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. 2021 Oct 3;10(10):2644.
doi: 10.3390/cells10102644.

Dysfunction of Glutamate Delta-1 Receptor-Cerebellin 1 Trans-Synaptic Signaling in the Central Amygdala in Chronic Pain

Pauravi J Gandhi  1 , Dinesh Y Gawande  1 , Gajanan P Shelkar  1 , Sukanya G Gakare  1 , Takaki Kiritoshi  2 , Guangchen Ji  2   3 , Bishal Misra  1 , Ratnamala Pavuluri  1 , Jinxu Liu  1 , Volker Neugebauer  2   3   4 , Shashank M Dravid  1
Affiliations

Dysfunction of Glutamate Delta-1 Receptor-Cerebellin 1 Trans-Synaptic Signaling in the Central Amygdala in Chronic Pain

Pauravi J Gandhi et al. Cells. .

Abstract

Chronic pain is a debilitating condition involving neuronal dysfunction, but the synaptic mechanisms underlying the persistence of pain are still poorly understood. We found that the synaptic organizer glutamate delta 1 receptor (GluD1) is expressed postsynaptically at parabrachio-central laterocapsular amygdala (PB-CeLC) glutamatergic synapses at axo-somatic and punctate locations on protein kinase C δ -positive (PKCδ+) neurons. Deletion of GluD1 impairs excitatory neurotransmission at the PB-CeLC synapses. In inflammatory and neuropathic pain models, GluD1 and its partner cerebellin 1 (Cbln1) are downregulated while AMPA receptor is upregulated. A single infusion of recombinant Cbln1 into the central amygdala led to sustained mitigation of behavioral pain parameters and normalized hyperexcitability of central amygdala neurons. Cbln2 was ineffective under these conditions and the effect of Cbln1 was antagonized by GluD1 ligand D-serine. The behavioral effect of Cbln1 was GluD1-dependent and showed lateralization to the right central amygdala. Selective ablation of GluD1 from the central amygdala or injection of Cbln1 into the central amygdala in normal animals led to changes in averse and fear-learning behaviors. Thus, GluD1-Cbln1 signaling in the central amygdala is a teaching signal for aversive behavior but its sustained dysregulation underlies persistence of pain. Significance statement: Chronic pain is a debilitating condition which involves synaptic dysfunction, but the underlying mechanisms are not fully understood. Our studies identify a novel mechanism involving structural synaptic changes in the amygdala caused by impaired GluD1-Cbln1 signaling in inflammatory and neuropathic pain behaviors. We also identify a novel means to mitigate pain in these conditions using protein therapeutics.

Keywords: CGRP; Cbln1; GluD1; PKCdelta; amygdala; glutamate; pain; parabrachial nucleus.

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Conflict of interest statement

Provisional patent (62963253, Applicant Creighton University, SMD).

