To understand vago-vagal reflexes, one must have an appreciation of the events surrounding the encoding, integration, and central transfer of peripheral sensations by vagal afferent neurons. A large body of work has shown that vagal afferent neurons have nonuniform properties and that distinct subpopulations of neurons exist within the nodose ganglia. These sensory neurons display a considerable degree of plasticity; electrophysiological, pharmacological, and neurochemical properties have all been shown to alter after peripheral tissue injury. The validity of claims of selective recordings from populations of neurons activated by peripheral stimuli may be diminished, however, by the recent demonstration that stimulation of a subpopulation of nodose neurons can enhance the activity of unstimulated neuronal neighbors. To better understand the neurophysiological processes occurring after vagal afferent stimulation, it is essential that the electrophysiological, pharmacological, and neurochemical properties of nodose neurons are correlated with their sensory function or, at the very least, with their specific innervation target.
- visceral afferents
to fully understand vago-vagalreflexes, one must appreciate all aspects of the reflex response from transduction of the sensory signal by receptors on vagal afferent nerve terminals to the transfer and integration of this peripheral signal centrally to the efferent vagal output and resultant response in the target organ. The initiation of vago-vagal reflexes, the receptors transducing the signals, and the mechanisms involved in their transduction by vagal afferent terminals has been dealt with in the previous article in this themes series by Powley and Philips (37a) and the events surrounding the central integration and efferent vagal response will be dealt with subsequently. This review will concentrate on the sensory component of vago-vagal reflexes at the level of the vagal afferent neurons in the nodose ganglion.
It has been known for quite some time that the esophageal distension produced by swallowing elicits a powerful reflex relaxation of the proximal stomach, which increases gastric volume and decreases intragastric pressure to allow efficient transfer of swallowed food into the stomach (7). Investigations by several laboratories have demonstrated that this gastroinhibition is dependent on an intact vagal nervous system (39). Further studies have revealed that stimulation of vagal afferents at different levels of the gastrointestinal tract elicits other vagally mediated gastrointestinal reflexes, including inhibition of food intake (45), inhibition of gastric emptying (40), motility (23), acid secretion (30), and pancreatic secretion (27). Vago-vagal reflexes are not, however, restricted to the gastrointestinal system. For example, activation of cardiac vagal afferent neurons elicits the Bezold-Jarish reflex comprised of an increase in cardioinhibitory parasympathetic activity and a decrease in sympathetic activity resulting in bradycardia and hypotension (4). Similarly, activation of respiratory vagal afferents evokes bronchoconstrictions (26).
ORGANIZATION OF VAGAL AFFERENT (SENSORY) NEURONS
The cell bodies of gastrointestinal, respiratory, and cardiac vagal afferent neurons are contained within the nodose and jugular ganglia. The central fibers of these bipolar neurons continue to ascend in the vagus nerve, they enter the brain stem through the solitary tract, and finally synapse on neurons in the nucleus of the nucleus of the solitary tract (NTS) in the medulla. Anatomical, chemical, and physiological studies have shown that at the level of the brain stem, gastrointestinal vagal afferent terminal fields have discrete localizations within the NTS (1, 31). Similarly, retrograde tracing studies have shown that gastrointestinal vagal parasympathetic motor neurons are organized in a viscerotopic mediolateral columnar arrangement that spans the entire rostrocaudal extent of the rat dorsal motor nucleus of the vagus (14). With such an advanced level of organization of vagal afferent nerve terminals, vagal efferent neurons, and vagal efferent nerves, it is reasonable to expect that vagal afferent neurons themselves would also exhibit some degree of organization. Indeed, within the nodose ganglion, neurons tend to be organized in a viscerotopic manner. Neurons that innervate the esophagus or the intestine are located rostrally, whereas neurons that innervate the stomach and pancreas are located more caudally; neurons that innervate the aortic depressor nerve are located rostrally within the nodose ganglion (46).
