1Division of Neurosurgery, Barrow Neurological Institute, Phoenix, Arizona 85013; and 2Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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ABSTRACT |
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Craig, A. D. and J. O. Dostrovsky. Differential Projections of Thermoreceptive and Nociceptive Lamina I Trigeminothalamic and Spinothalamic Neurons in the Cat. J. Neurophysiol. 86: 856-870, 2001. The projections of 40 trigeminothalamic or spinothalamic (TSTT) lamina I neurons were mapped using antidromic activation from a mobile electrode array in barbiturate anesthetized cats. Single units were identified as projection cells from the initial array position and characterized with natural cutaneous stimuli as nociceptive-specific (NS, n = 9), polymodal nociceptive (HPC, n = 8), or thermoreceptive-specific (COOL, n = 22; WARM, n = 1) cells. Thresholds for antidromic activation were measured from each electrode in the mediolateral array at vertical steps of 250 µm over a 7-mm dorsoventral extent in two to eight (median = 6.0) anteroposterior planes. Histological reconstructions showed that the maps encompassed all three of the main lamina I projection targets observed in prior anatomical work, i.e., the ventral aspect of the ventroposterior complex (vVP), the dorsomedial aspect of the ventroposterior medial nucleus (dmVPM), and the submedial nucleus (Sm). The antidromic activation foci were localized to these sites (and occasional projections to other sites were also observed, such as the parafascicular nucleus and zona incerta). The projections of thermoreceptive and nociceptive cells differed. The projections of the thermoreceptive-specific cells were 20/23 to dmVPM, 21/23 to vVP, and 17/23 to Sm, whereas the projections of the NS cells were 1/9 to dmVPM, 9/9 to vVP, and 9/9 to Sm and the projections of the HPC cells were 0/8 to dmVPM, 7/8 to vVP, and 6/8 to Sm. Thus nearly all thermoreceptive cells projected to dmVPM, but almost no nociceptive cells did. Further, thermoreceptive cells projected medially within vVP (including the basal ventral medial nucleus), while nociceptive cells projected both medially and more laterally, and the ascending axons of thermoreceptive cells were concentrated in the medial mesencephalon, while the axons of nociceptive cells ascended in the lateral mesencephalon. These findings provide evidence for anatomical differences between these physiological classes of lamina I cells, and they corroborate prior anatomical localization of the lamina I TSTT projection targets in the cat. These results support evidence indicating that the ventral aspect of the basal ventral medial nucleus is important for thermosensory behavior in cats, consistent with the view that this region is a primordial homologue of the posterior ventral medial nucleus in primates.
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INTRODUCTION |
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Lamina I of the trigeminal
and spinal dorsal horn is an integral component of the central neural
representation of pain and temperature sensations (for review:
Craig and Dostrovsky 1999). Lamina I receives input from
A
and C primary afferent fibers, including specific nociceptors and
thermoreceptors, and it is the major source of ascending output from
the superficial dorsal horn. Lamina I cells contribute about half of
the direct projections to thalamus from the dorsal horn in cats and
monkeys, and their projections in the trigeminothalamic and
spinothalamic (TSTT) tracts are behaviorally and clinically critical
for pain and temperature sensations.
The original physiological description of spinal lamina I neurons by
Christenson and Perl (1970) provided clear evidence that different response classes can be recognized. They described
nociceptive-specific (NS) cells responsive to noxious mechanical
stimulation or noxious heat or both and, in addition, thermoreceptive
cells responsive to cooling the skin with ethyl chloride spray, some of
which were specific, but some of which responded also to noxious
stimuli. Mosso and Kruger (1973)
reported similar
findings in the trigeminal dorsal horn. Lamina I TSTT projection cells
in the trigeminal dorsal horn were identified in the cat by
Dostrovsky and Hellon (1978)
, who reported corresponding
response characteristics and who documented thermoreceptive lamina I
units sensitive only to warming as well. Kumazawa et al.
(1975)
described similar cells in the spinal cord of the
monkey. Thermoreceptive and nociceptive lamina I TSTT projection
neurons have since been documented in the spinal cords of both cat
(Craig and Bushnell 1994
; Craig and Dostrovsky
1991
; Craig and Hunsley 1991
; Craig and
Kniffki 1985a
) and monkey (Dostrovsky and Craig
1996
). Consistent with the earlier descriptions, these studies
recognized three categories of lamina I TSTT cells: NS neurons,
thermoreceptive-specific neurons (WARM or COOL, formerly "COLD"),
and polymodal nociceptive neurons sensitive to noxious heat, noxious
pinch, and noxious cold (HPC). These categories not only have
qualitatively different response properties but also different
ascending conduction velocities and different susceptibilities to
descending or pharmacological modulation (Craig and Serrano
1994
; Dawson et al. 1981
; Dostrovsky et
al. 1983
). Evidence has recently been obtained that they can be
distinguished morphologically as well (Han et al. 1998
).
A natural inference is that these distinct classes of lamina I TSTT
neurons might have distinct projection targets in the thalamus. The
present study was motivated by the contrary observation (Craig
and Dostrovsky 1991; Dostrovsky and Broton 1985
)
that both COOL and NS lamina I TSTT cells can be antidromically
activated from the submedial nucleus (Sm) in the medial thalamus of the cat. Anatomical evidence indicates that Sm is one of three main sites
in cat thalamus in which ascending lamina I TSTT fibers terminate
(Craig 1987
, 1991
), in addition to the ventral aspect of
the ventroposterior complex (vVP) and the dorsomedial aspect of the
ventroposterior medial nucleus (dmVPM). The suggestion that the
different classes of lamina I TSTT cells might not have distinct
projections contrasted with the distinctive features of these different
classes and with the repeated observation that only
thermoreceptive-specific neurons can be recorded in the region of dmVPM
(Auen et al. 1980
; Landgren 1960
).
