HCN3 Ion Channels - Roles in Sensory Neuronal Excitability and Pain: HCN3 Ion Channels and Pain

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DOI: 10.1113/JP278211

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Citation for published version (APA): Lainez, S., Tsantoulas, C., Biel, M., & McNaughton, P. A. (2019). HCN3 ion channels - roles in sensory neuronal excitability and pain: HCN3 ion channels and pain. The Journal of Physiology, 597(17), 4661-4675. https://doi.org/10.1113/JP278211

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Download date: 02. Oct. 2021

DOI: 10.1113/JP278211

HCN3 ion channels: roles in sensory neuronal excitability and pain

Sergio Lainez1,*, Christoforos Tsantoulas1,3,*, Martin Biel2 and Peter A McNaughton1,3.

Running title: HCN3 ion channels and pain

1Wolfson Centre for Age-Related Research, King´s College London, Guy´s Campus, London SE1 1UL, UK.

2Center for Integrated Protein Science (CIPS-M) and Center for Drug Research, Department of Pharmacy, Ludwig-Maximilians-Universität Munchen, Munich, Germany.

3Correspondence should be addressed to Peter McNaughton, email: [email protected], or to Christoforos Tsantoulas, email [email protected].

* These authors contributed equally to this paper.

Keywords: HCN3, Ion channel, Neuron, Sensory, Nociception, Pain

Key points:

 HCN ion channels conducting the Ih current control the frequency of firing in peripheral sensory neurons signalling pain

 Previous studies have demonstrated a major role for the HCN2 subunit in chronic pain but a potential involvement of HCN3 in pain has not been investigated

 HCN3 was found to be widely expressed in all classes of sensory neurons (small, medium, large) where it contributes to Ih

 HCN3 deletion increased the firing rate of medium, but not small, sensory neurons

This is an Accepted Article that has been peer-reviewed and approved for publication in the The Journal of Physiology, but has yet to undergo copy-editing and proof correction. Please cite this article as an 'Accepted Article'; doi: 10.1113/JP278211.

 Pain sensitivity both acutely and following neuropathic injury was largely unaffected by HCN3 deletion, with the exception of a small decrease of mechanical hyperalgesia in response to a pinprick

 We conclude that HCN3 plays little role in either acute or chronic pain sensation

Summary

HCN ion channels govern the firing rate of action potentials in the pacemaker region of the heart and in pain-sensitive (nociceptive) nerve fibres. Intracellular cAMP promotes activation of the HCN4 and HCN2 isoforms, while HCN1 and HCN3 are relatively insensitive to cAMP. HCN2 modulates action potential firing rate in nociceptive neurons and plays a critical role in all modes of inflammatory and neuropathic pain, but the role of HCN3 in nociceptive excitability and pain is less studied. Using antibody staining, we found that HCN3 is expressed in all classes of somatosensory neurons. In small nociceptive neurons, genetic deletion of HCN2 abolished the voltage shift of the Ih current carried by HCN isoforms following cAMP elevation, while the voltage shift was retained following deletion of HCN3, consistent with the sensitivity of HCN2 but not HCN3 to cAMP. Deletion of HCN3 had little effect on the evoked firing frequency in small neurons, but enhanced the firing of medium-sized neurons, showing that HCN3 makes a significant contribution to the input resistance only in medium-sized neurons. Genetic deletion of HCN3 had no effect on acute thresholds to heat or mechanical stimuli in vivo, and did not affect inflammatory pain measured with the formalin test. Nerve-injured HCN3 KO mice exhibited similar levels of mechanical allodynia and thermal hyperalgesia to WT mice, but reduced mechanical hyperalgesia in response to a pinprick. These results show that HCN3 makes some contribution to excitability, particularly in medium-sized neurons, but has no major influence on acute or neuropathic pain processing.

Key points

HCN ion channels generate an inward current that can regulate action potential firing in somatosensory nerve fibres and can play an important role in pain sensation. The HCN1 isoform plays a limited role only in cold sensation following nerve injury. HCN2, on the other hand, is a key regulator of excitability in nociceptive nerve fibres, and controls the perception of inflammatory and neuropathic pain, but has no influence on acute pain sensation. Here we examine a potential role for the HCN3 isoform in neuronal excitability and pain. HCN3 is widely expressed in somatosensory neurons, and contributes to the regulation of firing of action potentials in medium-sized neurons, amongst which many have a nociceptive function. Genetic deletion of HCN3, however, had little impact on acute pain sensation, on inflammatory pain, nor on pain following nerve injury (neuropathic pain). We conclude that HCN3 does not play an important role in pain sensation.

Introduction

Ion channels belonging to the Hyperpolarization activated, Cyclic Nucleotide-gated ion channels family (HCN ion channels) have recently attracted interest as drivers of chronic pain (Chaplan et al., 2003; Brown et al., 2004; Luo et al., 2007; Momin et al., 2008; Emery et al., 2011; Acosta et al., 2012; Weng et al., 2012; Noh et al., 2014; Young et al., 2014; Tsantoulas et al., 2016; Tsantoulas et al., 2017). Genetic deletion or pharmacological block of HCN2 abolishes thermal hyperalgesia in inflammatory pain, and both thermal and mechanical hyperalgesia in neuropathic pain (Emery et al., 2011; Emery et al., 2012; Young et al., 2014; Tsantoulas et al., 2017). Genetic deletion or pharmacological inhibition of HCN1, on the other hand, has more limited effects on either inflammatory or neuropathic pain, although it does provide partial analgesia in some modalities of neuropathic pain, including in cold hypersensitivity triggered by oxaliplatin- or nerve injury-induced neuropathies (Momin et al., 2008; Tibbs et al., 2013; Resta et al., 2018). In the present study we sought to determine whether HCN3 ion channels play a role in acute, inflammatory or neuropathic pain.