Figures

Figure 1
Figure 1
Glutamate receptor delta 1 (GluD1) serves as a critical regulator at parabrachio-amygdalar synapses. (A) Schematic representation of parabrachio-amygdalar pathway. (i) Nociceptive information from periphery reaches laminae I and V spinal dorsal horn neurons. Ascending projections transmit nociceptive information via lateral parabrachial nucleus (PB) to the central amygdala (CeA). (ii and iii) Lateral and capsular divisions of the CeA (CeLC) receive glutamatergic inputs from PB-CGRP+ terminals. GluD1 is localized postsynaptically and Neurexin 1 (Nrxn) is the presynaptic partner of the intermediate protein Cbln1. (B) (i) Immunohistochemical analysis reveals enriched expression of GluD1 in CeLC. (ii) No labeling in GluD1 KO demonstrates antibody specificity. (iii) GluD1 shows perisomatic and punctate labeling on PKCδ+ neurons. (iv) Negligible co-labeling with SOM+ neurons. (v) Quantification of GluD1 colocalization in both neuronal populations reveals 93.49% ± 1.104% were PKCδ+, whereas only 0.788% ± 0.572% were SOM+ (n = 5 mice). (vi) The 3D reconstruction of GluD1 puncta on PKCδ+ neurons. (C) GluD1 localization to PKCδ+ neurons using the PKCδ-CFP-Cre reporter mouse line. (D) Higher colocalization (yellow arrows) of GluD1 with PB marker vGluT2 vs. BLA marker vGluT1 (vGluT1-GluD1 (n = 5 mice): 2.74 ± 0.93; vGluT2-GluD1 (n = 5 mice): 11.16 ± 0.48, **** p < 0.0001, two-tailed unpaired t-test). (E) GluD1 is postsynaptic to CGRP terminals. Confocal analysis and 3D reconstruction reveal close apposition of GluD1 with CGRP. (F) GluD1 and CGRP show colocalization in BNST. Immunohistochemistry for GluD1 and CGRP in BNST demonstrate that CGRP ensheathes perisomatic GluD1 with a basket-like structure, similar to the pattern observed in CeLC. (G) Selective reduction in vGluT2 puncta in CeLC in GluD1 KO mice (Two-way ANOVA, genotype F (1, 12) = 4.774, p = 0.022, vGluT1: WT vs. GluD1 KO: 166.750 ± 21.379 vs. 145.5 ± 22.29; vGluT2: WT vs. GluD1 KO: 239 ± 19.759 vs. 158.25 ± 13.665, Bonferroni’s multiple comparisons test, * p = 0.025, n = 4 mice per group). (H) GluD1 deletion leads to significant reduction in CGRP projection in CeLC. Immunohistochemistry for CGRP in WT and GluD1 KO exhibit significant reduction in CGRP in CeLC, suggesting an impairment in PB inputs. (WT (n = 5 mice) vs. GluD1 KO (n = 4 mice): 218.4 ± 33.0 vs. 91.1 ± 15.9, * p = 0.015, two-tailed unpaired t-test).
Figure 2
Figure 2
GluD1 is critical for regulating excitatory neurotransmission at parabrachio-amygdala synapses. (A) Whole-cell mEPSC recordings at −70mV (in the presence of picrotoxin and tetrodotoxin) from CeC neurons demonstrate significantly lower mEPSC frequency and amplitude in GluD1 KO (WT (n = 8) vs. GluD1 KO (n = 9): Frequency: 6.17 ± 0.83 Hz vs. 2.91 ± 0.96 Hz, * p = 0.022, two-tailed unpaired t-test with Welch’s correction; Amplitude: 16.78 ± 0.869 pA vs. 13.66 ± 1.124 pA, * p = 0.045, unpaired t-test). (B) No change in mIPSC frequency and amplitude in CeC due to deletion of GluD1 (Frequency: p = 0.74, Amplitude: p = 0.77; two-tailed unpaired t-test, n = 8). (C) Whole-cell evoked EPSCs from CeC neurons at -70 mV in the presence of picrotoxin with recording electrode in CeC (yellow asterisk) and stimulating electrode on PB fibers (red asterisk). The input–output curve shows significant reduction in evoked EPSC amplitude in GluD1 KO mice. (Two-way repeated measures ANOVA, genotype F (1, 28) = 23.03, p < 0.0001; Bonferroni’s multiple comparisons test: WT (n = 15) vs. GluD1 KO (n = 25); 800 µA current injection: 48.97 ± 7.39 vs. 17.16 ± 1.43, **** p < 0.0001; 1 mA current injection: 47.00 ± 6.40 vs. 24.46 ± 3.56, **** p < 0.0001).
