Combined Experiments with in Vivo Fiber Photometry and Behavior Tests Can Facilitate the Measurement of Neuronal Activity in the Primary Somatosensory Cortex and Hyperalgesia in an Inflammatory Pain Mice Model

Abstract

The pain matrix, which includes several brain regions that respond to pain sensation, contribute to the development of chronic pain. Thus, it is essential to understand the mechanism of causing chronic pain in the pain matrix such as anterior cingulate (ACC), or primary somatosensory (S1) cortex. Recently, combined experiment with the behavior tests and in vivo calcium imaging using fiber photometry revealed the interaction between the neuronal function in deep brain regions of the pain matrix including ACC and the phenotype of chronic pain. However, it remains unclear whether this combined experiment can identify the interaction between neuronal activity in S1, which receive pain sensation, and pain behaviors such as hyperalgesia or allodynia. In this study, to examine whether the interaction between change of neuronal activity in S1 and hyperalgesia in hind paw before and after causing inflammatory pain was detected from same animal, the combined experiment of in vivo fiber photometry system and von Frey hairs test was applied. This combined experiment detected that amplitude of calcium responses in S1 neurons increased and the mechanical threshold of hind paw decreased from same animals which have an inflammatory pain. Moreover, we found that the values between amplitude of calcium responses and mechanical thresholds were shifted to negative correlation after causing inflammatory pain. Thus, the combined experiment with fiber photometry and the behavior tests has a possibility that can simultaneously consider the interaction between neuronal activity in pain matrix and pain induced behaviors and the effects of analgesics or pain treatments.

INTRODUCTION

Pain sensation plays an important role in defense against threats to homeostasis and survival.¹⁾ By contrast, chronic pain, which is characterized by prolonged pain sensation, is caused by neuropathic or inflammatory changes. The QOL of patients is affected by chronic pain, which leads to mood disorders such as anxiety and depression.²⁾ Thus, chronic pain is still challenging to treat clinically. Traditional analgesics were used in patients with chronic pain. However, these medications commonly have limited effects against chronic pain symptoms. Previous studies on humans and rodents have reported that several brain regions, such as the anterior cingulate cortex (ACC), insular cortex, and primary somatosensory cortex (S1), which respond to pain stimulus, are activated under chronic pain conditions.^3,4) Therefore, it is essential to understand the mechanism of chronic pain in the brain that receives the sensory component of pain and contributes to mood disorders.

Several studies using in vivo two-photon imaging revealed that change of neuronal circuits which caused by both activation of excitatory neurons and astrocytes develop a chronic pain.^5–8) Moreover, the excitatory neurons in S1 cortex indicated that the evoked calcium responses for pressure stimulation into hind paw or spontaneous calcium transients were increased by sciatic nerve injury or administration of complete Freund’s adjuvant (CFA) which induced an inflammatory response into hind paw.^6,9–11) Thus, the mechanism of chronic pain induction in the S1 cortex is important.

Recently, to identify the involvement of neuronal activities in the pain matrix in the development of chronic pain symptoms such as the sensory and affective components of pain, combined experiment with the behavior tests and in vivo calcium imaging using fiber photometry was applied.^12–16) The effects of drugs such as dexmedetomidine and duloxetine into ACC in chronic or inflammatory pain model mice with expressed GCaMP were measured using this combined experiment. As the results, administration of abovementioned drugs reduced the calcium responses of glutamatergic neurons in ACC and showed the anti-anxiety like behaviors.^12,14) Moreover, other previous studies also reported that the change of neuronal responses in parabrachial nucleus, ACC, or basolateral amygdala could be monitored using in vivo fiber photometry system in several pain model mice.^13,15,16) Thus, these combined experiment of this optical system and behavior tests seemed to be useful to understand the mechanism of causing chronic pain or to examine the effects of analgesics or pain treatments.

