Identification of Different Types of Spinal Afferent Nerve Endings That Encode Noxious and Innocuous Stimuli in the Large Intestine Using a Novel Anterograde Tracing Technique

In mammals, sensory stimuli in visceral organs, including those that underlie pain perception, are detected by spinal afferent neurons, whose cell bodies lie in dorsal root ganglia (DRG). One of the major challenges in visceral organs has been how to identify the different types of nerve endings of spinal afferents that transduce sensory stimuli into action potentials. The reason why spinal afferent nerve endings have been so challenging to identify is because no techniques have been available, until now, that can selectively label only spinal afferents, in high resolution. We have utilized an anterograde tracing technique, recently developed in our laboratory, which facilitates selective labeling of only spinal afferent axons and their nerve endings in visceral organs. Mice were anesthetized, lumbosacral DRGs surgically exposed, then injected with dextran-amine. Seven days post-surgery, the large intestine was removed. The characteristics of thirteen types of spinal afferent nerve endings were identified in detail. The greatest proportion of nerve endings was in submucosa (32%), circular muscle (25%) and myenteric ganglia (22%). Two morphologically distinct classes innervated myenteric ganglia. These were most commonly a novel class of intraganglionic varicose endings (IGVEs) and occasionally rectal intraganglionic laminar endings (rIGLEs). Three distinct classes of varicose nerve endings were found to innervate the submucosa and circular muscle, while one class innervated internodal strands, blood vessels, crypts of lieberkuhn, the mucosa and the longitudinal muscle. Distinct populations of sensory endings were CGRP-positive. We present the first complete characterization of the different types of spinal afferent nerve endings in a mammalian visceral organ. The findings reveal an unexpectedly complex array of different types of primary afferent endings that innervate specific layers of the large intestine. Some of the novel classes of nerve endings identified must underlie the transduction of noxious and/or innocuous stimuli from the large intestine.     

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1''-dioctadecyl-3,3,3'',3''-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.     

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum–nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.     

The antennal lobe of Libellula depressa (Odonata,Libellulidae)

Here we describe the antennal lobe of Libellula depressa (Odonata, Libellulidae), identified on the basis of the projections of the afferent sensory neurons stemming from the antennal flagellum sensilla. Immunohistochemical neuropil staining as well as antennal backfills revealed sensory neuron terminal arborizations covering a large portion of the antennal lobe. No clear glomerular structure was identified, thus suggesting an aglomerular antennal lobe condition as previously reported in Palaeoptera. The terminal arbors of backfilled sensory neurons do, however, form spherical knots, probably representing the connections between the few afferent neurons and the antennal lobe interneurons. The reconstruction revealed that the proximal part of the antennal nerve is divided into two branches that innervate two spatially separated areas of the antennal lobe, an anterioventral lobe and a larger posteriodorsal lobe. Our data are consistent with the hypothesis that one tract of the antennal nerve of L. depressa contains olfactory sensory neurons projecting into one of the sublobes, while the other tract contains thermo-hygroreceptive neurons projecting into the other sublobe.     

Effects of scaphognathite nerve stimulation on the acutely deafferented crab ventilatory central pattern generator

1. Sensory axons from crab (Carcinus maenas) scaphognathites enter the thoracic ganglion primarily via the LNb branch of the levator nerve. The LNa branch of the levator nerve and the depressor nerve each contain relatively few sensory axons.
2. Acutely deafferented ventilatory central pattern generators show a free running burst rate which is lower than that observed in intact crabs. Electrical stimulation of the levator nerve, or of its LNb branch, increases the burst rate in a frequency dependent manner. Stimulation at high enough intensity to recruit afferents will restart a paused motor rhythm. Stimulation of the levator nerve with short pulse trains phase resets and can entrain the rhythm.
3. In addition to increasing the burst rate, LNb stimulation also causes a progressive elimination of motor neurons from the bursts as the stimulating frequency increases, probably due to depolarization of the 3 oval organ ‘giant’ afferent axons in this branch. Intracellular depolarization of single oval organ afferents will also inhibit some motor neurons as well as slow or stop the rhythm.
4. Continuous stimulation of the depressor nerve does not affect the ganglionic burst rate and this nerve contains only a few small diameter afferent axons; however, brief trains of stimuli can reset the rhythm in a phase-dependent manner.

