Tac1-expressing neurons in the central amygdala predominantly mediate histamine-induced itch by receiving inputs from parabrachial Tac1-expressing neurons

doi:10.21203/rs.3.rs-3939709/v1

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Tac1-expressing neurons in the central amygdala predominantly mediate histamine-induced itch by receiving inputs from parabrachial Tac1-expressing neurons

https://doi.org/10.21203/rs.3.rs-3939709/v1

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Itch is a distinct and bothersome sensation closely associated with a strong urge to scratch. Both the parabrachial nucleus (PBN) and the central amygdala (CeA) are responsive to itch stimuli and contain neurons that express tachykinin 1 (Tac1), which are known for their significant involvement in itch-induced scratching at both spinal and supraspinal levels. Significantly, the PBN neurons project their axons to form close connections with the CeA neurons. However, the role of the PBN^Tac1-CeA^Tac1 pathway in modulating itch remains to be determined. We utilized immunohistochemistry, fiber photometry, chemogenetic, and behavioral techniques to investigate the role of the PBN^Tac1-CeA^Tac1 pathway in itch. Our results indicate that neurons in the CeA can be more activated by acute itch than chronic itch. Notably, in response to acute itch stimuli, both CeA^Tac1 and PBN^Tac1 neurons are specifically activated by histamine (His)-induced itch. Furthermore, the Tac1-positive terminals from the PBN^Tac1 neurons formed close connections with CeA^Tac1 neurons. We also demonstrated that activating the PBN-CeA pathway using a chemogenetic approach could increase scratching behaviors in His-induced itch, other than chloroquine (CQ)-induced itch. Conversely, inhibiting the PBN-CeA pathway decreased scratching behaviors in mice with His-induced itch. Taken together, these results suggest that the PBN^Tac1-CeA^Tac1 pathway may play a specific role in modulating His-induced acute itch.

Parabrachial nucleus

Central amygdala

Itch

Tac1

Itch is an unpleasant sensation that triggers intense scratching behavior and aversion [1, 2]. Nowadays, only a limited number of patients with pruritus can find relief through antihistamine therapy. However, there are still many patients suffering from pruritus who lack effective treatment options. This may be associated with the distinct neural mechanisms underlying the different types of itching, including acute and chronic itch.

On the other hand, the investigation of the specific mechanism and proteins responsible for transmitting itch from the peripheral to the supraspinal level has focused on the spinal level, including the study of gastrin-releasing peptide (GRP) and the GRP receptor (GRPR) [3, 4]. Nevertheless, there has been comparatively less investigation into itch at the supraspinal level.

Tachykinin 1 (Tac1 gene) encodes the precursors of neurokinin A (NKA) and substance P (SP) [5]. In the dorsal root ganglion (DRG), SP plays a crucial role in the sensation of itch and pain. A recent report revealed that the activity of neurons expressing Tac1 in the lateral and ventrolateral periaqueductal gray (l/vlPAG) increases scratching behaviors during itch, thereby facilitating the processing of itch signals [6].

The spinoparabrachial pathway is a critical neural circuit through which itch signals are transmitted to the brain [7, 8]. The parabrachial nucleus (PBN) serves as the central relay for itch sensation, and its activity regulates the scratching response to itch. Within the PBN, neurons expressing calcitonin gene-related peptide (PBN^CGRP) can be activated by itch stimuli [9–11]. Notably, most PBN^CGRP neurons co-express Tac1 [5]. Therefore, we hypothesized that PBN^Tac1 neurons might be involved in regulating itch.

Functional magnetic resonance imaging (fMRI) studies have shown that the amygdala can be activated by itch stimuli that are histamine-dependent (represented by histamine, His) and histamine-independent (represented by chloroquine, CQ) [12, 13]. The central amygdala (CeA) is a critical part of the amygdala and is primarily composed of gamma-aminobutyric acid-ergic (GABAergic) medium-spiny neurons [14]. Anatomically, the structure of the CeA is divided into three parts: medial (CeM), lateral (CeL), and pericentral (CeC) [15–18]. Research has shown that Tac1⁺ neurons are also expressed in the CeA [19]. However, little was known about the role of CeA^Tac1 neurons in itch.

Both the PBN and CeA can be significantly activated by itch stimuli [20, 21]. Studies have shown that the majority of PBN^CGRP neurons co-express Tac1, can be activated by itch stimuli and project their axons mainly to the CeA [5, 10]. Therefore, these Tac1⁺ neurons located in the PBN might project to the CeA and be involved in the transmission of itch.

Therefore, we hypothesized that PBN^Tac1 neurons project to the CeA, and it is possible that CeA^Tac1 neurons may be involved in the sensation of itch. We used morphological, behavioral, and chemogenetic approaches to investigate the role of the PBN-CeA pathway on itch regulation and to differentiate between various types of itch.