The HCN family comprises four members (HCN1-4) that carry an inward current activated by hyperpolarization from the membrane resting potential. In both the heart and the nervous system the membrane current carried by HCN ion channels has a pace-making role, because the channels carry an inward current and are activated by membrane hyperpolarization following an action potential, leading to rebound membrane depolarization and initiation of a further action potential (Biel et al., 2009; DiFrancesco, 2010). A significant difference between members of the HCN family is that the voltage-dependence of activation of HCN2 and HCN4 is shifted in the positive direction on the voltage axis by direct cAMP binding to the C-terminus, but while both HCN1 and HCN3 contain a cAMP-binding domain, their voltage- dependence is largely insensitive to elevations of cAMP (Santoro et al., 1998; Ludwig et al., 1999; Mistrik et al., 2005). Thus the inward current carried by HCN2 and HCN4 can be enhanced by physiologically important transmitters which act to elevate intracellular cAMP levels, such as adrenaline in the heart (DiFrancesco, 2010) or prostaglandin E2 in nociceptive neurons (Emery et al., 2012). Expression of HCN1 and HCN4 underlies the pacemaker current (If) in the pacemaker region of the rodent heart (Baruscotti et al., 2011; Fenske et al., 2013), although species differences have also been noted (Thollon et al., 2007; Stillitano et al., 2008). In neurons, the main mediators of the hyperpolarisation-activated current, Ih, are HCN1, 2 and 3 (Momin et al., 2008).

HCN3 has been shown to be expressed in rat DRG neurons by RT-PCR, in situ hybridisation and immunocytochemistry (Chaplan et al., 2003; Kouranova et al., 2008; Cho et al., 2009), although in mice, by contrast, mRNA for HCN3 has been reported to be expressed at low levels (Moosmang et al., 2001; Schnorr et al., 2014). No overt phenotype is seen in HCN3-/- mice, which are viable and healthy throughout development from embryonic to adult stages (Fenske et al., 2011). No impairment in cardiac function is seen in HCN3-/- mice, and pacemaker activity is normal, although there is a change in the repolarization rate of the epicardial ventricular action potential, leading to an increase of both the T-wave amplitude and the QT duration in the ECG at low heart rates (Fenske et al., 2011). Recent work has also shown some changes in the fear responses of HCN3-/- mice (Stieglitz et al., 2017).

To investigate a possible role for HCN3 in pain, we examined in vitro electrophysiological and expression data in sensory neurons from HCN3 and HCN2-deficient mice. We also assessed the impact of deletion of HCN3 using in vivo models of inflammatory and neuropathic pain. We find that despite HCN3 being highly expressed in all classes of DRG neurons, and playing a role in modulating the excitability of medium diameter neurons, its deletion has little impact on pain sensation, as shown by the absence of any marked phenotype in inflammatory and neuropathic pain models in vivo.

Materials and Methods

Ethical Approval

All animal procedures conformed to institutional guidelines (approval reference PPL 70/7695) and the United Kingdom Home Office Animals (Scientific Procedures) Act 1986, and were in compliance with guidelines (Grundy, 2015). Mice were housed in groups of five, with ad libitum access to food and water. In order to perform Seltzer surgery, mice were anaesthetized with inhaled isoflurane and the respiratory rate was closely monitored thereafter to confirm depth of anaesthesia. Mice were culled according to Schedule 1 (Animals Act 1986) protocols; either via cervical dislocation for tissue collection, or via exposure to a rising concentration of carbon dioxide at the endpoint of behavioural experiments. Every effort was made to minimize animal suffering and the number of animals used.

Transgenic mice

Globally deficient HCN2-/- and HCN3-/- mice were generated as previously described (Mistrik et al., 2005; Emery et al., 2011; Fenske et al., 2011). Both male and female wild type (C57BL/6, Charles River) and knockout mice were used in experiments. Observers performing behavioural experiments were blinded to the genotype of the animals. Because HCN2 KO mice develop adverse phenotypes such as absence epilepsy at older ages (Ludwig et al., 2003), we used young (3-4 week old) mice for tissue extraction. Any HCN2 KO mice demonstrating repeated seizure activity were humanely culled.

DRG neuron culture

Cultures of WT and HCN2-/- and HCN3-/- mouse DRG neurons were prepared as follows. Briefly, 3-4 week old HCN2-/- mice and 1-2 month old HCN3-/- mice underwent cervical dislocation and decapitation and DRG were exposed by laminectomy. DRG from all spinal levels were collected and placed in cold Dulbecco’s modified Eagle’s medium (DMEM). After axon trimming, DRG were incubated for 1 hour at 37C in collagenase-supplemented DMEM (Worthington, 2mg/ml). After gentle centrifugation and aspiration of the collagenase solution, DRG neurons were resuspended in DMEM supplemented with 10% FCS, 1% Penicillin/Streptomycin and 1% L- glutamine pre-warmed to 37C. DRG were then mechanically dissociated by manual trituration and plated into 13mm culture dishes pre-coated with a mixture of poly-l-lysine (100g/ml) and laminin (20 g/ml) in PBS. Electrophysiological recordings were performed at room temperature (22C) 24 h after plating the neurons.

Electrophysiology

Solutions

Manual patch-clamp experiments were carried out using an extracellular solution containing (in mmol/L): 140 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose and 10 D-Mannitol (pH adjusted to 7.3 and osmolality 308 mOsm/L). The intracellular solution contained (in mmol/L):

140 KCl, 1.6 MgCl2, 2.5 MgATP, 0.5 NaGTP, 2 EGTA and 10 HEPES (pH adjusted to 7.25 and osmolality 290 mOsm/L). Forskolin (50 μmol/L, from 50mM stock in DMSO) was diluted in extracellular solution on the day of the experiment. All reagents were purchased from Sigma- Aldrich (St. Louis, MO).

Whole-cell patch-clamp recordings

DRG neurons of small (<20μm diameter), medium (20-30μm) or large (>30μm) size were chosen for electrophysiological recordings. Whole-cell patch-clamp recordings were performed using an Axopatch 200B patch-clamp amplifier. Borosilicate patch-clamp pipettes (Science Products GmbH, Germany) were pulled using a P-97 horizontal micropipette puller (Sutter Instruments, USA). Prior to use all pipettes were fire-polished with a Narishige MF-900 microforge, giving a final resistance between 2.5 and 3.5 M. Pipette offset was corrected after immersing the pipette tip in extracellular solution and before approaching the cell. Once a giga- seal was obtained between the pipette and the cell, capacitative transients were cancelled prior to achieving the whole-cell configuration. Series resistance was compensated by 40-70%. Cells were held at -60 mV in voltage-clamp mode. When working in current-clamp mode, the I-Clamp fast mode was used. Whole-cell current or voltage recordings were sampled at 20 kHz and low- pass Bessel filtered at 2 kHz. Data was acquired using Axon pCLAMP software version 10.4 and analysed offline with Clampfit 10.4 (Molecular Devices, LLC).