     

Deletion of Sema3a or plexinA1/plexinA3 Causes Defects in Sensory Afferent Projections of Statoacoustic Ganglion Neurons

Statoacoustic ganglion (SAG) neurons project sensory afferents to appropriate targets in the inner ear to form functional vestibular and auditory circuits. Neuropilin1 (Npn1), a receptor for class 3 semaphorins, is required to generate appropriate afferent projections in SAG neurons; however, the ligands and coreceptors involved in Npn1 functioning remain unknown. Here we show that both plexinA1 and plexinA3 are expressed by SAG neurons, and plexinA1/plexinA3 double mutant mice show defects in afferent projections of SAG neurons in the inner ear. In control mice, sensory afferents of SAG neurons terminate at the vestibular sensory patches, whereas in plexinA1/plexinA3 double mutants, they extend more dorsally in the inner ear beyond normal vestibular target areas. Moreover, we find that semaphorin3a (Sema3a) is expressed in the dorsal otocyst, and Sema3a mutant mice show defects in afferent projections of SAG neurons similar to those observed in plexinA1/plexinA3 double mutants and in mice lacking a functional Npn1 receptor. Taken together, these genetic findings demonstrate that Sema3a repellent signaling plays a role in the establishment of proper afferent projections in SAG neurons, and this signaling likely occurs through a receptor complex involving Npn1 and either plexinA1 or plexinA3.     

Target specificity and size of avian sensory neurons supported in vitro by nerve growth factor,brain-derived neurotrophic factor,and neurotrophin-3

To obtain insight into which subpopulations of sensory neurons in dorsal root ganglia are supported by different neurotrophins, we retrogradely labeled cutaneous and muscle afferents in embryonic day 9 chick embryos and followed their survival in neuron-enriched cultures supplemented with either nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3). We found that NGF is a wide survival factor for subpopulations of both cutaneous and muscle afferents, whereas the survival effects of BDNF and NT-3 are restricted primarily to muscle afferents. We also measured soma size in each neurotrophic factor. These new data show that BDNF- and NT-3–dependent cells appear to be a mixture of two populations of neurons: one small diameter and the other large diameter. In contrast, based on size alone, NGF-dependent cells appear to be a single population of only small-diameter neurons. Thus, BDNF and NT-3 may have some new, previously unreported effects on small-diameter afferent neurons. © 1994 John Wiley & Sons, Inc. 1994 John Wiley & Sons, Inc.     

Monoaminergic Modulation of Spinal Viscero-Sympathetic Function in the Neonatal Mouse Thoracic Spinal Cord

Descending serotonergic, noradrenergic, and dopaminergic systems project diffusely to sensory, motor and autonomic spinal cord regions. Using neonatal mice, this study examined monoaminergic modulation of visceral sensory input and sympathetic preganglionic output. Whole-cell recordings from sympathetic preganglionic neurons (SPNs) in spinal cord slice demonstrated that serotonin, noradrenaline, and dopamine modulated SPN excitability. Serotonin depolarized all, while noradrenaline and dopamine depolarized most SPNs. Serotonin and noradrenaline also increased SPN current-evoked firing frequency, while both increases and decreases were seen with dopamine. In an in vitro thoracolumbar spinal cord/sympathetic chain preparation, stimulation of splanchnic nerve visceral afferents evoked reflexes and subthreshold population synaptic potentials in thoracic ventral roots that were dose-dependently depressed by the monoamines. Visceral afferent stimulation also evoked bicuculline-sensitive dorsal root potentials thought to reflect presynaptic inhibition via primary afferent depolarization. These dorsal root potentials were likewise dose-dependently depressed by the monoamines. Concomitant monoaminergic depression of population afferent synaptic transmission recorded as dorsal horn field potentials was also seen. Collectively, serotonin, norepinephrine and dopamine were shown to exert broad and comparable modulatory regulation of viscero-sympathetic function. The general facilitation of SPN efferent excitability with simultaneous depression of visceral afferent-evoked motor output suggests that descending monoaminergic systems reconfigure spinal cord autonomic function away from visceral sensory influence. Coincident monoaminergic reductions in dorsal horn responses support a multifaceted modulatory shift in the encoding of spinal visceral afferent activity. Similar monoamine-induced changes have been observed for somatic sensorimotor function, suggesting an integrative modulatory response on spinal autonomic and somatic function.     

