Responsive regularity of neurons in the hindlimb region of primary somatosensory cortex to different temperature thermal needle stimulation of "Zusanli"(ST36) in mice.

OBJECTIVES

To study the regularity of central response to thermal needle stimulation of "Zusanli" (ST36) at different temperature, and to analyze the temperature difference of central responses.

METHODS

Six male C57BL/6j adult mice were used in the present study. For observing activities of neurons in the hindlimb region of left primary somatosensory cortex (S1HL, A/P=0.46 mm, M/L=1.32 mm, D/V=-0.14 mm) by using a fast high-resolution miniature two-photon microscopy (FHIRM-TPM), the mice were anesthetized with 3% isoflurane (inhalation), with its head fixed in a stereotaxic apparatus, then, adeno-associated virus (AAV-hSyn-GCaMP6f-WPRE-hGHpA, for showing intracellular calcium transients in neurons transfected) was injected into the left S1HL region using a micro-syringe after scalp surgical operation. The mice's right ST36 were stimulated using internal thermal needles with the temperature being 43 ℃, or 45 ℃, or 47 ℃, separately. Image J software and MATLAB 2020b software were used to process the image data of neuronal calcium activity (Ca signaling) in the left S1HL region, including the instant maximum calcium peak value (ΔF/F) in 2 s, instant calcium spike frequency in 2 s, short-term calcium peak value (ΔF/F) in 3.5 min, short-term calcium spike frequency in 3.5 min, calcium peak duration in 3.5 min, maximum calcium peak value (ΔF/F) at the 1 , 2 and 3 min, and calcium spike frequency at the 1, 2 and 3 min after thermal needle stimulation.

RESULTS

In comparison with the normal temperature needle stimulation, the instant intracellular maximum calcium peak value, instant calcium spike frequency, short-term maximum calcium peak value, short-term calcium spike frequency, and calcium peak duration of S1HL neurons in response to 43 ℃, 45 ℃ and 47 ℃ internal thermal needle stimulation of ST36 were significantly increased (<0.001, <0.01). Comparison among the 43 ℃, 45 ℃ and 47 ℃ thermal needle stimulation showed that the 45 ℃ thermal needle stimulation was obviously superior to 43 ℃ and 47 ℃ thermal needle stimulation in increasing instant calcium spike frequency, short-term calcium spike frequency and calcium peak duration of S1HL neurons (<0.001, <0.01). The 47 ℃ thermal needle stimulation was stronger than 43 ℃ and 45 ℃ thermal needle stimulation in increasing the instant maximum calcium peak value (<0.001). The maximum calcium peak value was apparently higher (<0.001) at the 2 min than that at the 1 and 3 min after 43 ℃, 45 ℃ and 47 ℃ thermal needle stimulation. No significant differences were found in the short-term maximum calcium peak value among the 3 thermal needle stimulation and in the calcium spike frequency among the 3 time points after 43 ℃, 45 ℃ and 47 ℃ thermal needle stimulation.

CONCLUSIONS

S1HL neurons respond to all 43 ℃, 45 ℃ and 47 ℃ thermal needle stimulation of ST36 in mice, while more actively to 45 ℃ thermal needle stimulation.