Voltage-dependent activation of Ih currents was assessed by calculating the midpoint activation voltage (V½) from tail currents recorded after applying a 1.5 s long voltage step to -140 mV, following a family of pre-pulse hyperpolarized voltage steps 1.5 s long from -140 to -40 mV (Δ =

20 mV). Maximal current density was measured following a step to -140mV. To calculate V½, plots of fractional activation vs voltage were fitted using a Boltzmann equation:

Where Vm is the membrane potential of the pre-pulse, V½ is the midpoint activation voltage, k is the slope factor, It is the current amplitude of the tail current recorded at -140 mV for a given pre-pulse and It (max) is the maximum current amplitude of the tail current. Neurons were classified as HCN-expressing if the bi-exponential inward current activated by a step to -140mV exceeded 50pA.

Data analysis

All data are presented as mean ± SEM. All results were analysed using GraphPad Prism version 6.0 (La Jolla, USA). Unpaired Student’s t-test and one-way ANOVA with repeated measures were used to determine statistical significance, as appropriate. Post-hoc analysis was carried out using the Bonferroni post-hoc test. P<0.05 was considered significant.

Surgery and behavioural testing

Mechanical pain threshold

Mechanical pain testing was conducted on 4-6 month old mice in a quiet temperature-controlled room with operators blinded to mouse genotype. Mechanical sensitivity was assessed using a dynamic plantar aesthesiometer (Ugo Basile, Italy) under computer control. Briefly, animals were placed in a ventilated plexiglass cage upon an elevated aluminium screen surface with 1cm mesh openings and allowed to acclimatise to their environment for 1h. Once exploratory behaviour had ceased, a 0.5 mm-diameter actuator filament delivered an increasing force (10 sec ramp, cutoff 5g) to the plantar surface of the hindpaw, until a reflex withdrawal response terminated the stimulation. Paw withdrawal thresholds were averaged over at least 3 measurements with 5 min intervals in between.

Thermal pain threshold

Heat pain thresholds were determined using the Hargreaves method (Hargreaves et al., 1988) on 4-6 month old mice. Briefly, each mouse was placed into a ventilated plexiglass cage on a glass floor. A thermal challenge from a calibrated (190mW/cm2) radiant light source (Ugo Basile, Italy) was applied to the hindpaw until a foot withdrawal was recorded. Withdrawal latencies were averaged from at least three consecutive tests, with a minimum of 5 minutes in between. A cut-off of 21 seconds was imposed to prevent the possibility of tissue damage.

Formalin test

The formalin test was conducted as previously described (Young et al., 2014) on 4-6 month old mice. Briefly, the plantar surface of the left hindpaw was injected with 20 μl of 4% formalin solution in saline using a 50 μL Hamilton syringe and a 30 gauge needle. Over the next 60 min, the time that the mouse spent licking, biting, or lifting the injected paw was recorded in 5- minute intervals. Pain responses during the early (0–10 min) and late (15–60 min) phases were analysed as previously described (Young et al., 2014).

Pinprick test

The pinprick test was performed as described by Jimenez-Diaz et al. (2008). Briefly, 1-2 month old KO and age-matched WT mice were placed in a ventilated plexiglass cage upon an elevated aluminium screen surface with 1cm mesh openings and habituated over a period of 2-3 consecutive days by recording a series of baseline measurements. To deliver the noxious stimulation, the point of a safety pin was applied to the lateral part of the plantar surface at an intensity sufficient to indent but not penetrate the skin. The duration of paw withdrawal was recorded with a minimum arbitrary value of 0.5 s for a brief normal response.

Randall Selitto test

Paw pressure withdrawal thresholds were determined using an Analgesy-Meter (Randall-Selitto test) from Ugo-Basile (Italy) on 1-2 month old KO and age-matched WT mice. Animals were habituated to the experimental room in their home cage for 15 min before the beginning of each testing session. Each animal was tested 4 times with a resting time of at least 5 min between each measurement to avoid sensitisation. Pressure at the moment of paw withdrawal was taken as a measure of pain threshold. No extra discs were added to the basic settings of the apparatus.

Partial sciatic nerve ligation (Seltzer model)

Partial sciatic nerve ligation (PSNL) was performed under isoflurane anaesthesia (administered at 3% and maintained at 2%). Briefly, the left thigh was shaved and sterilized. The sciatic nerve was then exposed at the upper level. The dorsal one-third to one-half of the sciatic nerve was exposed and tightly ligated at a site proximal to nerve trifurcation with a 5.0 vicryl suture (Harvard Apparatus). Finally, the overlaying muscle and skin were sutured and the animals were allowed to recover in a recovery chamber. Mechanical and thermal withdrawal thresholds were examined 1 and 3 days before surgery and on days 3, 7, 10 and 14 after surgery.

Immunohistochemistry

Tissue preparation

Mice (6-8 weeks old) were sacrificed. Lumbar (L4-L5) DRGs retrieved and post-fixed for 5 min at 4C in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.4. DRGs were then equilibrated in 20% sucrose in PBS at 4C overnight, before being embedded in O.C.T. (Optimum

Cutting Temperature) compound and rapidly frozen using liquid N2. Tissue sections were cut at 10μm thickness using a cryostat, and 5-10 sections were thaw-mounted onto Superfrost Plus glass slides (VWR).

