A genetically targeted ion sensor reveals distinct seizure-related chloride and pH dynamics in GABAergic interneuron populations

Abstract

Intracellular chloride and pH play fundamental roles in determining a neuron's synaptic inhibition and excitability. Yet it has been difficult to measure changes in these ions during periods of heightened network activity, such as occur in epilepsy. Here we develop a version of the fluorescent reporter, ClopHensorN, to enable simultaneous quantification of chloride and pH in genetically defined neurons during epileptiform activity. We compare pyramidal neurons to the major GABAergic interneuron subtypes in the mouse hippocampus, which express parvalbumin (PV), somatostatin (SST), or vasoactive intestinal polypeptide (VIP). Interneuron populations exhibit higher baseline chloride, with PV interneurons exhibiting the highest levels. During an epileptiform discharge, however, all subtypes converge upon a common elevated chloride level. Concurrent with these dynamics, epileptiform activity leads to different degrees of intracellular acidification, which reflect baseline pH. Thus, a new optical tool for dissociating chloride and pH reveals neuron-specific ion dynamics during heightened network activity.

Keywords: Biological sciences; Molecular neuroscience; Neuroscience; Techniques in neuroscience.

Graphical abstract

Figure 1

Cre-dependent ClopHensorN for targeting expression to genetically defined neuronal subtypes (A) The Cre-dependent ClopHensorN construct contained the inverted genetic sequence for the E²GFP-tdTomato fusion protein, flanked by loxP and lox2272 sites. The construct utilizes an elongation factor 1α promoter (EF-1α) and a WPRE to enhance expression. Cre recombinase catalyses the recombination of the floxed sequence, which allows for transcription of ClopHensorN. (B) High-titer AAV carrying the Cre-dependent ClopHensorN was delivered to organotypic hippocampal brain slices from Cre-expressing mice and, after three weeks in culture, ClopHensorN-expressing neurons were imaged using confocal microscopy. Simultaneous electrophysiological data were acquired via patch-clamp recordings from a nearby pyramidal neuron in current-clamp mode (IC₀). (C) Hippocampal slice from a CamK2a-Cre mouse containing ClopHensorN-expressing pyramidal neurons. Continuous white lines indicate the slice edge and dashed white lines delineate the pyramidal cell layer. (D) ClopHensorN signal from a representative pyramidal neuron, in which the tdTomato protein was excited at 561 nm (left), and the E²GFP protein was excited at 458 nm (middle), and 488 nm (right). (E and F) Equivalent images for a slice from a PV-Cre mouse (E) and the ClopHensorN signal from a representative PV interneuron (F). (G and H) Equivalent images for a slice from an SST-Cre mouse (G) and the ClopHensorN signal from a representative SST interneuron (H). (I and J) Equivalent images for a slice from a VIP-Cre mouse (I) and the ClopHensorN signal from a representative VIP interneuron (J).

Figure 2

ClopHensorN calibration (A) Calibration curves relating the pH sensitive ratio (F₄₈₈/F₄₅₈) of ClopHensorN to intracellular pH. Intracellular pH was systematically varied by controlling the extracellular pH in the presence of an ionophore cocktail. Data was fit using established equations^, and pK_a was estimated to be 7.14 (CI [7.1–7.17]). N = 40–60 cells at each pH value. Horizontal lines indicate mean, error bars indicate SEM, and dots indicate individual cells. (B) Calibration curves relating the chloride sensitive fluorescence ratio (F₄₅₈/F₅₆₁) to intracellular chloride concentration. Intracellular chloride was manipulated by changing the extracellular chloride in the presence of an ionophore cocktail. Data collected at pH 6 and 7 were fit using established equations as in “A”, and used to infer the relationship at pH 8. K_d was estimated to be 3.51 mM at pH 6, 5.66 mM at pH 7, and 27.12 mM at pH 8 (inset). N = 66 and 232 cells at each chloride value for pH 6 and 7, respectively. Horizontal lines indicate mean, error bars indicate SEM, and dots indicate individual cells.

