Hyperalgesia in mice lacking the Kv1.1 potassium channel gene

Neuroscience Letters 251 (1998) 121–124

Hyperalgesia in mice lacking the Kv1.1 potassium channel gene J. David Clark a,1, Bruce L. Tempel b ,* a

Department of Anesthesiology and Otolaryngology, The Virginia Merrill Bloedel Hearing Research Center, Seattle, WA 98195-7923, USA b Department of Head and Neck Surgery, University of Washington School of Medicine, Box 357923, Seattle, WA 98195-7923, USA Received 18 May 1998; received in revised form 17 June 1998; accepted 17 June 1998

Abstract Hyperalgesia and morphine induced antinociception were measured in mice lacking the gene for the Shaker-like voltage-gated potassium channel Kv1.1 alpha subunit. The effects of varying gene dosage were studied by comparing homozygous null (−/−) versus heterozygous (±) and wildtype (+/+) littermates. Hyperalgesia was measured using the paw flick assay, hot plate assay and formalin induced hind paw licking. It was observed that null mutant animals had significantly shorter latencies to response in the paw flick (36%) and hot plate (27%) assays while their licking times after hind paw injection of formalin was increased in both the first (74%) and second (65%) phases of the response compared to wildtype controls. Morphine induced antinociception in Kv1.1 null mutant animals was blunted. These studies indicate that Kv1.1 plays an important role in nociceptive and antinociceptive signaling pathways.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Potassium channels; Morphine; Hyperalgesia; Null mutant; Kv1.1

Potassium (K) channels are widely expressed in mammalian cells, and are recognized as controlling a diverse range of physiological processes. In neuronal cells, many receptor types including opiate, alpha-2-adrenergic and GABA-B have been shown to open K-channels thereby increasing potassium currents [1,8,9,16]. The specific molecular identities of the K channel genes underlying these currents have not been identified conclusively, but electrophysiological data indicate that members of the GIRK family of G-protein activated channels are involved [5,6]. More recent evidence suggests that several members of the Shaker-like (Kv1) class of voltage gated delayed rectifier K-channels could be involved in receptor mediated signaling pathways. For example, opiates mediate regulation of a delayed rectifier K channel in hippocampal neurons [12]. Still other data suggest a role for the delayed rectifier channels Kv1.5 and Kv1.6 in opiate signaling since their expression is strongly regulated by chronic exposure to opiates [7]. Galeotti et al. [2,3] have demonstrated that intrathecal injec* Corresponding author. Tel.: +1 206 6164696; fax: +1 206 6161828; e-mail: [email protected] 1 Present address: Department of Anesthesia, Stanford University, VAPAHCS, Palo Alto, CA 94304, USA.

tion of Kv1.1-specific antisense oligonucleotides reduce the antinociceptive potency of opiates, GABA-B agonists and tricyclic agents [2,3]. Using a Kv1.1 null mutant (knockout) mouse model [13] we demonstrate that Kv1.1 plays a central role in nociceptive pathways, and helps to mediate the analgesic effects of morphine. The Kv1.1 deletion mutation was established in embryonic stem cells of the 129Sv background. Chimeric mice were crossed to C3HeB/FeJ mice, and subsequently backcrossed for at least five generations to C3HeB/FeJ mice thus establishing a C3H congenic strain segregating for the Kv1.1 null allele. Litters from heterozygous intercrosses were genotyped at 1 week of age (see below). Animals aged 3.5–4.5 weeks were used in these experiments. Within individual experiments, all animals used were littermates. Behavioral testing was performed blind as to the genotype of individual mice. Mice were brought to the laboratory where behavioral tests were performed 30 min prior to testing to allow acclimation. For each litter of mice, DNA was isolated from tail clips after overnight digestion in proteinase K. An anchored 3primer PCR technique was used to determine genotype. A common reverse primer 5′ GCT TCA GGT TCG CCA CTC CCC 3′ along with Kv1.1-specific forward primer 5′ GCC