Figure 3
Figure 3
Inflammatory pain induces changes in GluD1, Cbln1, and GluA1 behavior. (A) GluD1 and PKCδ staining in CeLC was performed at 1 week after intraplantar CFA administration. Three-dimensional reconstruction and volume analysis of GluD1 elements in apposition with PKCδ+ soma was performed. Significant reduction in somatic GluD1 volume specifically in the right CeA in CFA mice (Two-way ANOVA, treatment x side interaction F (1, 17) = 10.03, p = 0.0056, treatment F (1, 17) = 7.08, p = 0.016, Saline (n = 4 mice left and 8 mice right) vs. CFA (n = 4 mice left and 5 mice right): left CeA: 9.71 ± 0.81 vs. 10.30 ± 2.01; right CeA: 12.85 ± 0.97 vs. 5.97 ± 0.56, Bonferroni’s post hoc test, *** p = 0.0005). (B) GluD1 and Cbln1 co-labeling in CeLC performed 1 week after intraplantar CFA administration. A substantial reduction in GluD1-Cbln1 colocalized puncta number in right CeA is observed (Two-way ANOVA, treatment F (1, 12) = 26.65, p = 0.0002, Right CeA: Saline (n = 4 mice): 25.5 ± 3.06 vs. CFA (n = 4 mice): 6.83 ± 0.24, * p = 0.024, Bonferroni’s post hoc test). Reduction in GluD1-Cbln1 colocalized puncta is also observed on left CeA (p = 0.037). (C) GluD1 and PKCδ co-labeling in CeLC performed 4 weeks after sham or SNL surgery in rats along with 3D reconstruction. Significant reduction is observed in perisomatic GluD1 volume in right CeA (Two-way ANOVA, treatment F (1, 12) = 14.66, p = 0.0024, Right CeA: Sham (n = 4 rat): 12.92 ± 2.77 vs. SNL (n = 4 rats): 3.17 ± 0.37, ** p = 0.007, Bonferroni’s post hoc test). No significant change in left CeA GluD1 volume (p = 0.19). (D) GluD1 and AMPA receptor subunit GluA1 co-labeling in CeLC performed 1 week after intraplantar CFA administration. An upregulation of surface GluA1 is observed after CFA treatment (Two-way ANOVA, treatment F (1, 12) = 11.06, p = 0.0060, Saline (n = 4 mice) vs. CFA (n = 4 mice): Right CeA: 110.48 ± 11.00 vs. 168.98 ± 15.44, * p = 0.018 Bonferroni’s post hoc test. No significant difference in left CeA (p = 0.26). (E) Upregulation of GluA1 subunit in CeLC of SNL mice (Two-way ANOVA treatment F (1, 12) = 11.37, p = 0.005, Sham (n = 4 rats) vs. SNL (n = 4 rats): Right CeA: 62.25 ± 5.32 vs. 112.89 ± 21.47, * p = 0.029, with Bonferroni’s multiple comparison’s post hoc test). (F) Chemogenetic modulation of PKCδ neurons using PKCδ-Cre mouse line. Representative image for site verification of DREADD injection. Activation of PKCδ+ neurons in GqDREADD injected normal animals using CNO (Sham-CNO) led to an increase in mechanical sensitivity (One-way repeated measures ANOVA, treatment F (1.50, 7.53) = 64.01, p < 0.0001; Sham-CNO (n = 6 mice); pre-CNO vs. 1 h post-CNO 6.373 ± 0.428 vs. 1.441 ± 0.365, *** p < 0.0001, Tukey’s post hoc test). No change was observed across different time points in the other two groups (n = 4 mice for CFA-vehicle and 4 mice for CFA-CNO). In contrast, in CFA injected mice, inhibition of PKCδ+ neurons in GiDREADD injected animals using CNO (CFA-CNO) significantly rescued mechanical hypersensitivity (One-way repeated measures ANOVA, treatment F (1.03, 4.13) = 12.88, p = 0.0215; CFA-CNO (n = 5 mice); pre-CNO vs. 1 hr post-CNO: 0.591 ± 0.040 vs. 3.289 ± 0.748, * p = 0.0464, Tukey’s post hoc test). No difference was observed across different time points in the other groups (n = 6 mice for CFA-vehicle and 4 mice for Vehicle-CNO).
Figure 4
Figure 4
