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Down-regulation of Kv4.3 channels and a-type K+ currents in V2 trigeminal ganglion neurons of rats following oxaliplatin treatment

Chemotherapy drugs such as oxaliplatin can increase nociceptive neuron excitability to result in neuropathic pain in orofacial and other regions in patients following chemotherapy. However, mechanisms underlying chemotherapy-induced increases of nociceptive neuron excitability are not fully understood. Kv4.3 channels are voltage-gated K+ channels mediating A-type K+ (IA) currents to control neuronal excitability. In the present study, we examined Kv4.3 channel expression on trigeminal neurons that innervate orofacial regions (V2 TG neurons) of rats using immunostaining method. We showed that strong Kv4.3 immunoreactivity (Kv4.3-ir) was present mainly in small-sized V2 TG neurons. The numbers of Kv4.3-ir positive V2 TG neurons were significantly reduced in oxaliplatin-treated rats, suggesting down-regulation of Kv4.3 channel expression on V2 TG neurons by the chemotherapy drug. Patch-clamp recordings from acutely dissociated rat V2 TG neurons showed that almost all nociceptive-like V2 TG neurons displayed IA currents with slow inactivation kinetics. The amplitudes of IA currents were significantly reduced in these nociceptive-like V2 TG neurons of oxaliplatin-treated group. Furthermore, we found that the excitability of nociceptive-like V2 TG neurons was significantly higher in the oxaliplatin-treated group than in the control group. These findings raise a possibility that down-regulation of Kv4.3 channels and IA currents in nociceptive V2 TG neurons is an underlying mechanism of oxaliplatin-induced orofacial neuropathic pain.


Introduction
Chemotherapy-induced peripheral neuropathy (CIPN) is a common and dose-limiting side effect for several essential chemotherapy drugs including oxaliplatin. 1 In patients with CIPN, neuropathic pain is often experienced in extremities, 2 but it can also occur in proximal body parts including the orofacial regions. 3,4 Neuropathic pain of CIPN is often manifested with exaggerated pain sensations triggered by innocuous mechanical stimuli (mechanical allodynia) and by innocuous or mild noxious cooling temperatures (cold allodynia/ hyperalgesia). The neuropathic pain in orofacial regions is a significant clinical issue due to its special location, severity, and lack of effective treatment. 5 Mechanisms underlying chemotherapy-induced neuropathic pain in orofacial regions are not fully understood. This lack of knowledge prevents us from identifying therapeutic targets for effective treatment of the neuropathic pain.
Orofacial regions are largely innervated by the maxillary (V2) branch of the trigeminal nerves. V2 trigeminal nerves of c-and Ad-fibers convey most nociceptive signals from orofacial areas to the central nervous system. The excitability of these nociceptive nerve fibers may become abnormally increased by chemotherapy drugs resulting in exaggerated pain induced by innocuous thermal and mechanical stimuli. Since the excitability of peripheral afferents including trigeminal nerves is largely controlled by voltage-gated K þ channels, 6 a reduction of the expression and/or functions of voltage-gated K þ channels in nociceptive V2 trigeminal neurons (V2 TG neurons) may lead to neuronal hyper-excitability and neuropathic pain such as orofacial mechanical and cold allodynia in CIPN. 7 Previous patch-clamp recordings made from dorsal root ganglion (DRG) neurons have shown that membrane depolarization evokes two major types of K þ outward currents, an inactivating K þ outward current and a non-inactivating K þ outward current. [8][9][10] The inactivating K þ outward current, also known as type-A current (I A ), can be further classified into fast inactivating current (I Af ) and slowly inactivating current (I As ) based on their inactivation kinetics. 8,10,11 It has been shown that I A currents become reduced following chronic compression of the DRG. 12 This change in I A currents is suggested to contribute to the enhanced neuronal excitability and the pain and hyperalgesia associated with chronic compression of the DRG. 12 A recent study also shows that I A currents in joint sensory neurons are reduced and excitability of these neurons are enhanced in a murine model of antigen-induced arthritis. 13 I A currents in DRG neurons are thought to be mediated by Kv1.4, Kv3.4, Kv4.2, and Kv4.3 channels. 14 Kv4.3 channels are found to be expressed in nociceptive DRG neurons that also express TRPV1 receptors and NaV1.8 channels. 15,16 Nociceptive DRG neurons also express Kv3.4 and Kv1.4 channels, 15,17 but have negligible expression of Kv4.2. 16 In a neuropathic pain model induced by spinal nerve ligation in rats, the protein levels of Kv3.4 and Kv4.3 in the DRG neurons are found to be greatly reduced. 18 It is currently not known whether Kv4.3 channels are expressed on V2 TG neurons to mediate I A currents in these cells. Furthermore, it is also not known whether Kv4.3 channel expression and I A currents may be altered under pathological conditions including CIPN. In the present study, we addressed these questions by immunostaining and patch-clamp recordings on acutely dissociated V2 TG neurons of control and oxaliplatin-treated rats.

