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Anterior nucleus of paraventricular thalamus mediates chronic mechanical hyperalgesia

Chang, Ya-Tinga,b; Chen, Wei-Hsinb; Shih, Hsi-Chienb; Min, Ming-Yuanc; Shyu, Bai-Chuangb; Chen, Chien-Changa,b,*

doi: 10.1097/j.pain.0000000000001497
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Pain-related diseases are the top leading causes of life disability. Identifying brain regions involved in persistent neuronal changes will provide new insights for developing efficient chronic pain treatment. Here, we showed that anterior nucleus of paraventricular thalamus (PVA) plays an essential role in the development of mechanical hyperalgesia in neuropathic and inflammatory pain models in mice. Increase in c-Fos, phosphorylated extracellular signal–regulated kinase, and hyperexcitability of PVA neurons were detected in hyperalgesic mice. Direct activation of PVA neurons using optogenetics and pharmacological approaches were sufficient to induce persistent mechanical hyperalgesia in naive animals. Conversely, inhibition of PVA neuronal activity using DREADDs (designer receptors exclusively activated by designer drugs) or inactivation of PVA extracellular signal–regulated kinase at the critical time window blunted mechanical hyperalgesia in chronic pain models. At the circuitry level, PVA received innervation from central nucleus of amygdala, a known pain-associated locus. As a result, activation of right central nucleus of amygdala with blue light was enough to induce persistent mechanical hyperalgesia. These findings support the idea that targeting PVA can be a potential therapeutic strategy for pain relief.

Paraventricular thalamus is a potential target for pain treatment. Paraventricular thalamus activation elicits mechanical hypersensitivity in naive mice. Conversely, inhibiting paraventricular thalamus activity abolishes the existing chronic pain.

aTaiwan International Graduate Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, Taiwan

bInstitute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

cDepartment of Life Science, Center for Neurobiology and Cognition Science, Institute of Zoology, National Taiwan University, Taipei, Taiwan

Corresponding author. Address: Institute of Biomedical Sciences, Academia Sinica, 128 Academia Rd Sec 2, Nankang, Taipei 11529, Taiwan. Tel.: 886-2-2652-3522. E-mail address: ccchen@ibms.sinica.edu.tw (C.-C. Chen).

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.painjournalonline.com).

Received February 11, 2018

Received in revised form December 12, 2018

Accepted December 14, 2018

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1. Introduction

Acute pain serves as an early-warning protective system for individuals to avoid noxious and harmful stimuli. In contrast, chronic pain is often no longer protective.6,52 Overall, 20% to 25% of the world's population has chronic pain.22,26 Pain-related diseases are among the top 10 leading causes of life disability.36 Given the impact of chronic pain on the quality of life, the unmet demand for pain management urges a better understanding of pain perception and chronic pain development.

Nociceptive signals that may be perceived as painful are conveyed by the peripheral nerve system (PNS) to the spinal cord or brainstem and then transduced into the brain. Chronic pain syndromes of different etiologies can activate neurons with distinct supraspinal projections and thus alter brain activities at diverse loci despite sharing several commonalities.1,3,21,43 Chronic pain is often accompanied by persistent high neuronal reactivity (usually hyperexcitability) in the central nervous system (CNS). This central sensitization53 is responsible for lowered threshold, increased response to stimuli, and enlarged projection field. Multiple brain regions, including the thalamus, are sensitized in people with chronic pain and experimental animals.3,32,38,53

The thalamus is an important brain region involved in nociceptive brain circuitry.1,33,52 Thalamic metabolites, structure, and neuronal activity are altered in individuals with pain and rodent pain models.21,24 Several thalamic subregions are involved in the pain pathway. Ventral posteromedial and ventral posterolateral thalamus neurons project to the primary sensory cortex for pain discrimination.16 The dorsal medial thalamus (MD) receives projections from cortex layer 5 and then projects the signals to associated regions, such as the anterior cingulate cortex (ACC).55 The thalamic reticular nucleus is involved in inhibitory regulation of the pain pathway.35,50

The anterior nucleus of the paraventricular thalamus (PVA) is located in the anterior midline thalamus. The midline thalamus receives various inputs and conveys integral signals to the limbic system for regulating physiological function.50 Anatomically, the PVA is also connected to regions involved in pain perception, including the ACC, central nucleus of amygdala (CeA),57 parabrachial (PB) area, and periaqueductal gray (PAG).29,39,41 We reported that Cav3.2-dependent activation of extracellular signal–regulated kinase 1/2 (ERK1/2) in the PVA modulates the initiation of acid-induced chronic muscle pain in mice.14,15 However, whether the PVA is a common locus involved in chronic pain of different etiologies is unclear. Clinically, many chronic pain disorders share the pathological features of neuropathic and inflammatory pain.31 Therefore, we tested whether the PVA is involved in neuropathic and inflammatory pain in mice. Here, we provide evidence to support the finding that PVA neuronal activity is essential for the development of persistent mechanical hyperalgesia in both neuropathic and inflammatory pain models.

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2. Materials and methods

2.1. Animals and pain models

All research conformed to the National Institutes of Health guidelines in accordance with the guidelines specified by the Institutional Animal Care and Utilization Committee, Academia Sinica. Female C57B6 mice of 8 to 12 weeks old were used. We used only female mice because clinical studies show higher pain prevalence in female than in male mice.19 All mice were housed in specific pathogen-free conditions in the Institute of Biomedical Sciences, Academia Sinica. Two animal models were chosen to induce mechanical hyperalgesia: (1) Formalin model: 10 μL of 5% formalin in phosphate-buffered saline (PBS) was injected subcutaneously into the plantar surface of the left hind paw9,10; (2) spared nerve injury (SNI) model45: the left common peroneal and sural nerves were dissected, and the tibial nerve was left intact.