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00516- 3

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TCT GAC AGT GAC CTC AGC 3′ (product 337 bp) and the neomycin resistance gene-specific forward primer 5′ CCT TCT ATC GCC TTC TTG ACG 3′ (product 475 bp) were added to 500 ng of DNA. After PCR amplification, products were analyzed using 1.5% agarose electrophoresis. In early experiments Southern blot analysis of DNA samples were carried out in parallel to validate the PCR based results. The rota rod test was used as a gross indicator of general neuromuscular function, balance and coordination. For this test mice were acclimated as usual, then placed on a rota rod (Model 720A; IITC, Woodland Hills, CA) turning at 15 rev./min. The number of falls in a 3-min period were counted [11]. Cutaneous hyperalgesia was measured by the method of Hargreaves et al. [4] using a cutaneous hyperalgesia meter (Model 336, IITC). After the 30-min acclimation period in the behavioral laboratory, mice were placed on a glass surface in a ventilated clear cylindrical enclosure of 10 cm diameter and 7 cm height and allowed to acclimate for an additional 10 min. Thermal nociceptive stimulation was then induced using a focused beam of light directed at the glabrous skin of a hindpaw while in contact with the glass surface. Stimulation was not continued past 25 s to avoid tissue damage. Paw flick latency was measured for both hind paws allowing 5 min between measurements. The hot plate assay was done as described by O’Callaghan and Holtzman [10] with equipment from IITC. The hot plate was set thermostatically at 52.5 ± 0.1°C. Mice were placed in a cylindrical clear enclosure on the hot plate and the time to licking of a hindpaw, the end-point of the assay, was measured with a stopwatch. Animals were left on the hot plate no longer than 60 s to prevent injury. After acclimation as described above, mice were injected with 20 ml of 1.0% formalin solution subcutaneously in the dorsal surface of the right hindpaw using a microsyringe and 30 gauge needle. The mice were then immediately placed in a clear enclosure 20 × 20 × 20 cm with a mirror underneath so that the hindpaw was visible at all times. Nociceptive behavior was defined as licking or biting of the hind paw [15]. Preliminary experiments demonstrated that mice, regardless of genotype, had the expected two phase response to formalin. In experiments where the analgesic effects of morphine were to be assessed, morphine or saline was administered subcutaneously 25–30 min prior to behavioral assay. Preliminary experiments demonstrated that regardless of mouse genotype, maximal morphine effects were seen at this time point at the doses used here. Results from the rota rod testing were used to evaluate neuromuscular function. No significant differences were observed between Kv1.1 genotypes using a protocol shown by others to be sensitive enough to detect differences in neuromuscular performance between wild type mice and mice deficient in the gene for dopamine D4 receptors [11]. The number of falls in 3 min averaged for at least seven

mice in each group were wild type 5.2 ± 0.6, heterozygous 5.4 ± 0.5, and null mutant 5.5 ± 0.7. We used several different pain assays to minimize the possibilities that observed differences in nociceptive response were modality specific or due to differences in motor function. The first assay used a thermal stimulus directed to the animal’s hindpaw in the form of focused light, the so-called paw flick assay (Fig. 1A). Using this assay it was observed that while wild type and heterozygous animals had indistinguishable latencies of 5.6 ± 0.4 versus 5.7 ± 0.3 s, that null mutant animals had a shorter latency, 3.6 ± 0.2 s. A second type of thermal stimulus paradigm, the hot plate assay, was also employed. The response of hindpaw licking in this assay relies on intact higher CNS function. Again, the wild type and heterozygous animals had similar latencies of 25.6 ± 1.7 versus 27.1 ± 1.6 s, respectively, while the null mutant animals had an average latency of 18.7 ± 1.5 s (see Fig. 1B). Finally, the formalin assay was used to provide an analysis based on a different type of stimulus, direct chemical stimulation of nociceptors during the first phase, and stimulation based at least partially on the release of inflammatory mediators during the second (Fig. 2A,B). Here it was observed that during both phases the null mutant (knockout) animals exhibited a greater amount of a pain behavior, lick-

Fig. 1. The relationship between genotype and hyperalgesia. (A) The results of the paw flick assay. Animals were allowed to acclimate to standing on a glass surface for 10 min prior to the use of a focused beam of light to cause noxious thermal stimulation. Each bar represents the mean ± SEM latency for at least 12 animals in each group. (B) The relationship between genotype and latency to paw licking in the hot plate assay. The latency in seconds to the observation of lacking of a hindpaw after placing the animal on a 52.5°C hot plate was measured. Data are the mean ± SEM for at least 15 animals in each group. *P , 0.05.

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ing and biting of the affected paw, than either the wild type or heterozygous animals. The total licking time for wild type animals during the first phase was 61 ± 7.4 s, for the heterozygous animals was 67 ± 7.3 s, and for the null mutant animals was 106 ± 8.7 s. Likewise, during the second phase response the total licking time for the wild type animals was 130 ± 22 s, for the heterozygous animals was 149 ± 24 s, and for the null mutant animals was 215 ± 20 s. The responses of heterozygous animals were not statistically different from wild type. The analgesic effects of subcutaneous morphine were examined in both wild type and knock out animals using the paw flick and hot plate assays. The effects of morphine were concentration dependent, prolonging paw flick latency in both wild type and null mutant mice (Fig. 3A,B). However, the extent to which a given dose of morphine would prolong paw flick latency varied between genotypes. For example morphine at 8 mg/kg prolonged paw flick latency from 5.9 ± 0.2 to 18.2 ± 3.5 s in wild type animals compared with a baseline of 3.5 ± 0.2 extended to 8.3 ± 1.2 s after the same dose of morphine in null mutant animals. Higher doses of morphine would reliably extend paw flick latencies past the arbitrary 25 s cutoff for both wildtype and null mutant animals. The hot plate assay gave similar results with morphine significantly prolonging the latency to hind paw licking regardless of genotype, but providing a greater degree of effect in the wild type animals. Specifically, wild type ani-

Fig. 2. The relationship between genotype and total paw licking time in the formalin assay. (A) The total hindpaw licking times for the various genotypes during the first 5 min following injection of 1% formalin. (B) The licking times for the same groups of animals during the 10–20 min interval following injection. Data are mean ± SEM for at least 10 animals in each group. *P , 0.05.