However, it remains unclear whether the abovementioned combined experiments can identify the interaction of neuronal activities in S1, which is a surface region of the pain matrix and pain behaviors. The current study used the combined method of fiber photometry and behavioral tests and examined the efficacy of this strategy in identifying the interaction between neuronal activity in S1 and the symptoms of chronic pain.

MATERIALS AND METHODS

Mice

All animal experiments were conducted based on the Guidelines for Animal Experimentation in Neuroscience of the Japan Neuroscience Society and were approved by the Experimental Animal Research Committee of Kanazawa University (#AP-183970). Effort was made to decrease the number of animals used and their suffering. The current study used 8–12-week-old adult male C57BL/6 mice (Sankyo Labo Service Corporation, Inc., Tokyo, Japan). All mice were group-housed (4 or 5 mice per KN-600 cage; cage dimensions [width × length × height]: 220 × 320 × 135 mm; Natsume Seisakusho Co., Ltd., Tokyo, Japan) under standard laboratory conditions (temperature: 23 ± 1 °C, relative humidity: 55 ± 5%, and a 12/12-h light/dark cycle) with access to tap water and food ad libitum.

Animal Model

Inflammatory pain was induced by subcutaneous injection of CFA into plantar surface of the right hind paw (50%, 10 µL; CFA, Sigma-Aldrich, St. Louis, MO, U.S.A.) under anesthesia with isoflurane.¹⁷⁾ Mice which were received saline into hind paw were as a control. On 3, 7, and 14 d after CFA or saline injection, in vivo calcium imaging using fiber photometry and the von Frey hairs test were conducted.

Virus Injection for in Vivo Fiber Photometry Calcium Imaging

The mice were anesthetized via the intraperitoneal injections of combined anesthetics (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol).¹⁸⁾ Craniotomy was performed above the hind paw area of the contralateral S1 (0.5-mm posterior and 1.5-mm lateral to the bregma). After craniotomy, the adeno-associated virus (AAV) encoding the synapsin promoter-driven calcium indicator protein GCaMP7f (pGP-AAV9-syn-jGCaMP7f-WPRE, 1 × 10¹³ vg/mL; Addgene #104488)¹⁹⁾ used for fiber photometry calcium imaging was pressure-injected at 300–500 µm from the cortical surface. pGP-AAV-syn-jGCaMP7f-WPRE was a gift from Dr. Douglas Kim. After virus injection, the cranial window was sealed with the skull, which was fixed using dental cement (Super Bond, Sun Medical, Shiga, Japan) and adhesive glue (Aron α, Toagosei, Tokyo, Japan). The fiber optic cannula (RWD Life Science Co., Ltd., Guangdong, Chain) was implanted 500 µm above the place and fixed on the skull by dental cement. Calcium imaging was conducted 3–4 weeks after surgery and on 3, 7, and 14 d after the administration of CFA using the fiber photometry system (as shown in “Fiber Photometry”) with stimulus (0.4-g von Fray filament).

Behavior Test Acclimation

Each mouse was acclimated to the testing room for at least 30 min before each behavior test.

von Frey Hairs Test

The mechanical thresholds of the right hind paw were measured by von Frey hairs test before CFA or saline injection and on 3, 7, or 14 d after the administration of these solutions. The mice were habituated for 30 min in a transparent box with a mesh floor. Six calibrated von Frey hairs (0.16-, 0.4-, 0.6-, 1.0-, 1.4-, and 2.0-g bending forces) were pressed perpendicularly against the plantar surface of the hind paw until slight buckling occurred to assess tactile allodynia or hyperalgesia. The experiment started by testing the response to the 1.0-g filament. If the mice had a paw withdrawal response, the filament with the next lowest force was tested. If the mice did not have withdrawal response to the 1.0-g filament, the filament with the next highest force threshold was used. These tests were conducted during daytime, and the trial ended when four stimuli were applied after the first positive response.

