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Atkinson Pain Research Laboratory, Barrow Neurological Institute, Phoenix, Arizona 85013
Submitted 22 November 2002; accepted in final form 8 January 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
Functional magnetic resonance imaging (Aziz
et al. 2000b; Binkofski et al.
1998), magnetoencepholography
(Furlong et al. 1998;
Hecht et al. 1999), and
positron emission tomography (Aziz et al.
1997; Ladabaum et al.
2001) have demonstrated activation in the corresponding region of
the human cerebral cortex in response to esophageal or stomach stimulation.
Thus there seems to be a region in the neighborhood of the primary
somatosensory cortex (S1) that receives sensory input from abdominal organs
and might be involved in conscious sensations from these organs. However, this
region has not been anatomically or functionally identified, and the afferent
pathway responsible for this abdominal activation has not been determined.
Furthermore, it contrasts with the standard view that visceral sensation
involves primarily the insular cortex
(Cechetto and Saper 1990).
Very recently, it was reported that the most lateral part of S1 in rats
receives input from vagal afferents (Ito
2002). This provided the first anatomically identified
demonstration of this putative visceral region in experimental animals. The
results suggested that this region is continuous with the intraoral trigeminal
representation in S1. However, these results did not provide a direct
comparison with human cortex because the organization of S1 cortex differs
substantially between rat and human. While the sensorimotor cortex in rats
seems to be a combined entity (or a mosaic) with respect to submodalities of
somatic input (Gioanni 1987),
the corresponding cortex in many mammals including humans has distinct
cytoarchitectonic areas that receive input from different submodalities. In
particular, area 3b receives cutaneous mechanoreceptive input and is
considered to be S1 proper, and area 3a receives proprioceptive input and is
considered an adjunct of motor cortex
(Jones 1985;
Kaas 1993; Weisendanger and
Miles 1982). To address the issue of an S1 visceral region across species, it
is crucial to clarify whether the vagal input arrives in area 3a, area 3b or
both. If vagal input is directed to area 3b, or S1, this would support the
implication of Penfield's homunculus that S1 is involved in the perception of
visceral sensations. On the other hand, if the vagal input is received by area
3a, a fairly different scheme of the visceral sensory system would emerge
because area 3a is distinct from area 3b not only in the sensory submodalities
represented but also in the cortical networks involved. In addition to
proprioceptive input, area 3a receives nociceptive afferent input
(Craig 1995;
Tommerdahl et al. 1996) that
may be important for perception (Perl
1984). Thus the distinction of the area of sensorimotor cortex
that receives vagal afferent input impacts the possible role of these regions
in motor control and sensation.
With regard to this issue, cats have several advantages; they have
distinctively different cytoarchitectonic areas
(Avendano and Verdu 1992;
Ghosh 1997;
Hassler and Muhs-Clement 1964;
Leclerc et al.1994) with
well-documented deep (area 3a) and cutaneous inputs (area 3b or S1 proper)
(Felleman et al.1983;
Jones and Porter 1980). The S1
representation of the trigeminal intraoral region has been mapped
(Iwata et al. 1985;
Taira 1987) and can guide
localization of the vagal region (Ito
2002). Finally and most importantly, the vagal-evoked potential in
the region of sensorimotor cortex has been repeatedly examined in the cat.
After Siegfried described the region for the first time
(Siegfried 1961), others
(Aubert 1970; Aubert and Legros
1971; Korn and Masson 1963; Massion et al.
1966) clearly differentiated the evoked potential in the lateral
sigmoid gyrus, at the lateral extent of sensorimotor cortex, from the evoked
potential focus in the orbital gyrus, which corresponds to the insular
visceral region of humans, monkeys and rats. Unfortunately, these prior
authors did not provide a cytoarchitectonic description, although they
described the waveform, amplitude, latency, or laterality in some detail. It
was even left undetermined whether the evoked potential focus was in the
somatosensory or motor portion of sensorimotor cortex.
In the present study, we focused on identifying the cytoarchitectonic
location of this vagal-potential region in cats. The site was localized with
microelectrode recordings from the pial surface and from the cortical depth to
demonstrate the generator site. Somatosensory receptive fields were mapped in
the nearby S1 to determine the relationship between the vagal input focus and
S1. Finally, the cytoarchitecture of the site was determined. It is located in
area 3a not 3b. A preliminary report of this study was made
(Ito and Craig 2002).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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The experimental protocol was approved by the Institutional Animal Care and Use Committee, and all procedures complied with the guiding principles for the care and use approved by the American Physiological Society.
Seven cats of either sex weighing 2.4–4.5 kg were used; three were anesthetized with chloralose and four with alphaxolone/alphadolone (Saffan, Glaxo). Chloralose anesthesia (60 mg/kg iv) was preceded by ketamine (50 mg im) and supplemented with intravenous pentobarbital sodium as necessary. Animals that received Saffan anesthesia (12–18 mg/kg iv) were first tranquilized with acepromazine (0.3 mg/kg im); Saffan was continuously supplemented with infusion pump (8–12 mg · kg–1 · h–1). In all cats, a bolus injection of dexamethasone (10 mg iv) was given. The neck, head, and face were shaved. A rectal thermistor was inserted, and an infrared lamp as well as a heated water pad was used to maintain core temperature at 37.5°C. Eye salve was applied, 0.5% bupivicaine was injected subcutaneously at incision sites, and a local anesthetic was sprayed in the ear canals. Blood pressure and heart rate were monitored with the catheter inserted in the right carotid artery (contralateral to the examined hemisphere). End-tidal CO2 was maintained at 3.5–4.5%. Depth of anesthesia was monitored with the width of the pupil and reflexive change in blood pressure and heart rate and also with reflexes to pinch and corneal contact before introduction of paralytic agent.
