Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Mar 30:16:863342.
doi: 10.3389/fncel.2022.863342. eCollection 2022.

Facial Stimulation Induces Long-Term Potentiation of Mossy Fiber-Granule Cell Synaptic Transmission via GluN2A-Containing N-Methyl-D-Aspartate Receptor/Nitric Oxide Cascade in the Mouse Cerebellum

Di Lu  1   2 , Peng Wan  3 , Yang Liu  1   2 , Xian-Hua Jin  3 , Chun-Ping Chu  4 , Yan-Hua Bing  1 , De-Lai Qiu  4
Affiliations

Facial Stimulation Induces Long-Term Potentiation of Mossy Fiber-Granule Cell Synaptic Transmission via GluN2A-Containing N-Methyl-D-Aspartate Receptor/Nitric Oxide Cascade in the Mouse Cerebellum

Di Lu et al. Front Cell Neurosci. .

Abstract

Long-term synaptic plasticity in the cerebellar cortex is a possible mechanism for motor learning. Previous studies have demonstrated the induction of mossy fiber-granule cell (MF-GrC) synaptic plasticity under in vitro and in vivo conditions, but the mechanisms underlying sensory stimulation-evoked long-term synaptic plasticity of MF-GrC in living animals are unclear. In this study, we investigated the mechanism of long-term potentiation (LTP) of MF-GrC synaptic transmission in the cerebellum induced by train of facial stimulation at 20 Hz in urethane-anesthetized mice using electrophysiological recording, immunohistochemistry techniques, and pharmacological methods. Blockade of GABAA receptor activity and repetitive facial stimulation at 20 Hz (240 pulses) induced an LTP of MF-GrC synapses in the mouse cerebellar cortical folium Crus II, accompanied with a decrease in paired-pulse ratio (N2/N1). The facial stimulation-induced MF-GrC LTP was abolished by either an N-methyl-D-aspartate (NMDA) receptor blocker, i.e., D-APV, or a specific GluNR2A subunit-containing NMDA receptor antagonist, PEAQX, but was not prevented by selective GluNR2B or GluNR2C/D subunit-containing NMDA receptor blockers. Application of GNE-0723, a selective and brain-penetrant-positive allosteric modulator of GluN2A subunit-containing NMDA receptors, produced an LTP of N1, accompanied with a decrease in N2/N1 ratio, and occluded the 20-Hz facial stimulation-induced MF-GrC LTP. Inhibition of nitric oxide synthesis (NOS) prevented the facial stimulation-induced MF-GrC LTP, while activation of NOS produced an LTP of N1, with a decrease in N2/N1 ratio, and occluded the 20-Hz facial stimulation-induced MF-GrC LTP. In addition, GluN2A-containing NMDA receptor immunoreactivity was observed in the mouse cerebellar granular layer. These results indicate that facial stimulation at 20 Hz induced LTP of MF-GrC synaptic transmission via the GluN2A-containing NMDA receptor/nitric oxide cascade in mice. The results suggest that the sensory stimulation-evoked LTP of MF-GrC synaptic transmission in the granular layer may play a critical role in cerebellar adaptation to native mossy fiber excitatory inputs and motor learning behavior in living animals.