Activation of thalamic ventroposteriolateral neurons by phrenic nerve afferents in cats and rats.

It has been demonstrated that phrenic nerve afferents project to somatosensory cortex, yet the sensory pathways are still poorly understood. This study investigated the neural responses in the thalamic ventroposteriolateral (VPL) nucleus after phrenic afferent stimulation in cats and rats. Activation of VPL neurons was observed after electrical stimulation of the contralateral phrenic nerve. Direct mechanical stimulation of the diaphragm also elicited increased activity in the same VPL neurons that were activated by electrical stimulation of the phrenic nerve. Some VPL neurons responded to both phrenic afferent stimulation and shoulder probing. In rats, VPL neurons activated by inspiratory occlusion also responded to stimulation on phrenic afferents. These results demonstrate that phrenic afferents can reach the VPL thalamus under physiological conditions and support the hypothesis that the thalamic VPL nucleus functions as a relay for the conduction of proprioceptive information from the diaphragm to the contralateral somatosensory cortex.     

Functional properties of interneurons of the masticatory reflex

Interneurons of the supratrigeminal nucleus, transmitting effects from the sensory and motor branches of the trigeminal nerve to motoneurons of the muscles of mastication were investigated. Two groups of interneurons with different functional connections were found. The first group (A) contains neurons excited during stimulation of the sensory branches and the motor nerve to the digastric muscle (A₁), neurons excited during stimulation of sensory branches and high-threshold afferents of the motor nerve to the masseter muscle (A₂), and neurons excited only by low-threshold afferents of the motor nerve to the masseter muscle (A₃). Neurons of the second group (B) were activated only by sensory fibers of the trigeminal nerve. It is postulated that interneurons of group A transmit inhibitory effects to motoneurons of antagonist muscles of the lower jaw. Group B interneurons participate in the transmission of excitatory influences to motoneurons of the digastric muscle.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 4, No. 2, pp. 150–157, March–April, 1972.     

Growth pattern and NGF-dependent survival of dorsal root ganglia neurons of distinct glyco-phenotype

Cell-surface glyco-phenotypes of dorsal root ganglia (DRG) neurons were specified with monoclonal antibodies (mABs) D1 and E1. D1 demarcated sensory afferents in skin but not muscle target. More than 90% of the drg neurons supported by nerve growth factor (NGF) in vitro were D1 positive (D1⁺). A fraction of these D1⁺ neurons, those of small to intermediate soma size, coexpressed a PNGase-sensitive glycoepitope E1, defined by mAB E1. In situ and in vitro, E1⁺/D1⁺ and E1⁻/D1⁺ neurons and nerve fibers were affiliated. After separation of the two glyco-phenotypes, NGF-dependent survival of E1⁻/D1⁺ neurons was no longer observed. Two interrelated concepts emerge from these findings: (a) NGFs survival functions for cutaneous sensory neurons are in part indirect and appear to be based on interneuronal cooperation for survival; and (b) interneuronal survival dependencies are likely to be a decisive factor governing nerve fiber assemblages. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 193–207, 1998     