Antibody incubation

Prior to staining, non-specific immunoreactivity was blocked by 1h incubation in PBS supplemented with 2% bovine serum albumin (BSA, Sigma), 10% normal donkey serum and 10% normal goat serum (Jackson ImmunoResearch). For immunohistochemistry (IHC), slides were washed in PBS and incubated overnight at 4C with the primary antibody in staining buffer [PBS supplemented with 0.3% Triton X-100, 2% BSA (Sigma), 4% normal donkey serum and 4% normal goat serum (Jackson ImmunoResearch)]. Primary antibodies used in this study and their respective dilutions were; rat anti-HCN3 (1:500, TLL6C5, Thermo Scientific); mouse anti-β3tubulin (1:1000, Promega); chicken anti-peripherin (1:500, Abcam); rabbit anti-NF200 (1:500, Sigma); mouse anti-Nav1.8 (1:500, Neuromab). The next day, slides were washed and incubated for 2 hrs with the secondary antibody in staining buffer. Secondary antibodies used were donkey anti-mouse IgG-conjugated Alexa Fluor 488, donkey anti-mouse IgG2-conjugated Alexa Fluor 488, goat anti-chicken IgG-conjugated Alexa Fluor 488, donkey anti-rabbit IgG conjugated 594, donkey anti-rat IgG-conjugated Alexa Fluor 647 (all 1:1000, Invitrogen). After a final wash samples were mounted with Fluorsave mounting medium (Calbiochem).

Image acquisition and analysis

Staining was visualised under a fluorescence Axioplan microscope (Zeiss) and images were acquired with a digital camera and Axiovision software. To minimise variability, all comparisons between staining of WT and KO tissue was performed at the same time and the same exposure was used during acquisition. Image analysis was carried out using ImageJ software. For each image, 3 background intensity measurements were taken and mean intensity of background (BG) as well as SEM were calculated. Next, regions of interest (ROIs) were manually drawn around cells; a neuron was only considered positively stained when its intensity was higher than 2BG + 6SEM. These objective criteria correlated well with subjective criteria of positively labelled cells. Measurements were conducted using a 25x objective from at least 5 sections per DRG (> 200 cell profiles/animal). Measurement of cell diameter was only carried out on cells that had a clearly visible nucleus.

Results

The HCN3 isoform is widely expressed in sensory neurons

We performed immunohistochemistry using a previously validated HCN3 monoclonal antibody in order to determine the expression pattern of HCN3 in WT DRG neurons (Stevens et al., 2001; Muller et al., 2003). We identified neurons using the pan-neuronal marker β3-tubulin. Strong HCN3 staining was detected in 61.0  1.0 % of all lumbar DRG neurons from WT animals (n=3) (Fig. 1, top). Similar fractions of small (56.5  7.3%, <20m dia) and medium-large (60.8  2.3%) neurons were HCN3-positive (Table 1). Conversely, 31.3 ± 2.8% of HCN3-positive neurons belonged to the small-diameter class, while 73.5 ± 3.2% were medium-large diameter. Antibody specificity was demonstrated by the absence of HCN3 staining in lumbar DRG neurons from HCN3-/- mice (n=3 mice) with no fluorescence observed above background levels (Fig. 1, bottom). The frequency of HCN3 expression across DRG neurons of different diameter is illustrated in the cell-size distribution graphs (Fig. 1).

We next examined co-localisation of HCN3 with phenotypic markers of DRG neuron subpopulations (Fig. 2). Three markers were used: peripherin, which stains small unmyelinated neurons; NF200, which stains a neurofilament protein present in medium-large myelinated neurons (Aβ and Aδ); and NaV1.8, a voltage-gated sodium channel which is found predominantly in small/medium size neurons with a nociceptive function. HCN3 was highly co-expressed with all markers examined (Table 2), as would be expected given the wide distribution of HCN3 that we previously documented. About one third of peripherin-positive small neurons were also found to be immunoreactive for HCN3. Notably, within this population, over 60% of nociceptive neurons (identified by NaV1.8 expression) expressed HCN3. HCN3 was detected in about one- third of NaV1.8-negative neurons (29.4  2.5%), the large majority of which were myelinated A- fiber neurons (86.2  2.4%). Taken together, the results from the immunohistochemical analyses demonstrate that HCN3 is abundantly expressed in DRG neurons, including in nociceptive and non-nociceptive neurons of all sizes. Moreover, our data in mouse is in agreement with previous reports showing a majority of rat DRG neurons expressing the HCN3 isoform (Chaplan et al., 2003; Kouranova et al., 2008; Cho et al., 2009), though it does not agree

with the low expression found previously in mouse (Moosmang et al., 2001; Schnorr et al., 2014).

Roles of HCN2 and HCN3 in Ih currents in sensory neurons

We next examined the contribution of HCN2 and HCN3 to Ih currents in DRG neurons. In DRG neurons from HCN2-/- mice, 97 out of 107 neurons from all diameters expressed significant

Ih (see Methods for criterion), indicating that isoforms other than HCN2 contribute to Ih. The

HCN1 isoform is mainly responsible for the fast-decaying Ih current seen in large diameter neurons, while HCN3 is likely to be responsible for the slower residual Ih currents seen in small and medium diameter DRG neurons after genetic deletion of HCN2 (Momin et al., 2008; Emery et al., 2011). In order to dissect the relative contribution of HCN3 and HCN2, we first investigated the effect of cAMP elevation, achieved by application of forskolin, on V½, the value of membrane voltage at which Ih is half-activated. In view of the insensitivity to cAMP of HCN1 and HCN3, and in view of the low expression of HCN4 in DRG neurons (Cho et al., 2009), a shift in V½ following exposure to forskolin is likely to be attributable mainly to expression of the cAMP-sensitive HCN2 ion channel.

In small diameter neurons (<20m), which give rise to slowly conducting, unmyelinated

C-fibres, most of which are nociceptive in function, a significant shift in V½ was observed in both WT and HCN3-/- neurons after forskolin application, and in both cases the shift was comparable (+5.47 ± 1.08 mV vs. +6.58 ± 1.36 mV, no significant difference, p=0.52). By contrast, the cAMP- mediated shift in HCN2-/- small neurons was not significantly different from zero (+2.37 ± 1.46 mV, p=0.26) (Fig. 3A&B and Table 3). These results support the hypothesis that the main cAMP- sensitive isoform in small neurons is HCN2, but that significant current is also carried by the cAMP-insensitive HCN3 isoform.