Open-Field Test (OFT)

The mice were placed in an open-field arena (45 × 45 × 40 cm) with dim illumination (approximately 20 lux), and the time spent in the center zone and total distance traveled were recorded for 10 min using a web camera (C270n, Logitech International, San Jose, CA, U.S.A.). These variables were analyzed using ImageOF (https://cbsn.neuroinf.jp/database/item/id/ImageOF).^20,21)

Elevated Plus Maze (EPM) Test

The maze was made of plywood, and it included two open arms and two identical closed arms (10 × 37.5 cm for each arm; a wall height of 15 cm for closed arms). It was placed 60 cm above the floor in a dimly illuminated room (approximately 30 lux). Each mouse was placed in the center area with its head toward one open arm, and it was allowed to explore the maze for 5 min while being recorded using a web camera. The time spent in the open arms and the number of entry times into the open and closed arms were measured and analyzed using ImageEP (https://cbsn.neuroinf.jp/database/item/id/ImageEP).^20,21) After each exploration, the OFT and EPM apparatus were cleaned using 70% ethanol.

The ImageJ (https://imagej.nih.gov/ij/) plugins and precompiled plugins for the OFT (Image OF) and the EPM (Image EP) are freely available on the Mouse Phenotype Database website (http://www.mousephenotype.org/software.html).^20,21)

Fiber Photometry

The fiber photometry system designed by Doric was used to detect the fluorescence of genetically encoded biosensor. The mice implanted with a mono fiber optic cannula (200 µm, 0.5 NA, RWD Life Science Co., Ltd,., Guangdong, China) was attached to the optical patch cords (200 mm, 0.48 NA, 1 m long; Doric, QC, Canada) via zirconia connectors (Sleeve_ZR_2.5_BK, Doric). The excitation wavelength for GCaMP7f as 465-nm blue light or the control wavelength for calcium-independent GCaMP7f fluorescence as 405-nm violet light was delivered via the patch cord. Excitation and control lights were generated from light-emitting diodes (LEDs; CLED_405, CLED_465, Doric). The fluorescence signal was detected using Fluorescence Mini Cube{FMC4-G2_IE(400-410)_E(460-490)_F(500-550)_S, Doric}. Then, the biosensor signal was converted to the electrical signal by an AD converter (Micro 1401 mkII, Cambridge Electronic Design Ltd., Cambridge, U.K.).

Analysis of Calcium Responses in S1 Neurons

To analyze calcium responses in S1 neurons, which expressed GCaMP7f evoked by stimulation with von Frey filament into the hind paw, the baseline intensity F₀ was obtained by measuring the average intensity values during the pre-stimulus period (2 s). The amplitude of GCaMP7f signals was calculated as ΔF/F₀ (ΔF = F − F₀). To calculate quantitative parameters such as amplitude and area under the curve (AUC) of calcium response, the average fluorescent data of 10 times measurements were used. The amplitude of calcium response was defined as the peak value of the average fluorescent data. The AUC was calculated using GraphPad Prism 9 (GraphPad software, Boston, MA, U.S.A.), with the averaged fluorescent data obtained immediately (von Frey filament) and up to 6 s. after stimulation.

The Labeling of Nuclei

To confirm the expression pattern of calcium sensor and location of cannula in S1 cortex, deeply anesthetized GCaMP7f expressed mice were transcardially perfused with 4% paraformaldehyde in 0.1-M phosphate buffer (PB), after finishing in vivo fiber photometry imaging study. Brains were excised and postfixed overnight with the same fixative. The tissues were cryoprotected with 30% sucrose prepared in 0.1-M PB at 4 °C and were rapidly frozen with Tissue-Tek O.C.T. Compound (Sakura Finetek Japan, Tokyo, Japan). The tissues were finally stored at − 80 °C. Brain sections (50 mm) were cut using a cryostat (Leica CM1950, Leica Biosystems Nussloch GmbH, Nussloch, Germany) and processed for staining nuclei of cells in brain slices using 4′,6-diamidino-2-phenylindole (DAPI, Nacalai Tesque, Kyoto, Japan) diluted in 0.1 M PB (2 mg/mL) for 5 min. After washing in 0.1 M PB, sections were mounted in VECTASHIELD mounting medium (Vector Laboratories, CA, U.S.A.). These slices were subsequently imaged under a fluorescence microscope (BZ-X700, Keyence, Osaka, Japan).