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2.2. Behavioral testing

The nociceptive behavior categories included flicking, lifting, and licking of the paw by the tested animal. Mice were selected randomly and tested blindly to pharmacological treatments or viral infections. After the working solutions were prepared, the vehicle and working solution were placed in a new container and labeled numerically by another researcher. The name cards of virally infected mice were changed by another researcher before submission for further experiments. The tester confirmed the treatment when the whole set of experiments was conducted. Experiments were conducted at room temperature (approximately 22°C), and the stimuli were applied only when the animals were calm but not sleeping or grooming.

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2.2.1. Spontaneous behavior

The occurrence of formalin-induced nociceptive behavior was recorded at the end of every 5 seconds (instantaneous sampling). The total events in every 5 minutes were accumulated for 1 hour. Successful induction of spontaneous behavior was monitored as a criterion for hyperalgesia experiments.

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2.2.2. Evoked nociceptive behavior

2.2.2.1. Mechanical hyperalgesia

Measurement of the mechanical withdrawal response of experimental mice involved using von Frey filaments as described previously.13 In brief, mice were habituated for at least 30 minutes before testing. Von Frey filaments with bending force from 0.16 to 2 g were applied in a progressively increasing manner. Then, a von Frey monofilament with 1-g bending force was selected for hyperalgesia experiments. The withdrawal frequency was calculated by the number of times the hind limb withdrew in 10 applications of 1-g-force monofilament. The interval between each monofilament applicant was at least 30 seconds until the mouse placed its 4 paws on the matrix evenly. Withdrawal frequency (%) = (times of withdrawal response/10 applications) × 100%. The average withdrawal frequency of hind limbs was calculated as a readout in the formalin model and naive mice experiment, including optogenetics and phorbol 12,13-dibutyrate (PDBu) infusion experiments. For the SNI model, the withdrawal frequency of each hind limb was calculated separately. The original values of the withdrawal ratio for all figures are in the supplementary table (available at http://links.lww.com/PAIN/A738).

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2.2.2.2. Thermal hyperalgesia

After the end of the von Frey test, mice were tested for thermal hyperalgesia on a 50°C thermal plate, and the withdrawal latency(s) was measured.

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2.2.2.3. Blue light–evoked mechanical hyperalgesia

At 5 to 6 weeks after viral infection, a 23-G guide cannula was implanted above the PVA, targeting the dorsal third ventricle (D3V). At 2 days after cannulation, animals were tested for mechanical sensitivity. With the animal briefly anesthetized with vaporized isoflurane, the optical fiber (UM22-200, 0.22NA. ∅200 μm; Thorlabs, Newton, NJ) connected to a Finer-Optic Rotary Joint directly linked to an light emitting diode driver (Doric Lenses, Quebec, Canada) was inserted into the cannula. The mouse was placed back in the same cubicle and received blue light stimulation (10 Hz; 50-millisecond [ms] pulse width; 6.9 mW) for 4 minutes. Mechanical sensitivity was tested during blue light stimulation. For repeated blue light stimulation, mice received blue light simulation for 5 minutes every consecutive day.

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2.3. Electrophysiology and blue light stimulation

2.3.1. In vivo electrophysiological recordings

Extracellular field potentials and the multiunit activities in PVA with sciatic nerve stimulation (SNS) were conducted as described (Cheng et al., 2017). In brief, the multichannel electrode probe (NeuroNexus, Ann Arbor, MI) was loaded in the PVA (posterior: 0.3 mm; left: 0.7 mm to the bregma) at 12° from the vertical line in anesthetized mice (1.5% isoflurane). A cuffed electrode was hooked on the left sciatic nerve to generate the contact current. The stimulation threshold was determined by a minimal pulse to induce toe prickling, and the applied current was 1- to 20-fold of the threshold. The component elicited by at least 5-fold of threshold was regarded as nociceptive response.11 After recording PVA multiunit activities, the optical fiber was loaded into the D3V (left: 1 mm; 3.2 mm ventral to the bregma) of adeno-associated virus–expressed humanized channelrhodopsin H134R mutant (AAV-hChR2)–infected mice at 15° from the vertical line. Five pulses of 5, 10, or 20 Hz blue light stimulation were applied. The sampling rate was 6 and 24 kHz for field potential and sweep spike unit, respectively. Data were processed in a multichannel data acquisition system (TDT, Alachua, FL). One of the 16 contact points of the electrode was amended as a lesion channel. The exact insertion site was identified by 2 electrical lesions (10-second pulse of 25 μA applied current). Post hoc Nissl or 4′,6-diamidino-2-phenylindole (DAPI) staining was used for identifying the lesion cites.

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2.3.2. Whole-cell recordings

At 6 to 8 weeks after intra–PVA AAV-hM4D or AAV-eYFP infection, mice were anesthetized with 1.5% isoflurane. Brains were quickly removed and immersed in the ice-cold artificial cerebrospinal fluid (in mM: 119 NaCl, 2.5 KCl, 26.2 NaHCO3, 1 NaH2PO4, 1.3 MgSO4, 11 glucose, and 2.5 CaCl2, pH 7.4, gassing with 5% CO2/95% O2). Slices of 300-μm thick were cut with a vibrating tissue slicer (D.S.K. Microslicer DTK-1000; Dosaka EM, Kyoto, Japan), then allowed to recover at room temperature (24°C-25°C) for 1.5 hours. We applied the current at the beginning of the recording to induce spontaneous action potential firing. Action potentials were recorded before and after 10 μM clozapine-N-oxide (CNO) perfusion using a glass pipette with a patch amplifier (Multiclamp 700 B; Axon Instruments, Molecular Devices, San Jose, CA) at room temperature (internal solution, in millimoles: 131 K-gluconate, 20 KCl, 10 HEPES, 2 EGTA, 8 NaCl, 2 ATP, 0.3 GTP, and 6.7 biocytin, 300-305 mOsm, pH 7.2). Data were discarded when the Rs varied by >20% from its original value during the recording. All signals were low pass filtered at a corner frequency of 1 kHz and digitized at 10 kHz using a Micro 1401 interface (Cambridge Electronic Design, Cambridge, United Kingdom). Data were collected with the use of Signal software (Cambridge Electronic Design).