Figure 3

Neuronal subtypes exhibit different baseline intracellular chloride and pH values (A) Intracellular chloride varies across neuronal subtypes, with PV interneurons and VIP interneurons exhibiting higher baseline chloride than CamK2a pyramidal neurons (χ²₍₃₎ = 19.43, p = 0.0002, Kruskal-Wallis test, followed by post-hoc Dunn’s multiple comparisons tests; CamK2a vs PV, p = 0.0001; CamK2a vs SST, p = 0.1493; CamK2a vs VIP, p = 0.0052; PV vs SST, p = 0.1521; PV vs VIP, p = 0.8035; SST vs VIP, p > 0.999). N = 24, 28, 32 and 49 neurons from 15, 26, 12 and 26 slices for CamK2a, PV, SST and VIP, respectively. Y axis is limited to 100 mM for plotting purposes. Bars and lines indicate median and IQR, dots indicate individual neurons. (B) Baseline intracellular pH varies across neuronal subtypes. No interneuron subtype was different from CamK2a pyramidal neurons, but within the interneuron subtypes, VIP interneurons exhibited a more acidic baseline pH than PV or SST interneurons (χ²₍₃₎ = 13.5, p = 0.0037, Kruskal-Wallis test, followed by post-hoc Dunn’s multiple comparisons tests; CamK2a vs PV, p = 0.8375; CamK2a vs SST, p = 0.3606; CamK2a vs VIP, p > 0.9999; PV vs SST, p > 0.9999; PV vs VIP, p = 0.0353; SST vs VIP, p = 0.0078). N = 28, 36, 35 and 57 neurons from 15, 26, 12 and 26 slices for CamK2a, PV, SST and VIP, respectively. Bars and lines indicate median and IQR, dots indicate individual neurons. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ∗∗∗ indicates p < 0.001.

Figure 4

The activity of GABAergic interneuron subtypes and pyramidal neurons is highly correlated during epileptiform activity (A) Cartoon illustrating simultaneous current clamp recordings from a PV interneuron (Pipette 1, P₁) and a nearby pyramidal neuron (P₂; left). Representative traces (right) from a PV interneuron (top) and a pyramidal neuron (bottom) pair during an ED. (B) Equivalent cartoon (left) and example traces (right) from an SST interneuron (top) and a pyramidal neuron (bottom) pair during an ED. (C) Equivalent cartoon (left) and example traces (right) from a VIP interneuron (top) and a pyramidal neuron (bottom) pair during an ED. (D) During EDs, the activity of PV (N = 24 EDs from 8 neuronal pairs in 8 slices), SST (N = 15 EDs from 8 neuronal pairs in 3 slices) and VIP (N = 28 EDs from 9 neuronal pairs in 6 slices) interneurons was highly correlated with the activity of nearby pyramidal neurons (interaction between neuron pair type and time window: F_(2,64) = 3.771, p = 0.0283, repeated measures two-way ANOVA followed by Sidak’s post-hoc multiple comparisons of baseline vs ED: PV-CamK2a, p < 0.0001; SST-CamK2a, p < 0.0001; VIP-CamK2a, p < 0.0001). Lines indicate median and IQR, dots indicate individual EDs. The degree of correlation with the pyramidal neuron’s activity was not different across the interneuron subtypes during EDs (Sidak’s post-hoc multiple comparisons, p > 0.05). ∗∗∗ indicates p < 0.001.

Figure 5

Monitoring chloride and pH dynamics in different neuronal subtypes during epileptiform activity (A) Cartoon of the experimental setup (top panel) and representative data for a CamK2a pyramidal neuron (bottom three panels). Network activity was always monitored via a current-clamp recording from a nearby pyramidal cell (black trace, second panel), while intracellular chloride dynamics (third panel) and pH dynamics (bottom panel) were determined for a ClopHensorN-expressing CamK2a pyramidal neuron. Data are shown for a representative 10 min period of the recording. Vertical dashed lines mark the start of EDs. (B) Equivalent cartoon and example data for a PV interneuron. (C) Equivalent cartoon and example data for an SST interneuron. (D) Equivalent cartoon and example data for a VIP interneuron.

