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NEUROSCIENCE
Sensory suppression during feeding
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*Department of Neurobiology, Pharmacology, and Physiology, and Committee on Neurobiology, University of Chicago, 947 East 58th Street, Chicago, IL 60637
Edited by Donald W. Pfaff, The Rockefeller University, New York, NY, and approved September 29, 2005 (received for review July 21, 2005)
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Abstract |
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homeostasis | nociceptive modulation | pain | raphe magnus | ON and OFF cells
The suppression of pain during feeding indicates the activation of endogenous pain modulatory pathways. The brainstem ventromedial medulla (VMM) is a critical area in the descending control of pain and is the final common pathway from the brain to the spinal cord (8-11). The VMM modulates pain bidirectionally: its activation can produce either pain facilitation or pain inhibition (10-16). The pain-facilitatory and pain-inhibitory effects are thought to be mediated by two populations of neurons with opposing responses to noxious stimulation and morphine (17-20). Cells activated by noxious stimulation are inhibited by opioids. These cells are classified as ON cells and are thought to facilitate nociception. Cells inhibited by noxious stimulation are excited by opioids and are termed pain-inhibitory OFF cells. These putative pain-facilitatory and pain-inhibitory neurons have spontaneous activity that varies across the sleep-wake cycle; most ON cells discharge in bursts during waking and are inactive during slow-wave sleep, and most OFF cells are continuously active during slow-wave sleep and sporadically active during waking (21, 22). Experiments 2-4 were designed to study the contributions of VMM ON and OFF cells to sensory suppression during feeding.
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Materials and Methods |
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Test Apparatus. Rats were tested in a Plexiglas box (25 x 45 x 35 cm) with a wire-mesh floor. A modified Hargreave's apparatus (23) located underneath the cage was used to deliver radiant heat to the hind paw. The heat intensity was set to elicit baseline withdrawal latencies of 3-4 s and terminated at a maximum duration of 8 s.
Experiment 1: Noxious Heat Stimulation During Quiet Waking, Grooming, and Eating. Five rats were tested on each of 2-3 days for paw withdrawal from noxious heat during quiet waking, grooming, and eating. Noxious heat stimulation was given at intervals >5 min. The total number of trials each rat received ranged from 41 to 60. Grooming of the face and head with the forepaws was used as a control for active movements. The rats were given laboratory chow (global 18% protein rodent diet, Harlan no. 2918; Teklad, Madison, WI), chocolate chips, and yogurt drops to eat.
Experiment 2: Cellular Discharge During Feeding. Surgery. A small craniotomy was performed in the area overlying VMM. For extracellular recording, a threaded microdrive base with a guide tube was implanted (anterior-posterior, -11.3 mm from bregma, at midline) for the introduction of tungsten recording electrodes (5 M
, A-M Systems, Carlsborg, WA). Stainless steel screws were placed in the frontal and parietal bones for differential electroencephalographic (EEG) recording, and stranded stainless steel microwires were sutured into the nuchal muscles and the femoris muscles of the right hind paw for electromyographic (EMG) recording. The EEG and EMG leads were attached to a microconnector (Omnetics, Minneapolis) and affixed to the skull. The rats were allowed at least 1 week to recover from surgery.
Data acquisition. A preamplifier within the commutator cable amplified the EEG and EMG signals 5-fold. These signals were then further amplified 10,000-fold by a differential ac amplifier (A-M Systems) and digitized at 1 kHz by a Power1401 interface (Cambridge Electronic Design, Cambridge, UK). The preamplifier buffered the unit signal at unity gain. The unit signal was then amplified 10,000-fold by an ac differential amplifier (Warner Instruments, Hamden, CT) and digitized at 20 kHz by the Power1401 interface. Extracellular recordings were taken from all units that provided stable recordings. For each isolated unit, an amplitude threshold was set by using SPIKE2 acquisition software (Cambridge Electronic Design). When the signal crossed this threshold, the time of that crossing was stored. In addition, 46 digitized points of the waveform were saved: 21 points before and 25 points after threshold crossing. Individual waveforms were reviewed offline, sorted according to amplitude and shape, and assigned to a particular unit by using a template-matching algorithm provided by SPIKE2.