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2.4. Drug administration and viral infection

The utility of PDBu, CNO, U0126, and U0124 was as previous described.15 Adeno-associated virus was obtained from the UNC Vector Core, University of North Carolina School of Medicine. In brief, 0.4 mM PDBu, 10 mM U0126, and 10 mg/mL CNO were dissolved in 100% dimethyl sulfoxide (DMSO) as stocks. Before being given to animals, drugs were diluted in saline to a concentration of 0.2 mM PDBu and 5 mM U0126/U0124, and 0.5 mg/mL CNO. Clozapine-N-oxide was diluted in H2O to a 1-mM concentration for whole-cell recording. An amount of 20 pmol PDBu, 1.5 nmol U0126 or U0124, or 0.2 μL AAV: rAAV5/CaMKII-hChR2(H134R)-enhanced Yellow Fluorescent Protein (eYFP)-EPRE (qPCR titer: 8.5 × 1012 vg/mL), rAAV8/CamKIIa-hM4D-mCherry (Dot Blot titer: 3.3 × 1012 vg/mL), rAAV5/CaMKII-eYFP (qPCR titer: 6.3 × 1012 vg/mL) was infused into the PVA (posterior 0.3 mm, ventral 3.5 mm to the bregma) directly or 2 days after cannulation. Clozapine-N-oxide was administered intraperitoneally (i.p.) to mice (3 mg/kg).

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2.5. In vivo transfection and neuron tracing

Expression of wheat germ agglutinin (WGA) protein in the brain can be used as an anterograde transneuronal tracer.7 We transfected 0.2 μL of 1 μg/μL of the plasmid CMV-WGA-mCherry in the right CeA (posterior 2.6 mm, right 1.7 mm, ventral 4.5 mm to the bregma) using T-Pro G-Fect Transfection Reagent (JT97-N004; T-Pro Biotechnology, New Taipei County, Taiwan) for 5 days.

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2.6. Immunostaining and cell counting

After mice were deeply anesthetized; PBS was perfused transcardially and fixed with 4% paraformaldehyde in pH 7.6 PBS in a sound-controlled environment. Brains were post-fixed overnight and stored at 4°C in 30% sucrose. Continual cryosections of 20- or 50-μm thick were mounted on slides for immunofluorescence staining. For phosphorylated ERK1/2 (pERK1/2) staining, a heat-induced epitope retrieval procedure with pH 6.4, 0.01M sodium citrate was required. Primary antibody against pERK42/44 (1:100, 4377; Cell Signaling, Danvers, MA), NeuN (1:500, MAB377; Millipore, Burlington, MA), or c-Fos (1: 400, sc-52 or sc-52 G; Santa Cruz Biotechnology, Dallas, TX) dissolved in blocking solution (3% normal goat serum or 5% bovine serum albumin) was applied overnight at 4°C. Continual paraffin sections of 5-μm thick were processed for immunohistochemical staining against pERK42/44 as described.13 Images were captured under a Zeiss Imager A1, Olympus microscope or Zeiss LSM700 confocal scanning laser microscope and analyzed using ZEN software. c-Fos–positive cells in PVA regions at 0.3, 0.4, 0.5, and 0.6 mm posterior to the bregma were counted using Image J, and the summation is shown in Figure 1. Paraventricular thalamus pERK1/2-positive cells were the average of positive cells counted from the PVA region at 0.3, 0.4, and 0.5 mm posterior to the bregma. For optogenetics stimulation and chemogenetic experiments, c-Fos–positive or pERK-positive cells were counted from PVA region at 0.4 to 0.5 mm posterior to the bregma using Image J.

Figure 1

Figure 1

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2.7. Statistical analysis

Paw withdrawal frequency and multiunit activities for different treatments across time and between groups were tested first by 1-way repeated-measures analysis of variance, followed by a posthoc test with the Holm-Sidak method between groups and paired t test across time. P < 0.05 was considered statistically significant. Data are presented as mean ± SEM.

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3. Results

To explore the role of the PVA in central sensitization in chronic pain, we first investigated any change in PVA neuronal activities by detecting c-Fos protein level, pERK1/2, and PVA neuronal activity responding to SNS in both formalin-induced inflammatory pain and SNI-induced neuropathic pain models.

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3.1. Neuronal activities increased in paraventricular thalamus in formalin-induced inflammatory pain model

Subcutaneous injection of formalin in the left hind paw of mice induced biphasic spontaneous responses (Fig. 1A) and significantly increased mechanical hypersensitivity lasting for at least 1 week9,20 (Fig. 1B). Nociception is the neural process of encoding noxious stimuli. If the PVA is truly involved in nociceptive circuitry and pain perception, we should be able to record PVA neuronal activity when the nociceptive inputs are transduced from the PNS. Thus, we recorded PVA neuronal activity with a multichannel electrode probe in naive and formalin-treated anesthetized mice with SNS46 (Fig. 1C). The exact channels inserted into the PVA (purple line in Fig. 1C) were identified by post hoc histological examination of lesion marks (Fig. 1D). Paraventricular thalamus multiunit activity responded to SNS in a strength-dependent manner (Fig. 1C, supplementary Fig. 1B, available at http://links.lww.com/PAIN/A738). These SNS-activated units were recorded in the PVA region mostly from 0.58 to 0.34 mm posterior to the bregma (supplementary Fig. 1A, available at http://links.lww.com/PAIN/A738). The evoked PVA multiunit sweep spike can be classified into 2 components: fast-responding component (fc) and late-responding component (lc) (Fig. 1E). fc spike units initiated from 100 to 400 ms and peaked from 200 to 300 ms after SNS in naive mice, whereas lc responded to SNS later than 400 ms. An increase in PVA neuronal activity under SNS in naive mice indicates that the PVA is involved in the nociception pathway. At 4 to 7 days after formalin treatment, a responding component reacting within 100 ms was observed when the stimulus strength was greater than ×5 threshold (Figs. 1C and E, supplementary Fig. 1B, available at http://links.lww.com/PAIN/A738). The units in fc increased in a strength-dependent manner in both naive and formalin-treated mice (Fig. 1C; supplementary Fig. 1B, available at http://links.lww.com/PAIN/A738). In formalin-treated mice, the summation of evoked units reacting within 400 ms after SNS started to show an increasing trend under the ×5 threshold and increased significantly under ×10 threshold SNS as compared with naive mice (Fig. 1F; supplementary Fig. 1B, available at http://links.lww.com/PAIN/A738). An increase in the multiunit response and the altered response pattern on multiunit recording in formalin-treated mice revealed a switch of firing pattern and enhanced neuronal excitability, which indicates a neuronal plasticity change in the PVA in formalin-induced hyperalgesic mice.