Recombinant Cbln1 administration into the central amygdala rescues behavioral hypersensitivity in an inflammatory pain model in a GluD1-dependent manner. (A) Experimental interventions. Mice underwent surgery for intra-CeA or intraventricular cannula implantation. After recovery, mechanical sensitivity was tested using von Frey test for paw withdrawal threshold under baseline condition followed by intraplantar injection of CFA. Effect of intra-CeA administration of Cbln1 (250 ng in 0.5 µL per side) was assessed. Paw withdrawal threshold following Cbln1 injection was measured at 6, 24, 48, 72, 96 h, and 1-week timepoints. (B) Alleviation of pain sensitivity after bilateral intra-CeA Cbln1 administration. Paw withdrawal threshold increased after a single Cbln1 injection and lasted up to a week; Two-way repeated measures ANOVA, treatment F (4, 22) = 68.54, p < 0.0001, Bonferroni’s post hoc test, WT-vehicle (n = 6 mice) vs. WT-Cbln1 (n = 7 mice); 6 h, * p = 0.012, 24 h–1 week, **** p < 0.0001. This improvement was not observed in GluD1 KO (GluD1 KO-vehicle (n = 4 mice), GluD1 KO-Cbln1 (n = 6 mice)). Cbln2 was not able to reverse the CFA-induced mechanical hypersensitivity (n = 4 mice). (B’) No change in mechanical threshold in von Frey analysis in control (non-CFA injected) paw. (C) Rescue of inflammatory pain by Cbln1 injection is lateralized. Recombinant Cbln1 first injected into the left CeA did not show any rescue in mechanical hypersensitivity. However, injection into the right CeA was able to rescue mechanical hypersensitivity; Two-way repeated measures ANOVA, treatment F (1, 9) = 44.15, p < 0.0001; Bonferroni’s post hoc test WT CFA-vehicle (n = 4 mice) vs. WT CFA-Cbln1 (n = 7 mice); 48 h and 72 h **** p < 0.0001. (C’) No change in mechanical threshold in von Frey analysis in control (non-CFA injected) paw. (D) D-serine (30 µg in 0.5 µL) opposes the antihyperalgesic effect of recombinant Cbln1 (250 ng in 0.5 µL). Two-way repeated measures ANOVA, treatment F (1, 6) = 5.35, p = 0.060, Bonferroni’s post hoc test WT-Cbln1 (n = 4 mice) vs. WT-Cbln1+D-serine (n = 4 mice); 24 h, ** p = 0.008; 48 h, *** p = 0.0001.
Figure 5
Figure 5
Recombinant Cbln1 administration normalizes synaptic dysfunction in the CeA in inflammatory pain. (A) Intracerebroventricular administration of Cbln1 (1.5 µg in 1.5 µL PBS) 48 h after CFA also attenuated mechanical hypersensitivity for at least one week. Two-way repeated measures ANOVA, F (3, 14) = 12.81, p = 0.0001, Bonferroni’s post hoc test, WT CFA-vehicle (n = 4 mice) vs. WT CFA-Cbln1 (n = 6 mice), ** p = 0.0051, **** p < 0.0001. No effect of Cbln1 was observed in GluD1 KO (n = 4 mice (vehicle), 4 mice (Cbln1)). (B) No change in mechanical threshold in von Frey analysis in control (non-CFA injected) paw. (C) Immunohistochemical analysis of right CeLC for the effect of ICV administration of recombinant Cbln1 on perisomatic GluD1 volume in the CFA pain model. Recombinant Cbln1 restored GluD1 levels in CFA mice compared to mice injected with PBS (One-way ANOVA, treatment F (2, 9) = 14.38, p = 0.009; Bonferroni’s multiple comparison; Saline-vehicle (n = 4 mice): 27.3 ± 1.53, vs. CFA-vehicle (n = 4 mice): 10.87 ± 1.94, ** p = 0.068; CFA-vehicle vs. CFA-Cbln1 (n = 4 mice): 30.3 ± 4.08, ** p = 0.0023). (D) Recombinant Cbln1 normalized surface GluA1 upregulation in the right CeLC in CFA pain model (One-way ANOVA treatment F (2, 9) = 9.23 p = 0.006; Bonferroni’s multiple comparison; Saline-vehicle (n = 4 mice) vs. CFA-vehicle (n = 4 mice): 112.9 ± 7.07 vs. 169.2 ± 6.83, * p = 0.013 and vs. CFA-Cbln1 (n = 4 mice): 114.2 ± 15.4, * p = 0.015).
Figure 6
Figure 6
