GFAP-NpHR mediated optogenetic inhibition of trigeminal nucleus caudalis attenuates hypersensitive behaviors and thalamic discharge attributed to infraorbital nerve constriction injury

Abstract

The significance of hyperactive astrocytes in neuropathic pain is crucial. However, the association between medullary astrocytes and trigeminal neuralgia (TN)-related pain processing is unclear. Here, we examined how optogenetic inhibition of medullary astrocytes in the trigeminal nucleus caudalis (TNC) regulates pain hypersensitivity in an infraorbital nerve (ION) constricted TN model. We used adult Sprague Dawley rats subjected to infraorbital nerve (ION) constriction to mimic TN symptoms, with naive and sham rats serving as controls. For in vivo optogenetic manipulations, rats stereotaxically received AAV8-GFAP-eNpHR3.0-mCherry or AAV8-GFAP-mCherry at the trigeminal nucleus caudalis (TNC). Open field, von Frey, air puff, and acetone tests measured pain behavioral flexibility. In vivo thalamic recordings were obtained simultaneously with optogenetic manipulation in the TNC. Orofacial hyperalgesia and thalamic hyperexcitability were both accompanied by medullary astrocyte hyperactivity, marked by upregulated GFAP. The yellow laser-driven inhibition of TNC astrocytes markedly improved behavioral responses and regulated thalamic neuronal responses. Halorhodopsin-mediated inhibition in medullary astrocytes may modify the nociceptive input transmitted through the trigeminothalamic tract and pain perception. Taken together, these findings imply that this subpopulation in the TNC and its thalamic connections play a significant role in regulating the trigeminal pain circuitry, which might aid in the identification of new therapeutic measures in TN management.

Keywords: Astrocytes; Medullary dorsal horn; Optogenetics; Trigeminal neuralgia.

Fig. 1

Virus expression in the TNC. A Schematic drawing of the site for virus injection B Coronal section of TNC, red dots showing the virus localization site C Representative fluorescent image showing the coronal section of TNC and virus localization, Scale = 1 mm. D, E DAPI (F, G) GFAP (H, I) eNpHR-mCherry (J, K) Merge images for colocalization of optogenetic virus expression in the TNC, Scales = 50 μm and 20 μm. Dashed white lines marked the laminar divisions in TNC

Fig. 2

Infraorbital nerve constriction induced hyperalgesia and upregulated GFAP expression in the TNC (A) Schematic diagram of infraorbital nerve constriction in rats. B Immunofluorescence staining showed that GFAP fluorescence intensity was significantly increased in the TNC of the TN group compared to that of the sham and naïve groups. Scale bar = 50 μm. C Quantification of GFAP immunostaining in the TNC of different groups. Data are presented as the mean ± SD, n = 5/group, two tailed Welch’s t-test, ****p < 0.0001 compared with the sham group. D Pictorial diagram of the behavior test for pain assessment (E) Representative trajectory of TN, sham and naïve groups in the OFT. F Number of explored areas and (G) mobility rate (in percentage) in each group before (baseline) and after (week 1 and week 3) surgery. H–L The mechanical pain threshold in the von Frey filament test in ipsilateral and contralateral whisker pads. J The thermal pain threshold. K-L The pressure threshold in air puff test in ipsilateral and contralateral whisker pads. ****, p < 0.0001, ordinary two-way ANOVA followed by Sidak’s multiple comparisons among groups. Data are displayed as the means ± SD, n = 20(TN), 20( sham), 5 (naïve)