If PVA activity is involved in persistent hypersensitivity, we should be able to detect c-Fos protein level change compared with the healthy control8,42 at the late phase after pain induction. At 1 week after formalin injection, c-Fos–positive neurons increased in number and distributed from 600 to 300 μm posterior to the bregma in the PVA, with only a few c-Fos–positive cells observed in the PBS control (Figs. 1G, a, b; supplementary Fig. 2A, available at http://links.lww.com/PAIN/A738). Increased c-Fos activity was also detected at an earlier time point: 2 days after formalin injection (supplementary Fig. 2B, available at http://links.lww.com/PAIN/A738). These data suggest that PVA neurons are activated during chronic pain progression.

During central sensitization, several signaling pathways play a role in mediating neuronal plasticity and circuitry rewiring in the pain pathways in the CNS, including the ERK pathway.31,53,54 We further tested whether PVA ERK1/2 activity, as indicated by pERK1/2, was involved in central sensitization in the formalin model. Phosphorylated ERK1/2 signals have been shown in the CeA at 3 hours after formalin injection.10 Here, we found increased pERK1/2 signals in the PVA at 3 hours and 3 days after formalin injection (Fig. 1H). These results indicate the involvement of PVA pERK1/2 activity during chronic pain progression in the formalin model.

In conclusion, PVA neuronal activity increased in response to SNS in naive mice, so the PVA is involved in the nociception pathway. Increased PVA neuronal excitability, a change in the multiunit response pattern, and increased c-Fos and pERK1/2 protein level in mice with formalin-induced persistent mechanical hyperalgesia indicate that PVA activity is involved in central sensitization in the formalin model.

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3.2. Neuronal activities increased in paraventricular thalamus in spared nerve injury–induced neuropathic pain model

We next tested whether the PVA is also involved in the SNI-induced neuropathic pain model. Spared nerve injury induces a unilateral mechanical neuropathic pain that lasts for several weeks,45 whereas the sham operation induced a modest postsurgical hypersensitivity for 2 days (Fig. 2A). At 2 weeks after SNI, multichannel recording of PVA neurons showed an increase in the multiunit response in fc and a new response component recording within 100 ms in the PVA in mice with SNI-induced persistent mechanical hyperalgesia similar to formalin-treated mice.15 This finding demonstrates a switch of firing pattern and enhanced neuronal excitability, indicating a neuronal plasticity change in the PVA in both formalin and SNI pain models.

Figure 2

Figure 2

The number of c-Fos–positive neurons in the PVA from 600 to 300 μm posterior to the bregma was higher at 2 weeks after SNI surgery than after sham operation (Fig. 2B; supplementary Fig. 2A, available at http://links.lww.com/PAIN/A738). Also, c-Fos signals were increased at the earlier time point after pain induction: 3 days after SNI (supplementary Fig. 2B, available at http://links.lww.com/PAIN/A738). Furthermore, pERK1/2 signals in the PVA were significantly higher in the SNI group than in the sham group at 3 days after surgery (Fig. 2C). These results support the involvement of PVA neuronal activity in progression of chronic mechanical hyperalgesia in both SNI and formalin models. As well, ERK1/2 signals might play a role in perception of PVA-mediated nociception in both inflammatory and neuropathic pain models.

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3.3. A time window to blunt existing chronic mechanical hyperalgesia by inhibiting paraventricular thalamus extracellular signal–regulated kinase activity

Although our findings suggest the involvement of PVA ERK1/2 activity in the development of mechanical hyperalgesia, the role of PVA ERK1/2 activity during chronic pain progression is unclear. Thus, we examined whether inhibiting ERK1/2 activity could alleviate the pain-like behavior at different stages in formalin- and SNI-induced mechanical hyperalgesia. In the formalin model, we infused U0126, an MEK1/2 inhibitor, or its inactive structural analogue U0124 into the PVA at 2 hours (2H), 1 day (1D), 2 days (2D), 3 days (3D), or 5 days (5D) after formalin injection. Intra-PVA infusion of U0126 but not U0124 at 2H after formalin injection attenuated the formalin-induced mechanical hyperalgesia (Fig. 3A1) and pERK1/2 signals in PVA at 3 hours after formalin injection (supplementary Fig. 3A, available at http://links.lww.com/PAIN/A738). This reduction in mechanical withdrawal frequency lasted for only several hours. The withdrawal response increased and did not differ between U0126 and U0124 control groups at 6 hours after U0126 infusion (Fig. 3A2). U0126 but not U0124 blunted the formalin-induced existing mechanical hyperalgesia when infused at 2D after formalin injection (Fig. 3A3). Also, infusion of U0126 at 1D, 3D, or 5D after formalin injection attenuated the formalin-induced mechanical hyperalgesia to a lesser extent than with 2D infusion (Fig. 3A4; supplementary Fig. 4A1, A2, A3, available at http://links.lww.com/PAIN/A738). Neither U0126 nor U0124 had any effect on thermal hyperalgesia induced by formalin injection (supplementary Fig. 5A, B, available at http://links.lww.com/PAIN/A738). We summarized the withdrawal frequency measured at day 7 after formalin injection from each group receiving intra-PVA U0126 infusion at various times (Fig. 3A4). At the 2D time window after formalin, chronic mechanical hyperalgesia was blocked and the withdrawal response was reduced to the basal level by inhibiting ERK1/2 activity in the PVA.