After cannulation of the trachea and the carotid artery, the cervical vagus nerves were bilaterally isolated. Each nerve was attached with a cuff electrode consisting of two pairs of platinum wires 30 or 50 mm apart; one pair was used for stimulation and the other for recording the compound action potential. The nerves were ligated distally to the electrode to avoid any vagal efferent-mediated responses confounding the cortical recording and to exclude possible contamination of sympathetic afferent activity (because the cervical vagus nerve may contain nerve fibers from the superior cervical ganglion).
Each animal's head was mounted in a stereotaxic frame so that the head was tilted with the left side up (only the left cerebral hemisphere was studied). The zygomatic arch, the postorbital processes, and the upper part of the mandible were removed. The eyeball was pulled ventral or, in one cat, removed. A craniotomy was made to expose the anterolateral cerebral cortex. Mineral oil or Tyrode's solution were often applied to keep the exposed area from drying out.
Vagal stimulation/recording
The vagus nerves were electrically stimulated with either a single rectangular pulse (duration: 0.3 or 2.0 ms) or a train of pulses (3 0.3-ms pulses at 1 kHz). The stimulus strength was initially adjusted to be supramaximal for A-delta fiber activation in the compound action potential recorded from the vagus nerve; it produced C-fiber activation as well (though the cortical response we recorded could not have resulted from C-fiber afferents based on the latency).
The cortical surface was photographed with a digital camera, and the recording sites were plotted on the printed photographs. The vagal-evoked field potential was recorded with both low- and high-resolution methods. A low-resolution map was made with a tungsten or platinum macroelectrode to isolate the focal region and differentiate it from the orbital focus. The dura was left intact. The electrode was moved in two dimensions on a 1 x 1-mm grid with a manipulator which was independent of the stereotaxic frame and aligned nearly parallel to the cortical surface. For mapping, the intensity of vagal nerve stimulation was increased so that it was twice that needed to produce a maximal cortical-evoked potential recorded at an arbitrary site near the coronal gyrus; it varied from 0.7 to 10.0 mA in the different cases.
In the four cats anesthetized with Saffan, high-resolution microelectrode
recordings were made after the rough mapping described in the preceding text.
The dura over the focal site was cut and reflected. A platinum-plated
tungsten-in-glass microelectrode (tip size: 
15 µm) was used to record
the evoked potential from the pial surface. The cortex was often moving (due
to respiration and arterial pulse pressure), so a slight pressure was usually
imposed on the surface by the microelectrode, which produced a variable degree
of dimpling. At the focal site, the laminar profile of the evoked potential
was examined with the same microelectrode by advancing it and penetrating the
cortex with steps of several hundred micrometers. The penetration angle was
oblique to the cortical surface because of the curvature of the target cortex
near the coronal sulcus.
The indifferent electrode was a silver wire wound in a disk shape or a stainless steel clip attached to nearby muscle. Signals were amplified with a band-pass of 1 Hz to 1 kHz for the macroelectrode and 10 Hz to 10 kHz for the microelectrode. Multi-unit spike activity was recorded with a band-pass of 300 Hz to 10 kHz. The signals were stored in a digital signal-processing system (CED Power 1401 and Spike 2 program). Field potentials were averaged and amplitude and latency were measured with this system.
Somatosensory mapping
The somatosensory mechanoreceptive representation (S1) was mapped to
determine its positional relationship with the vagal-evoked potential focus.
Because the detailed S1 map of the trigeminal intraoral region has been
established (Taira 1987), it
was sufficient to examine the receptive fields of middle layer neurons at
several arbitrarily chosen sites to extrapolate the location and extent of S1.
After the surface and depth recordings of the vagal potential were completed,
the dura was further opened widely to expose S1 posterior to the coronal
sulcus. The microelectrode was inserted to a depth of 1 mm, and
multi-unit responses were recorded from the middle cortical layers in response
to natural tactile stimulation of the head, oral, and forelimb regions. The
stimuli were tapping, brushing, or rubbing the skin and intraoral structures
and moving the jaw, forelimb, and toes to stimulate all available
mechanoreceptors, including in the pharynx and nares. Receptive fields were
recorded on standard drawings.
Histology
During the microelectrode recordings, several penetrations were marked with
lesions made by cathodal current (15–30 µA for 30 s). In the
macroelectrode recordings, several recording sites were marked with dye
deposits (Pontamine skyblue, 100 µA for 1 min under microscopic
observation) at the termination of the experiment. After the experiment, the
animals were injected with a lethal dose of barbiturate and the cortex was
removed. The tissue block was soaked in 10% Formalin for 1 wk, then in 30%
sucrose in Formalin for 1 wk, and then cut in serial 60-µm coronal frozen
sections and stained with thionin. Cytoarchitectonic areas were determined
using criteria based on Avendano and Verdu
(1992), Ghosh
(1997), Hassler and
Muhs-Clement (1964), and
Leclerc et al. (1994). Digital
photomicrographs were taken with a Leaf Microlumina, and the images were
processed with Adobe Photoshop for adjustment of brightness and contrast as
well as insertion of indications and anatomic landmarks.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Vagal stimulation produced a biphasic positive-negative potential over a
broad region of the sigmoid, coronal, and orbital gyri in
chloralose-anesthetized animals. The field was quite broad in
chloralose-anesthetized cats but more focused in Saffan-anesthetized cats (see
DISCUSSION). Even though the response was diffuse, there were two
sites with larger response amplitude than neighbors: one in the anterior part
of the lateral sigmoid gyrus and another more posterolaterally in the orbital
gyrus. The former region is the subject of the present study, whereas the
latter corresponds to the well-established "insular visceral sensory
cortex" (Clasca et al.