ELECTROPHYSIOLOGICAL PROPERTIES OF VAGAL AFFERENT NEURONS
A large body of work has been written about the electrophysiological properties of nodose ganglion neurons. From studies using extracellular recording techniques, it is known that vagal afferent neurons have a low level of spontaneous activity and that their action potential firing rate increases upon stimulation of the afferent terminal field. For example, intestinal vagal afferent neurons are excited by a variety of mechanical and chemical stimuli such as gastric or intestinal distension or in response to osmotic stimuli or perfusion of carbohydrates or lipids (18). Intracellular and whole cell patch-clamp recordings made from dissociated neurons revealed the nonuniform nature of vagal afferent neuronal active and passive properties (32, 44). Indeed, intracellular recordings from intact nodose ganglia indicated that some level of organization may exist with regard to electrophysiological properties, because neurons with particular attributes were found to have discrete localizations within the ganglion. In the guinea pig nodose ganglion, for example, a small subpopulation of neurons restricted to the midcaudal pole displays a slow afterhyperpolarization after action potential firing (44).
Unfortunately, the very nature of these blind recording techniques does not allow a distinction to be made among gastrointestinal, respiratory, and cardiovascular vagal afferent neurons within the nodose ganglion. By consequence, it has not been possible to state with absolute conviction what, if any, degree of functional specialization exists. That is to say it has not been possible to determine whether the observed differences in neuronal properties were related to the peripheral target organ of innervation or their sensory function, i.e., mechano-, osmo-, or chemosensitive.
Significant advances, however, have been made recently by combining electrophysiological recording techniques with neuronal tracing methods. The combination of these techniques has made it possible to record from dissociated vagal afferent neurons identified as per their target organ of innervation. In the rat, for example, we have shown that ∼80% of gastrointestinal vagal afferent neurons display phasic or rapidly adapting action potential firing properties, with the remaining 20% of neurons displaying tonic, or slowly adapting, action potential firing properties (6). Approximately 45% of gastrointestinal vagal afferent neurons display the hyperpolarization-activated nonspecific cation currentI H, whereas only 17% display the fast transient outward voltage-dependent potassium current, I A. The importance of the presence of these currents stems from their capability to determine the behavior of the neuronal membrane. In fact, the I H current has previously been identified in several neuronal systems as contributing to pacemaker activity (43) and in nodose neurons, has been shown to be an important target for modulation by neurotransmitters (25). The I A current acts to increase interspike interval and grade the rate of action potential firing (10). The presence of these currents in subpopulations of gastrointestinal neurons suggests that action potential firing rates may be modulated differently by various stimuli impinging on nodose neurons, either by neurotransmitters or neuromodulators or by frequency-dependent coding of vagal afferent receptor transduction.
Although relatively little is known about the properties of gastrointestinal vagal afferent neurons, far more information is available regarding the properties of cardiac and respiratory vagal afferent neurons. Baroreceptor neurons are by definition mechanosensitive neurons, yet recent work has only just started to identify the channels and mechanisms responsible for stretch sensitivity. Exposure to hypoosmotic solution increases the conductance in the cell bodies of aortic baroreceptor neurons (identified by a fluorescent tracer applied to the aortic arch) as well as in some, but not all, additional neurons from the nodose ganglia (11). This whole cell current has a reversal potential of approximately −11 mV and can be blocked by the mechanosensitive channel blocker gadolinium (11).
However, this gadolinium-sensitive current has some characteristics that do not correlate well with baroreceptor function, such as the long delay between stimulus and activation. For example, in vivo, the aortic arch multifiber baroreceptor activity responds dramatically within milliseconds to changes in pressure or stretch in the aortic arch (2). At the soma, the response to mechanical stimulation reaches its peak only after 2 s and the response to a single mechanical deformation persists for 30 s (12). However, in response to a hypoosmotic stimulus, the change in conductance at the soma of aortic baroreceptors does not occur immediately, but first occurs at 3 min after the stimulus, and the peak response occurs after 5–7 min (11). Other work also questions whether the gadolinium-sensitive currents at the soma are representative of the mechanosensitive mechanisms at the sensory endings. Although both the mechanically and osmotic stimulated current at the soma can be blocked by gadolinium when applied to the baroreceptor sensory endings, gadolinium has produced conflicting results. In one study (19), carotid sinus baroreceptor activation was reduced by gadolinium, whereas in another study (2), gadolinium had no direct effect on aortic baroreceptor mechanotransduction.