Fig. 3

GFAP expression and VPM thalamic recording in response to optic inhibition of TNC astrocytes. A-B Representative fluorescent image of GFAP expression in the TNC of TN/eNpHR- (A) and TN/eNpHR + (B) rats in response to the optic inhibition of TNC astrocytes. Scale = 100 μm and 20 μm. C Quantification of GFAP immunostaining in TNC of the TN/eNpHR- and TN/eNpHR + groups in the yellow laser on condition, n = 6/group, two-tailed Welch’s t-test, ****p < 0.0001 compared with another group. D Schematic diagram of in vivo optogenetic manipulation of TNC astrocytes with a yellow laser (590 nm) in conjunction with the thalamic recording (E) Coronal section of the VPM thalamus showing the recording site. F Mean firing rate from the VPM thalamus between the TN and sham groups. Two-tailed unpaired t-test with Welch’s correction, n = 16/group, ***P = 0.0002 compared to sham (G) Representative thalamic discharge (lower one presented in frequency, impulse per second) with raw traces (upper) during light-off and yellow laser-on conditions in the TN/eNpHR + group. H Mean firing rate (presented in Hertz) from VPM thalamus of TN/eNpHR + and TN/eNpHR- groups during light off and on conditions. Two-way repeated-measures ANOVA with Sidak’s multiple comparisons test, n = 8/group, *P = 0.0380 compared to light off condition of TN/eNpHR + (I) Mean firing rate (presented in Hertz) from VPM thalamus of sham/eNpHR + and sham/eNpHR- groups during light off and on conditions. Two-way repeated-measures ANOVA with Sidak’s multiple comparisons tests, n = 8/group, not significant, P = 0.4800 compared to the light-off condition of sham/eNpHR + . J Burst frequency (presented in Hertz) comparison between TN (TN/eNpHR + and TN/eNpHR-) and sham (sham/eNpHR + and sham/eNpHR-) groups in response to light stimulation on and off in TNC. Two-way repeated-measures ANOVA with Sidak’s multiple comparisons tests, n = 8/group, **P = 0.0017 compared to light off condition of TN/eNpHR + . Data are presented as the mean ± SD. K Representative burst traces of TN/eNpHR + rat from the VPM thalamus during light-off and light-on condition in TNC astrocytes

Fig. 4

Optogenetic inhibition of TNC astrocytes alleviates pain induced by ION constriction. A Schematic diagram of the optic modulation site in rat brain. B Light stimulation parameters during the behavior test. C Optic inhibition of TNC astrocytes increased mechanical withdrawal threshold in ipsilateral whisker pad during the von Frey filament test in TN/eNpHR + rats. Data are presented as the mean ± SD. Two-way ordinary ANOVA with Sidak’s multiple comparisons tests, n = 8/group, ***P = 0.0006 compared to light off condition of TN/eNpHR + rats. Two-way ordinary ANOVA with Sidak’s multiple comparisons tests, n = 8/group, ****P < 0.0001 compared to light on condition between TN/eNpHR + and TN/eNpHR- rats (D) Schematic diagram of rats subjected to von Frey filaments (upper) and acetone (lower) tests with a tethered system for optogenetic manipulations. E Astrocyte-specific optic inhibition of TNC decreased the number of episodes during the acetone test for cold allodynia in TN/eNpHR + rats. Data are presented as the mean ± SD. Two-way ordinary ANOVA with Sidak’s multiple comparisons tests, n = 8/group, **P = 0.0075 compared to light off condition of TN/eNpHR + rats. Two-way ordinary ANOVA with Sidak’s multiple comparisons tests, n = 8/group, *P = 0.0160 compared to light on condition between TN/eNpHR + and TN/eNpHR- rats. F The pressure threshold during the air puff test was improved during TNC astrocyte inhibition with a yellow laser in TN/eNpHR + rats. Two-way ordinary ANOVA with Sidak’s multiple comparisons tests, n = 8/group, ****P < 0.0001 compared to light off condition of TN/eNpHR + rats. Two-way ordinary ANOVA with Sidak’s multiple comparisons tests, n = 8/group, ****P < 0.0001 compared to light on condition between TN/eNpHR + and TN/eNpHR- rats. G Schematic diagram of rat subjected to open field test with a tethered system for optogenetic intervention (H, I) Representative trajectory and exploration heatmap of TN/eNpHR + rats during light on and off conditions. J, K The number of explored areas (***P = 0.0007) and mobility rate (***P = 0.0004, expressed as a percentage) were considerably improved in TN/eNpHR + animals under light-on conditions compared to those under light-off conditions. No significant differences were observed in the other groups. Data are presented as the mean ± SD. Two-way ordinary ANOVA with Sidak’s multiple comparisons tests, n = 8/group, (explored areas, ****P < 0.0001; mobility rate, **P = 0.0012) compared to light on condition between TN/eNpHR + and TN/eNpHR- rats

Fig. 5

P2X3 immunoreactivity in the TNC in response to astrocyte-specific TNC optogenetic inhibition. Representative immunohistochemistry images showing P2X3 expression in the TNC of (A, C) TN/eNpHR + (B, D) TN/eNpHR- animals in response to astrocyte-specific TNC optogenetic inhibition. Black arrowheads represent the P2X3 expressed areas in the TNC. (E) Quantification of P2X3 immunoreactivity in the TNC of TN rats with optogenetic inhibition with a yellow laser. Data are presented as the mean ± SD. Two-tailed Mann–Whitney test, n = 6/group, **P = 0.0043 compared to another group