Therefore we directly examined whether the thalamic projections of
lamina I TSTT neurons are related to their physiological response
characteristics by antidromically mapping the projections of single
identified units using a mobile array of electrodes that could
encompass all of the anatomical projection sites in the cat thalamus.
Our results confirm that lamina I TSTT neurons project mainly to these
three sites, and they provide evidence that thermoreceptive and
nociceptive lamina I TSTT cells do have different thalamic projection
patterns. Preliminary reports were made (Craig and Dostrovsky
1991
; Dostrovsky and Craig 1993
).
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METHODS |
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General procedures
All procedures were conducted in accordance with the guiding principles for the care and use of animals approved by the American Physiological Society, and the protocols were approved by the appropriate institutional animal care and use committees. Experiments were performed on 46 domestic cats (2.0-5.9 kg, mean = 3.3), approximately half male and half female. The animals were anesthetized with pentobarbital sodium (40 mg/kg ip), and a catheter in the right cephalic vein was used to administer intravenous supplements to maintain areflexia. The head, neck, back, face, and forelimb or hindlimb were shaved. Eye salve was administered, 0.5% marcaine was injected subcutaneously at all incision sites, cetacaine was sprayed in the ear canals, and a single intravenous injection of dexamethasone (10 mg) was administered. The left common carotid artery was catheterized to monitor blood pressure, and the trachea was cannulated. Paralysis was induced with pancuronium bromide (~0.4 mg/h), and a positive pressure respirator was used to maintain end-tidal CO2 at 3.5-4.5%. Anesthetic depth (areflexia) was verified every hour when the paralytic wore off. The animals were respired with 100% air or with 75% air and 25% O2. Core temperature was maintained at 37.5°C with a heating pad and a feedback-controlled infra-red heat lamp. The animal's head was mounted in a stereotaxic frame. A craniotomy was performed to provide access to the right thalamus. A laminectomy was performed to expose the spinomedullary junction (29 cases), the cervical enlargement (2 cases), or the lumbosacral enlargement (15 cases). The animals were suspended with vertebral clamps, and the cord surface was covered at all times with Tyrode's solution or artificial cerebrospinal fluid; in the lumbosacral cord, this was maintained at 38°C with a nichrome heating element. Agar (2.5%) was used for stabilization at the spino-medullary junction. A pneumothorax was performed if needed to reduce cord pulsations. At the end of the experiment, each animal was perfused with buffered 10% formalin. The recording segment and the thalamus were subsequently processed for standard histological examination by cutting serial 50-µm transverse frozen sections and staining with thionin. Photomicrographs were made with Kodak Technical Pan film or with a Leaf Lumina scanner and processed (contrast enhancement) with Adobe Photoshop.
Implantation of thalamic electrode array
Tungsten-in-glass microelectrodes (tip diameter ~25-40 µm)
were used to make single- and multi-unit recordings of somatosensory responses in the ventroposterior (VP) thalamus to determine the initial
placement of the antidromic stimulating electrodes. Electrode penetrations commenced anteriorly and laterally (AP9.5, ML6.5) to
identify the junction of the cutaneous and deep representations of the
forepaw and then moved posteriorly and medially to locate the small
(approximate diameter, ~0.25 mm) site at the dorsomedial aspect of
the face representation where units responding to cooling the
ipsilateral tongue can be found (Auen et al. 1980;
Landgren 1960
) and to identify as well the ventral
border of VP at the external medullary lamina. Based on these results,
the array of antidromic electrodes was implanted so that it was
situated 1 mm dorsal to the intended initial location (dmVPM, vVP, or
Sm); it was moved into the intended position after a lamina I unit was
isolated. The array consisted of seven (6 experiments) or eight (40 experiments) concentric bipolar electrodes evenly spaced at 1-mm
intervals in a mediolateral row. Custom-made Rhodes (Kopf Instruments,
Tujunga, CA) MCE-100X electrodes were used that were 45 mm in length
with an inner contact diameter of 25 µm and an outer contact 100 µm
higher that had a diameter of 150 µm; these were reinforced to within
5 mm of the tip with 0.35-mm diameter tubing to minimize deviation.
Monopolar search stimuli were delivered to each of the electrodes in
the thalamic array with the following parameters: 500 µA, 2-ms
duration, three or four pulses at 200-333 Hz, inner (or outer) contact
negative. The indifferent stimulus electrode was attached to the
incised cranial skin.
Characterization of lamina I TSTT units
Recordings were obtained from cells in the superficial dorsal
horn near the dorsal root entry zone with platinum-plated
tungsten-in-glass microelectrodes having tip sizes of ~15 µm.