Two kinds of peripheral afferent neurons in the earthworm reflex arc

• 1.1. Two kinds of neurons were identified in the body-wall longitudinal muscle layer of the earthworm, Amynthas hawayanus, by the simultaneous potential recording and Lucifer Yellow-CH injection method with a single microelectrode.
• 2.2. Both kinds of neurons have their somata, neuntes and longitudinal processes imbedded in the longitudinal muscle layer. Those with two circular processes extending into the third segmental nerve trunk are tentatively named “intra-nerve-trunk” neurons and those with four circular processes extending into four setae shafts are tentatively named “intramural” neurons.
• 3.3. Both kinds of neurons responded to electrical and mechanical stimuli applied in an afferent direction to them.
• 4.4. The “intra-nerve-trunk” neuron decreased its response amplitudes to these stimuli after the third nerve trunk was sectioned in correlation to the response amplitude decrease recorded from the nerve trunk after it was sectioned.
• 5.5. The response amplitude decrease due to denervation implies a nonlinear structure of the earthworm reflex circuits.
• 6.6. The “intramural” neurons are believed to be primary sensory neurons connected to the mechanoreceptors in the setae.

     

Muscle‐derived neurotrophin‐3 reduces injury‐induced proprioceptive degeneration in neonatal mice

During perinatal development, proprioceptive muscle afferents are quite sensitive to nerve injury. Here, we have used transgenic mice that overexpress neurotrophin‐3 (NT‐3) in skeletal muscle (myo/NT‐3 mice) to explore whether NT‐3 plays a neuroprotective role for perinatal muscle afferents following nerve injury. Measurements of NT‐3 mRNA using RT‐PCR revealed that levels of endogenous NT‐3 mRNA in wild‐type muscles remained constant during the first postnatal week following nerve crush or nerve section on postnatal day (PN) 1. In comparison, myo/NT‐3 mice had significantly elevated levels of NT‐3 mRNA that were maintained or increased following injury. To assess whether muscle‐derived NT‐3 could prevent injury‐induced neuronal death, neuron survival in the DRG was analyzed in mice 5 days after sciatic nerve crush on PN3. Retrograde prelabeling of muscle afferents and parvalbumin immunocytochemistry both revealed that overexpression of NT‐3 in muscle significantly reduced neuronal loss following injury. Similar neuroprotective effects of NT‐3 were observed in wild‐type mice injected with exogenous NT‐3 in the gastrocnemius muscles. To test whether NT‐3 could prevent muscle spindle degeneration, spindle number and morphology were assessed 3 weeks after sciatic nerve crush or section on PN1. No spindles were present in either wildtype or myo/NT‐3 muscles after nerve section, demonstrating that NT‐3 overexpression cannot maintain spindles following complete denervation. Moreover, NT‐3 overexpression could not prevent moderate spindle loss in muscle and did not stimulate new spindle formation following nerve crush. Our results demonstrate that in addition to its early actions on sensory neuron generation and naturally occurring cell death, NT‐3 has important neuroprotective effects on muscle afferents during postnatal development. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 198–208, 2002; DOI 10.1002/neu.10024     

Shh influences cell number and the distribution of neuronal subtypes in dorsal root ganglia

The molecular mechanisms responsible for specifying the dorsal-ventral pattern of neuronal identities in dorsal root ganglia (DRG) are unclear. Here we demonstrate that Sonic hedgehog (Shh) contributes to patterning early DRG cells. In vitro, Shh increases both proliferation and programmed cell death (PCD). Increasing Shh in vivo enhances PCD in dorsal DRG, while inducing greater proliferation ventrally. In such animals, markers characteristic of ventral sensory neurons are expanded to more dorsal positions. Conversely, reducing Shh function results in decreased proliferation of progenitors in the ventral region and decreased expression of the ventral marker trkC. Later arising trkA⁺ afferents make significant pathfinding errors in animals with reduced Shh function, suggesting that accurate navigation of later arising growth cones requires either Shh itself or early arising, Shh-dependent afferents. These results indicate that Shh can regulate both cell number and the distribution of cell types in DRG, thereby playing an important role in the specification, patterning and pathfinding of sensory neurons.     

