Using a between-subjects design and a single morphine challenge dose, 129S6/SvEv and 129P3/J strains exhibited analgesic tolerance to morphine in the hot plate and tail withdrawal assays following two separate regimens of chronic morphine administration. These results were surprising, given three reports utilizing three different methodologies and indicating no tolerance in the 129 strains (Crain & Shen 2000
; Kest et al. 2002
; Kolesnikov et al. 1998
). Because there is a reduced intensity of the tail withdrawal response under morphine, we thought that we might be measuring a weaker response than previous investigators and this might account for the discrepant result. As such, we used a lower challenge dose of morphine and measured both the first and second tail flick response following a morphine challenge; however tolerance was observed in both instances. It is possible that tolerance might not have been observed in 129S6/SvEv mice had we employed the same temperature (55ºC) and exact methodology as previous investigators with this particular strain (Crain & Shen 2000
). Indeed, when employing the same chronic regimen and method of tolerance assessment as one of the three studies, no tolerance was observed in the 129P3/J strain (Kest et al. 2002
). Last, we report for the first time that following chronic morphine administration, there was a dramatic increase in the number of tail flicks during the first second following the baseline nociceptive response in C57BL/6J, but not 129P3 mice. This strain-specific rapid tail fibrillation represents an additional adaptive behavioral response that can occur following chronic morphine and which may be heritable.
Although we replicated the lack of morphine tolerance in 129P3/J mice following a within-subjects design and cumulative dosing (Kest et al. 2002
), we were unable to reproduce the robust shift to the right in the dose-response curve of C57BL/6J mice that was previously reported, in part because on day 4, the mice did not reach comparable analgesia to day 1, even up to a cumulative dose of 108.8 mg/kg (data not shown), and thus, ED50
calculations between days 1 and 4 were not comparable. This may be due to the fact that in our hands, morphine was considerably less sensitive in this strain compared to the previous study (Kest et al. 2002
) and thus, perhaps maniuplating the starting dose or increment dose would have affected the degree of tolerance detected (Duttaroy et al. 1997
). Nevertheless, in C57BL/6J mice, we only saw a significant change in analgesic efficacy (i.e., tolerance) at the higher morphine doses, beginning at 66.8 mg/kg (). Given our effort to conduct the experiment exactly as previously described, the source of discrepancy is not clear. However, in the past, using C57BL/6J mice, we have constructed cumulative dose-response curves following a number of different chronic morphine regimens and cumulative dosing procedures and have never observed large shifts in the dose-response curve or ED50
values. Although using separate mice for each dose might alleviate the problem, due to the exponential costs that this would require, we frequently report a difference in analgesic efficacy following a single morphine challenge dose (Bryant et al. 2006
; Bryant 2005
; Eitan et al. 2003
A likely explanation for the lack of tolerance in the lower cumulative doses is that acute tolerance develops rapidly and progressively in both strains on both days 1 and 4, and thus, there is no difference in analgesia at the initial doses. This hypothesis is supported by the observation that a cumulative dose of 24.8 mg/kg produces approximately 30% MPE in C57BL/6J mice (), whereas roughly the same acute dose (25 mg/kg) produces almost 100% MPE in naive C57BL/6J mice (). Although not as drastic of a difference, this holds true for 129P3/J mice such that a cumulative dose of 6.6 mg/kg produces approximately 45%MPE () whereas a similar acute dose (7.5 mg/kg) in naive mice produces approximately 75–95%MPE (, ). An alternative explanation to acute tolerance is that repeated testing induces tissue inflammation in the tail which results in hyperalgesia on top of morphine analgesia and as a result, an apparent reduction in morphine analgesia.
