Initiation of calcium signaling by mechanical acupuncture

Abstract

The objective of this dissertation is to explore the fundamental acupuncture mechanism to find out if there is a cellular and molecular foundation. Our experiment was to search for a specific and well-defined systematic signaling network with a focus on calcium activities of the muscle and brain cells. The acupoint ST36 in the mouse’s hind limb was selected based on strong clinical data support in recently published research. In the case of the muscle connective tissues, we transfected the mouse hind limb muscle with calcium indicator. An acupuncture needle driven by a piezoelectric transducer was inserted into ST36. We focused a two-photon fluorescent microscope in vivo in the area near the needle insertion site and observed dynamic real-time Ca2+ activities. It was found that significant Ca2+ waves were generated. Regions with increasing Ca2+ concentration were observed to be mostly confined to a single main fiber and moving away from the vibrating needle. The ionic migration, constituting the initial stage of acupuncture signaling, appeared to be able to reach a nerve ending and the CNS.

In the second phase of our experiment, two-photon in vivo calcium imaging was used to investigate neuronal and astrocytic activities separately in mouse somatosensory cortex during mechanical acupuncture. We found that acupuncture stimuli at acupoint site increased Ca2+ responses of pyramidal neurons and astrocytes in primary somatosensory cortex (S1), and these neuronal and astrocytic responses were attenuated by an N-methyl-D-aspartate (NMDA) receptor antagonist, suggesting that the induced calcium activity is mainly dependent on glutamatergic transmission. Astrocytic Ca2+ responses were silenced by transient receptor potential ankyrin 1 (TRPA1) receptor antagonist, suggesting that the potentiation of astrocytic calcium was activated by acupuncture mechanically. We further chemogenetically inhibited pyramidal neurons and found that clozapine N-oxide (CNO) treatment suppressed neuronal calcium activities but induced stronger astrocytic Ca2+ response compared to saline control group upon acupuncture stimulation. These observations indicate that activities of neurons and astrocytes in CNS in response to acupuncture stimulation are interactive and possibly cooperative.

In conclusion, our findings collectively demonstrated the activation of Ca2+ migration in muscle and Ca2+ transients in both cortical neurons and astrocytes during acupuncture stimulation, providing the first piece of in vivo and real-time evidence for a communication network and neural activation induced by acupuncture.