1997). Stimulation of either right or left vagus nerves elicited
the evoked potential in the sigmoid gyrus. The contralateral (right) nerve or
the ipsilateral (left) nerve was primarily used to examine the evoked
potential with macroelectrode recording in three cats or one cat,
respectively, and in the other three cats, the evoked potentials on both sides
were examined. No systematic difference was observed in the focal sites or
amplitude related to the laterality of stimulation (see also
Aubert and Legros 1970).
The evoked potential on the sigmoid gyrus was focused near the anterior (lateral) tip of the coronal sulcus. Figure 1 shows a surface map of this potential field in a cat anesthetized with Saffan. The potential distribution was centered just anterior to the tip of the coronal sulcus. The gradient of the potential decay with distance from the focal site was relatively weak, and the potential field extended widely both medially and laterally to the sulcus. However, the potential amplitude was generally greater in the region medial to the sulcus than laterally, and it was clearly focused at a site anteromedial to the tip of the sulcus. In fact, in every animal (n = 7) the macroelectrode focal site was just medial to the anterior tip of the coronal sulcus, and no other potential focus was evident in the region just lateral to the coronal sulcus.
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Histological examination of sites marked with dye after macroelectrode
surface mapping in two cats showed that the center or the largest response
site of the evoked potential region appeared to be in area 3a (arrowhead,
Fig. 1C),
characterized both by a well developed granular layer and large pyramidal
cells in the fifth layer. This is consistent with the general concept in prior
histological studies (Avendano and Verdu
1992; Hassler and Muhs-Clement
1964) that the cortex adjacent to the anterior tip of the coronal
sulcus, including both medially and laterally adjacent regions, consists of
area 3a.
Microelectrode surface map
The vagal-responsive field examined with macroelectrode recordings from the intact dura was broad and poorly focused. By contrast, potential mapping from the pial surface with a microelectrode gave far better localization of the focal area. Figure 2 shows an example of such mapping. Although the surveyed area was smaller than that of Fig. 1, Fig. 2 still shows that negligible responses were obtained in the periphery of a very clear response focus. The large-amplitude evoked potentials were concentrated in a region just larger than 1 mm2. The difference in amplitude of the recorded potential between the response center and periphery was so dramatic that it was easy to demarcate the focus of the vagal-evoked potential region on the cortical surface. With microelectrode mapping, it was clearer that the vagal-responding region was restricted to the area medial to the coronal sulcus even though it closely adjoined the sulcus.
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In four cats anesthetized with Saffan, a complete microelectrode map was
recorded from the pial surface with contralateral vagal stimulation. The
latency and amplitude of the evoked potential were both relatively invariable
among cats; the average peak amplitude was 685 µV (range: 482–780),
and the mean value of the peak latency was 11.0 ms (range: 9.4–12.1).
Figure 3 illustrates the
distribution of vagal potentials relative to the coronal sulcus in all four
animals (Fig. 3,
A–D). The recording sites were spaced 0.5 mm
in general and 0.2 mm at the focal sites. In all four animals, the
largest evoked potentials were obtained from a stripe-like region rather than
a focal concentric region, and they were restricted to a small region
anteromedial to the coronal sulcus. Slight variations in peak amplitude at
adjacent points in the focal site were ascribed to variability in the contact
pressure of the microelectrode due to surface movement and dimpling.
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With the vagal-evoked potential focus well localized on the anteromedial
side of the anterior tip of the coronal sulcus, we investigated its
relationship with the physiologically determined S1 somatosensory
representation in three cats by making depth recordings with the
microelectrode. The region surrounding the anterior part of the coronal sulcus
was mainly examined, in accordance with the maps produced by Felleman et al.
(1983) and by Taira
(1987).
Figure 4 shows two examples. In
Fig. 4A, the most
anterior mechanoreceptive responses were obtained with stimulation of the
ipsilateral anterior tongue (site 1). More posteriorly, receptive fields on
the contralateral hard palate (site 2), lower teeth (site 3), upper gingiva
(site 4), and lip (site 5) were found in this order. Further posteriorly,
extraoral receptive fields were found, such as the subnostril regions (sites 6
and 7). In the case illustrated in Fig.
4B, the most anterior responsive site was found with
bilateral glottal stimulation (site 1); more posteriorly, the lip (site 2) and
facial responding sites (sites 3–5) were found. Outside this oral/facial
region, penetrations encountered neurons with receptive fields on the forelimb
(sites 6–8). All but one (site 7) of these sites were located lateral to
the coronal sulcus. In the three cats, the most anterior somatosensory
representation in the region of the coronal sulcus was the tongue
(Fig. 4A, site 1),
glottal region (Fig.
