Involvement of supralemniscal nucleus (B9) 5-HT neuronal system in nociceptive processing: a fiber photometry study

Nociception is important perception that has harmful influence on daily life of humans. As to main pain management system, some descending pathways are called descending antinociceptive systems (DAS). As main pathways of DAS, it is well known that dorsal raphe (B6/B7) - rostral ventromedial medulla (B3) - spinal dorsal horn includes serotonergic system. However, possible role of supralemniscal (B9) serotonin (5-HT) cell group in pain management is still open question. In this study, we measured activities of B9 5-HT neuronal cell bodies and B9 5-HT neuron-derived axons located in the locus coeruleus (LC) and ventral tegmental area (VTA), which are also main players of pain management, using fiber photometry system. We introduced the G-CaMP6 in B9 5-HT neurons using transgenic mice carrying a tetracycline-controlled transactivator transgene (tTA) under the control of a tryptophan hydroxylase-2 (TPH2) promoter and site-specific injection of adeno associated virus (AAV-TetO(3G)-G-CaMP6). After confirmation of specific expression of G-CaMP6 in the target population, G-CaMP6 fluorescence intensity in B9 group and LC/VTA groups was measured in awake mice exposed to acute tail pinch and heat stimuli. G-CaMP6 fluorescence intensity rapidly increased by both stimuli in all groups, but not significantly reacted by nonnociceptive control stimuli. The present results clearly indicate that acute nociceptive stimuli cause a rapid increase in the activities of B9-LC/B9-VTA 5-HTergic pathways, suggesting that B9 5-HT neurons play important roles in nociceptive processing.


Introduction
In clinical psychiatric medicine, clinicians take patients' various perceptions into consideration. Nociception is important perception that has harmful influence on daily life of human [1]. Pain symptoms with some physical and mental disorders has increasingly become a major social problem. In the psychiatric field, pain symptom is frequently emerged in somatoform pain disorder [2], major depressive disorder [3], neuropathic pain [4], and sleep-related disorder [5]. As to drug therapy of these diseases, serotonin noradrenalin reuptake inhibitor (SNRI), selective serotonin reuptake inhibitor (SSRI), tricyclic antidepressant (TCA) and anticonvulsant are prescribed [6]. Monoaminergic system of central nervous system (CNS) is thought to be involved in the etiology of those diseases. As to main pain management system, there are some descending pathways [7,8], which are called descending antinociceptive system (DAS). As main pathways of DAS, it is well known that the locus coeruleus (LC)spinal dorsal horn circuit includes the noradrenergic system, periaqueductal gray -rostral ventromedial medulla (RVM) -spinal dorsal horn includes serotonergic (5-HTergic) system [9,10]. Also, mesolimbic dopamine (DA) system affects regulating nociceptive activation [11,12]. Studies have demonstrated that monoaminergic pathways play roles in processing nociceptive information by electrophysiological methods. Activation of LC NA neurons and RVM serotonin (5-hydroxytryptamine, 5-HT) neurons in response to nociceptive stimuli have been indicated by some electrophysiological studies [13,14]. Also, ventral tegmental area (VTA) DA neurons play roles in regulating nociceptive information [12,15,16]. We recently certified that acute nociceptive stimuli rapidly increased the activities of LC noradrenalin (NA) neurons and RVM 5-HT neurons and VTA DA neurons in awake mice by using fiber photometry system [17,18].
5-HT has extensive innervation in CNS [19] and subsets of the 5-HT cells are designed as B1-B9 groups in a caudal to rostral direction [20]. The dorsal raphe nucleus (DR: B7 and B6), median raphe nucleus (MR: B8 and B5), and RVM (B3) are well known as major parts. However, supralemniscal (B9) 5-HT cell group located just dorsal to the medial lemniscus is less known [21] and has hardly been studied. In this study, we focused on the possible contribution of B9 5-HT system in pain processing because B9 5-HT cells project to LC and VTA [22], which are important nuclei in pain processing (see above). We have demonstrated fiber photometry system [17,18] for evaluating real time and cell type specific neuronal activity in awake mice with G-CaMP6 as a detector of Ca 2+ concentration in the neuron of interest. This system has high temporal resolution (< sec) and unaffected by metabolism as in microdialysis, a different character from other electrophysiological and chemical methods. Our study reported that acute nociceptive stimuli increased the activity of LC NA neurons or the activity of VTA DA neurons by adopting fiber photometry system. Therefore, we thought that it is meaningful to appraise the activities of B9-LC 5-HTergic pathway and B9-VTA 5-HTergic pathway in response to acute nociceptive stimuli.
We first introduced the G-CaMP6 in B9 5-HT neurons using transgenic mice carrying a tetracyclinecontrolled transactivator (tTA) transgene under the control of a tryptophan hydroxylase-2 (TPH2) promoter and site-specific injection of adeno associated virus (AAV-TetO(3G)-G-CaMP6). We confirmed the specific expression of G-CaMP6 in B9 neuronal cell body and axon located at the targeted sites (LC and VTA) using an immunohistochemical method. We measured the Ca 2+ signal of G-CaMP6 in these sites of awake mice while they were exposed to acute nociceptive stimuli.