Figure 3

Figure 3

In SNI models, intra-PVA infusion of U0126 15 minutes before surgery induced a transient reduction of SNI-induced mechanical hyperalgesia without affecting the development of chronic mechanical hyperalgesia (Fig. 3B1). Of note, pretreating the PVA with U0126 abolished the modest postsurgical hyperalgesia induced by sham operation (Fig. 3B1). We also infused U0126 into the PVA at 4 hours (4H), 2D, 3D, 5D, and 10 days (10 D) post SNI. Inhibiting PVA ERK1/2 activity 4H after surgery alleviated the mechanical response only transiently (supplementary Fig. 4B1, available at http://links.lww.com/PAIN/A738). Intra-PVA infusion of U0126 but not U0124 at 3D post SNI abolished the SNI-induced existing mechanical hyperalgesia and the effect lasted for at least 11 days (Fig. 3B2). To a lesser extent, intra-PVA U0126 infusion at 2D, 5D, or 10D also attenuated withdrawal frequency persistently (supplementary Fig. 4B2, B3, B4, available at http://links.lww.com/PAIN/A738). The SNI-induced mechanical hyperalgesia was not affected by infusion of the same dose of U0126 into the D3V, nor was the off-site control targeting the barrel cortex when U0126 was infused at 3D after SNI (supplementary Fig. 6A, 6B, available at http://links.lww.com/PAIN/A738). We summarized the withdrawal frequency measured at day 14 post SNI for each U0126 treatment group: 3D after SNI was the best time window to blunt SNI-induced existing mechanical hyperalgesia with 1 dose of intra-PVA U0126 infusion (Fig. 3B3). PVA pERK1/2 signals decreased after the end point of the behavior test when U0126 was infused at the time window in both models (supplementary Fig. 3B, available at http://links.lww.com/PAIN/A738). These results demonstrate a critical time window to blunt chronic mechanical hyperalgesia in both formalin and SNI models by inhibiting ERK1/2 activity in PVA.

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3.4. Inhibition of paraventricular thalamus neuron with DREADDs alleviates spared nerve injury–induced neuropathic pain and formalin-induced inflammation pain

Next, we examined whether inhibiting PVA neurons could attenuate chronic neuropathic pain and inflammatory pain. We used the technique of designer receptors exclusively activated by designer drugs (DREADDs) to inhibit PVA neuronal activity remotely using the AAV-CaMKII-hM4D-mCherry vector.2 To control for any off-target effect or any effects caused by the metabolic conversion of CNO to clozapine,23 we conducted the same experiments using a control eYFP (AAV-CaMKII-eYFP) vector expressed in the PVA. The expression of hM4D was visualized by fluorescent mCherry signal in the PVA 4 weeks after infection (Fig. 4A1). Adeno-associated virus–mediated expression of hM4D or eYFP had no effect on the basal mechanical response (supplementary Fig. 7B, available at http://links.lww.com/PAIN/A738). To confirm that activation of hM4D-expressing PVA (hM4D-PVA) neurons inhibits PVA neuronal activity, we recorded hM4D- and eYFP-expressing PVA (eYFP-PVA) neurons on brain slices in the presence of CNO, an hM4D agonist, 5 minutes after 10 μM CNO perfusion; the spontaneous action potentials of hM4D-PVA neurons slowed down and eventually stopped. Clozapine-N-oxide did not reduce the neuronal activity recorded from eYFP-PVA neuron (Fig. 4A2).

Figure 4

Figure 4

We then examined whether DREADDs-mediated inhibition of PVA neurons could attenuate chronic mechanical hyperalgesia in formalin and SNI models. The diagrammatic representation of vial injection sites within PVA was shown in supplementary Fig. 8A (available at http://links.lww.com/PAIN/A738). In the formalin model, i.p. administration of 3 mg/kg CNO at 2H after formalin injection (Fig. 4A3) led to a transient reduction of withdrawal frequency at 2 hours and lasted for 4 hours. We also activated hM4D with CNO to inhibit hM4D-PVA neurons at 2D post formalin injection, the time point to blunt chronic mechanical hyperalgesia when targeting the PVA ERK1/2 pathway. The withdrawal responses were decreased to the basal level in at 3 hours post CNO administration in the hM4D-PVA group but not in the eYFP-PVA group (Fig. 4A4, insert). Surprisingly, one dose of CNO alleviated the formalin-induced existing mechanical hyperalgesia (Fig. 4A4). In the SNI model, i.p. administration of CNO at 4H post SNI (Fig. 4B1) or 3D post SNI (Fig. 4B2) led to a transient or sustained inhibition of withdrawal frequency in the hM4D-PVA but not in the eYFP-PVA group. One dose of CNO alleviated the SNI-induced existing mechanical hyperalgesia for up to 11 days (Fig. 4B2). One reason for this long-lasting effect may be that inhibiting PVA neurons with hM4D at critical time points interrupted PVA ERK1/2 activity and abolished the chronic mechanical hyperalgesia induced by formalin and SNI surgery. Thus, we examined PVA pERK1/2 signals after behavior tests (Fig. 5). The pERK1/2 signals were lower for the hM4D-expressing than for the eYFP-expressing PVA neurons in both formalin (Fig. 5A) and SNI (Fig. 5B) models.