4B, site 1), or front teeth (not shown). Penetrations
were made further anterior to these sites and no further mechanoreceptive
responses were obtained. The most anterior mechanoreceptive region was
separated by ≥1 mm from the vagal response focus.
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On the other hand, multi-unit responses to proprioceptors of the forelimb
were found medial to the sulcus (Fig.
4B, sites 6 and 8), at the anteroposterior level of the
facial representation. They responded to claw movement or forepaw tap (cf.
Dykes et al. 1980). The area
just lateral to this deep input region and medial to the vagal site was
unresponsive to conventional stimulation, including press/pinch of the
contralateral cheek or intraoral palpation. In one animal
(Fig. 4A), evoked
potentials were obtained in response to stimulation of the ipsilateral
masseteric nerve in this region, just anterior and medial to the coronal
sulcus. Although the focal site was closer to the vagal site than the forelimb
response sites, it was still separate by 1 mm (posterior and medial) from
the vagal-evoked potential focus.
Because the cytoarchitectonic location of S1 is well established, we examined histologically only few of the recording sites of somatic receptive fields. They were found in either area 3b or 3a according to whether surface or deep receptive fields were recorded, respectively, as established in the literature.
Depth recording
The same microelectrode used for surface mapping of the vagal-evoked potential and for mapping of multi-unit mechanoreceptive responses was also inserted into the cortex in the vagal response region to determine whether the generator site could be demonstrated by a polarity reversal indicative of the current sink. Because the vagal region was quite well localized, only a few penetrations at the focal site were needed for this purpose. The penetrations were not always perpendicular to the cortical surface, so that even a penetration at the center of the focal site did not always record the deep negative potential with an amplitude comparable to the surface positivity. Nonetheless, in all three animals in which depth recordings were made, deep negative potentials with comparable latency were recorded, and reversals were observed in the cortical depth below the surface focal sites.
Figure 5 shows an example of depth recording at the penetration corresponding to the most posterior and lateral of the focal recording sites indicated by large dots in Fig. 3A (open arrow). The evoked field potential was recorded at five different depths, and a lesion was made at the deepest point (site 5). The surface positive potential became larger in the middle of the track (sites 2 and 3, corresponding to layer III of the cortex), and it reversed to a negative potential in the depth (sites 4 and 5, layers IV and V/VI border, respectively). Figure 5C also shows a multi-unit response recorded at site 2; many spikes responded to the stimulation with latencies between 8 and 22 ms, consistent with the onset and peak latencies of the field potential recorded at the same site of 5.8 and 10.4 ms, respectively. Multi-unit responses to vagal stimulation and polarity reversals of the evoked field potential were obtained in all three depth recordings (open arrows) located at the anterior, middle, and posterior parts of the surface vagal focus in this particular animal. Multi-unit responses in penetrations with polarity reversals were also obtained in one other cat. These results definitely demonstrated that there was a cluster of neurons activated at short latencies by vagal input in the center of the response focus determined by the surface mapping.
Cytoarchitecture
Figure 5D shows the entire electrode track for the penetration just described, from the entry (arrow) to the lesion (double arrowhead), in a Nissl-stained section. In this photomicrograph, the area demarcated by the four open arrows is recognizable as area 3a. Area 3a was most clearly distinguished when situated as the transitional area between areas 4 and 3b at more posterior levels (cf. Fig. 7). Area 4 was characterized by both the lack of layer IV and the presence of the dark-stained giant pyramidal cells in layer V. By contrast, area 3b was characterized by a thick layer IV and a cell-sparse stripe-like layer V. Area 3a was distinguishable as an area intercalated between these two areas with clear but less thick layer IV overlying giant pyramidal cells in layer V. Area 3a was also distinguishable from areas 3b and 4 by the sublamination of layers III and VI in the latter two areas. At more anterior levels, such as Fig. 5D, area 4 was replaced by area 6, which made the medial border of area 3a less clear. However, by tracing anterior from the more easily distinguished posterior levels, it was possible to follow and delineate area 3a with its characteristically well-developed granular layer and darkly stained large pyramidal cells in layer V. The penetration shown in Fig. 5D was entirely within the limits of area 3a. That is, the large positive potential in the upper layers, the smaller negative potential in the deeper layers, and the multi-unit responses in the middle layers were recorded in area 3a. In all four brains in which microelectrode mapping and depth recordings were made, large positive supragranular potentials and negative infragranular potentials were recorded within area 3a, as histologically verified.
Figure 6 shows the deep field potential recordings in another case in which penetrations were made at the two sites indicated by arrows in Fig. 3C. In this cat, the penetrations were nearly normal to the cortical surface. Figure 7 shows the reconstructed cytoarchitectonic limits of area 3a surrounding two microelectrode penetrations across a series of consecutive sections spaced 120 µm apart. The lesions marked in Fig. 7 were made at the approximate depth of the reversal of the field potential in each penetration. The lesions are evident in the accompanying photomicrographs of the indicated sections. Both penetrations were, like that shown in Fig. 5, just medial to the anterior tip of the coronal sulcus. This part of the cortex was occupied entirely by area 3a; the lateral border of area 3a with area 3b was at almost the same location in the sulcus throughout this level. The medial border of area 3a shifted significantly along the anteroposterior sequence. Posteriorly, the cortex medial to the coronal sulcus included, from medial to lateral, areas 4, 3a and 3b, and area 3a was wide at this coronal plane. On the other hand, anteriorly, as described earlier, area 4 was replaced by area 6, which extended ventrally, and area 3a became restricted to a narrow region just above the coronal sulcus or the dimple of its anterior tip. Thus the location of the two penetrations differed with respect to the extent of area 3a, though they were similar in position relative to the coronal sulcus.