Recordings were made in the trigeminal dorsal horn at the University of
Toronto, and recordings were made in the spinal cord at the Barrow
Neurological Institute. In general, whether in the trigeminal,
cervical, or lumbosacral dorsal horn, lamina I was recognized at depths
of 200-600 µm by the presence of units with slow, irregular ongoing activity; such activity was often inhibited by radiant heat and excited
by application of an innocuous cool stimulus to the face, forepaw, or
hindpaw, indicative of COOL cells (see following text). In the spinal
cord, lamina I is just below the Group I-II afferents that have
regular ongoing discharge. (Below this, we find a 200-µm-thick zone,
in the outer substantia gelatinosa, where our electrodes rarely pick up
unitary discharges, followed by field potentials and multi-unit
responses to low-threshold mechanical stimulation in inner lamina II
and laminae III-V.) The identification of TSTT projection neurons at
these depths provides assurance that the units were lamina I neurons
because TSTT neurons are rarely located in laminae II-III in the cat
(Zhang et al. 1996).
The microelectrode was moved so that a single unit was isolated on the
basis of spike amplitude, and then activation from the thalamic
electrode array was tested. The antidromic nature of each unit's
response was determined on the basis of a constant latency, a discrete
all-or-none threshold to stimulation, the ability to follow a 200- to
333-Hz train of at least three stimuli with constant latencies, and
collision by a closely preceding orthodromic spike (Fig.
1). All units that fulfilled these
criteria demonstrated a definitive critical interval for collision with orthodromic spikes when tested (Ranck 1975). The
orthodromic and antidromic spike waveform of every unit studied was
constantly monitored to ensure that recordings from the same single
unit were maintained throughout the characterization and antidromic mapping. Lesions were made at these recording sites (5-20 µA
cathodal current for 20-40 s), and every one found (40 in 46 cats) was located in lamina I of the trigeminal or spinal dorsal horn (Fig. 2).
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Antidromically activated single units were tested with natural
cutaneous stimulation, including innocuous and noxious mechanical (brush, cotton wisp, and flat forceps), innocuous cooling (beaker of
wet ice), innocuous warming (radiant heat lamp), noxious heat (heat
lamp), and noxious cold (ice-cold beaker) stimuli (Craig and
Dostrovsky 1991; Craig and Hunsley 1991
;
Craig and Serrano 1994
). The cells were classified as:
thermoreceptive-specific cells sensitive to cooling (COOL) or warming
(WARM); NS cells sensitive only to heat, pinch, or both; and HPC cells
sensitive to noxious heat, pinch, and noxious cold. Wide dynamic range
cells also occur in lamina I, but these rarely project to thalamus in the cat (Craig and Kniffki 1985a
; Craig and
Serrano 1994
; Hylden et al. 1986
).
The COOL cells were identifiable by irregular spontaneous activity that
could be silenced by innocuous radiant warming of the skin, by vigorous
excitation on contact or near contact with a cold object, and by their
insensitivity to mechanical stimulation with a probe warmed to neutral
skin temperature. They displayed graded responses to innocuous cooling
but did not show increased responses to noxious cold (see Fig. 1). Some
COOL units are weakly excited by noxious heat (reflecting the
"paradoxical" discharge of some cooling-specific primary afferents)
(see Hensel 1981) or phasically excited by a strong
pinch. The rare WARM cells (only 2 were encountered) were identified by
their response to innocuous warming, inhibition by cooling, and their
insensitivity to noxious or mechanical stimuli. In contrast, polymodal
nociceptive HPC lamina I TSTT neurons were identifiable by their lower
ongoing discharge (see Andrew and Craig 2001
), their
insensitivity to warming, their phasic excitation by innocuous cooling,
and their sustained, graded discharge to noxious cold, noxious heat,
and pinch. The thresholds of HPC cells to noxious cold vary (see
Craig and Serrano 1994
). They are insensitive to
innocuous mechanical stimulation with thermally neutral probes.
Finally, we characterized NS lamina I TSTT cells by their selective,
graded sensitivity to pinch or noxious heat (see Fig. 1) or
both pinch and heat. In the present experiments, we included as NS
cells two units that were briskly responsive to noxious heat and pinch
but appeared to have been sensitized by prior stimulation (one
responded intermittently to hair movement and another was sensitive to
a cotton wisp slowly drawn across the glabrous skin at the tip of the
nose but to no other innocuous stimuli).
Receptive fields (RFs) were determined by manual mapping with threshold
stimuli and indicated on figurines. Receptive fields were located on
glabrous or hairy skin of the face, the forepaw, or the hindpaw
according to the segmental location of the recording sites and were
consistent with prior descriptions (Craig and Dostrovsky 1991; Craig and Hunsley 1991
; Craig and
Kniffki 1985a
; Craig and Serrano 1994
;
Dostrovsky and Hellon 1978
). In the spinal experiments, a series of computer-controlled cooling and warming steps was applied
with a Peltier thermode (20 × 20 mm) placed on the RF (see Fig.
1) (Craig and Hunsley 1991
; Craig and Serrano
1994
).