Rat model of CIPN
Male Sprague-Dawley rats (300-450 g) were used. Animal care and use conformed to NIH guidelines for care and use of experimental animals. Experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. Animals were divided into two experimental groups, the CIPN group induced by oxaliplatin (oxaliplatin-treated group) and the control group. For the oxaliplatin-treated group, 19 rats were injected with oxaliplatin intraperitoneally at a dose of 2 mg/kg (200 ml each rat) for five consecutive days (an accumulative dose of 10 mg/kg). The rats in the control group were injected with the same amount of saline.

Immunohistochemistry
TGs were harvested from animals 28 to 30 days following oxaliplatin or saline injections and used for immunostaining to determine Kv4.3 expression. In brief, animals were anesthetized with ketamine/xylazine (100 mg/kg:10 mg/kg, i.p.), transcardially exsanguinated with 0.1 M phosphate-buffered solution (PB), and perfused with 4% paraformaldehyde (PFA) in 0.1 M PB solution. TGs were removed and placed in 30% sucrose for cryoprotection for two nights. The TGs were then embedded in OCTV R compound (Baxter Scientific) and cut on a cryostat (Leica Biosystems, Buffalo Grove, IL) into 10lm sections. The sections of TGs were thaw-mounted onto slides and allowed to air-dry. For each slide, three TG sections from saline-injected animals (control) and three TG sections from oxaliplatin-treated animals were mounted on the same slide. These TG sections were then encircled together with hydrophobic resin (PAP Pen-The Binding Site) so that the two groups were paired in immunostaining. The slide-mounted sections were rinsed with the BupHTM-Modified Dulbecco's Phosphate-Buffered Saline (PBS, ThermoFisher Scientific, Waltham, MA) three times, and then sequentially incubated at room temperature with ethanol solutions at the concentrations of 50% for 10 min, 70% for 10 min, and 50% for 10 min. The slides were rinsed three times with PBS and sections were incubated with 10% normal goat serum in PBS for 1 h at room temperature. The sections were incubated with a polyclonal rabbit anti-Kv4.3 antibody (1:2000 diluted with 5% normal goat serum in PBS; Abcam, Cambridge, MA) at 4 C for two nights. Following three rinses with PBS solution, the sections were incubated with a secondary antibody for one night at room temperature. The secondary antibody (1:1000 in 5% normal goat serum in PBS) was a goat anti-rabbit IgG conjugated with Alexa-594 (ThermoFisher Scientific, Waltham, MA). The sections were rinsed three times with PBS solution, cover-slipped with the Prolong Diamond Antifade Mountant medium (ThermoFisher Scientific). Slices were viewed under an upright fluorescent microscope (BX-43, Olympus, Tokyo, Japan) and images were captured using a CCD camera (DP80, Olympus, Tokyo, Japan).