Statistical Analysis

Statistical analyses were conducted using the Statistical Package for the Social Sciences software (IBM, Armonk, NY, U.S.A.) or GraphPad Prism 9 (GraphPad software, Boston, MA, U.S.A.). Data with two variables were analyzed statistically using unpaired t-tests. Meanwhile, data with three or more variables were analyzed using one-way ANOVA with the post-hoc Bonferroni’s multiple comparison tests. Behavioral data on changes in the mechanical threshold of the right hind paw were analyzed using two-way repeated-measures ANOVA, followed by the Bonferroni’s multiple comparison tests. The correlation coefficient between the mean value of neuronal calcium responses in S1 and mechanical threshold of hind paw, or anxiety like behaviors were calculated by Spearman’s rank-order correlation or Pearson correlation coefficient. In all cases, p values of < 0.05 were considered statistically significant. The exact details on the number of mice in this study are provided in the results of each experiment.

RESULTS

The Mice with Inflammatory Pain Presented with Hyperalgesia and They Were More Likely to Indicate Anxiety-Like Behaviors

To prepare the mice that will be included in the combination experiments with in vivo calcium imaging and measurement of mechanical thresholds into hind paw, adeno-associated virus (AAV9-hsyn-GCaMP7f) were injected into the left S1 cortex. Three weeks after viral injection, the mechanical thresholds in the right hind paw were measured using the von Frey hairs test before CFA was administered. On 3, 7, and 14 d after administering the inflammatory substance, the mechanical threshold in the CFA-injected hind paw significantly decreased compared with that in the saline-injected hind paw (Figs. 1A, B), similar to previous studies.^17,22)

Fig. 1. Mice with Inflammatory Pain Presented with Hyperalgesia and Had a Tendency of Pain-Related Anxiety-Like Behaviors

(A) The time schedule of the combined experiments with several behavior tests and fiber photometry in mice with inflammatory pain in which the calcium sensor was expressed in S1. FP = fiber photometry, VF = von Frey hairs test, OFT = open-field test, EPM = elevated plus maze test. (B) Changes in mechanical thresholds in the right hind paw after subcutaneous injection of CFA. *** p < 0.0001 by two-way repeated-measures ANOVA, followed by the Bonferroni’s test, F_{(3, 27)} = 10.59, *** p < 0.0001. n = 5–6 per group. Values were presented as mean ± S.E.M. (C) Typical tracks from each saline- or CFA-injected mouse in the OFT and EPM test. Red-dotted squares on the upper images represent the center region. Meanwhile, the black- and red-dotted bars on the lower images indicated the closed and open arms, respectively. (D) The results of OFT were shown in D. The time spent in the center region (left panel) and the total traveled distance (right panel) in saline- or CFA-injected mice. Values were presented as mean ± S.E.M. (E) Fig. 1E indicated the results of EPM. The duration of the open arms (left panel), and the number of entry into the open arms (middle panel). The number of entering closed arms between saline- and CFA- injected mice (right panel). * p < 0.05, unpaired t-test.

A previous study revealed that the mice induced with inflammatory pain presented with anxiety.¹⁷⁾ To examine whether the mice in this study also exhibited anxiety-like behaviors, routine assays (i.e., OFT and EPM) were performed on mice on 14 d after the subcutaneous injection of CFA or saline (Fig. 1C). Although the CFA-injected mice did not spend significantly less time in the center region of the open field, this group had a lower time in the open arms (Figs. 1D, E; Results of OFT: saline group: 49.7 ± 13.0 s; CFA group: 29.6 ± 2.1 s, Results of EPM: saline group: 87.2 ± 21.5 s; CFA group: 12.2 ± 4.6 s, n = 6–5 per group, the unpaired t-test, p < 0.05 for the saline group vs. the CFA group). Data were presented as mean ± standard error of the mean (S.E.M.). Therefore, based on the behavioral test results, the mice had induced hyperalgesia and were more likely to present with anxiety-like behaviors.