In medium diameter DRG neurons (20-30m), most of which give rise to thinly myelinated A-fibres with both nociceptive and non-nociceptive functions, the shift in the V½ of

Ih following elevation of cAMP (Table 3) was significantly smaller in HCN3-/- neurons than in WT neurons (+7.6 ± 1.36 vs +13.3 ± 1.29 mV, respectively; p=0.004, one-way ANOVA with

Bonferroni posthoc test). In HCN2-/- neurons a significant shift in V½ was still observed (+6.7 ± 2.2 mV), suggesting that some of these neurons may express HCN4 (Fig. 3C).

In large diameter WT DRG neurons (>30m), giving rise to fast conducting, thickly myelinated A-fibres, elevation of cAMP did not lead to a shift in V½, consistent with domination of Ih by the fast-activated and cAMP-insensitive HCN1 isoform (Momin et al., 2008). Similarly, no significant effect of cAMP elevation was observed after deletion of either HCN3 or HCN2 (Fig 3D).

The results above are consistent with co-expression of both HCN2 and HCN3 in small DRG neurons and suggest that genetic deletion of either might therefore reduce the maximal current amplitude. To test this, we applied a hyperpolarizing voltage step from a holding potential (Vh) of -60 mV, a membrane voltage at which all HCN isoforms are fully deactivated, to

-140 mV, at which full activation is achieved, and measured the maximum inward Ih current

(Table 3, top row and Fig. 4). No significant differences in maximal Ih density were seen in HCN3-/- neurons when compared to WT, regardless of neuronal size. Current densities following elevation of cAMP were also similar to those seen in untreated neurons and did not show any significant changes between WT and HCN3-/- (data not shown). Deletion of HCN2 caused some reduction of current density in small neurons but this effect did not reach statistical significance (Table 3 and Fig. 4). Surprisingly, in view of the evidence above that HCN2 plays at most a minor role in large neurons, deletion of HCN2 caused a significant reduction in current density in large neurons (Table 3 and Fig. 4). The overall absence of a prominent effect on current density in most sensory neurons (with the exception of HCN2 knock-out in large neurons), is likely due to compensatory upregulation of other isoforms following deletion of HCN2 or HCN3.

A third way in which the contribution of HCN isoforms can be assessed is through differences between Ih activation kinetics between WT neurons and neurons with an HCN gene deletion. Currents were recorded after application of a hyperpolarizing voltage step from a Vh of -60 mV to -140 mV and were fitted by a double exponential function. No change in slow or fast time constants was observed in small and large DRG neurons with deletion of either HCN2 or HCN3 (Table 3). However, in medium-sized neurons both the fast and the slow components of current activation were speeded by deletion of HCN3 but not HCN2 (see Table 3 and Fig. 5). Time constant values for the fast component were 194±28 ms (n=32) in WT and 94±7 ms (n=26) in HCN3-/- (p=0.0052), while the slow component time constants were 978±122 ms and 578±47 ms, respectively (p=0.011). These results support the idea that HCN3 is dominant in medium-sized neurons and has a slower time constant than HCN2. The results suggest that

HCN3 may play an important role in shaping the properties of Ih in medium DRG neurons.

HCN3 depresses the excitability of medium diameter DRG neurons

We next examined the excitability of small and medium diameter DRG neurons by recording their firing of action potentials in response to depolarizing current injection. Firing frequency was assessed by applying current injection steps of 1 s duration from 0 to 60 pA in 10 pA increments. When no firing was seen, a second set of steps from 60 to 120 pA was applied to medium DRG neurons, as these tend to show a highly variable firing threshold. Neurons showed a heterogeneous firing pattern as follows: silent (no firing), adapting responses (phasic) and finally tonic (continuous) firing. Only neurons exhibiting a tonic firing pattern were analyzed, as these are the DRG neurons that are likely to have the greatest physiological relevance for pain (Figure 6).

In small DRG neurons, 2 out of 22 HCN3-/- neurons showed phasic firing, with the remaining being tonic (representative traces shown in fig. 6A). There may be some tendency for a higher firing rate in HCN3-/- than WT, and lower in HCN2-/-, but any effect was small and was not significant with the numbers of neurons studied (Fig. 6B).

A higher proportion of medium-sized DRG neurons showed phasic firing following HCN3 deletion. Thus, in WT mice 12.5% (2/16 neurons) showed phasic firing, compared to 28.6% (6/21) in HCN3-/- and 12.5% (1/8) in HCN2-/- neurons. A significantly higher tonic firing frequency was seen at all current injections above 10pA in HCN3-/- DRG neurons compared to WT or HCN2-/- (Fig. 6C). Specifically, the mean firing rate with 60 pA current injection was 22.1±4.1, 13.75±2.34 and 7.9±1.7 Hz in HCN3-/-, WT and HCN2-/-, respectively.

Acute, inflammatory and neuropathic pain in HCN3-/- mice

We evaluated the role of the HCN3 isoform in acute nociceptive responses to both mechanical and thermal stimuli. Mechanical withdrawal thresholds in response to von Frey stimulation were similar in WT and HCN3-/- mice, with values of 3.91±0.14 g (n=9) and 3.61±0.13 g (n=9) in WT and HCN3-/-, respectively (not significant, p=0.128, Fig. 7A). Similarly, no significant difference was seen in thermal withdrawal thresholds, which were 6.160.63 s (n=9) and 7.890.76 s (n=9) in WT and HCN3-/- respectively (not significant, p=0.126, Fig. 7B). Given the altered excitability of medium-sized neurons we observed electrophysiologically following HCN3 deletion (Fig. 6C), we also conducted behavioural tests to specifically address the possibility of altered Aδ-fiber nociceptive signalling. Comparing WT and HCN3-/- mice in the paw pressure assay (Randall Sellito test) we found no significant differences in this pain modality (WT, 99.6±4.6 g vs HCN3-/-, 109.0±6.4 g, p=0.248, Student’s t-test; n=10/group; Fig. 7C).