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Fig. 3. The relationship between genotype and the efficacy of morphine in causing prolongation of paw flick latency. Morphine at the indicated dosages was injected subcutaneously 25 min prior to paw flick assay. Data are the mean ± SEM for at least 11 animals in each group. Statistical analysis was done to evaluate differences between wild type and null mutant mice receiving the same amount of morphine. *P , 0.05.

mals increased their hindpaw licking time from 25.8 ± 1.4 to 47.1 ± 4 s compared knockout animals which increased their baseline time of 19.3 ± 1.5 to 29.4 ± 2.2 s. In the hot plate assay only the lower 3 mg/kg dose of morphine was used because 8 mg/kg morphine caused the paw licking latencies to extend beyond the 60 s cutoff in both the wild type and null mutant groups. Taken together, the paw flick and hot plate data indicate that the effects of morphine are attenuated in the null mutant animals because latency to response to a noxious stimulus is prolonged for a greater period of time in wild type compared to null mutant animals. These findings support the conclusion that Kv1.1 null mutant mice have an increased sensitivity to noxious stimuli. The three principal observations leading to this conclusion are that (1) using a paw flick assay which involves a spinally-mediated reflex withdrawal of a hindpaw from a beam of focused light, Kv1.1 null mutants have shorter latencies, (2) using a hotplate assay in which animals exhibit a more complex licking behavior in response to a noxious thermal stimulus Kv1.1 null mutants have shorter latencies, and (3) using formalin to provide both chemical and inflammatory stimuli, Kv1.1 null mutants again exhibit more of a pain behavior (licking) than their wild type littermates. The consistent result of increased sensitivity to the noxious stimulus across the three separate assays speaks to the robust nature of this observation. It also demonstrates that the increased sensitivity is not modality specific, i.e. thermal, chemical and inflammatory nociceptive signaling systems seem to be affected by the gene deletion. While it is not known if other sensory functions such as vibratory or pressure sensation are affected, hearing in these mice is altered (unpublished observations), suggesting that the Kv1.1 plays a role in several separate types of sensory systems. Our observation that Kv1.1 null mutants have increased sensitivity to noxious stimuli is similar to the results of studies done on mu opiate receptor null mutant mice [14]. These results together with the results of a Kv1.1 antisense ‘knock down’ study [3] prompted us to look at the effects of

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an opiate, morphine, in our Kv1.1 null mutant mice. We observed that larger doses of morphine were needed to achieve similar paw flick latencies in null-mutant versus wild type mice. Hot plate data corroborated these results showing a decreased efficacy of morphine in prolonging the latency to paw licking in Kv1.1 null mutant versus wild type mice. We did not attempt to examine the analgesic effects of morphine using the formalin model. It is interesting that in the Kv1.1 null mutants that pain may be easier to elicit, and that prevention of pain with opiates may be more difficult. It is a common clinical observation that individuals with neuropathic pain can exhibit both lower pain thresholds as well as poor responsiveness to opiate therapy. These observations suggests that the Kv1.1 null mutant mice may provide a useful laboratory model for this type of pain. On a cellular level a plausible model to explain our observations might involve hyperexcitable sensory neurons. If a K channel involved in determining action potential threshold or repolarization were to be eliminated, the cell might be rendered hyperexcitable. The physiological consequences of hyperexcitability of sensory neurons could be increased sensitivity to noxious stimuli and less sensitivity to analgesics. In this model it is plausible but not necessary for the opiate receptor to couple directly to the Kv1.1 channel. Further studies aimed at identifying cells in nociceptive sensory systems that express Kv1.1 and examining how Kv1.1 expression changes with opiate tolerance or pain may help refine our model. This work was supported by a grant from the NIH (DC/ NS 02739 to B.L.T). J.D.C. was supported by a NIDA training grant (DA 07278). The authors would like to thank Ms. Linda Robinson for her assistance with animal care and genotyping experiments. [1] Andrade, R., Malenka, R.C. and Nicoll, R.A., A G protein couples serotonin and GABAB receptors to the same channels in hippocampus, Science, 234 (1986) 1261–1265. [2] Galeotti, N., Ghelardini, C., Capaccioli, S., Quattrone, A., Nicolin, A. and Bartolini, A., Blockade of clomipramine and amitriptyline analgesia by an antisense oligonucleotide to mKv1.1, a mouse Shaker-like K+ channel, Eur. J. Pharmacol., 330 (1997) 15–25. [3] Galeotti, N., Ghelardini, C., Papucci, L., Capaccioli, S., Quattrone, A. and Bartolini, A., An antisense oligonucleotide on the mouse Shaker-like potassium channel Kv1.1 gene prevents antinociception induced by morphine and baclofen, J. Pharmacol. Exp. Ther., 281 (1997) 941–949.

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