Mapping of antidromic activation sites
A map was made of the thalamic sites from which each unit could be antidromically activated by moving the array vertically in 0.25-mm steps at each of several anteroposterior planes. From its initial position, after unit isolation and characterization, the array was first moved up (dorsally) and then down (ventrally) in the same plane. More rostral planes were then examined, followed by more caudal planes. The anteroposterior planes were placed at intervals that varied from 0.75 to 1.5 mm but were usually evenly spaced at 1 mm. A total vertical extent of 7.0 mm was examined in each plane. At each 0.25-mm vertical step, monopolar search stimuli were delivered to each electrode in the array, and where the unit was antidromically activated, the threshold was determined with a constant current stimulator (WPI model A360). Collision was verified at the lowest threshold foci within each mapping plane. In three experiments, a unit was mapped on each side of the trigeminal dorsal horn, and in three other experiments two units were differentiated (with different spike shapes, RFs, and antidromic latencies), characterized and simultaneously mapped in one or more planes. Lesions were made by passing 20-30 µA for 30-40 s (cathodal) through the tips of the stimulating electrodes at strategic sites and at the unit recording location to enable reconstructions. Usually lesions were made at the bottom and the top of at least one electrode track in each plane, but a different pattern was used in each plane to facilitate histological reconstruction; lesions were also made at or just above critical low-threshold foci.
In general, monopolar stimuli were used as search stimuli for threshold mapping because this produced antidromic activation from a broad area and thereby provided assurance that projection areas between the electrodes or between mapping planes were not overlooked. In some experiments, however, monopolar stimulation was effective at very low currents, resulting in activation by the search stimulus from very wide areas and in less steep threshold gradients in the neighborhood of low-threshold foci; in these cases, bipolar stimulation between the inner and outer conductors of individual electrodes was used as an adjunctive measure to confirm localization of the low-threshold foci of activation. Bipolar stimulation resulted in higher thresholds that increased more sharply with increasing distance from the focus, presumably due to the more focused current spread, and thereby provided greater spatial definition of low-threshold foci. The adjunctive comparison of monopolar and bipolar stimulation thresholds often helped distinguish and cleanly separate neighboring antidromic projection foci.
Anatomical reconstructions
To reconstruct anatomical maps of the sites of antidromic
activation, each mapping plane was first identified histologically on
the basis of the pattern of the lesions made. The shrinkage within each
brain was estimated based on the distance between lesions made at the
top and the bottom of one electrode track (usually 10-15%). The
bottoms of the tracks of the individual electrodes were identified
within each plane, and vertical distances were measured from these
points, taking into account the shrinkage and the locations of other
lesions made at strategic sites. The antidromic activation sites and
the measured thresholds were then plotted on cytoarchitectonic drawings
made from the individual thalamic sections according to Craig
and Burton (1985), as in the example shown in Fig.
3. In most cases, the
sections were fairly well aligned with the electrode tracks so that at
least half of the vertical extent could be seen in one or two adjacent sections, but in some cases, the sections were misaligned due to the
plane of sectioning or due to progressive edema during the mapping
procedure. In these cases, portions of the electrode tracks were
visible on successive sections, and vertical depths from the marking
lesions were interpolated to the appropriate histological level.
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The antidromic mapping data were compiled for systematic analysis by
transposing the histological reconstruction of each individual unit
onto a standard series of coronal drawings of the cat thalamus made at
~0.5-mm intervals (the same series used by Norrsell and Craig
1999). In these standardized summaries, three different dot
sizes were used to represent the number of consecutive mapping points
at which thresholds
60 µA were measured: a small dot was placed if
only one point had a threshold
60 µA, a medium-sized dot was placed
if two consecutive points had such thresholds, and a large dot was used
to represent three such consecutive mapping points. The sizes of the
medium and large dots were such that the dot covered both or all three
of the low-threshold points. Comparison of the original reconstruction
for unit c13-11.1 (Fig. 3) with the standardized summary in
Fig. 4 shows that the
localization and the distinctness of the low-threshold projection foci
were preserved during this transfer.
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The final compilation of the antidromic mapping data was made by superimposing the standardized summary maps of all 40 thoroughly mapped cells, using a constant gray level for the threshold dots (in the program Adobe Photoshop), sorted according to cell type and RF location (Fig. 6). With this method, anatomical areas of overlap between the antidromic projection foci of these cells were signified with progressively darker gray levels. Statistical comparisons of the characteristics of different populations of neurons were made with the program CSS Statistica (Statsoft; Tulsa, OK).
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RESULTS |
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Characteristics of the total sample of antidromically activated lamina I TSTT units
The units selected for antidromic mapping were taken from a total
sample of 130 lamina I neurons that were identified from the initial
array position as TSTT cells by the demonstration of a high-frequency,
constant latency antidromic train response (Fig. 1). These lamina I
units were recorded in the trigeminal dorsal horn (nucleus caudalis) in
29 cats, in the cervical C8 segment in 2 cats,
and in the lumbar L7 segment in 15 cats. Of this
total sample, 89 lamina I TSTT units were recorded in the trigeminal
dorsal horn, and these had an average conduction velocity of 5.71 m/s
based on a mean latency of 5.25 ms (range 1.9-13.0) and a mean
distance of 30 mm. A total of 15 units recorded in the
C8 segment had an average conduction velocity of
3.41 m/s based on a mean latency of 30.8 ms (range 12-90 ms) and a
mean distance of 105 mm, and 26 units recorded in the
L7 segment had an average conduction velocity of
2.56 m/s based on a mean latency of 133 ms (range 38-360) and a mean
distance of 340 mm. The latencies recorded were the shortest obtained
from the site of the initial array placement in thalamus (targeted in
different cases at vVMb, Sm, or dmVPM; see following text). These
latencies and mean conduction velocities are consistent with prior
observations of lamina I trigeminothalamic and spinothalamic cells in
the cat (Craig and Kniffki 1985a; Dostrovsky and
Hellon 1978
). The present data at three spinal levels
demonstrate for the first time that there is a progressive decrease in
lamina I TSTT conduction velocities with increasing conduction distance.