We next examined inflammatory pain behaviour in the widely used formalin test. Formalin injection resulted in a typical biphasic nocifensive behavioural response, an early acute phase followed by a later inflammatory phase (Fig. 7D). No differences were observed between WT and HCN3-/- in either the acute or the inflammatory phases, suggesting that HCN3 plays no role in either acute or inflammatory pain.

Finally, the effects of HCN3 deletion on neuropathic pain behaviours following nerve injury were studied, using the Seltzer partial sciatic nerve ligation model. Three days after surgery, both WT and HCN3-/- mice showed a decrease in both von Frey and thermal thresholds in the ipsilateral hind-paw (Fig. 7E, F). A similar development of neuropathic pain, with no significant differences between WT and HCN3-/- mice, continued throughout the time-course of the experiment, indicating that HCN3 is not involved in the development of mechanical allodynia or thermal hyperalgesia. Finally, we used the pinprick test to assess development of mechanical hyperalgesia in the Seltzer model (Fig. 7G). Both WT and HCN3-/- mice developed mechanical hyperalgesia on the injured side, manifested as an increased duration of withdrawal response after noxious stimulation with a pinprick. This hypersensitivity was present from day 3 through to day 14, and was significant compared to either baseline or contralateral values (p<0.05, two-way RM ANOVA, n=8/group). Interestingly, the mechanical hyperalgesia in WT mice was significantly higher than HCN3-/- at all time points (3d, 3.23±0.56 vs 1.88±0.44 sec, p<0.001; 7d, 2.23±0.26 vs 1.48±0.27 sec, p<0.05; 14d, 2.69±0.36 vs 1.52±0.26, p<0.001).

Put together, the behavioural assays suggest that HCN3 deletion leaves acute and neuropathic pain processing largely unaffected, with the exception of a modest reduction of the development of mechanical hyperalgesia following nerve injury.

Discussion

The first description of Ih in primary afferent neurons was in the 1980s (Mayer & Westbrook, 1983), but the importance of the HCN family of ion channels in the development and maintenance of pain has only recently gained attention. Several studies have underscored the role of HCN ion channels in animal neuropathic and inflammatory pain models (reviewed in

Tsantoulas et al., 2016). The role played by the HCN2 isoform in nociceptive afferent fibres is critical, because neuropathic pain is completely abolished, in nerve injury, oxaliplatin and diabetic neuropathy models, by either pharmacological block of HCN2 or targeted deletion of

HCN2 in NaV1.8-expressing nociceptive neurons (Emery et al., 2011; Schnorr et al., 2014; Young et al., 2014; Tsantoulas et al., 2017). Deletion or pharmacological block of the HCN1 isoform has some analgesic effects in nerve injury models (Momin et al., 2008; Tibbs et al., 2013) as well as in cancer treatment-related neuropathy (Resta et al., 2018), but the effects are less prominent than those of deletion of HCN2. In the present study we used a mouse line with a genetic deletion of HCN3 (Fenske et al., 2011) to probe possible roles for HCN3 in pain. The HCN3-/- mouse is fertile and has a normal lifespan, with the only known differences from WT being a decrease in the epicardial action potential duration, with consequently an increase in the T wave amplitude at low heart rates (Fenske et al., 2011), and an altered fear response (Stieglitz et al., 2017).

Previous studies examining expression of HCN3 in rodent primary afferent neurons have given divergent results. While HCN3 expression in rat is widespread at both RNA and protein levels (Chaplan et al., 2003; Kouranova et al., 2008; Weng et al., 2012; Liu et al., 2015), no detectable HCN3 mRNA was found in mice (Moosmang et al., 1999). In the present study we used staining with a validated HCN3 antibody to demonstrate widespread expression of HCN3 in mouse DRG neurons, in agreement with the rat expression profile. Across all size classes, around 55-60% of neurons were positive for HCN3 (Table 1). More than 80% of large-diameter neurons, that subtend myelinated fibres, were positive for HCN3, while a smaller proportion (36%) of nociceptive (small unmyelinated) neurons were HCN3-positive (Table 2). Neurons positive for the DRG-specific Na channel isoform NaV1.8, most of which are nociceptive in function, also showed strong expression of HCN3 (63%).

Small neurons, amongst which nociceptors form the major class, show a prominent Ih current with a slow time constant that is not abolished by deletion of HCN2 and therefore may be carried by HCN3 (Emery et al., 2011). In agreement with previous work (Emery et al., 2011), deletion of HCN2 in small neurons not exposed to inflammatory mediators does not significantly affect evoked firing frequency. Deletion of HCN3 may have had a small effect in enhancing firing frequency in small neurons, but the effect was not significant with the sample size used. Experiments reported here support the idea that the cAMP-insensitive HCN3 is the main residual isoform following deletion of HCN2, because the voltage-dependence of Ih was not modulated by cAMP, while following deletion of HCN3 voltage-dependence was still observed (Fig. 3).

In medium-sized DRG neurons deletion of HCN2 had little effect on firing of action potentials in response to current injection, but deletion of HCN3 significantly increased the firing frequency. HCN3 contributes the majority of Ih current in medium-sized neurons , because

(i) the time constant of Ih is significantly speeded on deletion of HCN3 (Fig. 5), and (ii) injected current causes a higher firing rate when HCN3 is deleted, as expected if HCN3 is a significant contributor to the input resistance (Fig. 6). The enhanced firing frequency in medium-sized neurons, which predominantly subtend myelinated Aδ nerve fibres responding to noxious mechanical stimuli, suggested that HCN3 might play a role either in responses to acute noxious

mechanical stimuli, or in mechanical sensation following the induction of inflammatory or neuropathic pain.

To determine whether HCN3 does in fact play any role in pain sensation in vivo, we evaluated the effect of HCN3 deletion using acute, inflammatory and neuropathic pain models. There was no influence of HCN3 deletion on acute pain thresholds, either to mechanical or to heat stimuli. The behavioural response to formalin injection consists of an initial acute pain response, of licking and biting of the injected paw, followed by a slowly developing inflammatory pain response. Deletion of HCN2 has no effect on the initial phase but reduces the late phase, in agreement with other evidence showing that HCN2 contributes to inflammatory but not to acute pain sensation (Emery et al., 2011). Deletion of HCN3, in contrast, had no effect on either phase (Fig. 7), arguing against any contribution of the HCN3 ion channel to either acute or inflammatory pain sensation. Lastly, we used the Seltzer model of neuropathic pain, consisting of a partial ligation of the sciatic nerve (Seltzer et al., 1990), and tested for both thermal and mechanical hyperalgesia. Following surgery, HCN3-/- mice showed no difference from WT in the development of mechanical allodynia or thermal hyperalgesia (Fig. 7), as might have been expected given the limited role of HCN3 in modulating the firing properties of small neurons.

