Terahertz exposure enhances neuronal synaptic transmission and oligodendrocyte differentiation in vitro

Abstract

Terahertz (THz) frequency occupies a large portion of the electromagnetic spectrum that is between the infrared and microwave regions. Recent advances in THz application have stimulated interests regarding the biological effects within this frequency range. In the current study, we report that irradiation with a single-frequency THz laser on mice cortical neuron cultures increases excitatory synaptic transmission and neuronal firing activities. Microarray assay reveals gene expression dynamics after THz exposure, which is consistent with morphology and electrophysiology results. Besides, certain schedule of THz irradiation inhibits the proliferation of oligodendrocyte precursor cells (OPCs) and promotes OPC differentiation. Of note, the myelination process is enhanced after THz exposure. In summary, our observations suggest that THz irradiation can modulate the functions of different neuronal cells, with different sensitivity to THz. These results provide important understanding of the mechanisms that govern THz interactions with nervous systems and suggest THz wave as a new strategy for neuromodulation.

Keywords: Medical Physics; Neuroscience; Radiation physics.

Graphical abstract

Figure 1

Setup of a temperature-controlled THz exposure chamber (A) Schematic representation of the experimental setups. 1) THz QCL with stainless steel base, 2) CW/QCW current source, 3) OPA, 4) THz mirror, 5) THz lens, 6) support rod for optic mirror, 7) optical breadboard, 8) thermal controlled chamber, 9) heater controller, 10) mixed gas unit, 11) cell culture dish, 12) digital thermometer with 80PK-1 bead probe thermocouples, 13) THz sensor connected to THz power meter, and 14) shock-proof operating platform. (B) Quantification of the relative THz irradiation level with (After) or without (Before) the culture dish. Data are mean ± SEM relative to “Before” from three independent measurements. (C) Formula for calculating the height of culture dish (x) according to the defined spot area. R is defined as the radius of the THz lens (R = 12.7 mm), f is the effective focus length (f = 50 mm), and r is the radius of the defined spot area (r = 5 mm in our study). See also Figures S1 and S2.

Figure 2

THz exposure promotes neurite outgrowth and synaptogenesis (A) Diagram showing THz irradiation and succeeding analysis on neurons. (B) Quantitative real-time PCR for neurite growth genes, Tuj1 and Gap43, in THz-irradiated low-density neurons and control cultures. Data are mean ± SEM of transcript levels relative to control after normalization from three independent experiments each performed in triplicate. ∗p < 0.05, compared with control, Student's t test. (C) Quantitative real-time PCR for synapse-related genes, Homer1, Psd95, and Syn, in THz-irradiated neurons and control cultures. Data are mean ± SEM of transcript levels relative to control after normalization from three independent experiments each performed in triplicate. ∗∗, p < 0.01, compared with control, Student's t test. (D) Representative images of immunostaining of Tuj1 and Homer1 in 6-DIV low-density cultures after THz irradiation. Scale bar, 40 μm. (E) Quantification of the percentage of Homer1⁺ cells within Tuj1⁺ neurons in control and THz-irradiated cultures. Data are mean ± SEM (n = 3 independent experiments). ∗∗, p < 0.01 compared with control, Student's t test. (F) Representative images of immunostaining of Tuj1 and Homer1 in 6-DIV high-density cultures after THz irradiation. Scale bar, 40 μm. (G) Quantification of the density of Homer1⁺ spots within Tuj1⁺ neurons in control and THz-irradiated cultures. Data are mean ± SEM (n = 3 independent experiments). ∗, p < 0.05 compared with control, Student's t test. (H) Western blot assay for Syn showed increased expression in THz-irradiated neuron cultures. Histogram shows fold change measured by densitometry in the THz group relative to control after normalization to β-actin levels. Data are mean ± SEM (n = 3 independent experiments). ∗∗, p < 0.01, ∗∗∗, p < 0.001, Student's t test.

Figure 3

THz exposure promotes excitatory synaptic transmission (A) Representative traces of sEPSCs recording in control and THz groups. (B) The average frequency of sEPSCs from the THz group showed bigger value than that from the control group (Control: 1.6 ± 0.28 Hz, n = 15 cells; THz: 5.8 ± 1.06 Hz, n = 12 cells). Data are mean ± SEM, ∗∗∗, p < 0.001, unpaired t test. (C) The cumulative frequency distribution curve of sEPSCs frequency from the THz group shifted left compared with the curve of the control group. ∗∗∗∗, p < 0.0001, Extra sum-of-squares F test. (D) The average amplitude of sEPSCs from the THz group showed similar value with control group (Control: 35.3 ± 2.44 pA, n = 15 cells; THz: 45.1 ± 6.70 pA, n = 12 cells; unpaired t test, p = 0.15). Data are mean ± SEM. (E) The cumulative frequency distribution curve of sEPSCs amplitude from the THz group shifted right compared with the curve of the control group. ∗∗∗∗, p < 0.0001, Extra sum-of-squares F test. (F) Representative traces of sEPSCs synchronized events in the control and THz groups. (G) THz-irradiated neurons showed decreased inter-spike interval (ISI) of synchronized sEPSCs events compared with the control group (Control: 2,975 ± 192.1 ms, n = 15 cells; THz: 2,121 ± 340.1 ms, n = 12 cells). Data are mean ± SEM. ∗, p < 0.05, unpaired t test. (H) The area of synchronized sEPSCs events did not show significant difference between the THz and control groups (Control: −43,522 ± 9,437 pA·ms, n = 15 cells; THz: −33,858 ± 6,729 pA·ms, n = 12 cells). Data are mean ± SEM. See also Figure S3.