The conduction velocities of the characterized lamina I TSTT cells
differed according to their physiological class. For the entire sample
of 20 characterized lamina I TSTT cells recorded in the
L7 segment, where the differences were greatest,
the mean conduction velocities were: for NS cells, 1.83 ± 0.76 (SD) m/s, n = 5; for HPC cells, 3.24 ± 1.67 m/s,
n = 6; and for COOL cells, 5.78 ± 1.77 m/s,
n = 9. Despite the small numbers, these conduction velocities are pairwise significantly or nearly significantly different
(NS vs. HPC, P = 0.11; NS vs. COOL, P < 0.001; HPC vs. COOL, P < 0.02; t-tests),
consistent with the significant differences observed in prior samples
(Andrew and Craig 2001; Craig and Serrano 1994
).
Characteristics of the antidromically mapped lamina I TSTT cells
We selected 49 of the total sample of 130 identified lamina I TSTT cells for mapping with the mobile electrode array, and we mapped their thalamic projections in one to eight anteroposterior planes. The antidromic identification of each mapped unit as a TSTT cell was verified by demonstration of its critical collision interval (see Fig. 1). In each experiment, the array was implanted initially to stimulate one of the main lamina I projection sites. That is, each cell studied was selected because it was initially antidromically activated from vVP, dmVPM, or Sm. Accordingly, the initial location of the electrode array was varied in order that this selection would not produce a bias. The number of cells selected for antidromic mapping that were initially identified from each histologically verified location was: 16 from vVP, 13 from dmVPM, and 20 from Sm.
The final data set comprised 40 of the 49 antidromically mapped cells.
Two of the 49 units were discarded because of inadequate histology, and
7 were discarded because the histological reconstruction showed that
the region mapped had not encompassed all three major lamina I
termination sites; these units included primarily cells for which a map
had been obtained in only one or two planes. We initially focused on
COOL cells in this study (Craig and Dostrovsky 1991),
and of the 40 thoroughly mapped cells, 23 were thermoreceptive-specific (COOL or WARM) cells, while 8 were HPC cells, and 9 were NS cells. The
final sample of 40 well-mapped lamina I TSTT units included 28 trigeminothalamic cells and 12 spinothalamic cells recorded in the
L7 segment. The antidromic maps of these 40 cells
comprised an average of 5.6 planes (median 6.0).
The histological reconstruction of the complete mapping results from one representative experiment is shown in Fig. 3. In this experiment, the antidromic electrode array was initially positioned at AP 6.5, where electrodes 3 and 4 were meant to be situated at or near dmVPM, and from this site, we antidromically identified five lamina I trigeminothalamic COOL cells with RFs on the nose or the eyelids. The COOL cell (c13-11.1) that we selected for antidromic mapping was initially activated at a monopolar threshold of 270 µA on electrode 3 with an antidromic latency of 1.9 ms, and it had a large, well-isolated and stable action potential and a RF on the dorsal and ventral eyelids. Antidromic thresholds were mapped first at AP 6.5, then successively at AP 7.25, 8.0, 6.0, 5.25, and 4.25, using both monopolar and concentric bipolar stimulation.
This representative example shows the broad coverage and the clear localization of projection sites provided by antidromic mapping with the mediolateral array of electrodes. The unit was consistently activated by the 500-µA monopolar search stimulus from points rostrocaudally, mediolaterally, or dorsoventrally adjacent to the projection foci, indicating that the search coverage exceeded the 1-mm mediolateral spacing between electrodes, the 0.75- or 1.0-mm spacing between anteroposterior planes, and the 0.25-mm spacing between vertical test points. Nevertheless, clearly delimited foci with thresholds <20 µA were obtained. For example, at AP 5.25, the unit illustrated in Fig. 3 was antidromically activated with stimulus thresholds <200 µA over an extent of 2.5 mm from electrode 3, yet two clearly distinguishable low-threshold foci were defined, one at a depth of 4.5 mm (in dmVPM) and the other at 5.5 mm (in the ventral aspect of VMb, which is part of the vVP region). These two low-threshold foci had focal monopolar thresholds of 20 µA and were separated by a sequence of monopolar thresholds of 55, 125, and 60 µA at the intervening 0.25-mm steps. The localization of these foci was confirmed with the use of bipolar stimulation, with which these sites had focal thresholds of 35 and 45 µA and were even more clearly separated by a sequence of thresholds of 150, 230, and 90 µA at the intervening steps.
Low-threshold activation points with thresholds 60 µA with
monopolar stimulation were found for all of the 40 thoroughly mapped
cells. The average threshold at these foci was 20.5 µA ± 15.0 SD (range 2-60 µA), with a 25th percentile of 9.0 µA, a median of
17.0 µA, and a 75th percentile of 28.0 µA.
Different antidromic latencies were generally observed at different
projection foci, indicative of the different conduction times in
different collaterals. Slightly longer latencies were also recorded in
the periphery around each projection focus, consistent with longer
onset activation times associated with activation from a distance.
Significantly, at the projection foci of 22 cells we observed discrete
latency shifts at different stimulus intensities ("latency
jumping") as we have described before (Craig and Dostrovsky 1991). These shifts were as large as several milliseconds for spinal units; the largest was a shift from 52 to 45 ms at higher intensities at the projection focus in Sm for cell c40-1.