Patch-clamp recordings
Animals were euthanized by overdose of isoflurane 28 to 30 days following oxaliplatin or saline injections. TGs were then harvested from each animal. Under a dissection microscope, V2 and V1 parts of TG (V2 TG and V1 TG) were identified and a cut along the midline of TG was made to separate V2 part from the neighboring V1 region of the TG. The V2 TG was dissected out and incubated with dispase II (5 mg/ml) plus type I collagenase (2 mg/ml) in 2 ml bath solution at 35 C for 45 min. The bath solution was the same one used for cell perfusion in electrophysiology experiments (see below). After a rinse, V2 TGs were triturated to dissociate the neurons in the bath solution, and the dissociated V2 TG neurons were plated on glass coverslips coated with poly-D-lysine and maintained at room temperature. Whole-cell patchclamp recordings were performed within 1 to 4 h after cell plating.
The cells were perfused with normal bath solution flowing at 1 ml/min in a 0.5-ml recording chamber placed on the stage of an Olympus IX70 microscope. The bath solution contained (in mM) 150 NaCl, 5 KCl, 2 MgCl 2 , 2 CaCl 2 , 10 glucose, 10 HEPES, with pH of 7.35 and osmolarity of 330 mosM. The internal solution of electrodes contained (in mM) 135 K-gluconate, 0.5 CaCl 2 , 2 MgCl 2 , 5 KCl, 5 EGTA, 10 HEPES, 2 Na 2 -ATP, and 0.5 Na 2 -GTP, with pH of 7.35 and osmolarity of 330-335 mOsm. Recording electrode resistance was $5 MX. The junction potential was À15 mV and was adjusted during data analysis. The series resistance of each recording was below 30 MX and was not compensated. Recording signals were amplified with Axopatch 200B (Axon Instruments), filtered at 2 kHz, and sampled at 5 kHz using pClamp 10 (Axon Instruments). Unless otherwise indicated, all reagents were purchased from Sigma.
To determine membrane and action potential properties of V2 TG cells, recordings were performed under the whole-cell current-clamp mode.
Step current pulses were injected into cells through patch-clamp electrodes. The step currents were applied from À50 pA to 1975 pA in increments of 25 pA per step and the duration of each pulse was 1 s. To determine voltage-activated currents, recordings were performed under the whole-cell voltageclamp mode. V2 TG neurons were held at À75 mV in voltage-clamp experiments, and voltage steps were applied from À125 mV to 35 mV with increments of 10 mV each step and step duration of 500 ms. I A current components, which were predominant at the initial phase of outward currents, were not isolated from total outward currents in the present study. All recordings were performed at the room temperature of 24 C.

Data analysis
For the data of immunostaining on TG sections, Kv4.3immuno-positive and Kv4.3-immuno-negative neurons in V2 TG region were visually identified from the acquired fluorescent images. The percent of KV4.3-ir positive neurons was then calculated. For the data of electrophysiological recordings, membrane parameters and voltage-activated currents were analyzed using Clampfit 10 software. Unless otherwise indicated, data are reported as mean AE SEM, *p < 0.05, **p < 0.01, and ***p < 0.001, unpaired Student's t test.

Results
We explored whether there was a down-regulation of Kv4.3 channel expression in V2 TG neurons of rats 28 to 30 days following oxaliplatin treatment. In our previous studies, oxaliplatin treatment resulted in orofacial neuropathic pain that was manifested by orofacial mechanical and cold allodynia. 20 The reason for selecting the aforementioned time period in the present study was that our previous studies showed most prominent orofacial neuropathic pain during this time period. We focused on V2 TG neurons because orofacial regions are largely innervated by this branch of the trigeminal nerve system. In control animals, Kv4. We compared the percentage of Kv4.3-ir positive V2 TG neurons between control and oxaliplatin-treated groups (Figure 1(g)). In control group, about 20% of V2 TG neurons (20.3 AE 1.1%, 2083 neurons in 21 TG sections from seven rats, n ¼ 7) were Kv4.3-ir positive. In oxaliplatin-treated animals, about 10% V2 TG neurons (10.2 AE 0.6%, 1937 neurons in 20 TG sections from 7 rats, n ¼ 7) were Kv4.3-ir positive, significantly less than that of the control group (p < 0.001, Figure 1(g)).