2.1 Animals

This study was approved by the Ethics Committee of the Laboratory Animal Center at the Fourth Military Medical University. Mice were housed in a 12 h light/dark cycle (7 a.m. to 7 p.m.) with free access to water and food. The mice were kept at optimal temperatures (22–26°C) and humidity levels (40–70%), with no more than six mice in each cage. Adult male wild-type (WT) C57BL/6J mice were purchased from the Laboratory Animal Center of the Fourth Military Medical University. GAD67-GFP mice, Tac1-Cre mice, and Ai9-TdTomato mice were purchased from Jackson Laboratory (RRID: IMSR_JAX: 007673, RRID: IMSR_JAX: 021877, and RRID: IMSR_JAX: 007909, respectively) and used for the experiments. The mice were 6–12 weeks old and weighed between 20–30g. The figure legend in this article displays the number of mice in each group.

2.2 Stereotaxic injections

Mice were intraperitoneally injected with 2% pentobarbital sodium (40 mg/kg). The animals were then anesthetized and fixed on a stereotaxic instrument (RWD Life Science, Shenzhen, China). The craniotomy site was covered with sterile eye ointment, and the surgical area was disinfected using an iodophor disinfectant. The skull was exposed by making a midline scalp incision. Using a 1 µL Hamilton microsyringe, the virus was stereotactically injected at a rate of 15–20 nL/min, according to the coordinates. The needle was then left in place at the injection site for 10 minutes after the injection, and then it was subsequently removed. After the surgery, the incision was closed with sutures and sterilized using iodophor disinfectant. The mice were transferred to a heating pad and monitored until they fully recovered from anesthesia.

According to the mouse brain atlas, the injection coordinates are as follows: PBN: anterior posterior (AP), -5.11; medial lateral (ML), ± 1.21; dorso-ventral (DV), -3.4 mm. CeA: AP, -1.22; ML, ± 2.74; DV, -4.83.

For CeA^Tac1 neurons observations, we injected rAAV2/9-EF1a-DIO-mCherry-WPRE hGH pA (150 nL, 5 × 10¹² vg/mL, PT − 0013) into the right CeA of Tac1-Cre mice.

For fiber photometry of CeA^Tac1 neurons, we administered either the rAAV2/9-CAG-FLEX-jGCaMP7s-WPRE-SV40 pA (150 nL, 2 × 10¹² vg/mL, PT − 1421) or the control virus rAAV 2/9-EF1a-DIO-eGFP-WPRE hGH pA (150 nL, 2 × 10¹² vg/mL, PT − 0795) via injection into the right CeA of Tac1-Cre mice. The fibers (200 µm OD, 0.37 NA, Fiblaser, Shanghai, China) were implanted above the right CeA two weeks after the virus injection.

For the visualization of the PBN^Tac1-CeA^Tac1 pathway, the rAAV2/9-EF1a-DIO-eGFP-WPRE-hGH pA (150 nL, 2 × 10¹² vg/mL, PT − 0795) was delivered through injection into the right PBN of Tac1-Ai9 mice. In the Tac1-Ai9 mice, the tdTomato sequence was inserted into the tachykinin 1 locus of Tac1⁺ neurons to enable the expression of red fluorescence in the central nervous system (CNS).

To detect the neuronal activities of the PBN^Tac1-CeA pathway following modeling, the rAAV2/9-EF1a-DIO-mCherry-WPRE-hGH pA (150 nL, 5 × 10¹² vg/mL, PT − 0013) was administered via injection into the right PBN of Tac1-Cre mice for comparative modeling analysis.

For the chemogenetic manipulation of the PBN-CeA pathway, we administered injections of the following viral constructs: rAAV2/9-EF1a-DIO-hM3D(Gq)-eYFP-WPRE-hGH pA (150 nL, 2 × 10¹² vg/mL, PT − 0816), rAAV2/9-EF1a-DIO-hM4D(Gi)-eYFP-WPREs (150 nL, 5.04 × 10¹² vg/mL, PT − 0815), or the control virus rAAV 2/9-EF1a-DIO-eYFP-WPRE-hGH pA (150 nL, 5 × 10¹² vg/mL, PT − 0012). The PBN of C57BL/6J mice received bilateral injections of these constructs. Simultaneously, the rAAV2/R-hSyn-CRE-WPRE-hGH pA (150 nL, 2 × 10¹² vg/mL, PT − 0136) was bilaterally administered into the CeA.

All the viruses were purchased from BrainVTA Co., Ltd (Wuhan, China).

2.3 Chemogenetic manipulation

Behavioral experiments began three weeks after the injection of the chemogenetic virus, and the mice received intraperitoneal (i.p.) injections of clozapine (C6305, Sigma) at a dosage of 1 mg/kg [22]. Behavioral experiments should begin 30 minutes after the injection of clozapine and completed within 4 hours. Each behavioral experiment includes a 2- to 3-day interval between tests.

2.4 Fiber photometry

The rAAV2/9-CAG-FLEX-jGCaMP7s-WPRE-SV40 pA (150 nL, 2 × 10¹² vg/mL, PT − 1421, BrainVTA) was injected into the right CeA of Tac1-Cre mice. An optical fiber was implanted into the right CeA two weeks later. After one week of recovery, the fluorescence signals resulting from calcium-dependent (470 nm LED) and calcium-independent (410 nm LED) excitation were recorded using a fiber photometry system (Inper, Hangzhou, China). During the experiments, the lights in the behavioral chamber were turned off, and the environment was kept quiet. The mice were required to acclimatize in the behavioral chamber for 30 minutes before receiving intradermal (i.d.) injections. The acute pruritogens His or CQ were applied at the napes of mice to observe alterations in calcium signals.

