Rebuttal from Peter M. Lalley, Paul M. Pilowsky, Hubert V. Forster and Edward J. Zuperku

Our opponents contend that nanolitre microinjections into preBötC may not target critical preBötC neurons, and claim that drug diffusion from microdialysis probes located 0.5–1.1 mm from preBötC are superior (Montandon et  al. 2011). Microinjection studies rely on coordinates and functional properties of the preBötC (mixture of ventral respiratory column (VRC) I-and E-neurons) in addition to rapid onset tachypnoeic responses (Mustapic et  al. 2010) to d,l-homocysteic acid (DLH) microinjections (Schwarzacher et  al. 1995; Monnier et  al. 2003; Krolo et  al. 2005). With reverse microdialysis, the drug is continuously released and targets a large volume due to diffusion in a complex interstitial space, which is not localized to a specific target (Höcht et  al. 2013), e.g. only neurokinin-1 receptor-containing VRC neurons. To address this limitation ‘correlation maps’ based on probe distances from points in medullary 3-D space and corresponding response latencies were used to suggest the preBötC as the exclusive target. However, this method does not provide unequivocal results.

Several points of evidence from a number of studies reinforce the validity and accuracy of microinjections into targeted regions of the pons and medulla, and their local effectiveness. Extensive bilateral naloxone microinjections in the preBötC region were ineffective (n = 15, Mustapic et  al. 2010), but naloxone microinjections (∼540 nl each) into the parabrachial region produced rapid (30–40 s) and complete reversal of systemic opioid (remifentanil)-induced bradypnoea (Prkic et  al. 2012). Contrary to the opinion put forth by Montandon et  al. (2011), it is unlikely that this reversal was due to antagonism of endogenously released opioids since the amount of reversal matched the amount (72 ± 4%) of remifentanil-induced bradypnoea in both decerebrate (n = 9) and isoflurane-anaesthetized (n = 12) preparations. Furthermore, the parabrachial region is distinctly different from the rostral ventromedial medullary pain pathways (Phillips et  al. 2012). In contrast, Montandon et  al. (2011) dialysed naloxone (300 μm) for 45 min prior to i.v. fentanyl injections, allowing antagonist concentrations (>50 nm) to reach μ-opioid receptors at great distances.

The suggestion (Montandon et  al. 2011) that endomorphin-1 was injected into BötC in the Lonergan et  al. (2003) study, thereby causing tachypnoea, is incorrect. PreBötC was identified by field potential mapping and albumin–colloidal gold labelling at opioid injection sites. Moreover, in decerebrate dogs, all DAMGO (n = 16) preBötC microinjections produced tachypnoea.

Considering the limitations of reverse dialysis to target a specific site compared to the precision of microinjections, we conclude that preBötC μ-opioid receptors do not mediate the bradypnoea induced by systemic μ-opioid administration.

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Competing interests

None declared.