half-width, B) input resistance, C) initial instantaneous frequency, D) max instantaneous frequency, E) final instantaneous frequency, and F) FR accommodation ratio. Scatter plots include mean values ± SD. Download Figure 3-1, TIF file.
Table 3-1
Group comparison values with post hoc adjusted p-values for statistically significant properties based on AHP subtype. The correlated post-hoc test for comparing intrinsic properties from Table 3. Download Table 3-1, DOCX file.
Table 4-1
Group comparison values with post hoc adjusted p-values for statistically significant properties based on epilepsy subtype. The correlated post-hoc test for comparing intrinsic properties from Table 4. Download Table 4-1, DOCX file.
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