After the experiment concluded, perfusion was performed on all the mice. Only animals with brain regions expressing GCaMP7s and accurately localized fibers were included in the subsequent analysis. The change in fluorescence (ΔF/F) and the area under the curve (AUC) were calculated from 2 seconds before the start of scratching to 6 seconds after the onset of scratching. The data was processed using the Inper Studio data processing software.

2.5 Acute itch model and itch behavioral test

All behavioral tests on mice were conducted between 9 a.m. and 6 p.m. The hair on the backs of the mice's necks was shaved in advance. The mice were then placed in a rectangular opaque box measuring 15 × 10 × 25 cm³ for 3 consecutive days to acclimatize, with each session lasted 30 minutes at the same time each day. The device was cleaned with 75% alcohol to minimize the impact of any odor.

Mice were intradermally injected with 20 µL of His or CQ (both 10 µg/µL) into the midline of the nape using an insulin needle. After the injection, the mice were placed in a behavioral acquisition device for video recording. The number of scratches on the mice was recorded 30 minutes after the injection. A scratch was defined as the mouse lifting its hind paw to scratch the skin on the back of the neck before lowering the hind paw.

2.6 Chronic itch model

The skin on the napes and abdomen of the mice was shaved one day prior to the experiment, and the reagent 1-fluoro-2,4-dinitrobenzene (DNFB) was dissolved in a combination of olive oil and acetone. On the first day, 150 µL of 0.5% DNFB was applied to the abdomen for sensitization. After 3 days of sensitization, 150 µL of 0.2% DNFB was applied to the mice's napes for 2–3 weeks within the same time period.

2.7 Histology

Mice were injected with an overdose of pentobarbital sodium (100 mg/kg; i.p.). After being deeply anesthetized, the mice were perfused via the left ventricle with 50 mL of 0.01 M phosphate-buffered saline (PBS, pH = 7.4), followed by 150 mL of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH = 7.4).

Brains were removed after perfusion and fixed in 4% PFA for 4–6 hours. Subsequently, they were immersed in a 30% sucrose solution for 24 hours. Mice brains were cut into 30 µm transverse sections using a freezing microtome (CM1950; Leica, Heidelberg, Germany) and then arranged sequentially in six-well culture plates. Each six-well culture plate contained a one-sixth set of serial sections. The sections were subsequently stored in PBS at 4°C. Before conducting immunofluorescent histochemical staining or immunohistochemical staining, the sections must be blocked in 10% donkey serum for 30 minutes at room temperature. Then the sections should be incubated with the primary antibody at 4°C for 48–72 hours, followed by the secondary antibody at 4°C for 12–24 hours.

Immunostaining methods were used to assess dual-labeling of tdTomato/CGRP in the CeA. The primary antibody was goat anti-CGRP (1:500, ab36001, Abcam) and the secondary antibodies were alexa 488 donkey anti-goat (1:500, A31627, Invitrogen).

For c-FOS neurons induced by acute itch, mice were injected with 20 µL of saline, His, or CQ (10 µg/µL; i.p.) at the napes, and then placed in the original rearing cages for 2 hours before perfusion. The primary primary antibody was rabbit anti-c-Fos (1:500, ab222699, Abcam) and the secondary antibodies were alexa 488 donkey anti-rabbit (1:500, A21206, Invitrogen).

For c-FOS neurons induced by chronic itch, DNFB was applied to the napes of mice over a period of two consecutive weeks. DNFB was perfused two hours after application on the last day of chronic itch modeling. The primary primary antibody was rabbit anti-c-Fos (1:500, ab222699, Abcam) and the secondary antibodies were alexa 488 donkey anti-rabbit (1:500, A21206, Invitrogen).

Immunostaining methods were used to assess the triple-labeling of tdTomato/c-FOS /CGRP in the PAB and CeA. The primary antibodies used were rabbit anti-c-FOS (1:500, ab222699, Abcam) and goat anti-CGRP (1:500, ab36001, Abcam). The secondary antibodies used were alexa 488 donkey anti-rabbit (1:500, A21206, Invitrogen) and alexa 647 donkey anti-goat (1:500, ab15031, Abcam).

For the DAB reaction, the primary antibody used was rabbit anti-c-FOS (1:100, ab222699, Abcam). The secondary antibody was biotin-donkey anti-rabbit (1:200, AP182B, Merck Millipore, USA), and the tertiary antibodies were AB solution (1:200, PK − 6101, Vectorlabs, CA, USA) and DAB-nickel ammonium sulfate solution.

2.8 Fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) staining was performed according to the procedures described in previous studies [23]. Tac1-Ai9 mice were perfused and sectioned with enzymes-free throughout the experiments. The PB and PBS used were treated with 0.1% (v/v) diethyl pyrocarbonate (DEPC; DH098-2, Genview, FL, USA) overnight in a 37°C incubator. Autoclave the equipment before use.