The Fiber Photometry System Facilitated the Measurement of Calcium Responses in S1 Neurons for Mechanical Stimulus into the Hind Paw

To assess whether the calcium responses could be monitored in S1 neurons for stimulation with 0.4-g von Frey filaments or pinch stimulation to produce pain sensation with tweezers, in vivo fiber photometry recording was performed in mice injected with AAV-hsyn-GCaMP7f (Fig. 2A). The virus was locally injected into the hind paw region of the left S1 cortex, and the calcium sensor was expressed in neurons in layers 2/3 and 5 on 3 weeks after viral administration (Fig. 2B). To estimate moving artifacts, the filtered 405-nm signals were measured via stimulation with von Frey filaments. The changes in calcium signals were not observed after filament stimulation into the hind paw, thereby indicating that moving artifacts were limited in the in vivo fiber photometry system (Fig. 2C left). The fiber photometry system was used to record the calcium responses of S1 neurons with von Frey filament stimulation or pinch stimulations. Hence, the response patterns were likely to differ between each stimulation (Fig. 2C middle, right).

Fig. 2. The Fiber Photometry System Could Monitor Calcium Response for Normal- and Pain-Sensation in the S1 Neurons

(A) A schematic diagram of the in vivo fiber photometry system. (B) The typical images of GCaMP7f viral expression in S1 neurons. (C) Representative traces of calcium responses with von Frey filament stimulation under excitation at 405 nm (left). The calcium responses for von Frey filament (middle) or pinch stimulation in the hind paw (right). (D) Typical traces of calcium responses and heat maps with the von Frey filament before and after saline or CFA administration. The gray lines mean each value of calcium response for stimulation of von Frey filament. The black lines indicate averaged trace of each calcium response.

Next, to examine whether inflammatory pain induces change in the neuronal responses in S1, the calcium transient was monitored using fiber photometry before and after the administration of CFA into the hind paw. The enhancement of calcium responses to 0.4-g filament stimulation was not monitored 3 d after the administration of saline. However, the CFA-injected mice presented with enhanced calcium responses (Fig. 2D). The results obtained using the fiber photometry system were similar to those obtained with in vivo two-photon imaging.^9,10) Therefore, fiber photometry could monitor calcium transients.

Long-Term Repeated Calcium Imaging Revealed That Neuronal Calcium Responses in S1 Increased after the Administration of CFA into the Hind Paw

We performed repeated measurement of calcium responses in S1 before and on 3, 7, and 14 d after CFA injection (Fig. 3A). The average amplitude of calcium responses significantly increased on 3 and 7 d after the subcutaneous injection of CFA compared with that before administration (Fig. 3B; before saline injection, 0.030 ± 0.008; 3 d after saline injection, 0.024 ± 0.007; 7 d after saline injection, 0.033 ± 0.008; 14 d after saline injection, 0.028 ± 0.008; before CFA injection, 0.027 ± 0.009; 3 d after CFA injection, 0.052 ± 0.003; 7 d after CFA injection, 0.055 ± 0.004; 14 d after CFA injection, 0.045 ± 0.004; n = 6–5 per group, one-way ANOVA with the post-hoc Bonferroni’s test, F_{(3, 16)} = 4.53, p = 0.02: p < 0.05 before CFA injection vs. 3 or 7 d after CFA injection). Data were presented as mean ± S.E.M. On 3 d after the injection of each substance, the average amplitude of neuronal responses in CFA-injected mice was significantly higher than that in saline-injected mice (Fig. 3B; two-way RM ANOVA with the post-hoc Bonferroni’s test, F_{(3, 27)} = 3.62, p = 0.03: p < 0.05 3 d after saline injection vs. 3 d after CFA injection). By contrast, the AUC did not significantly differ between saline-injected mice and CFA-injected mice (Fig. 3C; one-way ANOVA with the post-hoc Bonferroni’s test, F_{(3, 16)} = 1.45, p = 0.26). Therefore, long-term repeated calcium imaging using fiber photometry can monitor neuronal responses in S1 before and after developing inflammatory pain.