In more recent work, DEG/ENaC proteins have been implicated in mechanotransduction. Subunits of these proteins have been localized to the baroreceptor sensory endings and the cell bodies of baroreceptor neurons in the nodose ganglia (13). Amiloride, which blocks ENaC channels, inhibited the increases in intracellular calcium in the soma of cultured baroreceptor neurons in response to mechanical stimulation (13). Application of benzamil, an amiloride analog, to the carotid sinus blocked the pressure-evoked increase in baroreceptor activity and blunted the reflex responses (13). However, the specificity of both amiloride and benzamil is uncertain, because these drugs also inhibit other electrically active mechanisms such as Na+/H+and Na+/Ca2+ exchangers.
PHARMACOLOGY OF VAGAL AFFERENT NEURONS
Despite being asynaptic (28), vagal afferent neurons have been shown to respond to a variety of neurotransmitters/neuromodulators (25, 33, 42, 44). The validity of claims of selective recordings from populations of neurons activated by peripheral stimuli may be diminished, however, by the recent demonstration that stimulation of a subpopulation of nodose neurons can enhance the activity of unstimulated neighbors (36) (see does modulation of ionic channels in nodose cell body correlate with transmitter release centrally?). Therefore, with the exception of a few studies in which the peripheral target of innervation was identified without doubt before electrophysiological recordings by the use of retrograde tracer application to the peripheral target organ (5,6, 16, 32), the overall problem still remains that one cannot be certain from what type of neuron (gastrointestinal, cardiac, or respiratory) one is recording.
Our recent studies have shown that striking regional differences exist with respect to some pharmacological properties of identified gastrointestinal vagal afferent neurons. For example, although the proportion of gastrointestinal neurons that respond to substance P with a membrane depolarization (∼20%) is independent of the region of innervation, the proportion of neurons that respond to serotonin [5-hydroxytryptamine (5-HT)] with a membrane depolarization is very much dependent on the region of innervation. Although the majority of neurons innervating the corpus or the duodenum (71 and 78%, respectively) respond to 5-HT, very few neurons innervating the fundus (31%), or especially the antrum/pylorus (14%), do so (6). These results support the observation that 5-HT released from enterochromaffin cells in the duodenum might be the principal sensory neurotransmitter relaying gastrointestinal information centrally. On the other hand, it seems quite unlikely that sensory transduction from the proximal stomach or the antrum/pylorus region will be relayed via 5-HT. Furthermore, regional differences also exist with respect to the type of response to 5-HT. We have observed three types of response to 5-HT in gastrointestinal vagal afferent neurons: 1) a fast transient 5-HT3-mediated response; 2) a slower, more maintained 5-HT2-mediated response; and 3) a mixed response with both fast (5-HT3) and slow (5-HT2) components (Fig. 1). Although the slow 5-HT2-mediated response was observed only rarely in nodose neurons innervating the intestine (<10%), it composed almost 50% of the responses observed in neurons innervating the stomach (6).