First, fluorescence-labeled antisense single-stranded RNA probes were synthesized using the Fluorescent RNA Labeling Kit (Roche Diagnostics, Basel, Switzerland). Brain sections were treated with 0.1 M DEPC-PB and 2% H₂O₂ diluted in 0.1 M DEPC-PB for 10 minutes each. The sections were treated with 0.3% Triton X-100 diluted in 0.1 M DEPC-PB for 20 minutes, followed by acetylation solution for 10 minutes. Finally, the sections were rinsed twice with 0.1 M DEPC-PB for 10 minutes each. The sections were prehybridized in a prehybridization buffer at 55°C for 1 hour. Tac1 riboprobes were then added to the prehybridization buffer at a final concentration of 1 µg/mL, and the reaction was allowed to proceed at 55°C for 24 hours. The next day, brain sections were placed in a wash buffer, rinsed twice for 20 minutes each at 58°C, and then sequentially incubated in ribonuclease A (RNase) buffer for 5 minutes at room temperature and with 20 µg/mL of RNase for 30 minutes at 37°C. The sections were rinsed twice for 20 minutes each at 37°C, first with 2 × SSC and then with 0.2 × SSC. The sections should be incubated in TBS (0.1 M Tris-HCl buffered 0.9% saline) for 1 hour. The primary antibodies, including Fab fragments POD-anti-Fluorescence (1:100, 11426346910, Roche Diagnostics), rabbit anti-RFP (1:500, ab62341, Abcam, MA, UK), and mouse anti-NeuN (1:500, mab377, Merck Millipore, USA), were added and incubated for 24 hours at room temperature. On the third day, the sections were rinsed twice with TNT (0.1 M Tris-HCl buffered 0.05% Tween 20) for 10 minutes each. The Tac1 mRNA signal was then amplified using TSA-Biotin solution. This was followed by the addition of the secondary antibodies: alexa 594 donkey anti-rabbit (1:500, ab141637, Invitrogen), alexa 647 donkey anti-mouse (1:500, A31571, Invitrogen), and FITC Avidin (1:500, A − 2001, Vectorlabs). The incubation period for these antibodies was 3 to 4 hours at room temperature.

2.9 Cell counting and statistical analyses

Observational and statistical analyses of the experiments were performed by counting the number of immunopositive neurons ipsilateral in the PBN and CeA from 10 randomly selected sections for each group of mice (3 mice in each group), as described in our previous publication [24]. For double-labeled neurons, the number of double-labeled neurons and c-FOS or tdTomato-positive neurons from each mouse was counted, and then the ratio of double-labeled neurons to c-FOS or tdTomato-positive neurons was calculated. Confirmation of positively labeled neurons using Image J software.

Statistical analyses were performed using GraphPad Prism 9 software. The raw data was analyzed using unpaired t-tests, one-way ANOVA, or two-way ANOVA for multiple comparisons. All data are expressed as the mean ± standard error of the mean (s.e.m.). The result was statistically significant at p < 0.05.

3.1 The distribution of CeA^Tac1 neurons, which may be involved in somatosensory neurotransmission

In the present study, our primary objective was to determine the distribution of CeA^Tac1 neurons. To specifically visualize Tac1⁺ neurons, we crossed Tac1-Cre mice with Ai9 reporter mice, resulting in the labeling of Tac1⁺ neurons with tdTomato. According to the cell counting results, Tac1⁺ neurons were differentially distributed in the rostral, middle, and caudal regions of the CeA, with a predominant distribution in the middle and caudal regions (Fig. 1a-b). Tac1⁺ neurons accounted for 10.9% of the rostral region, and 41.8% and 47.3% of the middle and caudal regions in the CeA, respectively (Fig. 1b). Our morphological evidence indicates that the distribution of CeA^Tac1 neurons in the Tac1-Ai9 mice corresponds to the distribution of the Tac1 mRNA signals in the Allen Brain Atlas (http://www.brain-map.org) (accessed January 1, 2024) and a previous report [19].

Next, we further validate the presence of CeA^Tac1 neurons in Tac1-Ai9 mice using FISH staining. Results showed the coexistence of Tac1 mRNA and tdTomato-positive cells from Tac1-Ai9 mice (Fig. 1c), indicating that the Tac1-Ai9 mice used in the present study are specifically designed to illustrate the distribution of CeA^Tac1 neurons. Therefore, we used Tac1 genetically modified mice (Tac1-cre mice and Tac1-Ai9 mice) for our subsequent experiments.

Importantly, the majority of CeA neurons are GABAergic [25]. To explore the relationship between Tac1⁺ neurons and GABAergic neurons in the CeA, we crossed GAD67-GFP and Tac1-Ai9 mice to generate GAD67-GFP/Tac1-Ai9 mice. In these mice, GFP labels the GAD67-positive neurons, while tdTomato labels the Tac1-positive neurons. The cell counting results showed that 51.3 ± 1.6% of Tac1⁺ tdTomato neurons showed GFP/tdTomato double-labeled (Fig. 1f-g), confirming that the majority of CeA^Tac1 neurons are GABAergic.