Fig. 3. The Enhancement of Calcium Response Was Monitored on 3, 7, and 14 d after CFA Administration Using Fiber Photometry

(A) Representative traces of calcium responses for stimulation with the von Frey filament before and 3, 7, or 14 d after saline (left) or CFA (right) administration. Gray traces indicate the mean value of calcium responses by stimulus of von Frey filament in each mouse which were administrated by saline or CFA. The black lines show the mean value of averaged responses in each mouse (gray lines). (B) The average amplitude of calcium responses for stimulation of von Frey filament into hind paw. ^# p < 0.05 by two-way repeated ANOVA, followed by the Bonferroni post-hoc tests, F_{(3, 27)} = 3.62, n = 5–6 mice per group. (C) The average area under curve (AUC) of calcium responses before and after administration of saline or CFA.

The in Vivo Fiber Photometry System Could Identify the Interaction between Neuronal Activity in S1 and Pain-Related Behaviors

The association between calcium signals in S1 neurons for stimulation with von Frey filament and pain-related behaviors in saline- or CFA-injected mice was compared. The scatterplot between the amplitude of calcium response and mechanical thresholds had a similar distribution before saline or CFA injection. By contrast, these scatterplots on 3, 7, or 14 d after the administration of CFA into the hind paw were clustered, and their distribution changed. Moreover, the scatter plots in 7 or 14 d after administration of CFA had a possibility to tend a negative correlation between neuronal calcium responses for von Frey filament stimulation and mechanical thresholds of hind paw which was CFA injected. (Fig. 4A; Pearson correlation coefficient for before saline injection, 3, 7 d after injection of saline, 3, 7, or 14 d after injection of CFA; Spearman’s rank-order correlation for before CFA injection or 14 d after saline injection). Next, the association between calcium response in S1 and pain-related anxiety-like behaviors was evaluated. However, the scatterplots did not show a particular distribution (Fig. 4B). The interaction between averaged amplitude of calcium responses 14 d after CFA or saline injection and duration in center region in OFT was calculated by Pearson correlation coefficient or Spearman’s rank-order correlation. The interaction between averaged amplitude of calcium responses 14 d after CFA or saline administration and duration in open arms in EPM was also evaluated by Pearson correlation coefficient.

Fig. 4. The Combined Experiment with Fiber Photometry and Behavior Tests Can Consider the Interaction between Neuronal Activity in S1 and the Mechanical Thresholds of the Hind Paw

(A) The scatter plots between the amplitude of calcium responses and the mechanical thresholds of the hind paw before and after saline or CFA administration (Spearman’s rank-order correlation or Pearson correlation coefficient). (B) The scatter plots for the association between the amplitude of calcium responses and anxiety-like behaviors on 14 d after saline or CFA administration (Spearman’s rank-order correlation or Pearson correlation coefficient).

DISCUSSION

In the current study, the fiber photometry system could detect neuronal responses in the S1 cortex with both light touch and pain stimulation from the periphery. Moreover, in vivo long-term calcium imaging using the abovementioned system could identify changes in neuronal calcium responses before and after developing inflammatory pain in the same animal. This system could reveal that the average amplitude of calcium responses inS1 on 3 d after CFA administration was significantly increased compared with saline injected mice group. Although no significant different in average amplitude of calcium responses were observed in mice which were on 14 d after injection of CFA compared with before injection of CFA and 14 d after administration of saline, our study also indicated that the neuronal responses showed the tendency of higher amplitude. Moreover, to measure the calcium responses more accurately on 14 d after administration of CFA, we need to decide the serotype or concentration of AAV which can express more stronger than its in this study, or to change more sensitive GCaMP-type calcium indicator such as jGCaMP8.²³⁾ In this study, to measure the neuronal activities in S1 via calcium sensor, we applied the AAV encoding hSyn promoter which express GCaMP into S1 cortex. However, it had a possibility that this AAV which can express the GCaMP in both excitatory and inhibitory neurons was difficult to exactly detect the pain-induced neuronal activities in S1 cortex. Thus, to monitor the calcium transients of excitatory or inhibitory neurons in S1 separately, we should change from used AAV in this study to AAVs which have other promoters such as calcium/calmodulin-dependent protein kinase II (CaMKII) or glutamic acid decarboxylase-65 (GAD65) promoter and consider the suitable AAV to monitor the neuronal activity using fiber photometry.