Such shifts were observed in each of the three main projection targets (dmVPM, vVP, Sm). These discrete latency shifts indicate that different
terminals were activated within the terminal arborization field of the
cell's axon (Lipski 1981
), which provides direct evidence supporting the conclusion that these antidromically mapped activation foci identify the terminal projection sites of these axons.
Distribution of antidromically mapped projection foci
Reconstructions of the projection maps of several
thermoreceptive-specific (COOL and WARM) cells and nociceptive (NS and
HPC) lamina I TSTT cells are shown at the standardized thalamic levels (see METHODS) in Figs. 4 and
5. These units are representative of the
major projection patterns, and they also show a variety of individual
ancillary projections. Both trigeminothalamic and spinothalamic cells
are illustrated; RF locations for each cell are denoted in the figures.
If a particular level of the thalamus was not mapped in an experiment,
then the image of that level is not included in the charts. The
fidelity of the transposition of individual histological
reconstructions onto the standardized series can be appreciated by
comparing the complete reconstruction of cell c13-11.1 in
Fig. 3 with its standardized representation in Fig. 4. Note that the
sizes of the dots in Figs. 4 and 5 represent the spatial extent of
low-threshold antidromic activation (60 µA), not absolute
thresholds (see METHODS). The projections of individual
cells varied, yet several patterns were apparent. The overall patterns
can be appreciated from the summary charts in Fig.
6, in which the projections of all 40 cells are superimposed.
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The predominant projection targets were vVP, Sm, and dmVPM, consistent
with anatomical observations based on anterogradely transported PHA-L
(Craig 1987, 1991
). Nearly all (37/40) of the thoroughly
mapped lamina I TSTT cells projected to vVP (including the ventral
aspect of VMb, the region of VPI, and the ventral aspect of VPL). Most
(32/40) projected to Sm. About half (21/40) projected to dmVPM.
The projections of the thermoreceptive-specific (COOL and WARM) lamina
I TSTT cells clearly differed from the projections of the nociceptive
(NS and HPC) cells. Whereas most cells in both populations projected to
vVP and to Sm, the thermoreceptive-specific cells had a characteristic
projection to dmVPM, and in contrast, almost none of the nociceptive
lamina I TSTT cells projected to dmVPM. Thus of the 23 thoroughly
mapped thermoreceptive-specific cells, 21 projected to vVP, 17 projected to Sm, and 20 projected to dmVPM. Of the nine thoroughly
mapped NS cells, all nine projected both to vVP and to Sm, but only one
projected to dmVPM. Similarly, of the eight thoroughly mapped HPC
cells, seven projected to vVP and six to Sm, but none projected to
dmVPM. The distinction between the projections of
thermoreceptive-specific cells and NS or HPC nociceptive lamina I TSTT
cells is statistically significant (P <105, General Linear
Model, CSS Statistica).
The characteristic projection of thermoreceptive-specific lamina I TSTT cells to dmVPM was focused at levels two and three in the standard series, as shown in Figs. 4 and 6. Corroborative evidence for this characteristic projection is provided by the observation that we consistently identified many additional COOL lamina I TSTT cells when searching for a TSTT unit to map with the array initially positioned at dmVPM, as noted in the detailed description of experiment c13 in the preceding text.
The thermoreceptive-specific and nociceptive lamina I TSTT cells also differed with respect to the distribution of their projections within vVP (Figs. 4 and 6). The projections of thermoreceptive-specific (COOL and WARM) cells were particularly concentrated within the medial portion of vVP. Nearly all of these cells projected to the ventrolateral aspect of VMb and/or the adjacent medial portion of VPI. There was even an indication of somatotopographic order because every trigeminothalamic thermoreceptive-specific cell (except the 2 that did not project to vVP) projected to this region at the two or three most posterior standard levels (Fig. 6), whereas every spinothalamic thermoreceptive-specific lamina I cell projected to this region at more anterior levels (especially levels 3 and 4).
In contrast, the projections of the nociceptive cells within vVP were more widely distributed throughout its caudal, rostral, medial, and lateral extents (Figs. 5 and 6). Lateral projection foci in the ventral aspect of VPL were regularly observed for nociceptive cells, regardless of RF location, but such projections were almost never observed for COOL cells. Some nociceptive cells projected throughout much of the mediolateral extent of vVP, whereas for others, discrete foci occurred in vVMb and vVPL (e.g., Fig. 5, HPC cell c40-1, NS cell c41-1). Of the 17 nociceptive (HPC and NS) cells, only 1 did not project to vVP [an HPC cell that projected only to caudal posterior nucleus (Po)].
More than two-thirds (17/23) of the thermoreceptive-specific cells and
nearly all (15/17) of the nociceptive cells also projected to Sm. The
single WARM cell that was mapped did not differ from the COOL cells in
this regard (Fig. 4). There was evidence of somatotopography in this
region as well (Fig. 6): lamina I trigeminothalamic cells (both
thermoreceptive and nociceptive) generally projected to the caudal pole
of Sm (level 3), whereas spinothalamic lamina I cells (both
thermoreceptive and nociceptive) generally projected to the rostral
half of Sm (level 4). This is consistent anatomically with the
rostrocaudal topographic organization of Sm (Craig 1987, 1991
; Craig and Burton 1985
).
