Impaired amygdala astrocytic signaling worsens neuropathic pain-associated neuronal functions and behaviors

Abstract

Introduction: Pain is a clinically relevant health care issue with limited therapeutic options, creating the need for new and improved analgesic strategies. The amygdala is a limbic brain region critically involved in the regulation of emotional-affective components of pain and in pain modulation. The central nucleus of amygdala (CeA) serves major output functions and receives nociceptive information via the external lateral parabrachial nucleus (PB). While amygdala neuroplasticity has been linked causally to pain behaviors, non-neuronal pain mechanisms in this region remain to be explored. As an essential part of the neuroimmune system, astrocytes that represent about 40-50% of glia cells within the central nervous system, are required for physiological neuronal functions, but their role in the amygdala remains to be determined for pain conditions. In this study, we measured time-specific astrocyte activation in the CeA in a neuropathic pain model (spinal nerve ligation, SNL) and assessed the effects of astrocyte inhibition on amygdala neuroplasticity and pain-like behaviors in the pain condition. Methods and Results: Glial fibrillary acidic protein (GFAP, astrocytic marker) immunoreactivity and mRNA expression were increased at the chronic (4 weeks post-SNL), but not acute (1 week post-SNL), stage of neuropathic pain. In order to determine the contribution of astrocytes to amygdala pain-mechanisms, we used fluorocitric acid (FCA), a selective inhibitor of astrocyte metabolism. Whole-cell patch-clamp recordings were performed from neurons in the laterocapsular division of the CeA (CeLC) obtained from chronic neuropathic rats. Pre-incubation of brain slices with FCA (100 µM, 1 h), increased excitability through altered hyperpolarization-activated current (I_h) functions, without significantly affecting synaptic responses at the PB-CeLC synapse. Intra-CeA injection of FCA (100 µM) had facilitatory effects on mechanical withdrawal thresholds (von Frey and paw pressure tests) and emotional-affective behaviors (evoked vocalizations), but not on facial grimace score and anxiety-like behaviors (open field test), in chronic neuropathic rats. Selective inhibition of astrocytes by FCA was confirmed with immunohistochemical analyses showing decreased astrocytic GFAP, but not NeuN, signal in the CeA. Discussion: Overall, these results suggest a complex modulation of amygdala pain functions by astrocytes and provide evidence for beneficial functions of astrocytes in CeA in chronic neuropathic pain.

Keywords: amygdala; astrocyte; behavior; electrophysiology; neuroimmune signaling; neuronal excitability; neuropathic pain; neurotransmission.

FIGURE 1

Temporal changes of GFAP mRNA and protein in the CeA of neuropathic rats. (A) Increased GFAP mRNA expression levels in the right CeA 4 weeks, but not 1 week, post-induction of SNL compared to sham control group. Importantly, no significant differences were observed between the two sham control groups (sham 1w vs. sham 4w). Bar histograms show means ± SEM. *,**p < 0.05, 0.01, two-way ANOVA with Bonferroni posthoc tests. Sham 1w, n = 7; sham 4w, n = 4; SNL 1w, n = 7; SNL 4w, n = 4. (B) Immunohistochemistry shows significantly increased GFAP (+) signal in CeC of chronic SNL rats compared to acute SNL or sham groups. A similar trend was detected in the CeL (C) though no statistical significance was reached (p = 0.0505). (D) Representative images of GFAP (+) signal in the CeA of different treatment groups. Bottom: representative sample area used for analysis. Scale bars, top, 200 μm; bottom, 10 μm *, **, p < 0.05, 0.01, one-way ANOVA Tukey’s posthoc tests. CeC and CeL: sham, n = 10, SNL 1w, n = 10, SNL 4w, n = 10 sections from three animals per group.

FIGURE 2

Facilitatory effects of astrocyte inhibition by fluorocitric acid (FCA) on CeLC neuronal properties in a neuropathic model. Neurons recorded from FCA-treated slices (100 μM, 1 h) showed increased neuronal excitability (A,B) induced by depolarizing current injections, while Ba²⁺ pretreatment had no effects compared to the control (ACSF) group (*, p < 0.05, two-way ANOVA with Bonferroni post hoc tests). Symbols show means ± SEM. ***, p < 0.001, two-way ANOVA. In the same neurons, FCA had no effects on rheobase (C) or AP threshold (D,E), but it decreased the depolarizing voltage sag (F,G) without affecting the I-V relationship (H,I) compared to the control (ACSF) treated cells. Bar histograms show means ± SEM. *, p < 0.05, unpaired student t-tests. Symbols show means ± SEM, two-way ANOVA. ACSF, n = 7; FCA, n = 8.