As apparent in Fig. 7 (B and C), both the entry point and the lesion of both penetrations were within area 3a. Because these two penetrations were made at the anterior and posterior parts of the surface potential region, these two sections probably correspond to the anterior and posterior limits of the vagal potential generator area in this brain. The anterior section was at almost the anterior limit of area 3a; in the section only 360 µm anterior, area 3a was no longer detectable. Thus, comparison with the extent of the vagal focal site in Fig. 3C suggests that, in this cat, the vagal afferent region occupied almost all of the most anterior (and lateral) part of area 3a, and it extended posteriorly in the most lateral (ventral) part of area 3a.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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),
suggested that visceral organs are represented in the primary somatosensory
cortex in the same manner as the cutaneous body surface. Early findings in
cats (Aubert 1970;
Siegfried 1961) and more
recent findings in rats (Ito
2002) seemed to support that scheme, but the present observations
in the more differentiated cortex of cats do not support that view. Because
areas 3a and 3b are distinguished in the sensorimotor cortex of primates
similarly to cats, the same visceral cortical organization could be present in
primates, including humans. Technical considerations
We mapped the evoked field potential. A field potential study is
advantageous for mapping a broad region because it examines mass activity.
Possible contamination by far field potentials
(Dong and Chudler 1984) was
mitigated by using subdural recording with a high-impedance electrode. The
response field was in each case restricted and focused, with a steep amplitude
gradient around the focal site (Fig.
2) that made it relatively easy to demarcate. Although it lay
adjacent to the coronal sulcus, the vagal response focus was not buried in the
sulcus. The depth recordings made at the focal sites demonstrated phase
reversal, a criterion for the presence of a current sink or generator site
within the cortex. Clusters of neurons that responded at comparable latency
indicated that the potentials represented neuronal activation at the site.
Therefore the focal sites revealed with microelectrode mapping apparently
represent dense vagal afferent input at the potential generating region.
The stimulating electrodes were embedded among neck musculature so that any
stimulus spread would have involved the neck muscles. The short latency of our
vagal potential is compatible with neck-muscle-evoked potentials. However, the
neck muscles are innervated by cervical spinal nerves and consequently
represented in the posterior sigmoid gyrus
(Barbas and Dubrovsky 1980),
posteromedial to the vagal focal region. We did not observe any potential foci
in that region during macroelectrode mapping. Furthermore, we stimulated the
masseteric muscle at the lateral side of the cranium, which is even more
anterior in the body, and its evoked potential site was distinctly posterior
and medial to the vagal-evoked potential site. Thus it is unlikely that the
present results included responses originating from afferents from surrounding
muscles.
We used two anesthetics, chloralose and Saffan. We initiated this study
with chloralose because it had been used in previous studies of vagal-evoked
potentials (Aubert 1970; Korn
and Massion 1964; Massion et al.
1966). However, under chloralose, the vagal-evoked potential field
was large and difficult to delimit just as somatic-evoked potentials are
(Harding et al. 1979). Under
Saffan anesthesia, the vagal-evoked potential had a comparable peak amplitude
but a more delimitable focus (Fig.
1). In addition, Saffan has less suppressive effects on autonomic
function than chloralose (Child et al.
1972; Ness and Gebhart
1988; Timms 1981),
and it does not produce the variable late-evoked potential components that can
confound studies under chloralose
(Boissonade and Matthews
1993). Thus, in contrast to prior studies of vagal-evoked
potentials under chloralose or pentobarbital, we found consistently short
response latencies under Saffan (see following text).
Composition of the vagus nerve: a mixture of "visceral" and "somatic" afferents?
While the cardiopulmonary and subdiaphragmatic branches of the vagus
consist entirely of afferents which innervate visceral organs that contain
smooth muscle, the cervical vagus nerve that we stimulated contains in
addition afferents that innervate oropharyngeal tissues that are characterized
by mixed smooth and striate muscle and so could be regarded as
"somatic." In particular, it contains afferents from the recurrent
laryngeal nerve that innervate the pharynx, larynx, trachea, and upper
(distal) esophagus. Our vagal stimulation must have activated these afferents
simultaneously, and consequently, the cortical potential we recorded probably
represents activation of both oropharygeal afferents as well as afferents from
cardiopulmonary and subdiaphragmatic visceral tissues. The vagalevoked
potential we recorded over the anterior sigmoid gyrus (sensorimotor cortex)
has essentially the same latency and shape as the potential that can be
recorded over the orbital gyrus (insular cortex; see
Aubert 1970; Korn and Massion
1964; Massion et al. 1966),
consistent with the view that similar or identical sets of afferents
contribute to both projection sites. A subsequent analysis of the sensorimotor
corticalevoked potential from cardiopulmonary and subdiaphragmatic vagal
afferents could directly validate this view; however, several additional
considerations support the inference that the evoked potential we recorded in
the anterior sigmoid gyrus includes activation by cardiopulmonary and
subdiaphragmatic vagal afferents and can be regarded as a putative visceral
activation site. First, the evoked potential focus we identified lies in area
3a, not 3b, indicating that it is not part of the mechanosensory
("somatic") afferent representation in cortex. Second, it lies at
the lateral part of the sensorimotor cortical strip; this is consistent with
the activation observed in human cortex by natural stimulation of both the
upper esophagus (which contains both striate and smooth muscle layers) and the
lower (proximal) esophagus (which contains only smooth muscle), as well as by
distension of the stomach and the colon (Aziz et al.