A previous study revealed that fiber photometry could record non-somatic calcium changes in structures such as dendrites and the dendritic spines.²⁴⁾ Moreover, in vivo two-photon calcium imaging showed that the calcium transients of these small structures significantly increased in mice with chronic pain.^25,26) Hence, based on our results, fiber photometry could also reflect changes in neuronal function during inflammatory pain.

Previous studies have reported that the in vivo fiber photometry system could monitor the deep brain regions such as the ACC and prefrontal cortex, which comprise the pain matrix.^12–16) The results of these studies suggested that combination experiments of in vivo fiber photometry and behavior tests enable us to understand the interaction between changes of neuronal activities in pain matrix which contribute to affective component of pain and pain modulation, pain induced anxiety, or cognition of pain. On the other hand, we also monitored changes of calcium responses in the S1 cortex before and after administration of CFA, resulting that averaged amplitude of calcium responses in S1 were significantly increased 3 or 7 d after injection of CFA, in present study. Thus, the fiber photometry system could measure neuronal function not only in the deep brain regions of the pain matrix but also the superficial region in vivo. Moreover, our study revealed that combination experiment of in vivo calcium imaging using fiber photometry and von Frey hairs test examine about changes of neuronal responses in S1 which mainly detects sensory component of pain from periphery, and transition of mechanical thresholds of hind paw, before and after causing inflammatory pain. The results of previous reports including our studies suggest that plastic changes in S1 cortex contributed to chronic pain or inflammatory pain.^{5–11,25,27)} Therefore, to investigate the interaction between neuronal activity in S1 and change of mechanical threshold of hind paw by above combined experiment may play important role to further understand the mechanism of causing chronic pain.

By contrast, this experimental method has limitations compared with in vivo multiphoton imaging. For example, to detect the calcium signals in the fiber photometry system, the fiber canula should be placed into the brain. Canula placement is associated with greater tissue damage and a risk of losing brain function. Multiphoton calcium imaging can measure the calcium signals at single cell resolution. Nevertheless, the fiber photometry system had a lower spatial resolution, and each neuronal calcium signal is challenging to monitor.

In conclusion, the combination experiments with in vivo fiber photometry system and several behavior tests have a possibility to identify the interaction between neuronal activity in pain matrix such as ACC or S1 and pain induced behaviors including emotion or sensation of pain, using scatter plots. Moreover, the combined experiment with fiber photometry and the behavior tests can simultaneously consider the effects of analgesics or pain treatments.

Acknowledgments

We thank Dr. Douglas Kim for generous gifts of plasmids for the AAV vector. We also thank Hiroshi Saitou (Technical Support Center in Kanazawa University), Tsuneo Nakamura (Engineering and Technology Department in Kanazawa University), Yoshitake Shiraishi (Engineering and Technology Department in Kanazawa University), and Rumi Shima for their invaluable suggestions and skilled technical support. This work was funded by the Nakatomi Foundation, the Kanazawa University CHOZEN project, JSPS KAKENHI Grant Number 19K16909, 23K08377 (to T.I.), 19K09323 and 22K09020 (to D.U.), and Research for collaboration with universities in Hokuriku 2021, 2022, 2023 (to T.I. and D.U.).

Conflict of Interest

The authors declare no conflict of interest.

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