FIGURE 3

Lack of effects of astrocyte inhibition by FCA on evoked synaptic responses in a neuropathic model. Pre-treatment of brain slices with FCA (100 μM, 1 h) had no effect on the peak amplitude, area under the curve (AUC) or decay time of mono-synaptic excitatory post-synaptic currents (EPSCs) evoked by the electrical stimulation of PB input to CeLC neurons (A–D) recorded in brain slices obtained from neuropathic animals. Additionally, no change was observed in the paired-pulse ratio at the PB-CeLC synapse (E,F). Similarly, pre-incubation with FCA (100 μM, 1 h) did not affect the peak amplitude, AUC or decay time of glutamate-driven inhibitory post-synaptic currents (IPSCs) evoked by the electrical stimulation of PB afferents onto CeLC neurons (G–J) in the neuropathic pain condition. No significant change was observed in the excitation/inhibition (E/I) ratio (K,L). Symbols show means ± SEM, two-way ANOVA. Bar histograms show means ± SEM, unpaired t-tests. ACSF, n = 7; FCA, n = 8.

FIGURE 4

Pronociceptive effects of astrocyte inhibition by FCA in CeA on neuropathic pain-like behaviors. (A) Mechanical thresholds (measured by electronic von Frey) of the left (injured), but not right, hind paw were significantly decreased 4 weeks after SNL surgery, confirming the neuropathic pain condition. Stereotaxic injection of FCA (100 μM, 1 μL) into the CeA significantly lowered mechanical withdrawal thresholds on the right hind paw and further decreased the thresholds on the left hind paw compared to vehicle (Veh) treatment. Bar histograms show mean ± SEM. ^##, ^### p < 0.01, 0.001 compared to baseline (pre-SNL surgery). *, p < 0.05, compared to vehicle, two-way ANOVA with Bonferroni’s multiple comparisons. Intra-CeA administration of FCA (100 μM, 1 μL) significantly decreased the withdrawal thresholds measured by compression (B) of the left (injured) and right paws and increased the audible vocalizations (C) evoked by noxious, but not innocuous, stimulation of the left (injured) and right hind paws, but had no significant effect on the ultrasonic vocalizations (D) though a trend for a facilitatory effect was observed for the noxious compression of the left (injured) hind paw. Injection of FCA (100 μM, 1 μL) into the CeA showed a non-significant trend to facilitatory effects on the facial grimace scale (E,G) and anxiety-like behaviors measured in the open field test (H–J) compared to vehicle. Bar histograms show mean ± SEM. *, **, ***p < 0.05, 0.01, 0.001 compared to vehicle, unpaired student t-tests. (A-G) Veh, n = 8; FCA, n = 8; (H-L) Veh, n = 7; FCA, n = 7.

FIGURE 5

Validation of astrocyte inhibition by FCA in CeA in neuropathic rats. Immunohistochemical analysis showed decreased percentage of astrocytic GFAP positive area, but not mean of positive signal, in the CeC (A,B) and CeL (C,D) after FCA (100 μM, 1 μL) injection into the CeA of SNL (4 weeks) rats compared to vehicle (Veh) group, consistent with astrocyte inhibition in the targeted area. NeuN staining showed no significant differences between the two groups (E–H), confirming the glia-specific effect of the pharmacological approach. Bar histograms show mean ± SEM. **, ***p < 0.01, 0.001 compared to vehicle, unpaired student t-tests. Veh, n = 5; FCA, n = 5 (I) Representative images of GFAP (+) staining in brain sections from Veh (left) and FCA (right) injected neuropathic rats. Scale bar = 200 μm.

FIGURE 6

Astrocyte signaling in the amygdala (CeA) in neuropathic pain. Astrocytic silencing increases the output of CeLC neurons through mechanisms involving decreased HCN signaling, resulting in increased neuropathic pain-related behaviors without affecting synaptic transmission at the PB-CeLC synapse. Therefore, endogenous astrocyte signaling may sustain protective neuronal functions. CeLC, laterocapsular division of the central nucleus of amygdala; PBN, parabrachial nucleus; HCN, hyperpolarization-activated cyclic nucleotide–gated (HCN) channels. Created with BioRender.com.