Keywords: NMDA receptor; cerebellum; in vivo electrophysiological recording; mossy fiber-granule cell synaptic transmission; nitric oxide; plasticity; sensory stimulation.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Facial stimulation at 20 Hz induced an MF-GrC LTP in the mouse cerebellar granular layer. (A) Upper: Representative extracellular recording traces showing a paired air-puff stimulation (10 ms, 60 psi, 50 ms interval) evoked MF-GrC synaptic responses before (pre) and after (post) the delivery of 20-Hz (240 pulses) air-puff stimulation. (B) Summary of data showing the time course of normalized amplitude of N1 in which the 20-Hz stimulation protocol (arrow) was delivered (filled circle) or not delivered (open circle) under control conditions. (C) Mean value (± SEM) with individual data showing the normalized amplitude before (pre) and after (post) the delivery of the 20-Hz stimulation protocol. (D) Mean value (± SEM) with individual data showing the normalized N2/N1 ratio before (pre) and after (post) the delivery of 20-Hz stimulation protocol. Note that the 20-Hz facial stimulation induced an LTP at MF-GrC synapse in the mouse cerebellum. *P < 0.05 vs. pre; n = 10 recordings from 10 mice.
FIGURE 2
FIGURE 2
NMDA receptor blocker, D-APV, abolished the facial stimulation-induced MF-GrC LTP in the mouse cerebellar cortex. (A) Upper: Representative extracellular recording traces showing a paired air-puff stimulation (10 ms, 60 psi, 50 ms interval) evoked MF-GC synaptic responses before (pre) and after (post) the delivery of 20-Hz (240 pulses) air-puff stimulation in the presence of D-APV (250 μM). (B) Summary of data showing the time course of normalized amplitude of N1 in which the 20-Hz stimulation protocol (arrow) was delivered in the presence of D-APV. (C) Mean value (± SEM) with individual data showing the normalized amplitude before (pre) and after (post) the delivery of the 20-Hz stimulation protocol in the presence of D-APV. (D) Mean value (± SEM) with individual data showing the normalized N2/N1 ratio before (pre) and after (post) the delivery of the 20-Hz stimulation protocol in the presence of D-APV. Note that blockade NMDA receptor abolished the 20-Hz facial stimulation-induced MF-GrC LTP in the mouse cerebellum. n = 9 recordings from 9 mice.
FIGURE 3
FIGURE 3
Blockade of GluN2A subunit-containing NMDA receptor blocker prevented the facial stimulation-induced MF-GrC LTP in the mouse cerebellar cortex. (A) Upper: Representative extracellular recording traces showing a paired air-puff stimulation (10 ms, 60 psi, 50 ms interval) evoked MF-GC synaptic responses before (pre) and after (post) the delivery of 20-Hz (240 pulses) air-puff stimulation in the presence of GluN2A subunit-containing NMDA receptor blocker, i.e., PEAQX (10 μM). (B) Summary of data showing the time course of normalized amplitude of N1 in which the 20-Hz stimulation protocol (arrow) was delivered in the presence of PEAQX. (C) Mean value (± SEM) with individual data showing the normalized amplitude before (pre) and after (post) the delivery of the 20-Hz stimulation protocol in the presence of PEAQX. (D) Mean value (± SEM) with individual data showing the normalized N2/N1 ratio before (pre) and after (post) the delivery of the 20-Hz stimulation protocol in the presence of PEAQX. Note that blockade GluN2A subunit-containing NMDA receptor abolished the 20-Hz facial stimulation-induced MF-GrC LTP in the mouse cerebellar granular layer. n = 7 recordings from 7 mice.
FIGURE 4
FIGURE 4
GluN2B subunit-containing NMDA receptor antagonist failed to prevent the facial stimulation-induced MF-GrC LTP in the mouse cerebellar granular layer. (A) Upper: Representative extracellular recording traces showing a paired air-puff stimulation (10 ms, 60 psi, 50 ms interval) evoked MF-GC synaptic responses before (pre) and after (post) the delivery of 20-Hz (240 pulses) air-puff stimulation in the presence of a GluN2B subunit-containing NMDA receptor antagonist, i.e., TCN (10 μM). (B) Summary of data showing the time course of normalized amplitude of N1 in which the 20-Hz stimulation protocol (arrow) was delivered in the presence of TCN. (C) Mean value (± SEM) with individual data showing the normalized amplitude before (pre) and after (post) the delivery of the 20-Hz stimulation protocol in the presence of TCN. (D) Mean value (± SEM) with individual data showing the normalized N2/N1 ratio before (pre) and after (post) the delivery of the 20-Hz stimuli protocol in the presence of TCN. Note that blockade GluN2B subunit-containing NMDA receptor failed to prevent the facial stimulation produced MF-GrC LTP in the mouse cerebellar granular layer. * P < 0.05 vs. pre; n = 8 recordings from 8 mice.
FIGURE 5
FIGURE 5
Blockade of GluN2C/D subunits-containing NMDA receptor did less effect on the facial stimulation-induced MF-GrC LTP in the mouse cerebellar granular layer. (A) Upper: Representative extracellular recording traces showing a paired air-puff stimulation (10 ms, 60 psi, 50 ms interval) evoked MF-GC synaptic responses before (pre) and after (post) the delivery of 20-Hz (240 pulses) air-puff stimulation in the presence of DQP-1105 (100 μM). (B) Summary of data showing the time course of normalized amplitude of N1 in which the 20-Hz stimuli protocol (arrow) was delivered in the presence of DQP-1105. (C) Mean value (± SEM) with individual data showing the normalized amplitude before (pre) and after (post) the delivery of the 20-Hz stimulation protocol in the presence of DQP-1105. (D) Mean value (± SEM) with individual data showing the normalized N2/N1 ratio before (pre) and after (post) the delivery of the 20-Hz stimulation protocol in the presence of DQP-1105. Note that blockade GluN2C/D subunits-containing NMDA receptor failed to abolish 20-Hz facial stimulation-induced MF-GrC LTP in the mouse cerebellar granular layer. * P < 0.05 vs. pre; n = 7 recordings from 7 mice.