Figure 6

Neuronal subtypes exhibit distinct chloride and pH dynamics during an ED (A) Cartoon (left) of the experimental setup for a CamK2a pyramidal neuron. Heat maps (right, upper row) show the membrane potential dynamics (Δ Vm), chloride dynamics (Δ Cl⁻), and pH dynamics (Δ pH) during all EDs for CamK2a neurons, relative to normalized ED length on the x axis. ED start is at 0% and ED end is at 100%. Plots (right, lower row) show the mean absolute membrane potential, and the median absolute chloride and pH across all CamK2a EDs, using the same x axis timescale. Shading depicts SEM for membrane potential and IQR for chloride and pH. N = 93 EDs from 23 CamK2a neurons in 15 slices. (B) Equivalent cartoon and data for all EDs in PV interneurons (N = 60 EDs from 28 PV interneurons in 26 slices). (C) Equivalent cartoon and data for all EDs in SST interneurons (N = 88 EDs from 32 SST interneurons in 12 slices). (D) Equivalent cartoon and data for all EDs in VIP interneurons (N = 123 EDs from 44 VIP interneurons in 26 slices).

Figure 7

Neuronal subtypes exhibit converging intracellular chloride concentrations and acidify during an ED (A) Subtypes differed in their chloride changes during an ED. EDs were divided into short EDs (less than 60s) and long EDs (greater than 60s), and compared to baseline levels before the ED and recovery levels after the ED. PV interneurons were the only subtype that did not exhibit a change in chloride (PV χ²₍₃₎ = 1.28, p = 0.7335, Kruskal-Wallis test). CamK2a neurons, SST interneurons, and VIP interneurons each exhibited a significant change in chloride during an ED (CamK2a χ²₍₃₎ = 19.89, p < 0.0002; SST χ²₍₃₎ = 31.75, p < 0.0001; VIP χ²₍₃₎ = 13.33, p < 0.004). Y axis is limited to 100 mM for plotting purposes. Bars and lines indicate median and IQR, dots indicate individual EDs. Asterisks indicate p values from post-hoc Dunn’s multiple comparisons tests comparing to baseline within each subtype. N = 93 EDs from 23 CamK2a neurons in 15 slices, 60 EDs from 28 PV interneurons in 26 slices, 88 EDs from 32 SST interneurons in 12 slices, 123 EDs from 44 VIP interneurons in 26 slices. (B) Subtype chloride levels converged during an ED. Chloride levels differed across the neuronal subtypes under baseline conditions (Baseline χ²₍₃₎ = 76.92, p < 0.0001, Kruskal-Wallis test) and during short EDs (Short ED χ²₍₃₎ = 51.21, p < 0.0001), but became indistinguishable during long EDs (Long ED χ²₍₃₎ = 1.46, p = 0.6918). Subtype differences were re-established after the EDs (Recovery χ²₍₃₎ = 74.65, p < 0.0001). Data replotted from “A” using same conventions. Asterisks indicate p values from Kruskal-Wallis tests comparing across subtypes. (C) All subtypes acidified during and ED, typically showing acidic shifts during even short EDs (CamK2a χ²₍₃₎ = 60.74, p < 0.0001, Kruskal-Wallis test; PV χ²₍₃₎ = 57.58, p < 0.0001; SST χ²₍₃₎ = 93.53, p < 0.0001; VIP χ²₍₃₎ = 76.57, p < 0.0001). Bars and lines indicate median and IQR, dots indicate individual EDs. Asterisks indicate p values from post-hoc Dunn’s multiple comparisons tests comparing to baseline within each subtype. (D) While all subtypes acidified during an ED, they exhibited differences in absolute pH (Baseline χ²₍₃₎ = 62.55, p < 0.0001, Kruskal-Wallis test; Short ED χ²₍₃₎ = 65.45, p < 0.0001; Long ED χ²₍₃₎ = 8.286, p = 0.041; Recovery χ²₍₃₎ = 49.97, p < 0.0001). Data replotted from “C” using same conventions. Asterisks indicate p values from Kruskal-Wallis tests comparing across subtypes. ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ∗∗∗ indicates p < 0.001.