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. 2022 Mar 3;42(9):1650–1665. doi: 10.1177/0271678X221080324

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© The Author(s) 2022

This article is distributed under the terms of the Creative Commons Attribution 4.0 License (https://creativecommons.org/licenses/by/4.0/) which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

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Figure 6. — Cortical neurons from epileptic human patients decrease their spontaneous EPSC frequency following HCAR1 activation. (a) HCAR1 mRNA detection in human brain sample from 17 different pharmacoresistant epileptic patients undergoing resective surgery. Brain samples were from both male (57%) and female (43%) patients with median age 18 years, and originated from frontal (n = 5), insular (n = 2), parietal (n = 1), and temporal (n = 9) cortex from both hemispheres. Basal expression of HCAR1 is normalized to a reference gene (β-actin). All samples were run in triplicate and data is shown as means±SD. (b) Maximum intensity projection of a biocytin-filled recorded human cortical neuron. (c) Example traces of sEPSCs recorded during baseline condition (black) and following application of 40 µM 3Cl-HBA (red). (d) Cumulative distributions of sEPSCs frequency were analyzed by using Kolmogorov-Smirnov test: P = 0.0005 versus HCAR1 activation. (e) Summary of recorded cells showing a decreased frequency of sEPSC by ∼40% induced by 3Cl-HBA compared to baseline (one sample t, n_{cells/patients} = 7/5, mean = 62.54 ± 10.89, P < 0.0001). (f) sEPSC amplitude showed no statistically significant changes between baseline and 3Cl-HBA application (one sample t, n_{cells/patients} = 7/5, mean = 87.65 ± 20.2, P = 0.13). Frequency and amplitude are shown in percentage compared to baseline. (g) Representative traces from neurons recorded before and after HCAR1 activation obtained from a series of current injections (−120 to 540 pA, 2 sec, 30 pA increments); the response to 540 pA current injection is shown. (h) Summary graph of the effect of HCAR1 activation on neuronal firing frequency following steps of current injection (n = 6). The firing frequency was calculated from the number of AP evoked by 540 pA current injection during 2 seconds. Individual data and significance is shown between the baseline and 3Cl-HBA conditions (paired t test, n_{cells/patients}= 6/5, Baseline: mean = 26.67 ± 14.62, 3Cl-HBA: mean = 23.5 ± 12.9, P = 0.035). (i) Example trace of calcium spiking activity from neurons in human acute slices with application of 3Cl-HBA. (j) Summary graph of spontaneous calcium spiking activity down-modulated by HCAR1 activation in cortical neurons from epileptic human patients (one-way ANOVA, n_{cells/patients}= 27/5, Baseline: mean = 2.6 ± 3.07, 3Cl-HBA: mean = 1.04 ± 1.37, Washout: mean = 2.21 ± 2.44, P = 0.013). The calcium spiking activity of individual cells is shown. Values are means ± SD; P < 0.05, **P < 0.01, ***P < 0.001 versus 3Cl-HBA application.