Animals
The tryptophan hydroxylase-2 tetracycline-controlled transactivator (TPH2-tTA) transgenic mice were used [18,23,24] (Fig. 1a). We have already confirmed the specificity of TPH2-tTA expression in the previous report [24]. Ten to fourteen-week-old mice were used in this study. All mice were kept on a condition of 12 h light/dark cycle (7:00 AM to 7:00 PM), the temperature of 24 ± 1°C, food and water were available ad libitum. All efforts were made to minimize animal suffering and discomfort; to reduce the number of animals used. All experimental procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Use Committee of Kagoshima University (MD17090).

Stereotaxic AAV injection
AAV vector production was performed by AAV Helper-Free system (Agilent Technologies, Inc., Santa Clara, CA, USA); their purification was as previously described [17,25,26]. Mice were anesthetized with 2-3% isoflurane using a vaporizer for small animals and fixed with stereotaxic instrument (ST-7, Narishige, Tokyo, Japan) with an aid of supportive ear bar (EB-6, Narishige) of which touching surfaces to the animal were covered with local anesthetic jelly (lidocaine, 2% Xylocaine AstraZeneca). Both eyes were preserved with vaseline, head hair was shaved using an electric hair shaver, and cranial dura mater was cut open with small scissors. We slowly sucked up AAV into a glass micropipette (1B150F-3, World Precision Instrument, Inc., Sarasota, FL, USA), which was connected to a nitrogen pressure source through polyethylene tubing and to an injection manipulator (I-200 J, Narishige). In this study, AAV-TetO(3G)-G-CaMP6 (Serotype: DJ; 1 μl/injection, 4 × 10 13 copies/ ml) and AAV-TetO (3G)-mCherry (Serotype: DJ; 1 μl/injection, 6 × 10 12 copies/ml) (Ohkura et al., 2012) ( Fig. 1a) were unilaterally injected into B9 site (Injection site was from bregma − 4.36 mm, lateral + 0.38 mm left side, and ventral − 5.08 mm from the cranium) (Fig. 1b). After AAV injection, the micropipette was left in place for 10 min before being slowly withdrawn; mice were given an antibiotic, penicillin G (40,000 U kg-1) through subcutaneous injection. After operation, each mouse was individually kept for 14 days (2 weeks) in normal breeding conditions (as mentioned in Animals section) because it is needed for mice to recover and it takes about 2 weeks for G-CaMP6 or mCherry to fully express (Fig. 1a).

In vivo fiber photometry system
We showed fiber photometry system in previous reports [17,18,25,26]. In this study, we adopted the fiber photometry system with two channels (Fig. 2a). In the first channel setting, the high-power LED driver (LEDD1B/ M470F3, Thorlabs, Inc., Newton, NJ, USA) continuously produces blue excitation light (470 nm, 0.5 mW at the tip of the silica fiber) and the light passes through the excitation bandpass filter (475 ± 12.5 nm) and reflected by dichroic mirror-1; into a silica fiber (diameter: 400 μm, numerical aperture = 0.6). The same fiber detects and collects the green fluorescent signal of G-CaMP6. The signal passes through dichroic mirror-1 and reflected by dichroic mirror-2 and passes through the bandpass emission filter (510 ± 12.5 nm) and guided to a photomultiplier tube (PMTH-S1-1P28, Zolix Instrument, Beijing, China). At the second channel setting, the high-power LED driver continuously produces yellow excitation light (590 nm) and the light passes through the excitation bandpass filter (590 ± 12.5 nm) and goes forward as well. The same fiber detects and collects the red fluorescent signal of mCherry. The signal was forward and passed through the bandpass emission filter (607 ± 12.5 nm) and guided to another photomultiplier tube. First channel was adopted for detecting the neuronal activity and the second channel was used as an indicator of total stability of the fiber photometry system because mCherry fluorescence doesn't reflect neuronal activity [26]. Both signals were digitized by an A/D converter (PowerLab8/35, ADInstruments Inc., Dunedin, New Zealand) and recorded by Labchart version-7 software (ADInstruments Inc.). Signals were collected at a sampling frequency of 100 Hz.