Abundant terminals positive for calcitonin gene-related peptide (CGRP) were densely expressed within the CeA, indicating a potential role in somatosensory processing [26, 27]. Consequently, we injected a cre-dependent anterograde virus (rAAV2/9-DIO-mCherry) into the right CeA of Tac1-Cre mice (Fig. 1d). After three weeks, immunohistochemical staining revealed that many CGRP-positive terminals made closed connects with Tac1⁺ neurons in the CeA (Fig. 1e). This evidence suggests that the group of CeA^Tac1 neurons may receive somatosensory inputs.

In conclusion, our findings indicate that Tac1⁺ neurons are predominantly located in the middle and caudal regions of the CeA and may play a role in somatosensory neurotransmission.

3.2 The CeA^Tac1 neurons primarily participate in His-induced itch

To investigate the role of the CeA in somatosensory neurotransmission, particularly in relation to itch, we utilized C57BL/6J mice as models to evaluate the effects of saline, acute itch (His, CQ), and chronic itch (DNFB). Research has demonstrated that c-FOS proteins are commonly acknowledged and utilized as markers of neuronal activity [28]. After successful animal modeling, the mice were perfused, and brain sections were subjected to DAB reaction for c-FOS and Nissl staining (Fig. 2a-b).

We noted that, apart from DNFB-induced chronic itch, the number of c-FOS-immunoreactive (-ir) neurons in the CeA increased in response to acute itch (saline vs. His: p < 0.0001, saline vs. CQ: p = 0.0022 < 0.01, saline vs. DNFB: p = 0.2310 > 0.05, Fig. 2c and Table 1). This indicates that neurons in the CeA may be significantly activated in response to acute itch stimuli (Fig. 2c), which is consistent with a previous report [21]. Notably, the cell counting results revealed a significantly higher number of c-FOS-ir neurons in the His model within the CeA compared to the other model groups (His vs. saline: p < 0.0001, His vs. CQ: p < 0.0001, His vs. DNFB: p < 0.0001, as shown in Fig. 2c and Table 1). The findings indicate that the activated neurons in the CeA were more closely associated with His-induced itch in various itch models.

Table 1

Number of c-FOS-ir neurons in the ipsilateral side of the CeA and PBN in C57BL/6J mice
	Saline	His	CQ	DNFB
CeA	5 ± 1	48 ± 4	22 ± 3	14 ± 2
PBN	17 ± 3	86 ± 8	55 ± 6	56 ± 5

Cell counts were performed on 10 sections of 30 µm thickness (3 mice in each model). These data were expressed as the mean ± s.e.m.. c-FOS-ir: c-FOS-immunoreactive, His: histamine, CQ: chloroquine, DNFB: 1-fluoro-2,4-dinitrobenzene.

Based on the evidence, further investigation was undertaken to explore the relationships between CeA^Tac1 neurons and itch. Tac1-Ai9 mice were used for establish the models of His, CQ, and DNFB, respectively. The mice underwent perfusion 2 hours after the successful establishment of the animal model. The immunohistochemical staining revealed the co-expression of c-FOS and tdTomato in the CeA, suggesting that CeA^Tac1 neurons were activated in response to both acute and chronic itch stimuli (Fig. 2e). In the CQ and DNFB groups, the percentages of c-FOS/tdTomato double-labeling in the tdTomato-positive neurons were 2.4 ± 0.6% and 3.7 ± 0.8%, respectively. In contrast, the percentage of neurons double-labeled for c-FOS/tdTomato in the tdTomato-positive neurons in the His model was 18.9 ± 2.5%, which was significantly higher compared to the other itch models (His vs. CQ: p < 0.0001, His vs. DNFB: p < 0.0001, Table 2). This indicates that within the CeA, the activated CeA^Tac1 neurons were more closely associated with His-induced itch.

Table 2

Number of c-FOS, tdTomato or c-FOS/tdTomato double-labeled neurons in the ipsilateral side of the CeA in Tac1-Ai9 mice
	His	CQ	DNFB
c-FOS	47 ± 3	14 ± 3	21 ± 2
tdTomato	86 ± 4	84 ± 6	79 ± 3
c-FOS + tdTomato	16 ± 2	2 ± 1	3 ± 1
c-FOS + tdTomato/c-FOS (%)	34.5 ± 4.3%	14.3 ± 2.1%	13.4 ± 2.0%
c-FOS + tdTomato/tdTomato (%)	18.9 ± 2.5%	2.4 ± 0.6%^****	3.7 ± 0.8%^****

Cell counts were performed on 10 sections of 30 µm thickness (3 mice in each model). These data were expressed as the mean ± s.e.m.. ^****p < 0.0001 as compared to His model. His: histamine, CQ: chloroquine, DNFB: 1-fluoro-2,4-dinitrobenzene.