Behavioral classification. Behavioral observations and/or EEG and EMG measures were used to determine wake and sleep states. State assignments were made continuously with no minimum bout length. Four states were recognized: active wake, quiet wake, slow-wave sleep, and paradoxical sleep. The rat was considered to be in active wake if he was engaged in active behaviors such as grooming, eating, drinking, urinating, defecating, or exploring. During such behaviors, the EEG was desynchronized and low in amplitude, and the EMGs showed both tonic and phasic activity. The rat was judged to be in quiet wake when he was sitting quietly with occasional head movements and postural adjustments. The EMG showed low tonic activity except during the brief, isolated moves. The EEG was desynchronized with short periods of synchronized, high-amplitude activity. Brief arousals from sleep during which the rat made slight postural adjustments before returning to sleep were also classified as quiet wake. During these microarousals, the EEG was desynchronized, and the nuchal and/or femoral EMG showed a short burst of phasic activity. The rat was considered to be in slow-wave sleep when he adopted a sleep posture and displayed primarily respiratory-related movements. In this sleep state, the EEG was synchronized and high in amplitude, and all EMG measures showed low tonic activity. During paradoxical sleep, the EEG was desynchronized and low in amplitude. Behaviorally, we observed and recorded on video tape "twitching" of whiskers, ears, and/or paws. Such activity in distal musculature was typically not evident in the nuchal EMG that was low to very low in amplitude during paradoxical sleep.
Electrophysiological and behavioral recordings. A microdrive-electrode assembly was connected to the threaded base on the rat's head. The rat was placed into the test cage and was able to move freely with free access to food and water. The recording electrodes were advanced manually into the VMM region until a unit was isolated. The rat's behavior was continuously monitored and videotaped. Some animals were tested with noxious thermal stimulation of the hind paw at intervals of >5 min. The recording depth for every unit was noted. At the end of each session, the cable and the microdrive-electrode assembly were removed. Rats were tested once a week. The final recording site from each animal was marked by applying a 20-µA anodal current for 4 min.
Histology. All rats were overdosed with sodium pentobarbital (i.p.) and perfused with a fixative containing 10% formalin in 0.1 M PBS (pH 7.4). The brainstem was removed and placed in 30% sucrose. Serial coronal sections (50 µm) were cut on a freezing microtome, mounted on slides, and stained with cresyl violet. The marked recording sites were examined microscopically and plotted onto standard sections. All other recording sites were calculated from the marked site. All recorded sites were within VMM.
Experiment 3: Effects of Air-Puff Stimulation on Eating-Induced Analgesia. Rats (n = 4) were tested for withdrawal responses to noxious radiant heat during quiet waking (no air, no eat), after air-puff stimulation (air, no eat), during eating (no air, eat), and during eating after air-puff stimulation (air, eat). On each of three days, rats were tested under all four conditions in random order. Air-puff stimulation averaged 7.4 ± 0.2 s in length at a rate of 5 liters/min directed at the rat's head. The rats were given chocolate chips to eat.
Experiment 4: Effects of Intra-VMM Muscimol on Eating-Induced Analgesia. Surgery, drugs, and histology. A 26-gauge stainless steel guide cannula (Plastics One, Roanoke, VA) was implanted above VMM (anterior-posterior, -11.3 mm; lateral, 0.0 mm; ventral, -7.7 mm relative to bregma) and cemented to the skull with dental acrylic. The internal microinjection cannula (33 gauge) extended 3 mm beyond the tip of the guide cannula and was connected to a swivel and a Hamilton 1-µl microsyringe via PE20 tubing. Muscimol (50 ng in 500 µl) or an equivalent volume of saline was delivered at a rate of 
0.5 µl/min. All injection sites were histologically verified to be within VMM (see Fig. 5B, which is published as supporting information on the PNAS web site).
Procedure. Rats were microinjected with muscimol (n = 3) or saline (n = 4) and tested for hind paw withdrawal from noxious radiant heat during eating and not-eating, with an interstimulus interval of 5 min. The testing order for eating and not-eating was counterbalanced across animals. The rats were given laboratory chow or chocolate chips to eat. On the second test day, rats received the reverse drug and were tested in the same eating/not-eating order as used on day 1.
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Data Analyses |
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set at a 0.05 level. Experiment 2. The discharge rate during each eating bout was compared with the discharge rates during periods of equivalent duration before and after each eating bout. An eating bout was considered to be the period during which a rat ate a single ingestant without interruptions of more than 4 s. This analysis was conservative because (i) an eating bout was frequently punctuated with brief pauses in eating that were accompanied by transient changes in cellular activity, and (ii) rats were generally asleep before eating, and most of the cells discharged infrequently during sleep.
Experiments 3 and 4. Repeated-measures ANOVAs were used to test for significant differences in withdrawal latencies and were followed by a post hoc Tukey's test for all pairwise comparisons. The level was set at 0.05.