Immunohistochemistry
To confirm AAV-induced expression of G-CaMP6 and mCherry in 5-HT neurons, after the experiments, mice were processed for immunostaining. Mice were deeply anesthetized with urethane (1.6 g/kg, i.p.) and transcardially perfused with 20 ml of phosphate buffered saline (PBS) and 20 ml of 4%-paraformaldehyde in PBS (Nacalai Tesque Inc., Kyoto, Japan). The brain was removed and post fixed in the same paraformaldehyde solution and soaked in 30% sucrose in PBS for 2 days. We formed serial 30 μm coronal sections including target sites (B9, LC, and VTA) with the cryostat (Cryotome FSE, Thermo Scientific, Yokohama, Japan). Every third section was adopted and floating immunohistochemical staining was performed. The sections were soaked in blocking  a Schematic representation of the fiber photometry system with two channels. b Target sites in this study: B9, LC and VTA. c The fluorescence signal intensity was abruptly increased when the position of the optical fiber tip was placed just above target site. d Schematic representation of the procedure of the recording. Fiber implantation was performed under isoflurane anesthesia. We set 3 h before the beginning of experimental sessions so that anesthesia would not affect experimental sessions. Total four stimuli (two kinds of acute nociceptive stimuli and two kinds of noninvasive control stimuli) were set in order from weaker stimulus to stronger stimulus as follows; the first is low temperature heat stimulus at 25°C, the second is a gentle touch, the third is acute heat stimulus at 55°C, and the last is acute mechanical tail pinch stimulus at the force of 400 g. Inter-stimulus interval was set as 30 min to reduce possible carryover effect from the previous stimulus guinea pig IgG (20,837,Biotium,1:200) in PBS for 2 h in a dark box. In some sections in LC, VTA, B9 were treated with anti-tyrosine hydroxylase (TH) antibody (AB152, Millipore, raised in rabbit, 1:500) and visualized with CF647 donkey anti-rabbit IgG (20,047, Biotium).
After incubation, the sections were washed once with PBS and mounted on microscope slides (PRO-02, Matsunami, Osaka, Japan) and covered with microcover glasses (C024601, Matsunami). We observed and imaged the sections under the fluorescence microscope (BZ-X700, Keyence, Osaka, Japan), and analyzed the images with Adobe Photoshop CC (Adobe Systems, Inc., San Jose, CA, USA). G-CaMP6 and mCherry were visible without immunostaining.

Acute nociceptive test
We applied two types of acute stress test showed in previous reports [17,18]. We applied acute tail pinch stimulus using a pinch meter (PM-201, Soshin-Medic, Chiba, Japan) and acute heat stimulus using a heating probe (5R7-570, Oven Industries, Inc., Mechanicsburg, PA, USA). Pinch stimulus was attached to the root of the tail for three seconds with a force of 400 g and heat probe set at 55°C was attached to the root of the tail for three seconds. We applied two noninvasive stimuli in the control group: gentle touch using a cotton stick and low temperature heat stimulus set at 25°C using the same heating probe (5R7-570).