The above morphological results above indicate that CeA^Tac1 neurons were significantly activated in the His-induced acute itch model, but not in the CQ-induced acute itch and DNFB-induced chronic itch. This suggests that CeA^Tac1 neurons may be specifically involved in His-induced acute itch. However, additional behavioral evidence is required to confirm the specific association between CeA^Tac1 and His-induced itch. Subsequently, an in vivo fiber photometric system was employed to observe Ca²⁺ signaling in freely awake mice. This was done to assess the activity of CeA^Tac1 neurons associated with scratching behavior induced by itch-inducing agents. The AAV2/9 viruses expressing GCaMP7s were administered through injection into the right CeA of Tac1-Cre mice, and optical fibers were subsequently implanted above the CeA (Fig. 2f). The controls received an injection of the rAAV2/9-DIO-eGFP virus. The increased fluorescence of GCaMP7s corresponded with the onset of scratching behaviors in both His and CQ-induced itch (Fig. 2k-l). No significant signal fluctuation was observed in the control group (Fig. 2m-n). Enhanced neuronal activity in the CeA was observed during His and CQ stimulation, as indicated by the area under the curve (AUC) (Fig. 2o). However, the neuronal fluctuations of CeA^Tac1 were more pronounced during His stimulation compared to CQ (His vs. CQ: p < 0.0001, Fig. 2o).

The collective findings suggest that neurons in the CeA can be activated significantly by acute itch. Subsequently, we demonstrated through morphological methods and in vivo fiber photometry recording that CeA^Tac1 neurons are more closely associated with His-induced acute itch.

3.3 The PBN^Tac1-CeA^Tac1 pathway may primarily participates in His-induced itch transmission

Studies have shown that the neurons in the CeA receive projections from the PBN [29, 30]. To determine the link between PBN^Tac1 and CeA^Tac1, we injected cre-dependent anterograde virus (rAAV2/9-DIO-eGFP) into the right PBN of Tac1-Ai9 mice (Fig. 3a). After 3 weeks, we observed that Tac1-positive terminals from the PBN formed close contact with Tac1-positive neurons within the CeA (Fig. 3d-f). This suggests that CeA^Tac1 neurons receive projections from the PBN^Tac1 neurons.

PBN serves as the central relay for itch [8]. To confirm the relationship between PBN and itch, we utilized C57BL/6J mice to model saline, His, CQ, and DNFB. The mice were perfused after the successful modeling, followed by DAB reaction for c-FOS and Nissl staining (Fig. 4a-b). The cell counting results indicated a significant increase in the number of c-FOS-ir neurons within the PBN in the itch models compared to the saline model (saline vs. His: p < 0.0001, saline vs. CQ: p = 0.0004 < 0.001, saline vs. DNFB: p = 0.0002 < 0.001, Fig. 4c and Table 1). In the PBN, in accordance with the CeA, compared with the CQ and DNFB groups, a higher number of neurons exhibited c-FOS-ir and were activated in the His group (His vs. CQ: p = 0.0040 < 0.01, His vs. DNFB: p = 0.0071 < 0.01, Fig. 4c and Table 1). This suggests that the PBN may be more closely associated with His-induced acute itch.

To further investigate the PBN^Tac1-CeA pathway in acute itch, we injected the rAAV2/9-DIO-mCherry virus into the right PBN of Tac1-Cre mice (Fig. 4d), and performed His and CQ modeling after 3 weeks (Fig. 4g, k). The number of c-FOS-positive cells within the PBN and CeA was counted. The cell counting results indicated that the number of c-FOS-ir cells in the His model of the PBN was higher than in the CQ model (His vs. CQ: p = 0.0020 < 0.01, Fig. 4n). Moreover, the number of c-FOS/viral-labeled tdTomato double-labeled neurons in the His model of the PBN was also higher than in the CQ model (His vs. CQ: p = 0.0062 < 0.01, Fig. 4o). As with the PBN, the number of c-FOS-ir cells was significantly higher in the His model than in the CQ model in the CeA (His vs. CQ: p < 0.0001, Fig. 4n). The results suggest that the PBN^Tac1 neurons, which project to the CeA^Tac1 neurons, may be closely correlated with His-induced acute itch.

Thus, our findings suggest that the PBN^Tac1-CeA^Tac1 pathway may predominantly be involved in the transmission of His-induced acute itch.

3.4 Chemogenetic activation/inhibition of the PBN-CeA pathway increases/decreases His-induced scratching behaviors

Our findings indicate that the CeA^Tac1-PBN^Tac1 pathway may primarily be involved in His-induced itch. Next, we would like to investigate the effects of modulating the PBN-CeA pathway on His-induced itch. Therefore, we performed stereotactic injections of rAAV2/9-DIO-hM3D(Gq)/hM4D(Gi)-eYFP or the control virus rAAV2/9-DIO-eYFP into the bilateral PBN of C57BL/6J mice. At the same time, the rAAV2/R-Cre was injected into the bilateral CeA (Fig. 5a).