The enhanced firing observed in medium-sized neurons when HCN3 is deleted suggests that HCN3 deletion could lower the threshold for activation and thus enhance the acute mechanical pain sensation that is mediated by this class of neurons. However, we found that there was no change in the acute pain threshold for mechanical stimuli in the form of either a pinprick or a compression of the paw (Randall Sellito test) in animals of the same age as those used for neuronal collection. Following induction of neuropathic pain, there was also no significant effect of HCN3 deletion in enhancing the response to compression, and in fact the trend of the data is in the opposite direction. HCN3 deletion did cause a significant change in the response to a pinprick, but in the opposite direction from that expected on the basis of the increased excitability of the medium-diameter class of neurons in culture; there was a modest reduction in the mechanical hyperalgesia triggered by nerve injury. The source of this discrepancy is not known but may be related to a different expression of ion channels such as other HCN isoforms in the neuronal cell body and in the axon terminal.

In summary, the present study adds to previous genetic approaches aiming to disentangle the roles of HCN isoforms in nociception. Previous work has shown that HCN1 is not involved in acute or inflammatory pain but may make some contribution to neuropathic pain induced by nerve injury or chemotherapy (Momin et al., 2008; Tibbs et al., 2013; Resta et al., 2018). The cAMP-modulated HCN2 isoform plays no role in acute pain sensation but is the main driver of both inflammatory and neuropathic pain (Emery et al., 2011; Young et al., 2014; Tsantoulas et al., 2017). Although HCN3 is abundantly expressed in DRG neurons of all classes and modulates the Ih current, particularly in medium-sized neurons, we show here that it does not play any major role in acute pain or in either the development or maintenance of inflammatory and neuropathic pain, with the exception of a modest influence on nerve injury- induced mechanical hyperalgesia. This study therefore further substantiates the idea that HCN2 is the most critical isoform implicated in inflammatory and neuropathic pain, and that development of HCN2-selective blocking drugs may provide an attractive route to analgesia.

Additional information

Funding: Supported by grants from the BBSRC (SL, BB/J009180/1) and the Medical Research Council (CT, MR/J013129/1) to PMcN and by the Deutsche Forschungsgemeinschaft (SFB 870) to MB.

Author contributions: SL, CT and PAM planned the study and wrote the manuscript. SL performed electrophysiology and in vivo experiments. CT carried out immunohistochemistry and in vivo experiments. MB and lab members derived HCN3-/- mice.

Conflicts of interest: The authors declare no conflict of interest.

All authors have approved the final version of the manuscript. Authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed

Table 1: Quantification of HCN3 expression in DRG neurons of different sizes

DRG neurons ± SE (%) Cell size HCN3(+) in each class Allocation of HCN3(+) within each class Small (<20μm) 56.5 ± 7.3 31.3 ± 2.8 Medium-large (>20μm) 60.8 ± 2.3 73.5 ± 3.2

Column 2 gives percentage of total number of neurons within each size class that were positive for HCN3; column 3 gives percentage of HCN3-positive neurons that fell within the given size class.

Table 2: Quantification of HCN3 co-localisation with neuronal markers in sensory neurons

DRG neurons ± SE (%) Marker HCN3(+) in each group Allocation of HCN3(+) within each group Peripherin 35.8 ± 1.2 46.4 ± 1.6 NF200 86.2 ± 2.4 53.5 ± 1.6

NaV1.8 63.3 ± 2.1 74.4 ± 1.2

Columns as in Table 1.

Table 3: Summary of electrophysiological properties of Ih in the three size classes of DRG neurons from WT and KO mice.

Wild-type HCN3-/- HCN2-/- Small Medium Large Small Medium Large Small Medium Large

Ih, pA/pF -10.1±0.9 -8.1±1.5 -24.7±2.8 -12.8±1.4 -9.7±1.6 -18.9±2.3 -7.4±0.7 -7.7±1.1 -13.1±1.7 (at -140mV) n=45 n=25 n=27 n=30 n=30 n=31 n=44 n=22 n=24 p - - - n.s. n.s. n.s. n.s. n.s. 0.003**

V½ (mV) -93.4±0.8 -98.4±0.9 -88.6±0.8 -93.2±0.9 -94.3±0.9 -87.6±0.9 -93.8±1.0 -99.2±1.4 -87.1±1.0 Control n=32 n=29 n=32 n=37 n=30 n=32 n=14 n=18 n=11

V1/2 (mV) -87.9±1.3 -85.1±1.6 -86.1±1.2 -86.7±1.7 -86.7±1.7 -84.1±1.2 -91.4±1.9 -92.5±2.8 -83.9±1.4 + Forsk n=27 n=21 n=18 n=23 n=21 n=21 n=12 n=11 n=11 p 0.014* <0.001*** n.s. 0.0016** <0.001*** n.s. n.s. 0.0091** n.s.

τf (ms) -100±7 -194±28 -72±5 -106±10 -94±7 -67±5 -110±11 -165±16 -65±5 (at -140mV) n=22 n=20 n=25 n=23 n=20 n=22 n=22 n=15 n=28 p-value - - - 0.961 0.005** 0.772 0.434 0.315 0.521

τs (ms) -611±67 978±122 -435±26 710±108 -578±47 -445±46 -692±59 -1029±112 -429±43 (at -140mV) n=22 n=20 n=25 n=23 n=20 n=22 n=22 n=15 n=28 p - - - n.s. 0.011* n.s. n.s. n.s. n.s.

