Activation of amygdala opioid receptors by electroacupuncture of Feng-Chi (GB20) acupoints exacerbates focal epilepsy

Abstract

Background: The effect of seizure suppression by acupuncture of Feng-Chi (GB20) acupoints has been documented in the ancient Chinese literature, Lingshu Jing (Classic of the Miraculous Pivot), however, there is a lack of scientific evidence to prove it. This current study was designed to elucidate the effect of electroacupuncture (EA) stimulation of bilateral Feng-Chi (GB20) acupoints on the epileptic activity by employing an animal model of focal epilepsy.

Methods: Administration of pilocarpine into the left central nucleus of amygdala (CeA) induced the focal epilepsy in rats. Rats received a 30-min 100 Hz EA stimulation of bilateral Feng-Chi acupoints per day, beginning at 30 minutes before the dark period and performing in three consecutive days. The broad-spectrum opioid receptor antagonist (naloxone), μ-receptor antagonist (naloxonazine), δ-receptor antagonist (naltrindole) and κ-receptor antagonist (nor-binaltorphimine) were administered directly into the CeA to elucidate the involvement of CeA opioid receptors in the EA effect.

Results: High-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints did not suppress the pilocarpine-induced epileptiform electroencephalograms (EEGs), whereas it further increased the duration of epileptiform EEGs. We also observed that epilepsy occurred while 100 Hz EA stimulation of Feng-Chi acupoints was delivered into naïve rats. EA-induced augmentation of epileptic activity was blocked by microinjection of naloxone, μ- (naloxonazine), κ- (nor-binaltorphimine) or δ-receptor antagonists (natrindole) into the CeA, suggesting that activation of opioid receptors in the CeA mediates EA-exacerbated epilepsy.

Conclusions: The present study suggests that high-frequency (100 Hz) EA stimulation of bilateral Feng-Chi acupoints has no effect to protect against pilocarpine-induced focal epilepsy; in contrast, EA further exacerbated focal epilepsy induced by pilocarpine. Opioid receptors in the CeA mediated EA-induced exacerbation of focal epilepsy.

Figure 1

Schematic representation of the experimental protocol. Closed bars indicate the dark period and open bars represent the light period of the 12:12 h light:dark cycle. Arrows depict the time of microinjections. R: EEG recording; Pilo: pilocarpine; EA: electroacupuncture; N: naloxone; μ-ant: μ-antagonist (naloxonazine); δ-ant: δ-antagonist (naltrindole); κ-ant: κ-antagonist (nor-binaltorphimine).

Figure 2

The focal epilepsy induced by microinjection of pilocarpine into the CeA. Panels A, B, C, D, E and F represent the EEG signals acquired from the electrodes of left frontal, left parietal, left occipital, right frontal, right parietal and right occipital cortices, respectively. The epileptiform EEGs were predominantly recorded from the electrode of left parietal cortex.

Figure 3

The effect of 100 Hz EA stimulation of bilateral Feng-Chi acupoints on the epileptic activities. Panels A, B, C and D respectively depict the EEG signals recorded from the naïve rats, the EA group, the pilocarpine group, and the PFS + EA + pilocarpine group, beginning from the dark onset of the dark period. Red lines indicate the time for pilocarpine administration (at the end of the 30-min EA stimulation). The blue boxes represent the epileptiform EEGs. Green arrowheads were the artifacts. The larger amplitudes, with EEG signals less than 2 mV, appeared in panels A, B and C were delta waves, which represent the state of slow wave sleep. Panels E, F, G and H were the enlarged figures which were respectively expanded the time scale from the panels A, B, C and D, and were recorded from the beginning of the dark period.

Figure 4

The summary of 100 Hz EA stimulation of bilateral Feng-Chi acupoints on the epileptic activities. Panel A depicts the results obtained from the dark period and panel B demonstrates the data acquired from the light period. The bars from the left to the right in both panels A and B represent the results obtained from the naïve rats, the EA group, the pilocarpine group, and the PFS + EA + pilocarpine group. In panel C, the black circles represent the values obtained from the naïve group, the black triangles depict the results of the EA group, the white circles demonstrate the values obtained from the pilocarpine group, and the white triangles indicate the data acquired from the PFS + EA + pilocarpine group. #: p < 0.05 vs. the naïve rats; ##: p < 0.01 vs. the naïve rats; **: p < 0.01 vs. the pilocarpine group.

Figure 5

The effects of naloxone, naloxonazine, naltrindole and nor-binaltorphimine on the 100 Hz EA-induced exacerbation of epileptic activities. Panels A, B, C, D and E respectively depict the EEG signals recorded from the PFS + EA + pilocarpine group, naloxone + EA + pilocarpine group, the naloxonazine + EA + pilocarpine group, the naltrindole + EA + pilocarpine group, and the nor-binaltorphimine + EA + pilocarpine group, beginning from the dark onset of the dark period. Red lines indicate the time for pilocarpine administration (at the end of the 30-min EA stimulation). The blue boxes represent the epileptiform EEGs. Green arrowheads were the artifacts. The larger amplitudes, with EEG signals less than 2 mV, appeared in panels B, C and D were delta waves, which represent the state of slow wave sleep. Panels F, G, H, I and J were the enlarged figures which were respectively expanded the time scale from the panels A, B, C, D and E, and were recorded from the beginning of the dark period.

Figure 6

The summary for the effects of naloxone, naloxonazine, naltrindole and nor-binaltorphimine on the 100 Hz EA-induced exacerbation of epileptic activities. Panel A depicts the results obtained from the dark period and panel B demonstrates the data acquired from the light period. The bars from the left to the right in both panels A and B represent the results obtained from the PFS + EA + pilocarpine group, naloxone + EA + pilocarpine group, naloxonazine + EA + pilocarpine group, naltrindole + EA + pilocarpine group and nor-binaltorphimine + EA + pilocarpine group. In panel C, the black circles represent the values obtained from the PFS + EA + pilocarpine group, the black triangles depict the results of the naloxone + EA + pilocarpine group, the white circles demonstrate the values obtained from the naloxonazine + EA + pilocarpine group, the white diamonds elucidate the results of the naltrindole + EA + pilocarpine group, and the white triangles indicate the data acquired from the nor-binaltorphimine + EA + pilocarpine group. **: p < 0.01 vs. the PFS + EA + pilocarpine group.