We compared total numbers of V2 TG neurons between control and oxaliplatin-treated groups to see if there was a net loss of total V2 TG neurons following oxaliplatin treatment. In the oxilaplatin-treated group, the average numbers of V2 TG neurons in each field were 97 AE 3.9 (20 sections of seven rats, n ¼ 7), not significantly different from those of control group (99 AE 4.2, 21 sections of seven rats, n ¼ 7).
We performed patch-clamp recordings from small-to medium-sized V2 TG neurons (diameter: 24 to 42 mm, Cell diameters (µm) 31.61 AE 0.73 mm, n ¼ 57) acutely dissociated from control and oxaliplatin-treated groups. Under the voltage-clamp mode, an outward current could be evoked when a depolarization step from À75 mV to À25 mV was applied to a V2 TG neuron (Figure 2(a) and (d)). In some V2 TG neurons, the voltage-activated outward current showed slow inactivation component (Figure 2(a)), consistent with I As subtype of I A currents reported previously in DRG neurons. 9 In the control group, most V2 TG neurons (21 of 29 cells, 72%) displayed I As currents and 28% of the V2 TG neurons (8 out of 29 cells) showed I Af currents (Figure 2 (g)). Interestingly, most V2 TG neurons that showed I As currents fired multiple action potentials in response to membrane depolarization (Figure 2 For the oxaliplatin-treated group, I As or I Af currents were also shown in V2 TG neurons (G-I). In contrast to the control group, significantly less V2 TG neurons (12 out of 28 cells) displayed I As currents and majority of V2 TG neurons (16 out of 28 cells) showed I Af currents in oxaliplatin-treated group (Figure 2(g)). Similar to control group, I As currents were associated with nociceptivelike and multiple action potential firing properties (Figure 2(h)) and I Af currents were associated with non-nociceptive and single action potential firing properties (Figure 2(i)).
We examined voltage-activated K þ outward currents in non-nociceptive-like V2 TG neurons to determine whether these currents were changed following oxaliplatin treatment (Figure 3). V2 TG neurons in this set of experiments all had narrow action potentials and their outward currents showed I Af current components without detectable I As current components. Current-voltage relationship (I-V curve) of voltage-activated K þ currents showed no significant difference in I Af current components between control group and oxaliplatin-treated group (Figure 3(a) to (c)). There was also no significant difference in non-inactivating outward current components (I K components) between the control group and the oxaliplatin-treated group (Figure 3(a), (b), and (d)). We compared I Af outward current components evoked at À25 mV between control and oxaliplatin-treated group. There was no significant difference in the amplitude between the two groups (Figure 3(e)). Similarly, amplitudes of I K current components evoked at À25 mV were also not significantly different between the two groups (Figure 3(f)).
Voltage-activated K þ currents were examined in nociceptive-like V2 TG neurons of the control and oxaliplatin-treatment groups. V2 TG neurons in this set of experiments all had broad action potentials. These V2 TG neurons all showed I As current components at initial time of voltage-activated outward currents. I-V curve showed a reduction of I As current components following oxaliplatin treatment (Figure 4 (a) to (c)). I-V curve also showed a reduction of noninactivating components of voltage-activated outward currents following oxaliplatin treatment (Figure 4(a), (b), and (d)). We chose I As current components evoked at À25 mV for a statistical comparison between the control and oxaliplatin-treated group. The amplitudes of I As were significantly smaller (p < 0.01) in oxaliplatintreated group (1402 AE 132 pA, n ¼ 12) than in the control group (2050 AE 142 pA, n ¼ 21, Figure 4(e)). Similarly, the amplitudes of non-inactivating components of voltage-activated outward currents evoked at À25 mV were significantly smaller (p < 0.01) in the oxaliplatintreated group (415 AE 91 pA, n ¼ 12) than in control group (824 AE 126 pA, n ¼ 21, Figure 4(f)).