In addition, ancillary antidromic activation sites were observed that were also consistent with the prior anatomical observations, and these too differed according to cell category. Projections to parafascicular nucleus (Pf, n = 5), to zona incerta (ZI, n = 2), and to supergeniculate nucleus (SG, n = 1) were observed for only a few of the 23 thermoreceptive cells, whereas projections to Pf (n = 7), to ZI (n = 7), to caudal Po (n = 9), to dorsal Po (n = 4), and to other sites [2 in VM, 2 in central lateral nucleus (CL), 1 in SG] were more common among the 17 thoroughly mapped nociceptive cells. Antidromic activation sites in caudal Po were found only for nociceptive units. It is noteworthy that projections to the dorsal portion of Po were generally located ~1 mm dorsal to the border of VP (e.g., unit c40-1, Fig. 5). In two cells, we encountered axon collaterals that ascended rostrally to join the optic tract (e.g., unit c13-11.1, Figs. 3 and 4), but we observed no collaterals within the hypothalamus.
Finally, the distribution of the ascending axons of thermoreceptive-specific and nociceptive cells also differed. Lamina I TSTT cells were antidromically activated at isolated sites in the mesencephalon, and these sites were marked with lesions in 13 cases. The ascending axons were activated with a median threshold of 40 µA (range 6-240 µA) and at latencies that were 0.2-0.9 ms shorter for trigeminothalamic cells and 2-6 ms shorter for spinothalamic cells than the shortest respective latencies observed from sites in the thalamus. For seven thermoreceptive cells, five sites were located in the medial half of the rostral mesencephalon (clustered near the border of the periaqueductal gray), and two were found in the lateral half (Fig. 7). For six nociceptive cells (3 HPC, 3 NS), all sites were found in the lateral half of the mesencephalon, clustered near the medial lemniscus in the classical location of the ascending spinothalamic tract. These differences are significant (Fisher exact test, P < 0.03).
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DISCUSSION |
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Technical considerations
The validity of these observations depends on certain considerations. First, antidromic mapping with the mediolateral array of electrodes provided sufficiently broad and complete coverage of the thalamus. The units were generally activated from broad areas by the 500-µA search stimulus, and adjacent activation points were usually observed from neighboring tracks. The array extended across nearly all of the thalamus, and the maps of the final data set of 40 thoroughly mapped cells included all three major anatomically identified lamina I TSTT termination sites, i.e., Sm, dmVPM, and vVP. The electrodes maintained nearly parallel vertical tracks, and the cytoarchitecture of the thalamus was recognizable despite multiple passes of the array. The array did produce compression and distortion of the thalamus in some cases, and progressive edema was also evident where mapping planes made later during an experiment showed curved trajectories. Thus reconstruction measurements were made from the bottoms of the tracks, which were always verified with lesions, and from additional marking lesions made at strategic locations (at or near low-threshold activation sites) during mapping.
Second, this method enabled the localization of projection sites.
Low-threshold antidromic activation foci were identified in each
experiment with a median threshold of 17 µA using electrodes with
25-µm tips. This implies a spatial resolving power of ~200 µm
(Ranck 1975), which justifies the mapping step size used
(0.25 mm). Antidromic activation was confirmed at all low-threshold sites by demonstration of a critical collision interval. The
low-threshold activation foci were clearly delimited by steeply
increasing thresholds at neighboring mapping points. These foci denote
terminal projection sites because we observed increases in activation
latencies, which indicates conduction slowing in terminal collaterals,
and because we observed the phenomenon of "latency jumping," which
indicates activation of different terminals within an arborization
field (Lipski 1981
). Significantly, the distribution and
topography of the projection sites identified in this study are
entirely consistent with the anatomically identified terminations of
lamina I TSTT neurons observed with anterograde labeling (Craig
1987
, 1991
).
Third, it should be noted that the simplified compilation method we
used did not depend on the representation of the exact numerical values
for thresholds at each point, in contrast with the technique for
plotting antidromic thresholds that we used for individual histological
reconstructions (e.g., Fig. 3) and that is commonly used by others
(e.g., Fields et al. 1995; Kitazawa et al.
1993
; Zhang et al. 2000
). Nonetheless, this
compilation method is empirical, and it enables comparison of the
histological localization of the projection sites with the spatial
resolution of 0.25 mm in these maps. This graphical method also
accommodated antidromic activation of axonal projections that
terminated between mapping planes. Finally, this method is spatially
consistent with the anatomically observed extent of the terminal arbors
of lamina I TSTT axons in these regions (Craig 1987
,
1991
).
Differentiation of the three major classes of lamina I TSTT neurons
Consistent with the description of three different physiological
response patterns by the earliest studies of lamina I neurons (Christenson and Perl 1970; Mosso and Kruger
1973
), we recognize three major and distinct classes of lamina
I TSTT projection neurons: NS, COOL or WARM, and HPC. These cell types
are distinguished by several features.
First, they have qualitatively different responses to natural cutaneous
stimuli in both cats and monkeys (Craig and Serrano 1994; Dostrovsky and Craig 1996
).
Thermoreceptive-specific (COOL and more rarely WARM) cells respond in a
graded manner only to innocuous thermal stimuli, and nociceptive NS and
HPC cells respond selectively to noxious thermal and mechanical
stimuli. The HPC cells differ from the NS cells in that they respond
also to noxious cold, and they differ from COOL cells in that they have
lower (colder) thresholds, are not inhibited by warming, and respond in
a graded manner to noxious heat and pinch.
