Isoflurane Alters Presynaptic Endoplasmic Reticulum Calcium Dynamics in Wild-Type and Malignant Hyperthermia-Susceptible Rodent Hippocampal Neurons

Isoflurane effects on presynaptic cytosolic Ca²⁺ influx and synaptic vesicle exocytosis are reduced in T4826I-RYR1 compared with wild-type mouse hippocampal neurons. a, Schematic diagram of the imaging protocol. Neurons were stimulated electrically with 20 action potentials (APs) at 20 Hz (for Ca²⁺ measurements using syn-GCaMP6f) or 100 APs at 10 Hz [for synaptic vesicle (SV) exocytosis measurements using syn-pH]. Representative average traces of (b) syn-GCaMP6f and (c) syn-pH responses to electrical stimulation in a T4826I-RYR1 neuron in control (black) and isoflurane (blue) conditions (50 boutons, DIV 16). Effects of isoflurane on peak (d) syn-GCaMP6f and (e) syn-pH measurements in T4826I-RYR1 malignant hyperthermia susceptible (MH) compared with wild-type (WT) mouse neurons normalized to their respective controls (d: p = 0.0013, unpaired t test, n = 6 WT, 10 MH; e: p = 0.0250, unpaired t test, n = 7 WT, 6 MH).

Isoflurane reduces resting endoplasmic reticulum Ca²⁺ concentration in rat hippocampal neurons. Baseline ER-GCaMP6-150 fluorescence was measured in wild-type rat neurons treated with isoflurane compared with a separate time control neuron [p = 0.0098, unpaired t test, n = 9 (left), n = 7 (right)].

Isoflurane reduces stimulation-evoked increases in endoplasmic reticulum Ca²⁺ concentration. a–c, Fluorescence images of a rat hippocampal neuron co-transfected with ER-GCaMP6-150 (green) and VAMP mCherry (red). Live-cell imaging measuring [Ca²⁺]ER (a) before and (b) during electrical stimulation. c, Snapshot of the presynaptic marker VAMP-mCherry. d, Schematic diagram of the protocol used in b, c. e, Representative average traces of ER-GCaMP6-150 fluorescence changes with 20 action potentials (APs) at 20 Hz stimulations for control, isoflurane, and washout conditions (n = 1, 50 boutons, DIV 16). f, Peaks of ER-GCaMP6-150 fluorescence over three stimulations of 20 AP each at 20 Hz for control (white circle) and isoflurane (blue circle) conditions. Control versus isoflurane, p = 0.0091; isoflurane versus washout, p = 0.0047; control versus washout p = 0.9967; one-way ANOVA, n = 8.

Isoflurane depression of baseline ER Ca²⁺ concentration was not sufficient to disrupt normalization using ΔF/F0. a, Schematic diagram of the protocol used in b, c. b, Representative average traces in arbitrary fluorescence units (AFU) of ER-GCaMP6-150 fluorescence changes with 20 action potentials (APs) at 20 Hz stimulations for control, isoflurane, and washout conditions (n = 1, 50 boutons, DIV 16). c, Arbitrary fluorescence unit (AFU) peaks of ER-GCaMP6-150 fluorescence over three stimulations of 20 AP each at 20 Hz for control (white circle) and isoflurane (blue circle) conditions. (control vs isoflurane p = 0.0002, isoflurane vs washout p = 0.0002, control vs washout p = 0.0425, one-way ANOVA, n = 8). d, Box and whisker plot comparing the same data set measured two ways: ΔF/F0 or total AFU of isoflurane-treated normalized to its respective control condition (p = 0.3457, paired t test, n = 8).

Reduction of stimulation-evoked increase in endoplasmic reticulum Ca²⁺ concentration by isoflurane is not dependent on reduced Ca²⁺ influx. a, Schematic diagram of the protocol used. Box and whisker plot comparing (b) syn-GCaMP6f or (c) ER-GCaMP6-150-transfected cells stimulated with 20 action potentials (APs) at 20 Hz with 0.30 (±0.11) mM isoflurane normalized to control with 1.2 mM Ca²⁺ Tyrode’s solution, or control with 1 mM Ca²⁺ Tyrode’s solution normalized to 1.2 mM Ca²⁺ Tyrode’s solution [b: p = 0.7963, unpaired t test, n = 8 (left), 5 (right); c: p = 0.0380, unpaired t test, n = 7 (left), 6 (right)].

Volatile anesthetics potentiate stimulation-evoked reduction of endoplasmic reticulum Ca²⁺ concentration in T4826I-RYR1 mouse neurons. a, Schematic diagram of the protocol. Neurons were stimulated electrically with 20 action potentials (APs) at 20 Hz. Representative average traces of T4826I-RYR1 malignant hyperthermia (MH) mouse neurons transfected with ER-GCaMP6-150 perfused with (b) 0.34 mM isoflurane, (c) 0.46 mM sevoflurane, or (d) 1 μm propofol. Peak ER-GCaMP6-150 effect of isoflurane, sevoflurane, or propofol on T4826I-RYR1 compared with wild-type (WT) mouse neurons normalized to their respective controls (e: p = WT isoflurane vs MH isoflurane: 0.0002, WT sevoflurane vs MH sevoflurane: 0.0295, WT propofol vs MH propofol: 0.9258, two-way ANOVA, n = 8, 7, 4, 6, 7, 8, respectively).

T4826I-RYR1 malignant hyperthermia susceptible mouse neurons remain stable and responsive over repeated stimulations. a, Schematic diagram of the protocol. Neurons were stimulated electrically with 20 action potentials (APs) at 20 Hz. Representative average traces of T4826I-RYR1 mouse neurons perfused with control solution transfected with (b) syn-pH, (c) syn-GCaMP6f, or (d) ER-GCaMP6-150.

Enhanced isoflurane-induced inhibition of stimulation-evoked presynaptic synaptic vesicle exocytosis, cytosolic Ca²⁺ concentration, and ER Ca²⁺ concentration in T4826I-RYR1 malignant hyperthermia susceptible mouse neurons is reversible. a, Schematic diagram of the protocol. Neurons were stimulated electrically with 20 action potentials (APs) at 20 Hz. Representative average traces of T4826I-RYR1 mouse neurons perfused with isoflurane transfected with (b) syn-pH, (c) syn-GCaMP6f, or (d) ER-GCaMP6-150.