Diameter classes of neurons: small < 20m; medium 20-30m; large >30m. The t-test was used to compare V½ between control and forskolin for each type of DRG. One Way ANOVA was used to compare Ih and  among different genotypes (WT, HCN3-/- and HCN2-/-) in each size class. ***, p<0.001; **, p<0.01; *, p<0.05; n.s., not significant.

Figure Legends

Fig 1. HCN3 expression profile in lumbar DRG neurons. Left image shows immunoreactivity for the pan-neuronal protein 3-tubulin. Right image shows immunoreactivity for HCN3 in the same field of view. Arrows denote examples of HCN3-positive neurons. Top row shows data from WT mice, bottom row shows data from HCN3-/- mice. Scale bar = 20m. Right, size- frequency histogram showing HCN3 expression as a function of DRG cell diameter in WT mice. HCN3+, cells with HCN3 immunoreactivity; HCN3-, cells that do not show HCN3 immunoreactivity.

Fig 2. HCN3 co-localisation with phenotypic markers in DRG neurons. Each row shows expression of molecular markers of DRG neuronal subpopulations (left image), HCN3 expression (middle image) and the overlay of both images to show co-localization (right image). Arrows denote examples of HCN3-positive neurons. HCN3 is expressed in 36% of DRG neurons positive for peripherin (top row, marking small unmyelinated neurons), 86% of NF200+ (middle row, marking medium-large myelinated) and 63% of NaV1.8+ (bottom row, marking small/medium sized nociceptive neurons). Scale bar = 20 μm.

Fig 3. Effect of deletion of HCN2 and HCN3 on V½ of Ih after FSK application in DRG neurons. A. In small neurons (<20 µm), the basal V½ from WT, HCN3-/- and HCN2-/- shows no major differences. Application of 50 µM FSK resulted in a significant depolarising shift in V½ of both WT and HCN3-/- neurons (V½=5.47 ± 1.08 mV vs. +6.58 ± 1.36 mV, no significant difference, p=0.52), but not in HCN2-/- (V½= 2.37 ± 1.46 mV, p=0.26). Solid lines show basal curves and dashed lines are FSK-treated cells. B. Histogram showing V½ in small DRG (<20 µm diameter). Application of forskolin results in a shift towards depolarized voltage potentials in WT and HCN3-/- DRG neurons, but not HCN2-/- neurons (One Way ANOVA, p values are shown for each pair of values). C. Histogram showing V½ in medium DRG (20-30 µm dia). Application of forskolin results in a shift towards depolarized voltage potentials in all conditions (One Way ANOVA, p values are shown for each pair of values). D. Large neurons (>30 m) show no significant shift in V½.

Fig 4. Effect of deletion of HCN2 or HCN3 on Ih amplitude in small, medium and large DRG neurons. A. Current density in small DRG neurons B. Medium DRG neurons. C. Large HCN2-/-

DRG showed a significant decrease in Ih compared to WT. Current density in large neurons was larger than small or medium diameter likely because of HCN1 expression. All data expressed as meanSEM; *(p=0.0034 vs WT, p=0.001 vs HCN3, One Way ANOVA).

Fig 5. HCN3 modulates Ih activation kinetics in medium DRG neurons. A, B. Scatter plots showing fast and slow time constants for WT, HCN3-/- and HCN2-/- mice. Deletion of HCN3 but not HCN2 results in faster activation kinetics. (Tau fast; *p=0.005 vs WT, Tau slow; *p=0.011 HCN3-/- vs WT and #p=0.023 HCN2-/- vs HCN3-/-). All data expressed as meanSEM. Time constant of activation after hyperpolarizing voltage step to -140 mV for 3s calculated by fitting

Ih with a double exponential equation.

Fig 6. HCN3 deletion enhances excitability of medium diameter DRG neurons. A. Representative traces showing firing responses to injection of 60 pA for 1 second from WT, HCN3-/- or HCN2-/- mice. B. AP frequency vs injected current for small DRG. Tendency for enhanced firing frequency (solid squares) in HCN3-/- and reduced frequency in HCN2-/- (not significant) C. Neurons from HCN3-/- mice exhibit higher firing frequencies compared to either wild type or HCN2-/- mice. Two-way ANOVA with repeated measures; *P<0.05; **P<0.01 compared to WT; #P<0.05; ##P<0.01 compared to HCN2-/-.

Fig 7. Acute, inflammatory and neuropathic pain responses in HCN3-/- mice. A, B, C: WT and HCN3-/- mice have indistinguishable acute mechanical (von Frey hair) heat (Hargreaves test) and paw pressure (Randall Selitto test) thresholds. D: No behavioural differences are seen in the formalin model of inflammatory pain. E, F: Development of mechanical allodynia and thermal hyperalgesia following nerve injury (Seltzer partial sciatic nerve ligation). Withdrawal thresholds of WT () and HCN3-/- () in response to mechanical (E) and radiant heat stimulus (F) applied to ipsilateral hind paw. G: Development of mechanical hyperalgesia to pinprick stimulus following injury. HCN3-/- mice showed a slightly reduced sensitivity compared to WT on the injured (ipsilateral) side. Ipsi; ipsilateral; contra, contralateral; *P<0.05 comparing genotypes, #P<0.05 comparing ipsi vs contra; n=8/group, two-way repeated measures ANOVA.

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Sergio Lainez

"Sergio Lainez obtained his Masters and PhD degrees in Biochemistry from University of Valencia. He studied the role of TRPV1 in pain using electrophysiology under the supervision of Dr Rosa Planells-Cases. Postdoctoral studies followed at Professor McNaughton’s lab, examining the role of HCN2/HCN3 in peripheral pain signaling. He has also been involved in areas such as renal and cardiac physiology and stem cell research, mainly working with ion channels and ancillary proteins. In 2017, he reinvented himself as a bid writer, working for a European

consultancy firm.”

Christoforos Tsantoulas

Christoforos Tsantoulas received his BSc in Biology from University of Athens in 2003. Following a Masters in Molecular Medicine at UCL, he completed his PhD on the role of Potassium Channels in Neuropathic Pain at King’s College London. After post-doctoral positions at Pfizer/Neusentis and University of Cambridge, he returned to King’s where he continues research on ion channels and peripheral pain modulation.