Experimental protocol
In this study, we recorded G-CaMP6/mCherry green/red fluorescence intensity of B9 5-HT neuronal cell body and the axon located in LC/VTA (projecting sites of B9 5-HT neurons, Fig. 2b) to acute nociceptive stimuli. Each mouse was individually kept for at least 14 days after injection of AAV (Fig. 1a). The mouse was again anesthetized with 2-3% isoflurane using a vaporizer for small animals and fixed with stereotaxic instrument (ST-7) with an aid of supportive bar (EB-6) of which touching surfaces to the animal was covered with local anesthetic jelly (AstraZeneca). Following experiments were conducted in head-fixed condition. The head hair was shaved using electrical hair shaver; cranial dura mater was cut open with small scissors. A silica fiber was slowly implanted into the places just above B9 (bregma − 4.36 mm, lateral + 0.38 mm left, and ventral − 5.08 mm from the cranium), LC (bregma − 5.34 mm, lateral + 0.80 mm left, and ventral − 2.60 mm from the surface of the brain) and VTA (bregma − 3.15 mm, lateral + 0.50 mm left, and ventral − 4.15 mm from the surface of the brain) (Fig. 2b). We monitored the fluorescence signal intensity throughout fiber implantation and confirmed that the fluorescence signal intensity increased abruptly when the optimal position of the fiber tip was placed just above the target site (Fig. 2c). Open space around the optic fiber was covered with an ointment to avoid possible drying. After the fiber was fixed and positioned to the optimal position, anesthesia was turned off; each mouse recovered from anesthesia. We set 3 h before the beginning of experimental sessions so that anesthesia would not affect experimental sessions. The mice were divided into three groups; B9 group (n = 6), LC group (n = 6) and VTA group (n = 6). Experimental sessions of each mouse consist of two kinds of acute nociceptive stimuli and two kinds of noninvasive control stimuli (total four stimuli). To reduce the effect from the previous stimulus, we set inter-stimulus intervals at 30 min; their stimuli were set in order of weaker to stronger stimulus as follows; the first is low temperature heat stimulus at 25°C, the second is a gentle touch, the third is acute heat stimulus at 55°C; the last is acute mechanical tail pinch stimulus at the force of 400 g (Fig. 2d). After experiments, mice were euthanized and processed for immunostaining.
The definition of neuronal activity characteristic index was set as follows: F: averaged fluorescent signal intensity value for three seconds just before each stimulus and defined as 100%; ΔF: (maximum fluorescent signal intensity value during each stimulus) -F; onset latency: time from the start of stimulus to the time when the fluorescence signal intensity exceeded the maximum value during the baseline period; peak latency: time from the start of stimulus to the time when the fluorescence signal intensity reach the maximum value.

Statistical analysis
Data analysis was conducted by two-way analysis of variance (ANOVA) with Sidak's test for post hoc analysis. Two factors on ΔF/F were modality (mechanical vs. thermal) and intensity (nociceptive vs. gentle control). Factors on latency were modality (mechanical vs. thermal) and brain area (B9, LC, or VTA). Values are expressed as the mean ± standard error of the mean (S.E.M). Probability values less than (p < 0.05) were considered statistically significant. The analyses were performed using GraphPad Prism version 7 (GraphPad software, San Diego, CA, USA).

Restricted expression of AAV-induced G-CaMP6/mCherry
Specific expressions of G-CaMP6/mCherry were confirmed in B9 5-HT neuronal soma (Fig. 3a, b). We found 23.3 ± 1.8 (n = 6, one representative slice per mouse) TPH-positive cells in the B9 and 88.3% of them also expressed G-CaMP6. All of the G-CaMP6 positive cells also expressed mCherry and 94.8% expressed TPH. Although anti-TPH antibody we used (AB1541) sometimes binds TH, distribution of anti-TH-positive structure in B9 did not overlap with that of G-CaMP6 (Fig. 3c). These expressions were also confirmed in B9 5-HTderived axons located in LC ( Fig. 4a and b) and VTA ( Fig. 4c and d). G-CaMP6/mCherry positive soma and axons were hardly observed outside SERT positive structures ( Fig. 4a and c). In LC and VTA, G-CaMP6/ mCherry double positive fibers were found not only near TH positive cell body (white rectangles in Fig. 4b and d) but also TH negative areas (yellow rectangles) within the nucleus. Therefore, fluorescence was detected in specific manner in B9/LC/VTA sites.
On the other hands, mCherry fluorescent intensity in B9 group and LC/VTA groups was not significantly different between stimulation intensities (nociceptive vs. gentle) and between modalities (mechanical and thermal) (B9 group intensity: When comparing response characteristics, onset latency was significantly different among 3 brain areas (F(2, 15) = 57.19, p < 0.001) and also between modality (F(1, 15) = 19.77, p = 0.0005). Sidak's multiple comparison revealed that onset latency in B9 was significantly  n = 6). c G-CaMP6/mCherry double positive cells did were not stained with anti-TH antibody immunostaining. Scale bar length:100 μm shorter than that in LC and VTA in both pinch and heat stimuli (Fig. 7a).