1997,
2000b;
Binkofski et al. 1998;
Furlong et al. 1998;
Hecht et al. 1999;
Ladabaum et al. 2001;
Lotze et al. 2001). This area
appears to be homologous to the site we identified in cat (see following
text). Third, a recent study suggests that the conscious sensation produced in
humans by distension of the upper esophagus, despite its potential designation
as somatic, differs significantly from the sensation produced by stimulation
of the overlying chest wall, in that it is diffuse, poorly localized, and
distinctly unpleasant, consistent with the sensations elicited from other
"visceral" organs (Strigo et
al. 2002). Fourth, single-unit recordings in the region of the
vagal-evoked potential site in sensorimotor cortex of the rat directly
indicate activation by afferent input from viscera, consistent with this
inference (Hanamori et al. 1998; Zhang and
Oppenheimer 1997). Finally, the vagal afferents that innervate the
upper trachea and esophagus have been regarded by some as somatic in part
because they terminate in the marginal layer of the trigeminal subnucleus
caudalis and the upper cervical dorsal horn (Contreras et al. 1982; see also
Panneton 1991); however, that
pattern of termination for visceral afferents is also present in the spinal
cord, where afferents that parallel sympathetic efferent fibers similarly
terminate in lamina I of the superficial spinal dorsal horn. Therefore whereas
the following comments refer empirically to the "vagal-evoked potential
site" in sensorimotor cortex, we infer that this represents a putative
visceral afferent activation site.
Comparison with prior vagal-evoked potential studies in cats
Our results confirmed and expanded the results of prior studies regarding
the vagal response focus in the sensorimotor cortex in cats. This site
corresponds to Siegfried's
(1961,
1962) "area A," in
contrast to "area B" in the orbital gyrus (insular cortex).
Subsequent studies (Aubert
1970; Aubert and Legros
1970; Korn and Massion 1964;
Massion et al. 1966) generally
located the region as lateral, instead of medial, to the anterior tip of the
coronal sulcus in their summary diagrams, although they described the area as
between the anterior end of the coronal sulcus and the presylvian sulcus just
as Siegfried did (Sigfried 1961). Our surface mapping always included the area
lateral to the coronal sulcus but never identified a potential focus there,
and the focal site was anteromedial to the coronal sulcus in all animals.
Furthermore, in none of the prior studies did the actual mapping data show the
focus lateral to the sulcus, and at least one figure (Fig. 6 of
Aubert and Legros 1970) clearly
illustrated the focal site anteromedial to the tip of the coronal sulcus; this
is consistent with our observations. Therefore despite the discrepancy with
their summary statements, it is apparent that the vagal response focus we
identified is the same as that observed in the prior studies and identified
first as area A by Siegfried
(1961).
The prior studies detailed the latencies of early and late vagal-evoked
potentials. The latency of the peak response in the present study
(9.4–12.1 ms) was comparable to that of the earliest response in the
prior studies (8–25 ms) (Aubert
1970; Aubert and Legros
1970; Korn and Massion 1964;
Massion et al. 1966), which is
attributable to myelinated vagal afferents. One study reported that both fast
and slowly conducting myelinated fiber groups contribute to the evoked
potentials in both the sensorimotor cortex and the insular cortex
(Massion et al. 1966).
Potentials at latencies attributable to unmyelinated vagal afferents were not
clearly distinguished in the present nor in the prior studies. Unmyelinated
C-fibers may be too variable (dispersed) in their conduction times to form a
coherent, observable evoked waveform
(Porter 1963), or they may be
more susceptible to anesthetic than myelinated afferents (cf.
Kalliomaki et al. 1993),
although vagal C-fiber responses have been observed in rat insular cortex
(Ito 1994).
Comparison with prior vagal-evoked potential studies in other species
In addition to cats, a vagal response focus has been observed in the most
lateral portion of sensorimotor cortex in rats, goats, and monkeys
(Ito 2002;
O'Brien et al. 1971;
Siegfried 1962). As in
Penfield's homunculus in human cortex, the vagal area was generally located
lateral to the intraoral representation in each of these species. This
correspondence suggests that the sensorimotor vagal area may be homologous
throughout mammals. Few studies have examined cytoarchitecture.
In rats, the vagal area was shown to occupy parietal "granular"
cortex, implying that it is within S1 (Ito
2002). However, the differentiation of sensorimotor cortex in the
rat is primordial, and there is overlap between the regions that receive input
from cutaneous and deep receptors (Gioanni
1987). That is, there is not a clear distinction between areas 3a
and 3b in the rodent. Although it has been suggested that the transitional
dysgranular part of S1 in rats receives deep input like area 3a of more
encephalic animals (Chapin and Lin
1984), this region was identified only in the spinal
representation. Thus the location of the vagal region in the granular cortex
lateral to the trigeminal region in the rat does not necessarily contradict
the localization of the vagal region in area 3a in the more differentiated
cortex of other mammals.
In macaque monkeys, the vagal-evoked potential was recorded in the inferior
precentral region at the lateral extreme of sensorimotor cortex
(O'Brien et al. 1971).