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Results |
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Most of the recorded cells (70%) were more active during waking than sleeping (49/67). Of those tested, most cells (70%) were excited by noxious heat stimulation (18/26). Fifty cells had one or both of these characteristics and were, therefore, classified as ON cells. More than half of the ON cells (n = 27) decreased their discharge rate during eating (Figs. 2 A and B and 3A). Two neurons displayed the physiological characteristics of pain-inhibitory OFF cells; one cell had sleep-active discharge, and the other cell was inhibited by noxious stimulation. These cells increased their discharge during eating and drinking (Fig. 3B). The locations of these ON and OFF cells are shown in Fig. 5A.
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Some of the VMM cells that were inhibited during eating were also recorded during drinking and micturition. Most ON cells (9/10) inhibited during eating were also inhibited when the rats drank water (Fig. 2C); the remaining cell was excited. Further, 13/14 ON cells inhibited during eating were also inhibited during micturition (Fig. 2D), with the remaining cell excited.
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Experiment 4. The results from experiment 3 are consistent with the idea that ON cell inactivation and OFF cell activation produce sensory suppression during eating. However, they do not directly implicate VMM cells. To test whether VMM neurons are necessary for sensory suppression during eating, VMM was inactivated by microinjection of the GABAA receptor agonist muscimol. Inactivation of VMM eliminated the suppression of motor withdrawals during feeding (Fig. 4B), demonstrating that VMM cell activity is critical to sensory suppression during eating.
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Discussion |
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Consistent with the behavioral data, ON cell activity decreased during eating of highly palatable sweet foods or sugar-free laboratory chow, but not during grooming. The changes in ON cell activity during eating appear to be behaviorally specific, occurring when specific movements, such as chewing and bringing the forepaws to the mouth, are centered within a feeding bout, but not when the same or similar movements are made during head and oro-facial grooming. We have also noticed no apparent change in ON cell activity during bruxism or chattering. Although chewing and bruxism produce different mandibular trajectories and activity patterns in the anterior temporalis muscles (30), both oral behaviors require activation of masticatory muscles. These observations suggest that decreases in ON cell activity during feeding are not due to the activation of masticatory muscles per se.
In further support for the idea that decreases in ON cell activity during feeding are not due to the activation of specific muscles or muscle groups, ON cell activity decreased during other homeostatic behaviors. Most of the ON cells tested decreased their discharge across multiple homeostatic behaviors: eating, drinking, and micturition. In anesthetized rats, decreases in ON cell activity and increase in OFF cell activation also occur during micturition (31). Because sensory suppression during feeding was reversed by VMM inactivation, VMM activity is necessary for eating-induced analgesia. Sensory suppression was also prevented by a stimulus that inhibits OFF cells and excites ON cells. Taken together with the electrophysiological data, this finding shows that the mechanisms underlying sensory suppression during eating involves decreases in VMM ON cell activity and increases in VMM OFF cell activity.
It is interesting that after preactivation of ON cells and preinhibition of OFF cells with an innocuous air puff, sensory suppression did not occur and rats interrupted feeding to respond to noxious heat. It is possible that our air-puff stimulation mimicked a situation frequently encountered in the natural environment where animals are extremely vulnerable to predation while feeding. Chipmunks that have heard a playback of conspecific alarm calls continue to feed but do so more slowly as they spend more time on vigilant behaviors (32). We speculate that predation-related stimuli activate ON cells and thereby evoke a state of vigilant eating without concurrent sensory suppression.
In sum, the present results provide direct evidence that VMM ON and OFF cells are critical to the completion of a homeostatic behavior. Because VMM stimulation modulates the cellular responses and behavioral reactions evoked by both noxious and innocuous inputs (33-35), a generalized decrease in reactivity to somatosensory stimulation may accompany feeding, allowing an animal to nourish itself without being distracted by every falling leaf or gust of wind. Taken together, our work has shown that decreases in ON cell firing and increases in OFF cell firing occur during sleep, eating, drinking, and micturition (21, 22, 31). All of these behaviors serve critical roles in maintaining homeostasis and survival. Thus, the inhibition of VMM ON cells and excitation of VMM OFF cells during homeostatic behaviors suppress sensory processing and thereby protect critical behaviors from disruption.
The activation of descending pain inhibition during eating and drinking provides a critical function for VMM cells in modulating sensory receptivity on a daily basis. VMM is situated in a phylogenetically ancient and conserved region of the hindbrain. Its anatomical location and evolutionary conservation is consistent with VMM having a fundamental and critical role in everyday, basic physiology. The suppression of incoming sensory input during homeostatic behaviors is a daily life-preserving function befitting VMM's medullary location.
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Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: VMM, ventromedial medulla; EEG, electroencephalographic or electroencephalogram; EMG, electromyographic or electromyogram.
To whom correspondence should be addressed. E-mail: p-mason{at}uchicago.edu.
© 2005 by The National Academy of Sciences of the USA
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