Figure 5

Figure 5

Thus, silencing PVA neuronal activity with Gi-coupled DREADDs efficiently attenuated existing mechanical hyperalgesia in neuropathic and inflammatory pain models. Inhibiting PVA neuronal activity at the critical time window could inhibit chronic mechanical hyperalgesia persistently. Taken together, these results suggest that PVA is a potential target for chronic pain relief.

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3.5. Repeated activation of the protein kinase C-extracellular signal–regulated kinase pathway in the paraventricular thalamus induces chronic mechanical hyperalgesia in naive mice

We reason that if ERK1/2 activity is essential for PVA sensitization and the development of chronic mechanical hyperalgesia, sustained behavioral alteration could be induced by direct activation of ERK1/2 signaling without peripheral insults. To test this idea, we used PDBu, a protein kinase C (PKC) agonist, to activate ERK1/2 in the PVA and then examined mechanical hyperalgesia of naive mice. Intra-PVA infusion of PDBu (20 pmole) led to ERK1/2 phosphorylation in PVA but not in amygdala (Fig. 6A). A single infusion of PDBu induced mechanical but not thermal hyperalgesia 3 hours after infusion (Figs. 6B and C), which suggests that ERK1/2 activity in PVA neurons is not involved in thermal hyperalgesia.

Figure 6

Figure 6

Next, we examined whether intra-PVA PDBu infusion could induce persistent pain-like behavior and found that the mechanical hyperalgesia induced by a single infusion of PDBu lasted less than 3 days (Fig. 6D, upper panel). Infusion of a second dose of PDBu at 3 days after the first infusion induced a second transient hyperalgesic event (Fig. 6D, middle panel). Of note, when we infused the second PDBu at 2 days after the first dose, the mice displayed a hyperalgesic response, but this time the response did not decrease to the baseline and the mechanical hyperalgesia persisted for up to 2 weeks (Fig. 6D, lower panel). Then, we infused PDBu (20 pmole/day) into PVA continuously using a 7-day osmotic pump, which induced a persistent mechanical hyperalgesia lasting for 2 weeks, even though the pump provided PDBu for only 7 days (Fig. 6E). The osmotic pump infusion site is shown in supplementary Fig. 8B (available at http://links.lww.com/PAIN/A738). Repeated infusion of PDBu into the CA1 region of the hippocampus had no effect on withdrawal response (supplementary Fig. 6C, available at http://links.lww.com/PAIN/A738). These results strongly suggest that repeated activation of the PKC-ERK pathway in the PVA is sufficient to induce mechanical hyperalgesia without peripheral insult in naive mice.

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3.6. Activation of paraventricular thalamus neuron with optogenetics induces mechanical hyperalgesia

Our data suggest that PVA neuronal activity likely facilitates the development of mechanical hyperalgesia. To further investigate the direct effect of PVA neuronal activity on the pain-like behavior, we expressed hChR2 (AAV5-CaMKIIα-hChR2(H134R)-eYFP) in the PVA. In the thalamus, CaMKIIα is mainly expressed in the midline thalamus17; thus, we can obtain good expression of hChR2 in the PVA (Fig. 7A). We used the sweep spike firing pattern shown in Figure 1B to identify the PVA multiunit response before optogenetic stimulation. Next, we inserted optical fiber into the D3V above PVA (Fig. 7A). Activation of hChR2 with 5-, 10-, and 20-Hz blue light repeatedly elicited the spike units in those channels within the PVA. The 10-Hz blue light stimulation could activate PVA units most steadily among channels and individuals (Fig. 7B). Then, we examined whether activation of PVA neurons by optogenetics could induce pain-like behavior under 10-Hz blue light stimulation. The viral infection in PVA had no effect on the basal response to mechanical stimulation (supplementary Fig. 7A, available at http://links.lww.com/PAIN/A738). The diagrammatic representation of vial injection sites within PVA and optical fiber insertion sites was shown in supplementary Fig. 9C (available at http://links.lww.com/PAIN/A738). Blue light stimulation evoked mechanical hyperalgesia in PVA-ChR2–expressing mice as compared with their own basal activity (Fig. 7C). In contrast, behavior did not differ between the basal activity and the mechanical hyperalgesia under the light stimulation in PVA-eYFP–expressing controls (Fig. 7C). One day after blue light stimulation, the hChR2 group still exhibited higher withdrawal response than the eYFP group and its own basal activity (Fig. 7C). The real-time readout of optogenetics stimulation demonstrates that once PVA neurons were activated, it promoted the development of mechanical hyperalgesia.

Figure 7

Figure 7

Because PVA activities increased in the later phase in chronic pain models, we tested whether activating PVA neurons was sufficient to induce long-term behavioral change. Mice were exposed to 10-Hz blue light for 5 minutes for 3 consecutive days (D0, D1, and D2). The hChR2-expressing mice exhibited higher mechanical withdrawal frequency after blue light stimulation than did eYFP-expressing controls. The enhanced withdrawal response lasted up to 12 days even when there was no more blue light stimulation after the 3-day protocol (Fig. 7D). We also found increased c-Fos signals in the PVA of those mechanical hyperalgesic mice at the end of behavioral test (Fig. 7E). These results suggest that PVA neurons indeed participate in the nociception pathway and may also be involved in top-down regulation of chronic pain development.

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3.7. Paraventricular thalamus receives innervation from central nucleus of amygdala

How PVA neurons receive the nociceptive inputs transduced from the PNS is unclear. One potential upstream loci projecting into the PVA in nociception transduction is the CeA. To test this possibility, we transfected the plasmid-expressing WGA and mCherry fusion protein (WGA-mCherry) into the right CeA (rCeA) (Fig. 8A). We detected red punctate signals (WGA-mCherry) colocalized with NeuN in the PVA at 5 days after the in vivo transfection (Fig. 8A), which indicates whether PVA potentially receives signals from the rCeA directly or indirectly. To further verify any direct innervation from the CeA to PVA, we delivered AAV-eYFP into the rCeA. The infected rCeA neurons showed strong eYFP signals, and eYFP-positive signals were observed in PVA (Fig. 8B).

