Ultrasound Stimulation Modulates Voltage-Gated Potassium Currents Associated With Action Potential Shape in Hippocampal CA1 Pyramidal Neurons

Figures

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  FIGURE 1. Schematic of the experimental system and the whole-cell recording of pyramidal neurons in the hippocampal CA1 slices. (A) Schematic of the entire experimental system and a cross-sectional diagram of the exposure setting with the actual dimensions. (B) Photomicrograph of a coronal section of hippocampal CA1 slice and a typical pyramidal neuron for the whole-cell recording. (C) Action potential in response to prolonged depolarizing current injection is one important feature to identify pyramidal neuron. Moreover, intracellular injection with 0.25% biocytin shows the morphology of a typical CA1 pyramidal neuron.

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  FIGURE 2. The effect of ultrasound stimulation on voltage-gated potassium currents of pyramidal neurons in CA1 hippocampus slices. (A) Representative outward potassium currents recorded in the control (black) and ultrasound stimulation (red). The currents were elicited by a protocol in which cells were depolarized to a variety of potentials (–60 to +60 mV) from a holding potential of –70 mV and the currents could be blocked by 30 mM TEA-Cl and 1 mM 4-AP (blue). (B) The individual current traces correspond to the currents measured at voltage steps of –20, 0, 20, and 40 mV in control (black), US (red) and blocker (blue) group. (C) I–V curves of outward potassium currents showed that ultrasound stimulation increased the outward potassium currents (∗p < 0.05). (D) The same relative changes of the outward potassium currents at different voltage steps were observed.

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  FIGURE 3. The effect of ultrasound stimulation on evoked firing of pyramidal neurons in CA1 hippocampus slices. (A) Representative voltage traces of evoked action potentials in response to current injections (100, 200, and 300 pA) in control (balck) and ultrasound stimulation (red). Dashed lines located at resting membrane potential (RMP) of neurons. (B) Collected data on the slope of firing frequency-currents relationship (F-I slope) showed that ultrasound stimulation significantly increased the firing frequency of pyramidal neurons (∗∗∗p < 0.001). Slope was determined by linear fit from 0 to 300 pA. (C) The effect of ultrasound stimulation on RMP was significantly decreased (∗∗p < 0.01).

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  FIGURE 4. The effect of ultrasound stimulation on action potential (AP) properties of pyramidal neurons in CA1 hippocampus slices. (A) Superimposed voltage traces in response to sustained threshold current (50 pA) and above threshold current (200 pA). Threshold current is defined as the minimum amount of current injection required for neuron to generate action potential. In contrast to the voltage traces of control (black), ultrasound stimulation (red) failed to generate action potential in the threshold current injection and generate more action potential in 200 Pa current injection. (B) Representative voltage trace of single AP in control (black) and US (red) group evoked by sustained above threshold current injections (200 pA). AP properties were further calculated and plotted as a function of potassium currents. (C–F) A population of 12 neurons showing the increment of threshold current (C) and AP fall rate (E) while the reduction of AP half-width (D) and AHP peak time (F) by ultrasound stimulation (∗∗∗p < 0.001, paired t-test).

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  FIGURE 5. The effect of ultrasound stimulation on passive properties of pyramidal neurons in CA1 hippocampus slices. (A) An example of representative voltage trace of pyramidal neuron in response to a small hyperpolarizing current (−20 pA). A single exponential was fitted to a 10 ms time window of voltage trace to calculate passive membrane properties, membrane capacitance (CM), membrane input-resistance (RM) and membrane time constant (TM). Ultrasound stimulation had little effect on CM (B, Control: 31.58 ± 2.22 pF; US: 29.82 ± 3.13 pF, p = 0.297, paired t-test). However, ultrasound stimulation caused a reduction in RM (C, Control: 259.75 ± 17.24 MΩ; US: 205.89 ± 14.11 MΩ, ∗∗∗p < 0.001, paired t-test) and TM (D, Control: 8.07 ± 0.59 ms; US: 5.98 ± 0.62 ms, ∗∗p < 0.01, paired t-test).

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  FIGURE 6. The underlying mechanisms of ultrasound stimulation on modulation of potassium channels in pyramidal neurons. (A) A new recording chamber was developed to isolate ultrasound waves which consisting of a cavity and sputtered to a lithium niobate substrate. (B) Representative voltage traces recorded from pyramidal neurons in response to a sequence of sustained currents injection (100, 200, and 300 pA). The result showed that the isolated ultrasound has no effect on the activity of neurons. (C) Temperature elevation was monitored during ultrasound stimulation in recording chamber using a thermal infrared imager. (D) The temperature profile showed that the temperature elevation was relatively small (less than 0.74 ± 0.09°C) during 30 s ultrasound stimulation.