Literature Review: Nausea is a condition described in various ways, depending on the individual describing the condition.2 Emesis is a hallmark standard for measuring degree of nausea, however many people experience debilitating nausea while lacking emesis. There are many different stimuli prompting nausea including, unexpected motion signals, toxins, migraines, gastrointestinal diseases, pregnancy, and psychological triggers.1
Motion sickness resulting in nausea occurs due to differences between anticipated sensory signals and the experienced sensory signals. Amplitude of motion typically does not contribute to motion sickness, such that the mismatch between expected and actual sensory input is the primary cause of motion sickness.3
Multiple approaches have shown that several brainstem areas contribute to the generation of nausea and vomiting, even though there are different environmental triggers. The lateral tegmental field (LTF), nucleus tractus solitarius (NTS), and the parabrachial nucleus (PBN) serve to integrate nauseagenic and emetic signals.3
Emesis requires coordinated contractions of the diaphragm and abdominal muscles. Under standard physiological conditions the diaphragm and abdominal muscles alternate contractions during respiration, but during vomiting these muscles co-contract to generate the pressure necessary to expel contents in the stomach from the body. The LTF, NTS, and PBN likely integrate emetic signals to conduct the appropriate muscle contractions for emesis. Since a variety of stimuli trigger emetic responses but the LTF, NTS, and PBN coordinate vomiting from each trigger, a “final common pathway” hypothesis has been proposed.7
It has been demonstrated that the vestibular system plays an integral role in causing motion sickness, a specific form of nausea. Individuals with bilateral labyrinthine deficits are not as susceptible to motion sickness as intact individuals.4 Specifically, the inferior and caudal medial vestibular nuclei instigate emesis via signals to the LTF and NTS.6
A transneuronal retrograde study shows the vestibular system has the anatomical capacity to receive visceral signals.5 However, it has not been demonstrated that vestibular nucleus neurons actually process visceral signals. These unique connections permit the possibility of visceral gastrointestinal inputs modulating vestibular signals contributing to nausea or emesis during periods of motion sickness. Copper sulfate (CuSO4) is an emetic agent that can be delivered to experimental animal models intragastrically. When infused, it activates gastrointestinal (GI) afferents indicating irritation of the stomach lining, likely via chemoreceptors and pain receptors.8
Experimentally, CuSO4 is useful because it can be delivered intragastrically and removed as needed, eliminating the stimulation of GI afferents when removed since it is not readily absorbed into the bloodstream.9 This work was conducted to determine the effects of intragastric infusion of CuSO4 on inferior caudal medial vestibular nucleus neurons during spontaneous firing and vestibular stimulation.
Methods: Twelve total laboratory bred cats of both sexes weighing 2.0-3.4kg were used as an animal model. Animals were anesthetized with 5% isoflurane in O2 then maintained at 1.5-2.5% isoflurane, and placed on a hydraulic servo-controlled tilt table with a stereotaxic frame. Blood pressure was measured via a transducer placed through the femoral artery into the abdominal aorta. Anesthesia was adjusted to maintain blood pressure around 100 mmHg and prevent active movement from the animal. Animals were tracheotomized, and a femoral vein was cannulated to allow the administration of drugs. Atropine sulfate and dexamethasone were provided every six hours in an effort to reduce airway secretions and brain swelling. Phenylephrine was used as needed to combat hypotensive episodes. To vertically align the vertical semicircular canals, the head was pitched down 30°. Hip pins supported the animal’s body to ensure it remained secured to the table during full body rotations. Both carotid arteries were dissected and ligated. An esophagostomy permitted an intragastric catheter for delivering CuSO4 when needed. The animal was decerebrated at the level of the midcolliculus. The caudal brainstem was exposed by an occipital craniotomy, and the caudal region of the cerebellum was removed to allow visualization of brainstem landmarks (such as the obex). A heating pad and heat lamp were used to maintain core body temperature at 37-38°C, measured via a rectal probe. Anesthesia was removed upon completion of surgical procedures. Vecuronium bromide was administered every 20 minutes at 0.1mg/kg intravenously to keep animals paralyzed. An artificial ventilator was used to maintain the animal’s end tidal CO2 at about 4%. Following completion of data acquisition Euthasol Euthanasia Solution was administered to euthanize the animal. Recordings were conducted with 4-6 MΩ tungsten microelectrodes. A Cambridge Electronic Design 1401 data collection system and the Spike2 computer program was used to electronically