Regulation of L-type Calcium Channels in Low-Intensity Pulsed Ultrasound (LIPUS) Excitation of Cultured Hippocampal Neurons

Background

In recent years, ultrasound stimulation has been widely used as a non-invasive technique to regulate neuronal activity both in vivo and in vitro. However, the potential mechanism of neural modulation effects induced by low-intensity pulsed ultrasound (LIPUS) remains unclear. This paper aims to reveal this mechanism by studying the excitatory effects of LIPUS on spontaneous activity and intracellular calcium (Ca2+) homeostasis in hippocampal neurons. The study found that LIPUS increases intracellular Ca2+ concentration by promoting L-type calcium channels (LTCCs), thereby activating Ca2+-dependent signaling pathways such as the CaMKII-CREB pathway, which in turn regulates gene transcription and protein expression.

Research Source

This paper was written by a research team led by Fan Wen-Yong, with authors from the Department of Physiology and Neurobiology, School of Life Sciences, Fudan University, the Institute of Acoustics, Department of Physics, Tongji University, Zhejiang Provincial People’s Hospital, and other institutions. The article was published online in Neuroscience Bulletin in March 2024.

Research Process

1. Experimental Setup

The research team established a LIPUS stimulation device, combined with whole-cell patch-clamp recording and Ca2+ imaging techniques to stimulate and observe cultured hippocampal neurons. LIPUS used a frequency of 1 MHz, a pulse repetition frequency of 0.3 Hz, and a duty cycle of 10%. The experiments included electrophysiology experiments, Ca2+ imaging experiments, and protein and mRNA expression experiments, involving piezoelectric transducers of different sizes (16 mm and 25 mm).

2. Hippocampal Neuron Culture

The experiment used hippocampal neurons from 1-2 day old newborn Sprague Dawley rats for cell isolation and culture. The culture medium was Neurobasal A, supplemented with 1% B27, 1% glutamine, and 1% penicillin-streptomycin.

3. Ca2+ Imaging

Cells used the Ca2+ probe Fura-2-AM to monitor real-time changes in intracellular Ca2+ concentration. Experiments showed that LIPUS stimulation significantly increased intracellular Ca2+ concentration, mainly through Ca2+ influx rather than release from stored Ca2+.

4. Electrophysiology Experiments

LIPUS significantly increased the frequency and amplitude of spontaneous action potentials (sAP) and spontaneous excitatory synaptic currents (sEPSC) in hippocampal neurons. These phenomena lasted for more than 10 minutes, indicating that LIPUS has a strong excitatory effect on neurons in a short time.

5. Protein and mRNA Expression

Western blot and PCR experiments were used to analyze the effects of LIPUS on Ca2+-dependent signaling pathways (such as CaMKIIα and CREB). Results showed that LIPUS increased phosphorylated CaMKIIα and nuclear translocation of CREB, while the expression of CaV1.2 and CaV1.3 proteins and their mRNA did not change significantly within 24 hours.

Research Results

1. LIPUS Stimulates Spontaneous Neuronal Activity

LIPUS stimulation significantly increased the frequency and amplitude of spontaneous action potentials and spontaneous excitatory synaptic currents in hippocampal neurons. The mechanism may be related to enhanced signal transmission between synaptic connections, indicating that ultrasound can enhance connectivity between neurons.

2. Mediation of Calcium Ion Influx through LTCCs

LIPUS increased intracellular Ca2+ concentration through LTCCs and significantly altered the electrophysiological characteristics of LTCCs, including hyperpolarization of activation potential and depolarization of inactivation potential. These changes indicate that LIPUS increases neuronal excitability through these channels.

3. Activation of Calcium-Dependent Signaling Pathways

The increased Ca2+ influx through LTCCs by LIPUS activated the CaMKII-CREB pathway. In vitro experiments confirmed that LIPUS increased phosphorylated CaMKIIα and maintained a higher level of neuronal excitation for a longer period through the nuclear translocation of CREB.

4. Long-term Effects of LIPUS on LTCCs

Although LIPUS significantly altered the function of calcium channels, it had no significant effect on the mRNA and protein expression levels of LTCCs within 24 hours. This suggests that LIPUS mainly achieves neural excitation by regulating channel activity rather than quantity.

Conclusion

This paper reveals for the first time the mechanism by which low-intensity pulsed ultrasound regulates Ca2+ influx in hippocampal neurons through LTCCs, thereby activating the CaMKII-CREB signaling pathway. This finding has important value for ultrasound technology in neural regulation and its clinical applications.

Research Highlights

The highlight of this study is the revelation of the specific mechanism by which LIPUS increases intracellular Ca2+ influx in neurons through LTCCs, and the first demonstration of the activating effect of LIPUS on the CaMKII-CREB signaling pathway. This paper provides theoretical basis and experimental data support for future applications of ultrasound technology in neural regulation.

Significance and Value

As a non-invasive technology, the potential of LIPUS in neuroscience research and clinical applications is continuously emerging. The research in this paper not only helps to understand the specific mechanism of ultrasound regulation of neurons but also provides a theoretical basis for developing new neural regulation technologies in the future. In addition, LIPUS has broad application prospects in treating neurodegenerative diseases such as Parkinson’s disease and stroke.

Through the research in this paper, the development and application of LIPUS technology can be further promoted, providing new ideas and methods for neuroscience research and clinical treatment.