Figure 8

Figure 8

Next, we examined whether the connection from the rCeA to PVA participates in nociception circuitry by stimulating hChR2-expresssing rCeA terminals in the PVA with blue light and measuring the mechanical withdrawal response (Fig. 8C). The diagrammatic representation of vial injection sites within rCeA and optical fiber insertion sites was shown in supplementary Fig. 8D (available at http://links.lww.com/PAIN/A738). After the first blue light stimulation on rCeA-hChR2–expressing nerve endings in PVA, the rCeA-hChR2–expressing mice showed slightly increased withdrawal frequency. On the next day, a second episode of blue light stimulation elicited stronger withdrawal response but the response reduced one day later. However, repeated blue light stimulation, 5 minutes of 10-Hz blue light each day for 4 consecutive days, induced chronic mechanical hyperalgesia that lasted for another 10 days (Fig. 8C). At 90 minutes after the fourth blue light stimulation, the number of c-Fos–positive neurons was identified adjacent to the hChR2-expresssing rCeA processes in the PVA, which suggests that PVA neuronal activity increased after the blue light stimulations (Fig. 8D; supplementary Fig. 9, available at http://links.lww.com/PAIN/A738). The number of c-Fos–positive neurons was also increased in the PVA at 10 days after the fourth blue light stimulation (Figs. 8E and F). These results suggest that PVA receives inputs from the rCeA, and this rCeA–PVA connection contributes to mechanical hyperalgesia in naive mice.

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4. Discussion

In this study, we demonstrated that PVA neurons play an essential role in the development of chronic mechanical hyperalgesia in neuropathic and inflammatory pain models. We showed enhanced PVA neuronal activities, indicated by increased c-Fos staining, hyperexcitability, and firing pattern changes using multichannel recording in formalin- and SNI-induced hyperalgesic mice. Direct activation of PVA neurons using an optogenetics approach or pharmacological activation of the PKC-ERK signal pathway repeatedly was sufficient to induce chronic mechanical hyperalgesia in naive mice without peripheral injury. We also demonstrated that PVA receives innervation from the rCeA, and the rCeA–PVA connection facilitates persistent mechanical hyperalgesia. Furthermore, pharmacological inactivation of PVA ERK1/2 signal pathway or direct inhibition of the PVA neuronal activity using the DREADDs technique completely blunted chronic mechanical hyperalgesia with inhibitions applied at the critical time point in each model. Our data suggest that targeting PVA neurons at the proper time alleviates the preexisting mechanical hyperalgesia persistently in mice.

The initiation and maintenance of chronic pain involves PNS and CNS reorganization in both human and animals.26,31,32,49 Central sensitization involves alterations from molecule to networks contributing to functional and structure plasticity: the increase in response intensity and frequency at the synapse, spinogenesis, alterations in neuron and microglia interactions, neural circuit rewiring, and modulated transcription and translation.6,53 Our data demonstrate that PVA neurons are activated in formalin and SNI models by enhancing the immunoactivity of c-Fos protein and show an increased multiunit response to SNS in the PVA after chronic mechanical hyperalgesia developed in formalin and SNI models. In vivo recording demonstrated the enhanced PVA neuronal excitability and the firing pattern change in these hyperalgesic mice (Fig. 1).15 Activation of the Cav3.2 T-type calcium channel, and NMDA, orexin, corticotropin-releasing factor, and dopamine receptors, all proteins expressed in the PVA can lead to a ERK1/2 cascade.5,13,18,30,48

We recorded a fast response component of PVA multiunit activity elicited within 100 ms post SNS in formalin (current study) and SNI models.15 This fast component disappeared in U0126-treated SNI mice.15 In total, 21% of PVA neurons are identified as burst firing neurons.56 Burst firing is one of the signatures of T-type calcium channels (T-channels) expressing neurons. Our previous study showed that the Cav3.2 T-channels are involved in PVA-regulated initiation of acid-induced chronic mechanical hyperalgesia.13 Further investigation is needed to confirm whether Cav3.2 T-channels are involved in PVA sensitization and regulation of PVA ERK1/2-dependent persistent mechanical hyperalgesia in formalin and SNI models.

Paraventricular thalamus is situated in the midline thalamus from 0.2 to 0.7 mm posterior to the bregma in mice; the relevant stereotaxic coordinates in rat is the rostral portion of the paraventricular thalamic (−1.2 to ∼2.2 mm). Anatomical studies showed that PVA could receive projections from many pain-related loci, including CeA, PB, medial prefrontal cortex (mPFC), ACC, MD, and PAG.34 We reported that inhibition of ERK1/2 phosphorylation in the PVA prevented repeated acid-induced chronic mechanical hyperalgesia, whereas pERK1/2 signals were still activated in the CeA by the second acid injection.13 Hence, the PVA might be downstream to the CeA for nociception transduction.

We found both WGA-mCherry and eYFP-positive signals in the PVA, which suggests that the PVA receives innervation from the rCeA. Repeat activation of hChR2-expressing rCeA nerve endings in the PVA increased c-Fos level at 90 minutes and 10 days after stimulations, and the persistent mechanical hypersensitivity lasting for at least 10 days, which indicates a functional switch of PVA neurons. CeA neuronal activity was increased in SNL, formalin, and acid-induced muscle pain models,10,14,37,47 and 95% of CeA neurons are inhibitory neurons.40 Nevertheless, our data show that the connection from the rCeA to PVA facilitates persistent mechanical hyperalgesia. Both excitatory and inhibitory neurons are expressed in the PVA.5,18,56 We also detected strong c-Fos–positive signals in PVA regions when we activated these rCeA innervations in the PVA using optogenetic tools (supplementary Fig. 9, available at http://links.lww.com/PAIN/A738). Interneurons in the PVA may be involved in receiving rCeA inputs and regulate the function of the PVA. The midline thalamus functions in regulating physiological function by receiving various inputs and conveying the integral signals to the downstream nuclei.50 As a substructure of the midline thalamus, PVA might also receive nociception inputs from various brain regions other than the CeA.

