Response from Sean M. Ward and Kenton M. Sanders

We characterized electrical slow wave activity in human stomach using intracellular electrical recording and suggested that previous studies may have under-estimated slow wave frequency (Rhee et al. 2011), because of the reliance on extracellular recording to monitor electrical activity in patients (e.g. Hinder & Kelly, 1977; O'Grady et al. 2010). We had shown previously that biopotentials recorded from gastric muscles with extracellular electrodes are largely movement artifacts due to muscle contractions and not reflective of authentic slow wave activity (Bayguinov et al. 2011). In his Letter to the Editor, Dr O'Grady and colleagues discount our results as artifacts and suggests that prostaglandin synthesis in gastric muscles caused abnormally high slow wave frequency in vitro (O'Grady, 2012). While no paper addresses all potential criticisms, our study controlled for the chronotropic effects of prostaglandins. We believe our paper makes an important contribution and raises questions about whether current evaluations of electrical rhythmicity in human patients are accurate when assessments are made with extracellular recording. Our findings also raise important questions about electrogastrography (EGG), which is used to measure gastric slow waves and diagnose electrical arrhythmias in patients. The value of these tests is questionable if this technique does not accurately record gastric electrophysiological events. O'Grady et al. (2012) suggest that slow wave frequencies are elevated abnormally by positive chronotropic effects imposed by prostaglandins in gastric muscles in vitro. In support of this hypothesis they cite a study of feline stomach in which in vivo recordings were compared with recordings from gastric muscles after excision and dicing of the stomach into many pieces (Xue et al. 1995). O'Gradyet al. misstate the findings of this study when they say that indomethacin blocked the increase in slow wave frequency only when applied immediately after tissue dissection. In fact, Xue et al. report (on p. 161): “Krebs solution containing indomethacin (10 μm) was added to the water bath after the frequency had increased to a maximum (2 h after isolating the segments). The high slow wave frequency returned to the in vivo level (17.3 to 4, and 12.2 to 4 cpm, respectively) after 60 min” (Xue et al. 1995). We performed equivalent tests on human muscles (Rhee et al. 2011, see Supplemental Fig. 1C). Actually, the issue of prostaglandins was the first question we addressed after observing the frequency of slow waves in human muscles. We were aware of the chronotropic effects of prostaglandins in vitro because one of us (K. M. Sanders) published the first study documenting this phenomenon in canine muscles 30 years ago (Sanders & Szurszewski, 1981), and we have investigated the chronotropic actions of prostglandins in several subsequent studies of gastric muscles (e.g. Sanders, 1984; Forrest et al. 2009). In our recent study, indomethacin had only minor effects on slow wave frequency (Rhee et al. 2011) and did not ‘restore’ slow wave frequencies to values considered acceptable by O'Grady and Colleagues. We also tested other chronotropic factors that might have influenced slow wave frequency but our observations did not conform to the dogma that O'Grady et al. seem to view as unassailable. There are problems using studies such as Xue et al. 1995 to refute our findings because that study employed extracellular recording to monitor slow wave activity. Extracellular electrical recordings are likely to be contaminated by muscle contractions (Bayguinov et al. 2011). When contractions are inhibited downstream of electrical slow waves, the biopotentials, typically claimed to be ‘slow waves’, disappear. Xue et al. 1995 made no attempt to suppress mechanical activity in excised muscles, so their conclusions about slow waves and slow wave frequency must be viewed with caution. Prostaglandins have dramatic effects on contractions of gastric muscles, and endogenous prostaglandins exert opposite effects in the proximal and distal stomach. In hundreds of hours of intracellular recording (e.g. Sanders & Szurszewski, 1981; Sanders, 1984; Forrest et al. 2009; Rhee et al. 2011) slow waves didn't ‘disappear’ and ‘reappear’ in response to changes in prostaglandin synthesis as was reported in various gastric regions and during different recording circumstances by Xue et al. 1995. Swings in contractile force and inhibitory effects in distal stomach and excitatory effects in corpus, rather than appearance and disappearance of slow waves, are likely to explain the mysterious occurrences and evanescence of ‘slow waves’ in various parts of the stomach during the course of their recordings. We recorded electrical slow waves in human gastric fundus (Rhee et al. 2011), but no one seems to find these events in the fundus with extracellular recording techniques (although small events are apparent in human gastric fundus in recordings of Hinder & Kelly, 1977). O'Grady et al. suggests that prostaglandins are responsible for the ‘appearance’ of slow waves in human fundus in vitro. They again misquote Xue and coworkers (1995) looking for support for this claim, but in fact the question of slow waves in the fundus in vitro was not addressed in that study. In reading the historical motility literature, we note that few investigators have questioned whether muscle movement is a significant contaminant in extracellular recordings. O'Grady and colleagues assume that since extracellular recording has been used for so long by so many luminaries in motility research, controls, such as movement suppression, are unnecessary and they want us to accept the findings of these studies (and his; O'Grady et al. 2010) on faith. We do not discount all findings of previous investigations of gastric electrical rhythmicity, as O'Grady et al. claim, but our observations raise questions about the ‘standard model’ of gastric electrophysiology that has been developed from extracellular electrical recording and/or observation of movements. O'Grady et al. point to MRI studies as proof of the 3 cpm electrical rhythm in human stomach, but MRI measures movements and movements may be the source of the 3 cpm biopotentials recorded with extracellular electrodes (Hinder & Kelly, 1977; O'Grady et al. 2010). Movement artifacts may also be a factor in magnetic field measurements of gastric activity because the source of the magnetic field might move during contraction in relation to the SQUID device (Bradshaw et al. 2006). Our recent findings challenge a bedrock technique of gastrointestinal (GI) motility research (Bayguinov et al. 2011) and current dogma on human gastric electrophysiology (Rhee et al. 2011). In the majority of previous studies, important control experiments were either not possible when classical studies were performed or ignored. Techniques now exist to isolate the electrical and mechanical components of smooth muscle activity; muscle contraction can be inhibited without affecting electrical slow waves (Bayguinov et al. 2011). We also understand the limitations on resolving field potentials imposed by: (i) the morphology of GI muscles, (ii) the limited mass of regenerative tissue within GI muscles (<10% of cells are interstitial cells of Cajal; ICC), (iii) the mechanism of slow waves, and (iv) the current sink imposed by smooth muscle cells on active regeneration of slow waves in ICC. Thus, it is irresponsible for investigators to ignore obvious controls for extracellular recording. We should also note that investigators not so heavily invested in extracellular recording in human patients have found it easier to accept the necessity for proper controls when interpreting data derived from extracellular recording in the GI tract.