Discussion
The results of this study clearly demonstrated that acute nociceptive stimuli rapidly affected the activity of B9 5-HT neuronal cell bodies and B9 5-HT nerve axons located in LC and VTA in conscious mice adopting fiber photometry system. Recent tracer studies revealed B9-LC/B9-VTA 5-HT neuronal pathways [22]. B9 5-HT cell group comprises approximately 20% of the total mesopontine 5-HT neurons [21,27], nevertheless has been much less studied compared to the wealth of studies on the DR, MR, and RVM groups. To our knowledge, our data using the fiber photometry system are the first report that measured the activities of B9 5-HT neurons during aversive stimuli and that showed possible role of B9 5-HT neurons in pain processing.
In addition, this is the first report that measured the activities of B9 5-HT nerve axons located in LC and VTA. The present results showed that the activity of B9-LC 5-HT pathway and B9-VTA 5-HT pathway were rapidly increased by acute nociceptive stimuli. The results of onset latency showed that in B9 was significantly shorter than those in LC or VTA in both pinch and heat stimuli (Fig. 7a). This result was in line with our hypothesis that the activities of B9 5-HT neuronal soma propagate to LC and VTA through B9 5-HT-derived axons (Fig. 8). Our previous studies using fiber photometry system showed that acute nociceptive stimuli rapidly increased the activities of LC NA neurons and VTA DA neurons [17,18]. Some studies have reported the activation of LC NA neurons using microdialysis [14] or electrophysiological recording [28,29]. Other studies have reported that nociceptive stimuli affected mesolimbic DA system [30,31] and mesocortical DA system [32,33]. In this regard, it is considered that B9 5-HT neuronal projection to LC affected the activity of LC  (Fig. 4b) and the DA neurons in VTA (Fig. 4d). Although we have revealed possible flow of pain information from B9 to LC and VTA, we need more studies to reveal physiological importance and impact of this pathway in pain regulation.
We confirmed the expression of G-CaMP6/mCherry in B9 5-HT neurons in TPH2-tTA mice injected with AAV-tetO-GCaMP6/mCherry by immunohistochemical method. In a previous study, we showed the expression of G-CaMP6/mCherry in RVM/DR 5-HT neurons in TPH2-tTA mice using the same AAV [18]. Our present results coincide with the previous studies showing dense collections of 5-HT cells in B9 [21,34]. Taken together, our method using AAV seemed applicable to study activity of any 5-HT neurons in the CNS.
Emerging evidence has pointed to anatomical and functional heterogeneity within the brainstem 5-HTergic cell groups [35,36]. Nevertheless, the function of B9 5-HT neurons has been largely unknown, except for studies reporting that B9 5-HT neurons played prominent roles in shaping aggression [37] and regulating affect and stress responsivity [36,38]. DAS, including LC, is considered to be involved in pain-emotion symptoms [28,39] and VTA involved in mood symptom [40,41] and fear [42]. An anatomical study demonstrated that B9 5-HT neurons project to the hypothalamus, cortex, and hippocampus that are related to some psychiatric symptoms [43]. Taking these observations together, our results suggest that B9-LC/B9-VTA 5-HT neuronal pathways may be related to pain-emotion symptoms.
In clinical psychiatric medicine, the center of therapeutic strategy is drug therapy and many psychiatric-related drugs affect the state of monoamines in CNS. SNRI, SSRI and TCA for pain treatment act on synapses of 5-HT neurons in CNS and it takes at least a few weeks to relieve pain symptoms [44]. In case of patients in the psychiatric field with pain symptoms receiving these drug therapy, the state of neuronal activity in a Limitations in this study are as follows. We did not adopt psychiatric-related drugs, therefore did not appraise how these drugs affect the activities of B9 5-HT neurons. Also, our protocol is only acute nociceptive system. Therefore, in the future, the protocol including psychiatric-related drugs and chronic nociceptive system will be adopted. Fiber photometry system measures activities of ensemble average of the labeled neurons but not single unit activity. Combination of advantages of multiple methodologies are needed.
In conclusion, the results of this study suggest that acute nociceptive stimuli cause a rapid increase in the activities of B9-LC/B9-VTA 5-HTergic pathways and suggest that B9 5-HT neurons play important roles in nociceptive processing in the CNS.
Additional file 1: Figure S1. Confirmation of fiber implantation tracking. Fiber track was located just above B9, LC and VTA.