First, we injected saline into the necks of mice and observed the number of scratches to assess spontaneous itch and exclude ineligible mice. The inclusion criterion for our subsequent experiments using mice was less than 20 spontaneous scratches in 30 minutes. After excluding the mice that did not meet the criteria, there were 6 mice in each group (Fig. 5e). Next, we separately injected mice with His and CQ to observe the role of the PBN-CeA pathway in modulating itch. Chemogenetic inhibition of the PBN-CeA pathway significantly reduces the number of scratching behaviors induced by His in mice that received the hM4Di virus after clozapine injection, compared with the eYFP group (hM4Di-Clozapine vs. eYFP-Clozapine: p = 0.0004 < 0.001, Fig. 5e). Conversely, activation of the PBN-CeA pathway significantly increased the number of scratching behaviors induced by His (hM3Dq-Clozapine vs. eYFP-Clozapine: p = 0.0001, Fig. 5e). For CQ-induced itch, neither activation nor inhibition of the PBN-CeA pathway resulted in significant changes (hM3Dq-Clozapine vs. eYFP-Clozapine: p = 0.8787 > 0.05, hM4Di-Clozapine vs. eYFP-Clozapine: p > 0.9999, Fig. 5e).

In conclusion, the results suggest that activating or inhibiting the PBN-CeA pathway may increase or decrease the number of scratching behaviors caused by His-induced itch. Specifically, the PBN-CeA pathway can modulate the transmission of His-induced itch.

In this study, we propose that the PBN^Tac1-CeA^Tac1 pathway primarily contributes to the transmission of His-induced itch. We found that the compared with CQ-induced acute itch and DNFB-induced chronic itch, injection of His activates CeA^Tac1 neurons significantly during scratching behaviors. The CeA^Tac1 neurons that receive PBN^Tac1 projections are more closely associated with His-induced itch. Inhibition of the PBN-CeA pathway reduced scratching behaviors in mice induced by His itch other than CQ.

Since PBN^Tac1 neurons receive direct input from spinal neurons, they serve as a crucial relay hub for transmitting itch information to the supraspinal level [7, 31]. Previous research has demonstrated that PBN^Tac1 neurons are activated in response to noxious stimuli, such as hot plate and inflammatory pain [31]. In the present study, we observed a high number of PBN^Tac1 neurons expressing c-FOS proteins in itch models. This suggests that PBN^Tac1 neurons are not only involved in transmitting pain signals but also play a significant role in signaling itch.

There are two distinct populations of PBN^Tac1 neurons that can project to the CeA and to the reticular formation in the medulla (MdD), respectively [5]. The axon terminals of PBN^Tac1 neurons extend to the CeM and CeL regions of the CeA, forming the PBN-CeA pathway [5]. In the present study, we observed that PBN^Tac1 neurons project to the CeA, and Tac1-positive terminals from the PBN were in close contact with CeA^Tac1 neurons. Studies have shown that the majority of CeA neurons are GABAergic and are involved in modulating itch through local GABAergic inhibition [25, 32]. Our morphological evidence indicates that 51.3 ± 1.6% of CeA^Tac1 neurons showed double labeling with GFP and tdTomato. Furthermore, the injection of His enhanced the activities of CeA^Tac1 neurons in fiber photometry monitoring. This suggests that CeA^Tac1 neurons receiving PBN^Tac1 afferents may be involved in the sensation of itch through GABAergic inhibition.

In some instances, itch stimuli may interact with the perception of pain during scratching. The results of the present study indicate that CeA^Tac1 neurons may be involved in the perception of itchiness, particularly in His-induced acute itch, but not in CQ and DNFB-induced itch. However, we suspect that the activation of CeA^Tac1 neurons during itching does not necessarily exclude the possibility that certain subpopulations of CeA^Tac1 neurons could also respond to other stimuli, such as pain during scratching behaviors. Therefore, further research is needed to clarify the distinction in regulating signaling for pain and itch in CeA^Tac1 neurons.

Although pain and itch are distinct sensations, we must acknowledge the complex interaction between them. Pain inhibits itching, and pain circuits transmit itch signals, so pain circuits could provide the basis for advancing our understanding of itch circuits [7, 33]. Pain and itch signals from the spinal cord can be transmitted upward through the PBN to the CeA [9, 34, 35]. Research has shown that the activation of the PBN-CeA pathway exacerbates neuropathic pain [34, 36, 37]. Given that the PBN-CeA pathway is involved in the regulation of pain, it is possible that the same pathway may also modulate itch. To confirm this possibility, we demonstrated through chemogenetic and morphological methods that inhibiting the PBN-CeA pathway reduces scratching behaviors in His-induced acute itch. This suggests that the PBN-CeA pathway may play a role in regulating His itching, and it could be one of the crucial pathways for itch signaling at the supraspinal level of the central nervous system.