Recombinant Cbln1 rescues hypersensitivity and averse-affective behaviors in a neuropathic pain model. (A) Schematic representation of SNL model. The left L5 spinal nerve was ligated. Cbln1 (500 ng in 1 µL) or PBS was injected into right CeA of SNL rat. (B) Intra-CeA Cbln1 showed reduction in noxious stimuli induced audible vocalization in SNL rats; n = Sham-vehicle (6 mice), Sham-Cbln1 (5 mice), SNL-vehicle (7 mice), SNL-Cbln1 (7 mice) (Two-way ANOVA treatment F (3, 103) = 120, p < 0.0001; Bonferroni’s multiple comparison test, SNL-vehicle vs. SNL-Cbln1: Day 1: 6.948 ± 0.793 vs. 4.273 ± 0.410, ** p = 0.0012; Day 2: 6.711 ± 0.796 vs. 4.076 ± 0.299, ** p = 0.0015; Day 3: 6.853 ± 0.606 vs. 4.226 ± 0.251, ** p = 0.0016; Day 7: 6.973 ± 0.712 vs. 4.604 ± 0.202, * p = 0.0126). (B’) Injection of recombinant Cbln1 into the right CeA reduced audible vocalization to innocuous stimuli in SNL rats (Two-way ANOVA treatment F (3, 103) = 204.5, p < 0.0001; Bonferroni’s multiple comparison test, SNL-vehicle vs. SNL-Cbln1: Day 2: 2.130 ± 0.171 vs. 1.497 ± 0.068, * p = 0.0284; Day 7: 2.258 ± 0.191 vs. 1.343 ± 0.145, ** p = 0.0015; n = 5–7 rats per group). (C) Intra-CeA Cbln1 reduced ultrasonic vocalization in SNL rats (Two-way ANOVA treatment F (3, 103) = 119.3, p < 0.0001; Bonferroni’s multiple comparisons test, SNL-vehicle vs. SNL-Cbln1: Day 2: 5.845 ± 0.765 vs. 3.974 ± 0.351, * p = 0.0323; Day 3: 6.213 ± 0.868 vs. 3.907 ± 0.199, ** p = 0.0041; Day 7: 6.221 ± 0.709 vs. 4.211 ± 0.212, * p = 0.0340; n = 5–7 rats per group). (C’) Ultrasonic vocalization to innocuous stimuli (Two-way ANOVA treatment F (3, 103) = 172.1, p < 0.0001; Bonferroni’s multiple comparison test, SNL-vehicle vs. SNL-Cbln1: Day 3: 2.070 ± 0.226 vs. 1.515 ± 0.073, * p = 0.0408; Day 7: 1.968 ± 0.143 vs. 1.333 ± 0.115, * p = 0.0256; n = 5–7 rats per group). (D) Reduction in mechanical thresholds in the von Frey test were mitigated by intra-CeA Cbln1 (Two-way ANOVA treatment F (3, 103) = 147.1, p < 0.0001; Bonferroni’s multiple comparison test, SNL-vehicle vs. SNL-Cbln1: Day 2: 3.781 ± 0.615 vs. 7.001 ± 1.111, * p = 0.0498; n = 5–7 rats per group). (E) Open arm entries in the elevated plus maze test were also increased by Cbln1 in SNL rats suggesting anxiolytic effect in neuropathic pain (Two-way ANOVA treatment F (3, 40) = 18.36, p < 0.0001; Bonferroni’s multiple comparison test, SNL-vehicle vs. SNL-Cbln1: Day 7: 14.078 ± 4.842 vs. 31.997 ± 6.443, * p = 0.0399; n = 5–7 rats per group). (F) Recombinant Cbln1 restored downregulated GluD1 in CeLC in SNL rats. Immunohistochemical analysis of perisomatic GluD1 levels in right CeA was performed in tissue from SNL rats injected with PBS or recombinant Cbln1. Recombinant Cbln1 restored GluD1 levels in SNL rats to the levels of sham rats. (One-way ANOVA F (2, 9) = 7.49, p = 0.012; Tukey’s post hoc test, Sham-vehicle (n = 4 rats): 9.54 ± 1.57 vs. SNL-vehicle (n = 4 rats): 4.53 ± 1.08, * p = 0.035; SNL-Vehicle vs. SNL-Cbln1 (n = 4 rats): 10.51 ± 0.69; * p = 0.014). (G) Recombinant Cbln1 normalized surface GluA1 levels in SNL rats. Immunohistochemical analysis was performed in SNL rats injected with either PBS or recombinant Cbln1 for AMPA levels. Cbln1 injection reduced the upregulated GluA1 expression in SNL rats. SNL (One-way ANOVA F (2, 8) = 27.17, p = 0.0003; Sham-vehicle (n = 4 rats): 58.35 ± 7.51 vs. SNL-vehicle (n = 4 rats): 110.92 ± 3.70, **** p < 0.0001; SNL-vehicle vs. SNL-Cbln1 (n = 4 rats): 91.48 ± 2.40, * p = 0.049).
Figure 7
Figure 7