Thus it would appear that the 5-HT-mediated responses of gastrointestinal vagal afferent neurons show regional-dependent variations in both the frequency of responding neurons as well as in the receptor subtypes involved. Responses of nodose neurons to 5-HT appear to be open to modulation. In guinea pig airway-sensitive vagal afferent neurons, for example, a line of research has demonstrated that vagal afferent neurons can be sensitized to nociceptive neurotransmitters such as neurokinins. Although naive airway-sensitive neurons are insensitive initially to neurokinins, after exposure to inflammatory antigens such as serotonin, histamine, bradykinin, or prostaglandins, subsequent reapplication of neurokinin induces a membrane depolarization, i.e., exposure to inflammatory mediators uncovers previously silent receptors, and this period of sensitization can last for periods from hours to days (34, 44). Our recent studies in identified rat gastrointestinal neurons have shown that previously silent neurokinin receptors can be uncovered in response to application of serotonin in a small proportion of neurons. Unlike guinea pig airway-sensitive neurons, where this sensitizing action of 5-HT has been shown to involve activation of 5-HT3 receptors (33), the unmasking of neurokinin receptors on gastrointestinal vagal afferent neurons appears to involve activation of 5-HT2 receptors (Fig.2). Given the apparent regional specialization with regard to serotonin receptor distribution, i.e., the predominance of 5-HT2-mediated responses in gastric rather than intestinal neurons, this may indicate a more prominent role of sensitization in gastric neurons.
Another example of the plasticity of nodose neuronal properties is illustrated with the recent finding that after the induction of experimental gastric ulcers, vagal afferent neurons become more excitable (16). With the use of injections of acetic acid into the stomach wall as a means of inducing gastric ulcers, it has been shown that in nodose neurons identified as projecting to the stomach, there are significant changes in the characteristics of voltage-sensitive sodium currents. In fact, after induction of gastric ulcers, the TTX-resistant sodium current is enhanced, whereas the TTX-sensitive sodium current remains inactive for a shorter period of time than in control. The consequence of these alterations in sodium currents is that gastric nodose neurons become more excitable (16).
The neurochemical phenotype of vagal afferent neurons is also subject to alteration after tissue insult. Although it is known that allergic inflammation of guinea pig airways increases the levels of sensory neuropeptides in nodose neurons (8), a recent study demonstrated with electrophysiological and immunohistochemical techniques that after inflammation, there is a profound alteration in the neurochemical coding of vagal afferent neurons. Specifically, after inflammation, mechanosensitive, low-threshold Aδ fibers express substance P or calcitonin gene related polypeptide that may be released centrally from vagal afferents (35). This alteration in the neurochemical phenotype of mechanosensitive neural elements would imply that simple mechanical deformations, which are nonnoxious in the normal state, would potentially result in the release of neuropeptides traditionally associated with noxious stimuli, inducing a potential hypersensitivity to deformation after inflammation.
DOES MODULATION OF IONIC CHANNELS IN NODOSE CELL BODY CORRELATE WITH TRANSMITTER RELEASE CENTRALLY?
One important question to be addressed regarding vagal afferent sensory neurons is, do the ionic channels and their modulation in the nodose cell body correlate with transmitter release from these neurons in the NTS? Calcium influx through voltage-gated calcium currents is necessary for the release of neurotransmitters from synaptic endings, but it cannot be assumed that the calcium currents at the soma represent those involved in transmitter release. In many neurons, the pharmacological profile of calcium currents at the soma does not match the channels responsible for transmitter release (22, 37). The types of voltage-gated calcium currents at the soma of identified aortic baroreceptors have been characterized and are comprised of a small T-type current. Larger, high-threshold calcium currents that are comprised mostly of N-type currents with a small P-type component are also present (32). To determine the calcium channels responsible for transmitter release at their synaptic endings, the nerve fibers of these neurons were stimulated in the solitary tract to evoke postsynaptic responses in NTS neurons that receive these monosynaptic inputs. The N-type calcium channel antagonist (ω-conotoxin-GVIA) blocked most of the excitatory synaptic neurotransmission to NTS neurons, whereas a smaller component was sensitive to the P-type antagonist agatoxin-IVA (32). Therefore, in aortic baroreceptor neurons, the calcium currents at the soma highly correspond to the calcium channels responsible for transmitter release at their synaptic terminals in the NTS (32).