We determined whether membrane excitability of nociceptive-like V2 TG neurons was increased following oxaliplatin treatment. V2 TG neurons in this set of experiments all had broad width of action potentials, multiple action potential firing pattern, and I As current components in voltage-activated outward K þ currents. Rheobase for initiating action potential firing in these nociceptive-like V2 TG neurons was significantly lower (p < 0.05) in oxaliplatin-treated group (108 AE 11 pA, n ¼ 9) than in control group (175 AE 21 pA, n ¼ 14, Figure 5  ( Figure 5(g)) were not found to be significantly different between the control and oxaliplatin-treated groups.

Discussion
In the present study, we have demonstrated that Kv4.3 channels are expressed mainly in small-sized V2 TG neurons of rats. We show that treatment of animals with the chemotherapy drug oxaliplatin leads to a significant down regulation of Kv4.3 channel expression in V2 TG neurons. We have further found that oxaliplatin treatment results in a significant reduction of I As components of voltage-activated K þ outward currents. These changes in Kv4.3 channels and I As currents are accompanied by an increase of membrane excitability in nociceptive-like V2 TG neurons. To our knowledge, this is the first report of such changes in V2 TG neurons. These findings may provide new insights into mechanisms underlying chemotherapy-induced neuropathic pain in orofacial regions. The oxaliplatin treatment regimen used in the present study has been demonstrated to be a reliable rat model of  CIPN in our previously studies. 20,21 We have previously shown that the oxaliplatin treatment regimen leads to mechanical and cold allodynia in orofacial regions. 20,21 Oxaliplatin is the first line chemotherapy drug for advanced colorectal cancer and it is also an essential chemotherapy drug for several other types of cancers. 1 Most cancer patients treated with oxaliplatin develop CIPN with symptoms including mechanical allodynia and cold allodynia/hyperalgesia. 22 Clinically, CIPN can occur in proximal body parts including the orofacial regions 3,4 in addition to the extremities. 2   . (e) Amplitudes of I As outward current components evoked by a voltage step from À75 to À25 mV in nociceptive-like V2 TG neurons. Open bar, control group (n ¼ 21); closed bar, Oxa-treated group (n ¼ 12). (f) Amplitudes of noninactivating outward current components evoked by a voltage step from À75 to À25 mV in nociceptive-like V2 TG neurons. Open bar, control group (n ¼ 21); closed bar, Oxa-treated group (n ¼ 12). Data represent mean AE SEM, *p < 0.05, **p < 0.01, comparing between control group and oxaliplatin-treated group, two-way ANOVA (c and d), or unpaired Student's t test (e and f).