Figure 4

THz exposure enhances firing properties of delay firing mode neurons (DFNs) (A) Representative traces of current clamp recording from a DFN. (B–D) THz-irradiated neurons showed similar value of the resting membrane potential (B), the membrane resistance (C), and the membrane capacitance (D) with the control group (RMP: control, −47.2 ± 4.86 mV, n = 12 cells; THz, −46.6 ± 4.68 mV, n = 12 cells. unpaired t test, p = 0.93; Rm: control, 276.1 ± 35.40 MΩ, n = 12; THz, 284.7 ± 38.99 mV, n = 12. unpaired t test, p = 0.87; Cm: control, 49.0 ± 5.09 pA, n = 12; THz, 44.5 ± 6.47 pA, n = 12. unpaired t test, p = 0.59). Data are mean ± SEM. (E) THz-irradiated neurons showed bigger slope of delta voltage-holding current relationship compared with the control group (Slope_control = 0.14, Slope_THz = 0.23). Data are mean ± SEM. ∗, p < 0.05, F-test. (F) THz-irradiated neurons showed smaller rheobase compared with the control group (Control: 203.3 ± 31.68 pA, n = 12 cells; THz: 112.7 ± 22.65 pA, n = 11 cells). Data are mean ± SEM. ∗, p < 0.05, Two-tailed unpaired t test. (G–J) THz-irradiated neurons showed similar value of the AP threshold (G), the first spike delay time (H), AP half-width (I), and the AP peak (J) with the control group. (K-L) THz-irradiated neurons generated more spikes compared with control neurons at the same stimulation of current injection. Data are mean ± SEM. ∗∗∗∗, p < 0.0001, two-way ANOVA; p < 0.05 started at sti. Intensity >170 pA, post hoc Sidak's multiple comparisons test. See also Figures S4 and S5.

Figure 5

THz irradiation alters gene expression dynamics in neurons (A) Heatmap of microarray data from neurons show categories of differentially expressed genes between control and THz groups. Each treatment was repeated three times. (B) Volcanic plot of the differentially expressed genes in THz-irradiated neurons and control neurons. (C) Gene ontology analysis of top 20 upregulated gene groups in THz-irradiated neuron cultures. (D) Gene ontology analysis of top 20 downregulated gene groups in THz-irradiated neuron cultures. (E) Quantitative real-time PCR for representative genes revealed by microarray in THz-irradiated neurons and control cultures. Data are mean ± SEM of transcript levels relative to control after normalization from three independent experiments each performed in triplicate. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001, compared with control, Student's t test.

Figure 6

Long-term THz irradiation inhibits OPC proliferation (A) Diagram showing short-term THz irradiation on OPCs for immunostaining assay. (B) Representative images of immunostaining for BrdU and Olig2 in 3-DIV OPCs after short-term THz irradiation. Scale bar, 50 μm. (C) Percentage of BrdU⁺ cells among Olig2⁺ cells in cultures from control or THz-irradiated groups. Data are mean ± SEM (n = 3 of independent experiments each group). n.s, Student's t test. (D) Diagram showing long-term THz irradiation on OPCs for immunostaining assay. (E) Representative images of immunostaining for BrdU and Olig2 in 3-DIV OPCs after long-term THz irradiation. Scale bar, 50 μm. (F) Percentage of BrdU⁺ cells among Olig2⁺ cells in cultures from control or THz-irradiated groups. Data are mean ± SEM (n = 3 of independent experiments each group). ∗∗, p < 0.01, Student's t test.

Figure 7

Long-term THz irradiation enhances OL differentiation and myelination (A) Diagram showing long-term THz irradiation and succeeding analysis for OL. (B) Representative images of immunostaining for CNPase and MBP at 5 DIV after THz irradiation. Scale bar, 50 μm. (C) Percentage of CNPase⁺ and MBP⁺ cells in OL cultures from control and THz-irradiated groups. Data are mean ± SEM (n = 3 of independent experiments each group). ∗∗, p < 0.01, Student's t test. (D) Quantitative real-time PCR of Mbp and Cnp, in THz-irradiated OLs and control cultures. Data are mean ± SEM of transcript levels relative to control after normalization from three independent experiments each performed in triplicate. ∗∗, p < 0.01, compared with control, Student's t test. (E) Diagram showing long-term THz irradiation on neuron-OL co-cultures for immunostaining. (F) Representative images of immunostaining for Tuj1 and MBP at 14 DIV co-cultures after THz irradiation. Scale bar, 50 μm. (G) Quantification of the length of myelin in defined area between the control and THz groups. Data are mean ± SEM (n = 3 independent experiments). ∗∗∗, p < 0.001, compared with control, Student's t test. See also Figure S6.