Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca2+ channel

Abstract

The importance of voltage-dependent Ca2+ channels (VDCCs) in pain transmission has been noticed gradually, as several VDCC blockers have been shown to be effective in inhibiting this process. In particular, the N-type VDCC has attracted attention, because inhibitors of this channel are effective in various aspects of pain-related phenomena. To understand the genuine contribution of the N-type VDCC to the pain transmission system, we generated mice deficient in this channel by gene targeting. We report here that mice lacking N-type VDCCs show suppressed responses to a painful stimulus that induces inflammation and show markedly reduced symptoms of neuropathic pain, which is caused by nerve injury and is known to be difficult to treat by currently available therapeutic methods. This finding clearly demonstrates that the N-type VDCC is essential for development of neuropathic pain and, therefore, controlling the activity of this channel can be of great importance for the management of neuropathic pain.

Fig. 1. Generation of mice lacking the α₁2.2 subunit of the N-type VDCC. (A) A simplified restriction map around exon 1 of the Cacna1b gene and structure of the targeting vector. The coding region of exon 1 is boxed. neo, PGK-neo cassette; DT-A, diphtheria toxin A fragment gene; B, BglII; E, EcoRI; H, HindIII; N, NotI; S, SstI; X, XbaI. (B) Southern blot analysis of tail DNA. DNA was digested with SstI and the blot was hybridized with a probe shown in (A). The 7.0 kb band is derived from the wild-type allele (WT), and the 5.3 kb band from the targeted allele (Mut). (C) RT–PCR analysis. cDNA derived from brain total RNA was used as a template. A fragment of 330 bp is diagnostic of normal Cacna1b expression. M, 100 bp ladder (Gibco-BRL). (D) Northern blot analysis. Poly(A)⁺ RNA (4 µg) from mouse brains was loaded in each lane. The blot was probed with a Cacna1b cDNA fragment (∼0.7 kb) corresponding to the cytoplasmic loop between repeats II and III of α₁2.2. A GAPDH probe was used as loading control (Sabath et al., 1990). (E) Immunoblot analysis. Membrane proteins (100 µg/lane) from mouse hippocampi were probed with a rabbit polyclonal anti-α₁2.2 (α_1B) (upper) or anti-α₁2.3 (α_1E) antibody (lower). No α₁2.2 protein is detected in Ca_v2.2–/– mice. On the other hand, α₁2.3 protein is expressed at a similar level in all three genotypes. +/+, wild-type; +/–, heterozygote; –/–, homozygous mutant.

Fig. 2. Ba²⁺ currents in DRG neurons from Ca_v2.2+/+ and Ca_v2.2–/– mice and effects of toxins on the Ba²⁺ currents. (A) Current–voltage relationships in Ca_v2.2+/+ and Ca_v2.2–/– DRG neurons. Representative current traces of DRG neurons from Ca_v2.2+/+ (top left) and Ca_v2.2–/– (top right) mice are shown. Current–voltage relationships in both genotypes of DRG neurons are shown in the bottom panel. Open circle, Ca_v2.2+/+; closed circle, Ca_v2.2–/– (n = 4–7). Currents were elicited by a series of voltage steps from –60 to +50 mV (with 10 mV increments) delivered at 10 s intervals with holding potential at –70 mV. (B) Time course of inhibition of the peak Ba²⁺ currents by 1 µM ω-conotoxin GVIA (GVIA) and 0.2 µM ω-agatoxin IVA (IVA) in Ca_v2.2+/+ (top) and Ca_v2.2–/– (bottom) DRG neurons. Peak Ba²⁺ currents, activated by 50 ms depolarization from –70 mV to –10 mV every 10 or 20 s, were plotted against time. Note that the effect of ω-conotoxin GVIA is indistinguishable from the current run-down in Ca_v2.2–/– DRG neurons.