Convergent responses to laryngeal and tongue stimulation were obtained in the
same region, anterior to S1, so the authors viewed this region as motor cortex
(O'Brien et al. 1971).
However, others have regarded the inferior precentral cortex as area 3
extending anterior and lateral from the tip of the central sulcus and
providing either the sensory representation of the ipsilateral intraoral
region (Manger et al. 1996) or
a taste-related region (Benjamin et al.
1968; Ogawa et al.
1985,
1989;
Pritchard et al. 1986). Thus
the possibility remains that the vagal potential site in monkey sensorimotor
cortex is also located in area 3a.
In humans, direct electrical stimulation of the lateral S1 region elicited
conscious alimentary sensation (Penfield
and Rasmussen 1950). Esophageal (Aziz et al.
1997,
2000b;
Binkofski et al. 1998;
Furlong et al. 1998;
Hecht et al. 1999), gastric
(Ladabaum et al. 2001) or
rectal (Lotze et al. 2001)
distension elicited activation in functional imaging studies in the same
general region around the lateral end of the central sulcus. The pathway
responsible for this cortical activation, and whether it represents motor or
sensory activity, has not been determined. Although these alimentary
structures are dually innervated by cranial and spinal nerves
(Collman et al. 1992), a vagal
contribution is strongly suggested by the sequence of cortical topography,
consistent with the present findings.
Thus in all representative mammals that have been investigated, an area at the lateral end of sensorimotor cortex has consistently been observed that could represent vagal afferent activity. However, the cytoarchitectonic location of the vagal response site has not been previously resolved.
Cytoarchitectonic location of the vagal response focus
The present results indicate that the region responsive to vagal input is
entirely outside the S1 representation, including the representation of the
intraoral structures. The somatosensory map of the cat S1 along the coronal
sulcus is well established (Felleman et
al. 1983; Taira
1987). The most anterior and lateral portion represents the
ipsilateral and contralateral intraoral mechanoreceptors, and then the lips
and extraoral structures are represented progressively more posteriorly and
medially (Taira 1987). Our
recordings in S1 reproduced this pattern, and furthermore, we found pharynx-
and glottal-responding neurons anterolateral to the representation of the
tongue (Fig. 4B).
Nonetheless, the vagal-responsive region was distinctly anterior and medial to
the S1 representation, and a region with no somatic response was intercalated
between the vagal region and the S1 representation.
The vagal response focus was anterior and medial to the anterior tip of the
coronal sulcus, and cytoarchitectonic studies and our observations indicate
that it lies within area 3a. Indeed, Hassler and Muhs-Clement
(1964) showed the anterior and
lateral border of area 3a crossing the coronal sulcus to include even the
region lateral to the anterior part of the coronal sulcus. Avendano and Verdu
(1992) construed this lateral
extension only at the most anterior tip of the coronal sulcus. Our reading of
the present material was even more conservative, and area 3a appeared to be
restricted entirely to the medial bank of the coronal sulcus and the lateral
sigmoid gyrus.
Lesions made at microelectrode penetrations in which field potential reversals and multi-unit responses were observed were all histologically identified within area 3a. This indicates that the current sink producing the surface positive potential is definitely located in area 3a not in area 3b. Although depth recordings were made only in the neighborhood of the focal site, there is no reason to expect additional generator sites that are not revealed by surface potentials. It is possible that vagal input may also activate neurons outside the borders of area 3a, but the main focus is certainly within area 3a. At the anterior end of area 3a where it is narrow (Figs. 5 and 7B), all of area 3a appears to be occupied by the vagal-responsive region. More posteriorly, at the level of Fig. 7C, the vagal-responsive region is restricted to the most lateral part of area 3a. Thus, the present observations indicate that the vagal response focus occupies the most anterolateral part of area 3a in the cat.
Input source
Area 3a in cat receives its principal proprioceptive and vestibular inputs
from the thalamic region that lies along the border between the ventral
posterior and the ventrolateral nuclei, that is, between the lemniscal
somatosensory relay and the cerebellar motor relay
(Dykes et al. 1986;
Jones and Porter 1980;
Kaas 1993). Activation of
group I inputs, for example, evokes in area 3a a so-called thalamocortical
primary response with an initially positive surface wave that reverses
polarity in deep layers (Landgren and
Silfvenius 1969; Odkvist et
al. 1975). Iwata et al.
(1985) described a
masseteric-nerve-evoked potential, which reversed to a deep negative component
at a depth of 1.5–2.5 mm, in the rostral part of cat area 3a, consistent
with our observations. This depth profile is in good agreement with that of
the present vagal potential, consistent with the hypothesis that the vagal
input to area 3a arrives by a direct thalamocortical projection like the
proprioceptive or vestibular input. The presence of sacral visceral (pelvic
nerve) input to neurons in the ventral periphery of the thalamic ventral
posterior complex (Brüggemann et al.
1994), from which projections to area 3a also arise
(Craig and Kniffki 1985),
supports this possibility as well.
The anterolateral region of area 3a receives a massive input from the
(visceral) granular insular cortex (Clasca
et al. 2000). However, that projection terminates in layers I,
III, and VI, which differs from the middle layer focus of the vagal-evoked
potential that we observed. In addition, previous work demonstrated that the
rat S1 vagal potential is independent of the insular input
(Ito 2002). Thus, at present
it seems likely that the vagal input to area 3a ascends by a direct thalamic
pathway. Retrograde labeling data are needed to address this issue.