FIGURE 6
FIGURE 6
A selective positive allosteric modulator of GluN2A subunit-containing NMDA receptor, i.e., GNE-0723, produced MF-GrC LTP and occluded the 20-Hz stimulation protocol-induced LTP of MF-GrC synaptic transmission. (A) Upper: Representative extracellular recording traces showing a paired air-puff stimulation (10 ms, 60 psi, 50 ms interval) evoked MF-GC synaptic responses before (pre-GNE), after (post-GNE) application of GNE-0723 (10 μM; gray), and after delivery of the stimulation train (poststimuli). (B) Summary of data showing the time course of normalized amplitude of N1 before and after application of GNE-0723 (10 μM; gray) and after delivery of the stimulation train (arrow). (C) Mean value (± SEM) with individual data showing the normalized amplitude before (pre-GNE) and after (post-GNE) application of NMDA and after the stimulation train (poststimuli). (D) Mean value (± SEM) with individual data showing the normalized N2/N1 ratio before (pre-GNE) and after (post-GNE) application of GNE-0723 and after delivery of the stimulation train (poststimuli). Note that perfusion of GNE-0723 receptor produced an MF-GrC LTP and occluded the 20-Hz facial stimuli-induced LTP of MF-GrC synaptic transmission. * P < 0.05 vs. pre; n = 6 recordings from 6 mice.
FIGURE 7
FIGURE 7
Inhibition of nitric oxide synthase abolished the facial stimulation-induced MF-GrC LTP in the mouse cerebellar cortex. (A) Upper: Representative extracellular recording traces showing a paired air-puff stimulation (10 ms, 60 psi, 50 ms interval) evoked MF-GrC synaptic responses before (pre) and after (post) delivery of 20-Hz (240 pulses) air-puff stimuli in the presence of nitric oxide synthase inhibitor, i.e., L-NNA (200 μM). (B) Summary of data showing the time course of normalized amplitude of N1 in which the 20-Hz stimuli protocol (arrow) was delivered in the presence of L-NNA. (C) Mean value (± SEM) with individual data showing the normalized amplitude before (pre) and after (post) the delivery of the 20-Hz stimulation protocol in the presence of L-NNA. (D) Mean value (± SEM) with individual data showing the normalized N2/N1 ratio before (pre) and after (post) the delivery of the 20-Hz stimulation protocol in the presence of L-NNA. Note that inhibiting nitric oxide synthase abolished the 20-Hz facial stimulation-induced MF-GrC LTP. n = 7 recordings from 7 mice.
FIGURE 8
FIGURE 8
Application of NO donor produced MF-GrC LTP, which prevented the 20-Hz stimulation protocol-induced LTP of MF-GrC synaptic transmission. (A) Upper: Representative extracellular recording traces showing a paired air-puff stimulation (10 ms, 60 psi, 50 ms interval) evoked MF-GC synaptic responses before (pre-SNAP) and after (post-SNAP) application of NO donor, i.e., SNAP (500 μM; gray), and after delivery of the stimulation train (poststimuli). (B) Summary of data showing the time course of normalized amplitude of N1 before and after application of SNAP (100 μM; gray) and after delivery of the stimulation train (arrow). (C) Mean value (± SEM) with individual data showing the normalized amplitude before (pre-SNAP) and after (post-SNAP) application of SNAP and after the stimulation train (poststimuli). (D) Mean value (± SEM) with individual data showing the normalized N2/N1 ratio before (pre-SNAP) and after (post-SNAP) application of SNAP and after delivery of the stimulation train (poststimuli). Note that perfusion of NO donor produced an LTP of MF-GrC synaptic transmission and prevented the 20-Hz facial stimuli-induced MF-GrC LTP. * P < 0.05 vs. pre; n = 9 recordings from 9 mice.
FIGURE 9
FIGURE 9
GluN2A immunoreactivity was expressed in GrCs of the mouse cerebellar cortical lobule Crus II. (A) A digital micrograph shows the confocal image of DAPI (blue) in the mouse cerebellar lobule Crus II. DAPI is a blue nucleic acid dye that preferentially dyes the dsDNA of cells. (B) Higher magnifications of the boxed area in (A). (C) Higher magnifications of the boxed area in (B) showing GluN2A subunit-containing NMDA receptor immunoreactivity expressed in GL (red; arrows). ML, molecular layer; PCL, Purkinje cell layer; GL, granular layer.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Akazawa C., Shigemoto R., Bessho Y., Nakanishi S., Mizuno N. (1994). Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats. J. Comp. Neurol. 347 150–160. 10.1002/cne.903470112 - DOI - PubMed
    1. Bhattacharya S., Khatri A., Swanger S. A., DiRaddo J. O., Yi F., Hansen K. B., et al. (2018). Triheteromeric GluN1/GluN2A/GluN2C NMDARs with unique single-channel properties are the dominant receptor population in cerebellar granule cells. Neuron 99 315–328. 10.1016/j.neuron.2018.06.010 - DOI - PMC - PubMed
    1. Bing Y. H., Zhang G. J., Sun L., Chu C. P., Qiu D. L. (2015). Dynamic properties of sensory stimulation evoked responses in mouse cerebellar granule cell layer and molecular layer. Neurosci. Lett. 585 114–118. 10.1016/j.neulet.2014.11.037 - DOI - PubMed
    1. Bliss T. V., Collingridge G. L. (1993). A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361 31–39. 10.1038/361031a0 - DOI - PubMed
    1. Bredt D. S., Glatt C. E., Hwang P. M., Fotuhi M., Dawson T. M., Snyder S. H. (1991). Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase. Neuron 7 615–624. 10.1016/0896-6273(91)90374-9 - DOI - PubMed

LinkOut - more resources