Integration of Sensory Quanta in Cuneate Nucleus Neurons In Vivo

Discriminative touch relies on afferent information carried to the central nervous system by action potentials (spikes) in ensembles of primary afferents bundled in peripheral nerves. These sensory quanta are first processed by the cuneate nucleus before the afferent information is transmitted to brain networks serving specific perceptual and sensorimotor functions. Here we report data on the integration of primary afferent synaptic inputs obtained with in vivo whole cell patch clamp recordings from the neurons of this nucleus. We find that the synaptic integration in individual cuneate neurons is dominated by 4–8 primary afferent inputs with large synaptic weights. In a simulation we show that the arrangement with a low number of primary afferent inputs can maximize transfer over the cuneate nucleus of information encoded in the spatiotemporal patterns of spikes generated when a human fingertip contact objects. Hence, the observed distributions of synaptic weights support high fidelity transfer of signals from ensembles of tactile afferents. Various anatomical estimates suggest that a cuneate neuron may receive hundreds of primary afferents rather than 4–8. Therefore, we discuss the possibility that adaptation of synaptic weight distribution, possibly involving silent synapses, may function to maximize information transfer in somatosensory pathways.     

Morphology and Nanomechanics of Sensory Neurons Growth Cones following Peripheral Nerve Injury

A prior peripheral nerve injury in vivo, promotes a rapid elongated mode of sensory neurons neurite regrowth in vitro. This in vitro model of conditioned axotomy allows analysis of the cellular and molecular mechanisms leading to an improved neurite re-growth. Our differential interference contrast microscopy and immunocytochemistry results show that conditioned axotomy, induced by sciatic nerve injury, did not increase somatic size of adult lumbar sensory neurons from mice dorsal root ganglia sensory neurons but promoted the appearance of larger neurites and growth cones. Using atomic force microscopy on live neurons, we investigated whether membrane mechanical properties of growth cones of axotomized neurons were modified following sciatic nerve injury. Our data revealed that neurons having a regenerative growth were characterized by softer growth cones, compared to control neurons. The increase of the growth cone membrane elasticity suggests a modification in the ratio and the inner framework of the main structural proteins.     

The African wave-type electric fish,Gymnarchus niloticus,lacks corollary discharge mechanisms for electrosensory gating

Gymnarchus niloticus, a wave-type African electric fish, performs its jamming avoidance response by relying solely upon afferent signals and does not use corollary discharges from the pacemaker nucleus in the medulla which generates the rhythmicity of electric organ discharges. This is in sharp contrast to the mode of sensory processing found in closely related African pulse-type electric fishes where afferent signals are gated by corollary discharges from the pacemaker for the distinction of exafferent and reafferent stimuli. Does Gymnarchus still possess a corollary discharge mechanism for other behavioral tasks but does not use it for the jamming avoidance response? In this study, I recorded from and labeled medullary neuronal structures that either generate or convey the pacemaker signal for electric organ discharges to examine whether this information is also sent directly to any sensory areas. The pacemaker nucleus was identified as the site of generation of the pacemaking signal. The pacemaker neurons project exclusively to the lateral relay nucleus which, in turn projects exclusively to the medial relay nucleus. Neurons in the medial relay nucleus send unbranched axons to the spinal electromotoneurons. These neurons are entirely devoted to drive the electric organ discharges, and no axon collaterals from these neurons were found to project to any sensory areas. This indicates that Gymnarchus does not possess the neuronal hardware for a corollary discharge mechanism.     