Fig. 3. Anxiety-related behaviors of wild-type (+/+), heterozygote (+/–) and homozygous mutant mice (–/–). (A and B) Elevated plus-maze test for a total of 5 min. The number of entries into each arm (A) and total time spent in each arm (B) are summarized for each genotype. Open columns: open arms; filled columns: closed arms. Entries into open arms are significantly increased in homozygous mutant mice compared with wild-type (P <0.01) and heterozygous mutant mice (P <0.05). +/+, n = 20; +/–, n = 22; –/–, n = 7. (C and D) An open-field test for a total of 2.5 min. Total locomotion time (C) and total path length (D) are summarized for each genotype. Homozygous mutant mice show significantly increased path length compared with heterozygous mutant mice (P <0.05). +/+, n = 20; +/–, n = 22; –/–, n = 9. (E) Startle responses to various intensities of sound pulses. Stimuli of 105 dB (open columns), 115 dB (gray columns) and 120 dB (filled columns) were given. Homozygous mutant mice show significantly decreased responses to sound stimuli of 115 and 120 dB compared with wild-type mice (P <0.05 for both sound stimuli). +/+, n = 11; +/–, n = 10; –/–, n = 7.

Fig. 4. Acute nociceptive responses of wild-type (+/+), heterozygote (+/–) and homozygous mutant mice (–/–). (A) Fifty percent hindpaw withdrawal thresholds to stimulation with von Frey hairs (+/+, n = 21; +/–, n = 29; –/–, n = 15). (B) Tail flick thresholds to noxious mechanical stimuli (+/+: n = 9; +/–, n = 15; –/–, n = 11). (C) Tail flick latencies to noxious heat (48–49°C: +/+, n = 22; +/–, n = 31; –/–, n = 17). Homozygous mutant mice show significantly prolonged latency compared with wild-type (P <0.05) and heterozygous mutant mice (P <0.01). (D) Hindpaw withdrawal latencies to noxious thermal stimuli with a low (infrared intensity 10, open columns) and high intensity (infrared intensity 40, filled columns). +/+, n = 21; +/–, n = 29; –/–, n = 15. (E) Hindpaw licking and/or jumping latencies in the hot plate tests (+/+, n = 21; +/–, n = 29; –/–, n = 15) at 50°C (open columns), 52°C (gray columns) and 55°C (filled columns).

Fig. 5. Nociceptive responses to noxious chemical stimulation of cutaneous or visceral tissue. (A and B) Formalin-evoked hindpaw licking behavior. (A) Open columns represent total time for licking and/or biting in phase 1 (1–7 min after injection); filled columns, total licking and/or biting time in phase 2 (10–47 min after injection). +/+, n = 20; +/–, n = 27; –/–, n = 14. (B) The licking and/or biting time is plotted against the time after formalin injection. Circles, wild-type; squares, heterozygote; triangles, homozygous mutant. Responses in the early half of phase 2 (phase 2A, 10–27 min after injection) were almost completely suppressed in the homozygous mutant. ** represents a significant difference at P <0.01 between +/+ and –/–, and # represents a significant difference at P <0.05 between +/– and –/– at each time point. (C) Visceral nociceptive response (abdominal writhes) produced by intraperitoneal injection of 0.6% acetic acid (+/+, n = 8; +/–, n = 5; –/–, n = 6). (D) Effects of sensitization by a noxious visceral conditioning stimulus on the formalin-evoked somatic nociception. In wild-type mice, which had received a noxious visceral stimulus (0.6% acetic acid injection) 3 weeks before, the phase 2 response (filled column) was considerably reduced compared with the control (gray column). The phase 2 response in homozygous mutant mice after sensitization was significantly (P <0.05) facilitated compared with that of naive homozygous mutants. +/+, n = 6; +/–, n = 4; –/–, n = 6. Data of naive mice (columns with broken lines) are presented for comparison.

Fig. 6. Mechanical allodynia and thermal hyperalgesia induced by spinal nerve ligation. Fifty percent hindpaw withdrawal thresholds to stimulation with von Frey hairs (A) and hindpaw withdrawal latencies to thermal stimuli (B) are plotted against the days after the operation. Circles, uninjured side; triangles, operated side. +/+, wild-type; +/–, heterozygote; –/–, homozygous mutant. Homozygous mutants developed an attenuated post-operative decrease in the threshold and the latency, which are clearly observed in wild-type and heterozygous mutant mice as neuropathic pain symptoms. +/+, n = 9–13; +/–, n = 11–14; –/–, n = 5–8.