record single unit activity, blood pressure, and table position sampled at 25,000 Hz, 100 Hz, and 100 Hz respectively. Brainstem landmarks were used to determine placement of the microelectrodes for recording neural activity. Once the electrode was manually positioned over the likely area of the inferior and medial vestibular nucleus, its movement through the brainstem was controlled with a hydraulic Kopf micropositioner to permit fine adjustments to isolate single units. Vestibular nucleus neurons were found by searching with a 0.5 Hz pitch and roll plane rotation, typically at an amplitude of 5° (clockwise wobble stimulus). To achieve the clockwise wobble stimulus the table synchronized a pitch plane sine wave motion and a roll plane cosine wave motion. Full body rotations simultaneously around the pitch and roll axes increased the likelihood of activating vestibular afferents, so neurons in the vestibular nucleus would demonstrate modulated firing rates accordingly. The animal moved in a nose-down, right ear-down, nose-up, left ear-down pattern during the clockwise wobble stimulus. Starting the pitch plane movement as a cosine wave motion, and the roll plane movement as a sine wave motion delivered a counter clockwise wobble stimulus to the animal. Units were considered vestibular responsive if the unit firing rate demonstrated modulation to the clockwise or counter clockwise wobble stimuli (modulation was defined as >0.5 signal to noise ratio). If a unit did not show modulation in response to either wobble stimulus it was discarded and not considered a candidate for further testing. If a unit responded to either wobble stimulus then it was a candidate for further testing. Vestibular responsive units then underwent a series of isolated pitch and roll plane tilts ranging from 0.2-1 Hz at amplitudes of 2.5-5° and 0.05-0.1 Hz at amplitudes of 5-10°. The variety of tilt frequency and amplitude provided insights into the different response dynamics of each unit. The segment of the tilt where unit firing rate showed maximum modulation was defined as the response vector orientation. The response vector orientation was determined by the unit responses to wobble stimuli, and then crosschecked with the isolated pitch and/or roll plane tilts. After completing a series of vestibular responsiveness tests, 83mg CuSO4 solvated in 10 mL of water was pushed into the stomach through the intragastric catheter. Spontaneous unit activity was recorded for one minute prior to CuSO4 injections, and then five minutes post-injection. The same series of tilts performed before the CuSO4 injection was then repeated, but now with the CuSO4 present in the stomach. Once the vestibular trials were completed, the CuSO4 was removed from the stomach, and the stomach was flushed six times with distilled water (10mL each time). At the conclusion of a recording session for an animal, 200-μA of negative current was passed through the recording electrode for 60 seconds to lesion at known coordinates. Once the animal was properly euthanized, the brainstem was excised and placed in 10% formaldehyde. Once fixed, a freezing microtome was used to transversely cut the brainstem into 100μm thick slices that were then mounted on microscope slides and stained with thionine before being coverslipped. The known lesion site coordinates, micropositioner depths, and recording track coordinates were used to determine the relative location of each recorded unit. Reconstructions of the recording sites and unit locations were made with computer-generated drawings overlaid on photographs of the slides. Following data acquisition, each run was sorted using Spike2 to ensure an accurate spike count. Histograms were generated for each rotation trial as an average spike count during the stimulus period binned at 500 bins per cycle. The least-squares minimization technique was used to fit sine waves to each neuronal response for each run.10 The resulting sinusoid was analyzed to determine the phase shift from the stimulus sinusoid (phase), and the amplitude relative to the stimulus sinusoid (gain). Then the signal-to-noise ratio was calculated to determine if responses were significant.10 When responses were consistent across trials, only contained a prominent first harmonic, and the signal-to-noise ratio was larger than 0.5 then they were deemed significant. Prism 6 software was used for statistical analyses, and if p<0.05 then results were considered statistically significant. Data consisting of two variables were compared with a Mann-Whitney test. Data consisting of multiple variables were compared with a nonparametric one-way ANOVA. The effect of CuSO4 on the phase and gain of a unit was analyzed with a two-way ANOVA. The number of units affected by a stimulus under two separate conditions were compared with chi squared tests.
Results: The responses of 47 neurons in the vestibular nucleus were tested for the effect of CuSO4 on spontaneous activity and full body rotations. Data from all animals were pooled for analytical purposes, and there was a median of three units tested per animal.