Because activity from the chorda tympani and the vagus nerves are similarly
processed in the brain stem (Nomura and Mizuno
1981,
1983), the cortical projection
of the chorda tympani, the main taste nerve, must be compared with that of the
vagus. Like the vagus, the chorda tympani activates multiple sites in the
cortex of both cat and monkey. In the cat, chorda-tympani-evoked potentials
were reported in the orbital gyrus and the coronal gyrus
(Burton and Earls 1969) and in
the monkey, in the inferior precentral area of the sensorimotor cortex and the
opercular/insular cortex (Benjamin and
Burton 1968; Benjamin et al.
1968; and Ogawa et al.
1985). The orbital/insular area receives input from the thalamic
taste area (monkey: Pritchard et al.
1986; cat: Ruderman et al.
1972; Yasui et al.
1987) and is regarded as primary gustatory cortex. In the monkey,
a collateral ("sustaining") projection from the thalamic taste
relay to the sensorimotor cortical areas was proposed
(Benjamin and Burton 1968; see
Pritchard et al. 1986),
although in the cat, taste neurons were not observed in the coronal region
(Cohen et al. 1950), and
instead, antero-grade tracing indicated projections from the thalamic taste
relay to orbital, perirhinal, and infralimbic cortices
(Yasui et al. 1987). Thus, the
data indicate that the multiple thalamocortical projections of the chorda
tympani parallel those of the vagus; however, it remains to be determined
whether the vagal and gustatory inputs to the orbital/insular and sensorimotor
cortices ascend from common sets of thalamic neurons.
On the possible role of area 3a in visceral motor and sensory functions
Stimulation in the somatic portion of area 3a evokes striate muscle
contractions (Preuss et al.
1996; Widener and Cheney
1997; Wu et al.
2000), and stimulation of this region can also have autonomic
effects. In cats, it can change respiratory rhythm
(Kaada 1951) via the phrenic
as well as the recurrent laryngeal nerves (Bassal and Bianchi
1981,
1982). Thus area 3a appears to
participate in visceral motor control via both sympathetic and parasympathetic
pathways as well as in somatic motor control
(Jones and Porter 1980;
Wiesendanger and Miles 1982).
Stimulation in the lateral precentral region of the monkey cortex evokes
swallowing (Martin et al.
1999) or changes in heart rate
(Hast et al. 1974), although
whether that region is cytoarchitectonically area 3a remains to be
resolved.
It has not yet been determined whether area 3a is involved in the conscious
perception of any sensory event. Activity in muscle afferents is essential for
the sense of joint position (Clark et al.
1985; Gandevia
1985; Goodwin et al.
1972), so area 3a, as the primary cortical target of deep receptor
afferents, may be involved in this sensation
(Jones and Porter 1980),
although it is heavily interconnected with area 2 posterior to S1
(Porter 1991). As part of the
same cytoarchitectonic area, the vagal afferent projection field in the
lateral portion of area 3a could share a role with the insular cortex in
sensations such as "sinking feeling," "sick feeling,"
"choking sensation," "nausea," and "shaking in
the heart," descriptors evoked by direct electrical stimulation of the
corresponding human cortex (Penfield and
Rasmussen 1950).
Cortical homeostatic afferent network
In addition to the region at the lateral end of sensorimotor cortex, two
other cortical regions are activated by vagal afferent input, namely, the
insular and the cingulate cortices, which also receive vagal input by way of a
direct thalamic pathway (Bachman et al.
1977; Cechetto and Saper
1987; Hallowitz and MacLean
1977). The insular cortex has direct interconnections with both
area 3a and with the cingulate cortex (cat:
Clasca et al. 2000). These
three regions apparently constitute the main visceral afferent activation
sites in the cortex and form together a visceral afferent cortical network.
This interpretation is supported by the consistent activation of all three
areas in imaging studies of visceral stimulation in the human brain
(Aziz et al. 2000a). Notably,
the same three regions (area 3a, the insular and cingulate cortices) also
constitute the main regions activated in the human cerebral cortex by painful
stimulation (see Craig 2002),
and prior evidence in the cat indicates that area 3a also receives nociceptive
thalamocortical input (Craig and Kniffki
1985). Thus the present findings are consonant with the
fundamental concept that pain and visceral sensation are different aspects of
a common homeostatic afferent system that activates limbic sensory (insular)
cortex, limbic motor (cingulate) cortex, and a particular portion of
sensorimotor cortex (area 3a) (Craig
2002). The role of area 3a in this network remains to be
resolved.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Support for this laboratory was provided by National Institutes of Health Grant NS-25616 and the Atkinson Pain Research Fund administered by the Barrow Neurological Foundation. S.-I. Ito was on leave from Kumamoto University.
Present address of S.-I. Ito, Dept. of Physiology, Shimane Medical University, 89-1 En-ya, Izumo City, Shimane 693-8501, Japan.
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FOOTNOTES |
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Address for reprint requests: A. D. Craig, Atkinson Pain Research Laboratory, Division of Neurosurgery, Barrow Neurological Institute, 350 W. Thomas Rd., Phoenix AZ 85013 (E-mail: bcraig{at}chw.edu).
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, tactile
responses; 
proprioceptive responses; 
, strong vagal-evoked
potentials. Activation from the forepaw occurred by mechanoreceptors on the
glabrous skin of the digit (7) or movement of the claw (6, 8).