We replicated the strain-specific spontaneous hyperalgesia resulting from chronic morphine that occurs in the tail withdrawal assay in C57BL/6J, but not 129P3/J mice (Bryant et al. 2006
; Bryant 2005
; Kest et al. 2002
) (). Additionally, C57BL/6J, but not 129P3/J mice, exhibited a large increase in the frequency of tail flicks during the first second following the first baseline nociceptive response (). To our knowledge, this sharp increase in the number of flicks is the first piece of evidence to suggest a change in the intensity of the nociceptive response following chronic morphine. Following a subsequent morphine challenge (), we did not observe any flicking during the second following the first flick in either strain. However, given the spontaneous hyperalgesia and the accompanied rapid flicking in C57BL/6J mice receiving chronic morphine, it is possible that there is a strain-specific increase in intensity of the response under morphine. In support, there is a strong relationship between the intensity of the nociceptive stimulus and the intensity of the electromyogram response of the extensor caudae medialis muscle of the tail (Tsuruoka et al. 1988
). Similar to an increase in stimulus intensity, chronic morphine also shortens the latency to the nociceptive response (). Interestingly, strain differences in the firing rate of cutaneous nociceptors to thermal stimulation have recently been reported with C57BL/6 mice exhibiting the highest frequency, in particular, at 49ºC (Lawson 2006
). Future studies using electrophysiological and electromyogram techniques will determine if there are strain-dependent changes in the frequency and intensity of the nociceptive response and whether these changes correlate with spontaneous morphine-induced hyperalgesia and tolerance.
In the hot plate assay, the development of analgesic tolerance to morphine was blocked in 129S6/SvEv and CD-1 mice by chronic co-administration with the non-competitive NMDA receptor antagonist MK-801 (), as previously reported with CD-1 mice in the tail flick assay (Elliott et al. 1994
; McLemore et al. 1997
) and with C57BL/6J mice using the same regimen and hot plate assay (Bryant et al. 2006
). Data from a previous study suggested that 129S6/SvEv mice had an NMDA receptor defect based on the observation that exogenous NMDA administration facilitated the development of morphine tolerance only in the outbred CD-1 strain (Kolesnikov et al. 1998
). An important difference between the previous study and the present one is that we used the hot plate assay and they used the tail flick assay. One drastic example of how these two pain assays can differ in terms of modulation of morphine tolerance comes from our recent observation that under the same chronic regimen (but in male C57BL/6J mice), MK-801 attenuated the development of morphine tolerance in the hot plate assay, while in the tail withdrawal assay, it actually facilitated
it (Bryant et al. 2006
). Thus, that an NMDA receptor antagonist attenuated morphine tolerance in the hot plate assay does not eliminate the possibility that 129S6/SvEv mice have dysfunctional NMDA receptors. However, because of the mere fact that these mice exhibit tolerance in the tail withdrawal assay (), if this strain has a defect in NMDA receptor function, it is not relevant to morphine tolerance under these conditions.
Chronic administration of MK-801 with morphine produced a significant increase in baseline latency in both 129S6/SvEv and CD-1 strains (), as previously reported in male C57BL/6J mice (Bryant et al. 2006
). Furthermore, chronic MK-801 per se prolonged the subsequent analgesic effect of a challenge morphine dose in 129S6/SvEV mice (), while having no effect in CD-1 mice (). One or both of these effects could contribute to the modulation of morphine tolerance in 129S6/SvEv mice. Enhanced morphine analgesia following chronic NMDA receptor antagonism has been reported previously (Dunbar & Yaksh 1996
). The strain dependency of the prolongation of morphine analgesia following chronic NMDA receptor antagonism suggests that the effect depends on the genotype. A complete strain survey examining the modulation of acute morphine analgesia and tolerance by chronic NMDA receptor antagonism will be necessary to determine the heritability of these traits. Possible mechanisms could include an upregulation of functional opioid receptors or enhanced binding of morphine as occurs with the mu opioid receptor-selective peptide agonist DAMGO following co-administration with an NMDA receptor antagonist (Wong et al. 1996
We conclude that the detection of morphine analgesic tolerance in 129 strains depends on the method of tolerance assessment. Utilizing a between-subjects design and a single challenge dose of morphine, comparable tolerance was observed between 129P3/J and C57BL/6J mice. This suggests that the degree and pattern of heritability of morphine tolerance across mouse strains previously reported (Kest et al. 2002
) may look completely different when employing this type of design and this may have implications for gene mapping studies where previous attempts to identify quantitative trait loci with morphine tolerance have been unsuccessful (Kest et al. 2004