channel expression has been observed in nociceptive DRG neurons in a neuropathic pain model induced by spinal nerve ligation in rats. 18 This change in Kv4.3 expression in nociceptive DRG neurons has been suggested to be an underlying mechanism leading to hyperexcitability of nociceptive DRG neurons and pathological pain. 18 Thus, down-regulation of Kv4.3 channel expression in V2 TG neurons may also lead to hyperexcitability of nociceptive V2 TG neurons and pathological pain in orofacial regions. Our patch-clamp recordings from acutely dissociated V2 TG neurons show two subtypes of I A currents, I As and I Af currents, in V2 TG neurons. This is consistent with previous observations of the two subtypes of I A currents in DRG neurons. 8-10 V2 TG neurons with I As currents appear to be mostly nociceptive neurons since their action potential width was broad. Previous studies in DRG neurons have shown that some cells co-express both I As and I Af currents, 8,10 raising a possibility that some of our nociceptive-like V2 TG neurons with I As currents may also contain some degree of I Af currents. In contrast to V2 TG neurons with I As currents (or I As plus I Af currents), V2 TG neurons with only I Af currents fire narrow-width action potentials, a non-nociceptive Control Oxa AP numbers at 2X Rheobase (g) Figure 5. Increases of membrane excitability in nociceptive-like V2 trigeminal ganglion neurons following oxaliplatin treatment. (a) Sample traces show action potential firing in responses to rheobase (dark) and 2Â rheobase (gray) depolarizing current steps in a nociceptive-like V2 TG neuron of control group. The rheobase is 200 pA. Resting membrane potential is À61 mV. (b) Similar to (a) except the recording was made from a nociceptive-like V2 TG neuron of oxaliplatin-treated group. The rheobase is 75 pA. Resting membrane potential is À54 mV. (c to g) Summary data of rheobase (c), resting membrane potential (d, RMP), input resistance (e), action potential threshold (f), and action potential numbers at 2Â rheobase (g) of nociceptive-like V2 TG neurons. Open bars, control group (n ¼ 14, c to g); solid bars, oxilaplatin-treated group (n ¼ 9, c to g). Data represent mean AE SEM, *p < 0.05, comparing between control group and oxaliplatin-treated group.
neuron property. This is consistent with a previous study in DRG neurons showing that DRG neurons with I Af currents are usually the neurons without an actionpotential shoulder. 8 We show that I As current components, but not I Af current components, are significantly reduced in the oxaliplatin-treated group. The reduction of I As currents in nociceptive-like V2 TG neurons is consistent with the down-regulation of Kv4.3 expression shown in our immunostaining results. Thus, down-regulation of Kv4.3 expression should at least partially account for the reduction of I As currents in our V2 TG neurons following oxaliplatin treatment. In nociceptive DRG neurons, previous studies have shown that a number of other voltage-gated K þ channels including Kv3.4, Kv1.4, and Kv4.2 channels are also expressed on nociceptive DRG neurons. 15,17 Therefore, it will be interesting to determine in the future whether these I A types of K þ channels may be expressed on V2 TG neurons and undergo down-regulation following oxaliplatin treatment. In our nociceptive-like V2 TG neurons, we have found that the non-inactivating components of voltageactivated outward currents were also significantly reduced. These non-inactivating current components could be part of I As currents and become downregulated. Alternatively, the non-inactivating outward current components could be mediated by other voltage-gated K þ channels and down-regulated in nociceptive-like V2 TG neurons following oxaliplatin treatment.
Since I As currents are activated at voltages near resting membrane potentials, these low-threshold voltageactivated K þ currents would serve as a brake to counteract membrane depolarization and thereby control neuronal excitability. The reduction of I As currents in nociceptive-like V2 TG neurons in the oxaliplatintreated group would compromise this excitabilitycontrolling mechanism. Consistently, we have found that a reduction of I As currents is accompanied by an increase of excitability in nociceptive-like V2 TG neurons in the oxaliplatin-treated group. In our recent study, we have shown that controlling V2 TG neuron excitability is also mediated by I m currents of KCNQ2/3 channels, another type of low-threshold voltage-activated K þ currents. 20,21 Furthermore, our recent study has found that oxaliplatin treatment leads to downregulation of KCNQ2/3 channels in V2 TG neurons and orofacial mechanical allodynia. 20 Thus, V2 TG neuron excitability is controlled by different types of low-threshold voltage-gated K þ channels and their down-regulation by chemotherapy drugs may be a common mechanism underlying chemotherapy-induced orofacial neuropathic pain.

Author contributions
JGG conceived and designed the experiments and wrote the paper. VV performed electrophysiology experiments, and JL performed immunostaining experiments.

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by NIH grants DE018661 and DE023090 to J.G.G.