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

Abstract

Our opponents contend that nanolitre microinjections into preBotC may not target critical preBotC neurons, and claim that drug diffusion from microdialysis probes located 0.5–1.1 mm from preBotC are superior (Montandon et al. 2011). Microinjection studies rely on coordinates and functional properties of the preBotC (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 (Hocht 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 preBotC 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 preBotC 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 BotC in the Lonergan et al. (2003) study, thereby causing tachypnoea, is incorrect. PreBotC was identified by field potential mapping and albumin–colloidal gold labelling at opioid injection sites. Moreover, in decerebrate dogs, all DAMGO (n = 16) preBotC microinjections produced tachypnoea. Considering the limitations of reverse dialysis to target a specific site compared to the precision of microinjections, we conclude that preBotC μ-opioid receptors do not mediate the bradypnoea induced by systemic μ-opioid administration.