Recombinant Cbln1 rescues hyperexcitability of CeC neurons in the neuropathic pain model. (A) Recombinant Cbln1 reduced hyperexcitability (F-I relation) in CeC neurons in brain slices from SNL rats. Current clamp recording from CeC neurons with membrane potential set at -60 mV. Current injections were performed to generate action potentials. SNL increased the excitability and Cbln1 was able to rescue hyperexcitability in SNL model (Two-way repeated measures ANOVA, treatment F (3, 34) = 5.6, p = 0.003, Tukey’s multiple comparison, SNL-vehicle (n = 11 neurons from 5 rats) vs. Sham-vehicle (n = 9 neurons from 4 rats): 80 pA p = 0.026, 100 pA p = 0.0045, 120 pA p = 0.0011, 140 pA p = 0.0005, 160 pA p = 0.0005, 180 pA p = 0.0009, 200 pA p = 0.0005, 220 pA p = 0.0014, 240 pA p = 0.0013, 260 pA p = 0.0027; SNL-vehicle vs. SNL-Cbln1 (n = 9 neurons from 5 rats): 200 pA p = 0.039, 220 pA p = 0.037, 240 pA p = 0.0025, 260 pA p = 0.0007; Sham-Cbln1 (n = 9 neurons from 5 rats) vs. SNL-vehicle: 200 pA p = 0.031, 220 pA p = 0.037, 240 pA p = 0.034, 260 pA p = 0.021). (B) Excitability of individual cells in various Sham and SNL, control and Cbln1 groups. Note the reduced variance of individual cell excitability in SNL rats compared to other groups. (C) Property of CeC neurons under different conditions. A change in rheobase was observed by Cbln1 administration in Sham animals as well as due to SNL. One-way ANOVA F (3, 35) = 7.14, p = 0.0007; Tukey’s multiple comparison, Sham-vehicle (n = 9 neurons from 4 rats) vs. Sham-Cbln1 (n = 10 neurons from 5 rats), * p = 0.0231, Sham-vehicle vs. SNL-vehicle (n = 11 neurons from 5 rats), *** p = 0.0006, Sham-vehicle vs. SNL-Cbln1 (n = 9 neurons from 5 rats), ** p = 0.0053.
Figure 8
Figure 8
Recombinant Cbln1 in CeA increases mechanical hypersensitivity in normal animals. (A) Local deletion of GluD1 in the CeA using AAV constructs. Conditional deletion of GluD1 from CeA was achieved using Cre-lox strategy. Mice were injected with with AAV9-hsyn-eGFP or AAV9-hsyn-eGFP-Cre, bilaterally into CeA. Deletion of GluD1 from CeA was confirmed by immunohistochemistry. (B) Deletion of GluD1 from the CeA does not lead to significant change in mechanical hypersensitivity. (C) Impaired averse fear learning in mouse with GluD1 ablation in the CeA. Significant deficits in fear acquisition at 4th CS US (Two-way repeated measures ANOVA, F (1, 10) = 6.31, p = 0.031; Bonferroni’s post hoc test, 4th CS-US: AAV-control (n = 7 mice) vs. AAV-Cre (n = 5 mice): 54.764 ± 4.765 vs. 26.66 ± 6.665, *** p = 0.0008) as well as in retrieval 24 h later (AAV-control vs. AAV-Cre: 40.47 ± 5.282 vs. 11.46 ± 4.619, ** p < 0.005, Unpaired t-test with Welch correction). (D) Mice underwent cannulation surgery for the implantation of bilateral CeA cannulas. Effect of recombinant Cbln1 (250 ng in 0.5 µL) injected into CeA of naïve mice. After measuring basal mechanical hypersensitivity, Cbln1 was administered intracranially into the CeA and mechanical sensitivity (von Frey test) was measured in the right hind paw at 3 h, 6 h, 24 h, and 1 week. Significant increase in mechanical sensitivity is observed from 3 h to 24 h. (Two-way repeated measures ANOVA, treatment F (3, 15) = 8.77, p = 0.0009; Bonferroni’s post hoc test, WT-vehicle (n = 4 mice) vs. WT- Cbln1 (n = 5 mice): 3 h, 5.371 ± 0.223 vs. 1.553 ± 0.110, **** p < 0.0001; 6 h, 5.549 ± 0.253 vs. 1.418 ± 0.114, **** p < 0.0001; 24 h, 6.347 ± 0.341 vs. 2.088 ± 0.217, **** p < 0.0001). No change was seen in GluD1 KO mice after Cbln1 administration (n = vehicle (5 mice), Cbln1 (5 mice)). (E) Recombinant Cbln1 led to a significant reduction of perisomatic GluD1 at 6 h timepoint predominantly in right CeA compared to vehicle (PBS) injected mice. (Two-way ANOVA treatment F (1, 10) = 19.45, p = 0.0013; Bonferroni’s post hoc test, WT-vehicle (3 mice) vs. WT-Cbln1 (4 mice): Right CeA: 29.74 ± 3.08 vs. 8.72 ± 1.57, ** p = 0.0048; Left CeA: 24.31 ± 3.91 vs. 12.82 ± 4.89, p = 0.10).
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