Octopaminergic modulation of the femoral chordotonal organ in the stick insect

The modulatory action of DL-octopamine on the multicellular femoral chordotonal organ (fCO) of the stick insect Cuniculina impigra was examined using extracellular recordings from the fCO nerve and intracellular recordings from single sensory neurons. To determine the octopaminergic effect on position, velocity and/or acceleration sensitivity of mechanoreceptors direct mechanical stimulations with defined parameters were applied to the fCO apodeme. The spontaneous activity in the fCO nerve was enhanced in a dose-dependent manner by octopamine (threshold at 5 × 10^?7 M). This was based on enhanced activity of position sensitive neurons as the fCO activity for all position stimuli was shifted to higher values. Intracellular recordings of single sensory cells showed that velocity-sensitivity of single sensory cells was not altered by octopamine. Similarly, the response of fCO afferents to ramp-and-hold stimuli revealed that acceleration sensitivity was unaffected by octopamine. The observed alterations in the fCO activity indicate that responses to static stimuli are enhanced while responses to motion stimuli are not affected by octopamine. These findings suggest that the octopaminergic modulation of the fCO may affect the animals' posture and those leg movements that rely on position information.     

Presynaptic selection of afferent inflow in the spinal cord.

The synaptic effectiveness of sensory fibers ending in the spinal cord of vertebrates can be centrally controlled by means of specific sets of GABAergic interneurons that make axo-axonic synapses with the terminal arborizations of the afferent fibers. In the steady state, the intracellular concentration of chloride ions in these terminals is higher than that predicted from a passive distribution, because of an active transport mechanism. Following the release of GABA by spinal interneurons and activation of GABA(A) receptors in the afferent terminals, there is an outwardly directed efflux of chloride ions that produces primary afferent depolarization (PAD) and reduces transmitter release (presynaptic inhibition). Studies made by intrafiber recording of PAD, or by measuring changes in the intraspinal threshold of single afferent terminals (which is reduced during PAD), have further indicated that muscle and cutaneous afferents have distinctive, but modifiable PAD patterns in response to segmental and descending stimuli. This has suggested that PAD and presynaptic inhibition in the various types of afferents is mediated by separate sets of last-order GABAergic interneurons. Direct activation, by means of intraspinal microstimulation, of single or small groups of last-order PAD-mediating interneurons shows that the monosynaptic PAD elicited in Ia and Ib afferents can remain confined to some sets of the intraspinal collaterals and not spread to nearby collaterals. The local character of PAD allows cutaneous and descending inputs to selectively inhibit the PAD of segmental and ascending intraspinal collaterals of individual muscle spindle afferents. It thus seems that the intraspinal branches of the sensory fibers are not hard wired routes that diverge excitation to spinal neurons, but are instead dynamic pathways that can be centrally controlled to address information to selected neuronal targets. This feature appears to play an important role in the selection of information flow in muscle spindles that occurs at the onset of voluntary contractions in humans.     

Superior laryngeal and hypoglossal afferents tonically influence upper airway motor excitability in anesthetized rats.

Upper airway (UA) muscle activity is stimulated by changes in UA transmural pressure and by asphyxia. These responses are reduced by muscle relaxation. We hypothesized that this is due to a change in afferent feedback in the ansa hypoglossi and/or superior laryngeal nerve (SLN). We examined 1) the glossopharyngeal motor responses to UA transmural pressure and asphyxia and 2) how these responses were changed by muscle relaxation in animals where one or both of these afferent pathways had been sectioned bilaterally. Experiments were performed in 24 anesthetized, thoracotomized, artificially ventilated rats. Baseline glossopharyngeal activity and its response to UA transmural pressure and asphyxia were moderately reduced after bilateral section of the ansa hypoglossi (P < 0.05). Conversely, bilateral SLN section increased baseline glossopharyngeal activity, augmented the response to asphyxia, and abolished the response to UA transmural pressure. Muscle relaxation reduced resting glossopharyngeal activity and the response to asphyxia (P < 0.001). This occurred whether or not the ansa hypoglossi, the SLN, or both afferent pathways had been interrupted. We conclude that ansa hypoglossi afferents tonically excite and SLN afferents tonically inhibit UA motor activity. Muscle relaxation depressed UA motor activity after section of the ansa hypoglossi and SLN. This suggests that some or all of the response to muscle relaxation is mediated by alterations in the activity of afferent fibers other than those in the ansa hypoglossi or SLN.