Habituation, desensitization and sensitization of the Hering–Breuer reflex in normal and Mecp2−/y knockout mice

In an interesting study published recently in The Journal of Physiology,Stettner et al. (2007) reported that Mecp2−/y knockout (KO) mice devoid of the X-linked methyl-CpG binding protein 2 gene demonstrated prolonged and highly variable postinspiratory vagal efferent activity that correlated with the characteristic breathing arrhythmias in these mutant animals. Furthermore, glutamate microinjections into the pontine Kolliker–Fuse nucleus evoked significantly longer apnoeas with enhanced postinspiratory vagal discharge compared with wild-type (WT) controls. Finally, the authors provided data to suggest that repetitive electrical stimulation of vagal afferents also elicited significantly longer Hering–Breuer reflex (HBR)-like apnoeas in the Mecp2−/y KO mice without any sign of ‘desensitization’ of the reflex. We applaud the authors' elaboration of impaired vagal-pontine control of the respiratory rhythm in Mecp2−/y KO mice. However, we feel obliged to point out that their notion of differing ‘desensitization’ of the vagally induced HBR apnoeas in wild-type and Mecp2−/y KO animals is problematic and is at variance with current understanding of non-associative learning in brain pathways in general and in vagal-pontine mediation of the HBR in particular. Comparison with previous related studies is important because not only does it help to put the significance of the work in perspective, but in this case, it also reveals major discrepancies in interpretation of the results that point to a very different conclusion. For many decades, non-associative learning in the parlance of the behavioural neuroscience literature has been generally thought of as a dual process (habituation or sensitization) corresponding to activity-dependent down- or up-regulation of the response to a continuous or repetitive stimulus (Groves & Thompson, 1970). The notion of desensitization as a novel (and non-trivial) form of non-associative learning was first introduced in this Journal by Siniaia et al. (2000) precisely in the context of fictive HBR modulation of the respiratory frequency (particularly expiratory duration (Poon et al. 2000)) induced by low-intensity, high-frequency vagal electrical stimulation. This notion has led to a general framework of non-associative learning with four base modes (habituation, desensitization, primary sensitization and secondary sensitization), which has proved to harmoniously unify the functional characteristics of non-associative learning reported in many brain systems across animal phyla and sensory modalities (Poon & Young, 2006). Conventionally, habituation is characterized as (among other functional criteria) a down-regulating adaptation with short-term memory (Groves & Thompson, 1970; Poon & Young, 2006). Importantly, the latter remains latent post-stimulation and is activated (recalled) only when stimulated again – a process which has been termed ‘input gating’ (Young et al. 2003; Poon & Young, 2006). Desensitization is distinguished from habituation by the explicit expression of post-stimulation memory rebound and recovery, as desensitization (i.e. secondary habituation) is not subject to input gating. The exponential down-regulation effects of habituation and desensitization have been likened to those of a monophasic or biphasic (without or with memory rebound) neural differentiator or high-pass filter (Poon & Siniaia, 2000; Poon et al. 2000; Poon & Young, 2006). In the vagally induced HBR, desensitization was abolished after lesioning of the Kolliker–Fuse nucleus or systemic administration of the non-competitive NMDA receptor antagonist MK-801 (Poon et al. 2000; Siniaia et al. 2000). A working scheme of vagal-pontine mediation of the HBR and its habituation and desensitization has been proposed previously (Poon & Siniaia, 2000; Siniaia et al. 2000; Poon, 2004; Song & Poon, 2004). The authors of this paper stated that ‘All WT preparations showed a clear desensitization of the HBR in response to repetitive vagal stimulation’. However, from the presented data (Fig. 7A in Stettner et al. 2007) it appears that the shortening of the apnoea duration after repetitive stimulation reflected habituation (in the classical sense) of the HBR instead of desensitization, since a memory rebound was not evident at the cessation of vagal stimulation. Indeed, if anything, the expiratory duration remained longer immediately after both the 1st and 15th stimulation trial compared with resting conditions, showing the memory effects of concomitant secondary (not primary; see Poon & Young, 2006) sensitization of the HBR instead. This post-stimulation sensitization memory appeared to be accentuated in the Mecp2−/y KO animals, as indicated by the sustained apnoeas extending well beyond the vagal stimulation period (Fig. 7B and C). The mechanism of this peculiar secondary sensitization (instead of desensitization) of the HBR in wild-type and Mecp2−/y KO animals is unclear but could potentially result from activation of rapidly adapting pulmonary stretch receptor fibres or even cardiopulmonary C-fibres, as such secondary sensitization effects were not elicited by low-intensity, high-frequency (15–40 μA, 40–80 Hz) vagal electrical stimulation in rats (Poon et al. 2000; Siniaia et al. 2000). The authors also used a repetitive vagal stimulation protocol to induce non-associative learning of the HBR, apparently because the evoked apnoea was not habituated within the 10 s stimulation period due to the strength of the stimulation. Such a repetitive stimulation protocol is routine in studies of non-associative learning in invertebrate sensorimotor pathways to allow for refractory (‘output gating’) of the resultant motor response (Poon & Young, 2006). Since the mammalian respiratory motor response is non-refractory, a continuous (and sufficiently long) vagal stimulus should be as effective as a repetitive one in discerning habituation, sensitization and desensitization (Young et al. 2003), but neither protocol was found to induce secondary sensitization when vagal stimulation was kept at low intensities to obviate artifacts and nerve fatigue in rats (MacDonald et al. 2004). These caveats aside, the authors have provided convincing evidence of deranged vagal-pontine modulation of the respiratory rhythm in Mecp2−/y KO mice, which, when viewed in the proper light, reveals diminished habituation and accentuated secondary sensitization of fictive HBR. The potential roles of pontine postinspiratory activity in mediating the abnormal habituation, desensitization and sensitization of the HBR and resultant breathing arrhythmias remain to be clarified, although neurons with postinspiratory (early expiratory) discharges have been previously identified in the vicinity of the Kolliker–Fuse nucleus in the rat (Song et al. 2006). This non-associative learning perspective should help illuminate the pathogenesis of breathing abnormalities in these mutant animals or those with DNA hypomethylation (Fan et al. 2001), and in patients with Rett syndrome caused by mutations of the MECP2 gene.