We also found that activation of PVA neurons by PDBu infusion is sufficient to induce acute or persistent mechanical hyperalgesia depending on the duration of PDBu administration. Activation of PKC-ERK pathway by PDBu in the CeA leads to enhanced acute mechanical hypersensitivity in mice.10 Our group recently showed that intrathecal administration of PDBu in primed mice induced persistent mechanical hyperalgesia.12 However, induction of persistent pain-like behavior via activation of the PKC–ERK pathway in the brain has not been reported. Here, we showed that infusion of 20 pmole PDBu into the PVA also induced a transient increase in the withdrawal response to mechanical stimulation for 2 days in naive mice. Of note, infusion of a second dose of PDBu in the PVA while the mouse still displayed hypersensitivity led to moderate chronic mechanical hyperalgesia. Moreover, continuous infusion of PDBu induced a persistent mechanical hyperalgesia similar to that observed in neuropathic or inflammatory pain models. This striking effect of PDBu infusion suggests that PKC–ERK pathway in PVA is critical for the development of chronic mechanical hyperalgesia. This notion is further supported by our results showing the increase of PVA ERK1/2 activity in formalin and SNI models. Differential enhancement of mechanical hyperalgesia in naive mice depends on how frequently PVA neurons receive PDBu infusion, which indicates that the PVA probably functions as an activity-dependent modulator in regulating mechanical hyperalgesia.

Blockade of ERK1/2 activation by the MEK inhibitor U0126 in the amygdala and PVA reduces formalin-induced mechanical hyperalgesia transiently10 and acid-induced mechanical hyperalgesia,14 respectively. In this study, we showed that inhibition of PVA ERK1/2 activity at different times after pain induction reduced the mechanical hyperalgesia to different degrees in formalin and SNI models. Inhibiting PVA ERK1/2 activity within 24 hours after pain induction transiently reduced mechanical hyperalgesia, whereas inhibiting the activity later than 24 hours after pain induction provoked a long-term alleviation on formalin- and SNI-induced mechanical hyperalgesia. Inhibition of PVA ERK1/2 activity at the critical time points, 2 days after formalin injection or 3 days after SNI surgery, abolished chronic mechanical hyperalgesia. We recently reported that intra-PVA infusion of U0126 at 3 days after SNI surgery reversed the SNI-induced hyperexcitability and firing pattern switch of PVA neurons.15 Thus, inhibiting PVA ERK1/2 activation at the critical time probably disrupts the PVA neuronal plasticity switch. These findings suggest that PVA ERK1/2 activity plays an essential role in the development and maintenance of persistent mechanical hyperalgesia.

Extracellular signals induce ERK1/2 signaling cascades, and a dynamic or sustained activation of ERK1/2 leads to different determinations of cell functions. Transient increased ERK1/2 activity increases the transcription of immediate early genes, such as c-Fos. A sustained increase in ERK1/2 activity enhances transcriptional and translational events and persistent change in neuronal activity.27,31,44 Differential temporal regulation of PVA ERK1/2 activation may be the mechanism underlying the critical time window for the abolishing persistent mechanical hyperalgesia as demonstrated in this study. During central sensitization, ERK activation leading to modulated transcription and translation contributes to spinogenesis and synaptic protein turnover in the CNS.28 Our data showed that the best time point to block PVA ERK1/2 activation was 2 days for the formalin model and 3 days for the SNI model. Hence, translation modulation might contribute to the sustained ERK1/2 activation and the development of this time window. However, anatomical studies showed that PVA could receive projections from many pain-related loci, including the CeA, PB, mPFC, ACC, MD, and PAG.34 The PVA also projects to the ACC, basolateral amygdala, PAG, and most strongly to the nucleus accumbens.25 The PVA obviously has a strong neural connection with prelimbic circuitry, which is involved in emotion and decision-making pathway. The involvement of the prelimbic circuitry in chronic pain has been demonstrated in animal models by optogentic, electrophysiological, and genetic approaches and in patients with functional magnetic resonance imaging.4,48,51 Whether the connection between the PVA and mPFC or nucleus accumbens contributes to chronic pain modulation needs further investigation.

Overall, we found that PVA neurons receive innervation from the rCeA and engage in pain circuitry involved in persistent mechanical hyperalgesia in formalin-induced inflammatory and SNI-induced neuropathic pain models. A time window was identified for the long-term relief of mechanical hyperalgesia via inhibiting PVA ERK1/2 activity or PVA neuronal activity. Inhibiting PVA may be a potential therapeutic strategy for pain relief.

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Conflict of interest statement

The authors have no conflict of interest to declare.

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Acknowledgements

The authors thank the Pathology Core and the Common Facilities Core Laboratory at the Institute of Biomedical Sciences, Academia Sinica, Taiwan, and the Neuroelectrophysiology Core at Neuroscience Program of Academia Sinica, Taiwan. This work was supported by grants from MOST, Taiwan (NSC 100-2311-B-001 -002 -MY3, MOST 105-2314-B-001 -003 -MY3), and the Academia Sinica, to C.-C. Chen.

Author contributions: Y.-T. Chang, B.-C. Shyu, M.-Y. Min, and C.-C. Chen designed the research. Y.-T. Chang, W.-H. Chen, and H.-C. Shih performed the experiments. Y.-T. Chang, W.-H. Chen, and C.-C. Chen analyzed the data and Y.-T. Chang and C.-C. Chen wrote the paper.

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Supplemental digital content

Supplemental digital content associated with this article can be found online at http://links.lww.com/PAIN/A738.

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Supplemental video content

Video content associated with this article can be found online at http://links.lww.com/PAIN/A739.

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Keywords:

PVA; Hyperalgesia; Persistent pain; Central sensitization; ERK

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