It therefore seems reasonable to extrapolate that modulation of voltage-gated calcium currents at the soma of aortic baroreceptors would predict modulation of neurotransmission from baroreceptors to NTS neurons at the first synapse of the baroreflex pathway. Recent work with both opioid and metabotropic glutamate receptor agonists support this conclusion. μ-Opioid agonists inhibit voltage-gated calcium currents in the soma of baroreceptor neurons (20). Consistent with this inhibition at the soma, μ-opioid agonists depress glutamate-mediated excitatory postsynaptic potentials in NTS neurons evoked by stimulation of the solitary tract (38). Activation of metabotropic glutamate receptors has been shown to suppress voltage-gated calcium currents and, in particular, the ω-conotoxin-GVIA-sensitive N-type voltage-gated calcium currents in nodose neurons (21). Consistent with this inhibition at the soma, activation of metabotropic glutamate receptors inhibited the excitatory postsynaptic responses in NTS neurons evoked upon stimulation, and this inhibition was mediated, in part, by ω-conotoxin-GVIA-sensitive N-type calcium currents (17). Recent work has confirmed and extended these results by determining that groups II and III, but not group I, metabotropic glutamate agonists inhibit the activation of NTS neurons evoked upon stimulation of the solitary tract (9).
DOES MODULATION OF IONIC CURRENTS AT CELL BODY ALTER AFFERENT ACTIVITY?
Although there are no synaptic contacts in the nodose ganglia, vagal afferent neurons have been shown to respond to a variety of neurotransmitters/neuromodulators (3, 25, 33, 42, 44). Despite being asynaptic, a recent report (36) has demonstrated that stimulation of a subpopulation of nodose neurons can enhance the activity of its unstimulated neighbors. Part of this cross-activation may be due to a diffusible messenger or transmitter release, because it can be blocked by the nonselective calcium channel antagonist cadmium and evokes a decrease in membrane resistance and current that reverses at approximately −25 mV (36). In other nodose neurons, however, an excitation occurred that was not associated with a resistance change and could have been due to activity-dependent changes in extracellular potassium concentrations (36). Potential diffusible mediators of the cross-excitation include serotonin. Serotonin is released from cell bodies in the nodose ganglia, and this release is calcium dependent but not inhibited by blocking TTX-sensitive sodium channels (15). Serotonin has been shown to depolarize nodose neurons (6, 33), but in other work, it has been shown to inhibit the excitation caused by hypoosmotic mechanical stress (29). The ability of nodose neurons to influence the activity of unstimulated neighbors may be a mechanism behind some examples of pathological disturbances in one organ affecting another organ. It is well known, for example, that a clear relationship exists between patients with airway hyperresponsiveness and gastroesophageal reflux disease (24), or irritable or inflammatory bowel disease (41). The ability of nodose neurons to communicate with each other may suggest that some of these effects may occur at the level of the vagal afferent neurons themselves.
In summary, clearly, vagal afferent neurons have nonuniform properties, and there is enough evidence emerging to state with a reasonable degree of confidence that the electrophysiological properties of vagal afferent neurons are specialized to their sensory function and/or target organ of innervation. To better understand the neurophysiological processes occurring after vagal afferent stimulation, it is essential that the electrophysiological, pharmacological, and neurochemical properties of nodose neurons are correlated with their sensory function or, at the very least, with their specific innervation target. Recent studies of the neurobiology of nodose neurons has helped advance our understanding of three major functions of these neurons: sensory transduction, modulation of their activity at the soma, and synaptic neurotransmission from their synaptic endings in the medulla. However, further work is clearly needed to 1) correlate mechanosensitive channels identified at the soma with sensory transduction at the sensory endings;2) identify paracrine messengers that are released and modulate ionic currents and activity at the soma; and 3) characterize the modulation of calcium channels that regulate transmitter release at the synaptic endings in these visceral afferent neurons.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-34944–17 (to Michigan Gastrointestinal Peptide Research Center) and DK-55530 and by National Heart, Lung, and Blood Institute Grants HL-59895 and HL-49965.
Due to space limitations, the authors were unable to provide an exhaustive reference list.
Address for reprint requests and other correspondence: K. N. Browning, 6520 MSRB1, 1150 West Medical Center Drive, Ann Arbor, MI 48109–0682 (E-mail:).
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