Itch is a more overwhelming sensation than pain, and so far, we have only explored a small part of the mechanisms of itch [1]. Currently, antihistamines are only effective for a small proportion of patients experiencing pruritus [38]. Therefore, there is still a lack of effective treatment for the majority of patients with pruritus. This may be related to the different mechanisms of the various types of itching. As we know, in acute itch, His acts through histamine H1 and H4 receptors to activate transient receptor potential vanilloid 1 (TRPV1) ion channels, thereby inducing itch; CQ-induced itch is specifically mediated by Mas-related G protein-coupled receptor A3 (MrgprA3) coupled to transient receptor potential ankyrin 1 (TRPA1) [39–41]. As demonstrated in this study, the use of chemogenetic methods to inhibit the PBN-CeA pathway reduced scratching behaviors induced by His itch, but not by CQ. Morphological findings also indicate that Tac1-positive terminals from the PBN^Tac1 made contacts with more FOS-positive neurons within the CeA in the His model compared to the CQ model. This suggests that CeA^Tac1 neurons receiving PBN^Tac1 projections may be more closely associated with His itch.

There are fewer studies on the underlying mechanisms of acute and chronic itch at the supraspinal level compared to the spinal level. In addition, the distinction between His and CQ has also been unclear, requiring further study. Taken together, our results reveal the specific roles of the PBN^Tac1-CeA^Tac1 pathway in His-induced itch, which might offer an effective intervention strategy for treating and distinguishing different itch-related diseases.

Author Contributions: Conceptualization: Y-N. Zhang, Z-Z. Kou; Investigation: Y-N. Zhang, S-J. Shi; E. Mao; Resources: J. Chen, M. Tian, X. Wang, Y-H. Zhou, Y-L. Chen, F-S. Huang; Writing - Original Draft: Y-N. Zhang, Z-Z. Kou, Z-P. Cai, Y-Q. Li; Writing - Review & Editing: Y-N. Zhang, Z-Z. Kou, Z-P. Cai, Y-Q. Li; Funding Acquisition: Z-Z. Kou, Z-P. Cai, M. Tian, F-S. Huang, Y-Q. Li. All authors have read and agreed to the published version of the manuscript.

Funding: This work was supported by grants from the National Natural Science Foundation of China (Nos. 82130034 and 82221001 to Y-Q. Li, 81971035 to Z-Z. Kou), STI2030-Major Projects (No. 2021ZD0204403 to Y-Q. Li), the Innovation Capability Support Program of Shaanxi (No. 2021TD-57 to Y-Q. Li), Li Yunqing Expert Workstation of Yunnan Province (No. 202005AF150014 to Y-Q. Li), the Swedish Society of Medicine (SLS-246981 to F-S. Huang), Natural Science Foundation of Shaanxi (2024JC-YBQN-0881 to M. Tian), and the Scientific Research Fund of Baotou Medical College (2020LH03001).

Data Availability: The data provided in this study are available on request from the corresponding author. These data are not publicly available due to the privacy of other scientific studies.

Conflict of interest: The authors have no conflicts of interest to declare.

Ethical approval: The study was approved by the Ethics Committee of the Laboratory Animal Center at the Fourth Military Medical University (ethics approval number: IACUC-20210356) to minimize animal suffering. The care and use of experimental animals strictly followed institutional guidelines and government regulations.

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No competing interests reported.

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Tac1-expressing neurons in the central amygdala predominantly mediate histamine-induced itch by receiving inputs from parabrachial Tac1-expressing neurons

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Animals

2.2 Stereotaxic injections

2.3 Chemogenetic manipulation

2.4 Fiber photometry

2.5 Acute itch model and itch behavioral test

2.6 Chronic itch model

2.7 Histology

2.8 Fluorescence in situ hybridization

2.9 Cell counting and statistical analyses

3. Results

3.1 The distribution of CeA^Tac1 neurons, which may be involved in somatosensory neurotransmission

3.2 The CeA^Tac1 neurons primarily participate in His-induced itch

3.3 The PBN^Tac1-CeA^Tac1 pathway may primarily participates in His-induced itch transmission

3.4 Chemogenetic activation/inhibition of the PBN-CeA pathway increases/decreases His-induced scratching behaviors

4. Discussion

Declarations

References

Additional Declarations

Status:

Version 1

Tac1-expressing neurons in the central amygdala predominantly mediate histamine-induced itch by receiving inputs from parabrachial Tac1-expressing neurons

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Animals

2.2 Stereotaxic injections

2.3 Chemogenetic manipulation

2.4 Fiber photometry

2.5 Acute itch model and itch behavioral test

2.6 Chronic itch model

2.7 Histology

2.8 Fluorescence in situ hybridization

2.9 Cell counting and statistical analyses

3. Results

3.1 The distribution of CeATac1 neurons, which may be involved in somatosensory neurotransmission

3.2 The CeATac1 neurons primarily participate in His-induced itch

3.3 The PBNTac1-CeATac1 pathway may primarily participates in His-induced itch transmission

3.4 Chemogenetic activation/inhibition of the PBN-CeA pathway increases/decreases His-induced scratching behaviors

4. Discussion

Declarations

References

Additional Declarations

Status:

Version 1

3.1 The distribution of CeA^Tac1 neurons, which may be involved in somatosensory neurotransmission

3.2 The CeA^Tac1 neurons primarily participate in His-induced itch

3.3 The PBN^Tac1-CeA^Tac1 pathway may primarily participates in His-induced itch transmission