Second, they have significantly different conduction velocities
(Craig and Kniffki 1985a; Craig and Serrano
1994
; Dostrovsky and Craig 1996
). As confirmed
in the present experiments, the NS cells conduct very slowly, many in
the range of unmyelinated axons, whereas the HPC and COOL cells conduct
progressively faster, consistent with small myelinated fibers. Larger
samples of ~200 lumbosacral lamina I TSTT cells support the
statistical significance of the pairwise differences observed in the
present sample (Andrew and Craig 2001
). These data
confirm earlier results indicating that the axons of trigeminothalamic
lamina I cells conduct on average about twice as fast as the axons of
spinothalamic lamina I cells, but the present comparison of trigeminal,
cervical, and lumbosacral lamina I TSTT cells indicates that the longer
axons are progressively thinner.
Third, they respond differently to descending and pharmacological
modulation; in particular, COOL cells are not inhibited by brain stem
stimulation, whereas NS cells are (Dawson et al. 1981;
Dostrovsky et al. 1983
) and COOL lamina I TSTT cells are not inhibited by systemic or topical morphine, whereas both NS and HPC
lamina I TSTT cells uniformly are (Craig and Hunsley
1991
; Craig and Serrano 1994
).
The present results add significantly to these distinctions by showing in cats that thermoreceptive-specific (COOL and WARM) lamina I TSTT cells have a significantly different projection pattern within the thalamus than NS or HPC cells. Nearly all thermoreceptive-specific cells projected to dmVPM, but in contrast almost no NS or HPC cells did. Many NS and HPC cells, but few thermoreceptive-specific cells, had ancillary projections to Po, ZI, and other sites. In addition, COOL cell axons ascended within the medial mesencephalon and terminated medially within vVP, whereas NS and HPC cell axons ascended in the lateral mesencephalon and terminated more broadly within vVP.
Thus the present findings clearly indicate that the axons of these
lamina I TSTT cells are differentially distributed. This anatomical
difference is consistent with the hypothesis that the different
physiological classes of lamina I TSTT cells are morphologically distinct. This hypothesis has recently received direct support from
observations of the somata of these cells, based on intracellular labeling in cats, that indicate that NS lamina I cells are fusiform neurons, COOL cells are pyramidal neurons, and HPC cells are multipolar neurons, as viewed in horizontal sections (Han et al.
1998; see also Light and Willcockson 1999
).
Additional evidence supports this anatomical and physiological
correspondence in the primate (Craig et al. 1999
;
Yu et al. 1999
). Our physiological observation that NS
cells have very slow conduction velocities also supports these findings
because Golgi studies reported that fusiform lamina I cells have
unmyelinated axons but that pyramidal and multipolar cells have
myelinated axons (Gobel 1978
; Lima and Coimbra
1986
). Thus the present findings add to considerable evidence
indicating that the three major lamina I TSTT physiological cell
classes that we recognize are robust and biologically relevant.
It is important to note that these classes do not include all lamina I
TSTT cells. The lamina I TSTT projection seems to constitute an
interoceptive afferent pathway that provides distinct
modality-selective sensory channels representing the physiological
condition of all tissues of the body (Craig and Dostrovsky
1999; Craig et al. 2000
). Some lamina I TSTT
cells are selectively responsive to deep (muscle, joint) input
(Craig and Kniffki 1985a
), and some are selectively responsive to chemical stimulation with histamine or mustard oil (Andrew and Craig 2001
). Lamina I neurons that are
selectively viscero- or metabo-receptive or responsive to C-fiber
mechanoreceptive input probably also exist, although such cells have
not been adequately documented yet (Cervero and Tattersall
1987
; Light and Willcockson 1999
;
Rosas-Arellano et al. 1999
; Urban and Gebhart
1999
; Vallbo et al. 1999
; Wilson and Hand
1997
).
Thalamic projection targets of the different classes of lamina I TSTT cells
The ascending axons of lamina I spinothalamic cells are
concentrated in the middle of the lateral funiculus in cats and
primates (Craig 1991, 2000
; Ralston and Ralston
1992
; Zhang et al. 2000
), where lesions in
humans disrupt pain and temperature sensation and where lesions in cats
interrupt innocuous thermosensory behavior (Norrsell 1979
,
1989
). Thermoreceptive-specific lamina I TSTT cells form a
unique ascending thermosensory pathway and display graded responses
that correlate with reports of innocuous thermal sensibility
(Craig and Bushnell 1994
; Davies et al.
1983
; Hensel 1981
). In primates and humans, the
thermosensory lamina I pathway is relayed by the thalamic nucleus VMpo
(the posterior part of the ventral medial nucleus) and terminates in
the dorsal margin of insular cortex (Craig et al. 1994
,
2000
; Davis et al. 1999
; Dostrovsky and
Craig 1996
). In cats, recent behavioral evidence indicates that
the homologous portion of thalamus is the caudoventrolateral aspect of
the basal part of the ventral medial nucleus (VMb); only a lesion of
this part of the thalamus produces a measurable disruption of a cat's
discriminative thermosensory behavior (Norrsell and Craig
1999
). Significantly, ventral VMb also projects to the insular
cortex (Clascá et al. 1997
; Vahle-Hinz and
Oertle 1993
; see also Yasui et al. 1987
). Our
present findings provide direct evidence that thermoreceptive-specific
lamina I TSTT cells terminate in this same part of the thalamus, and
thus the present functional anatomic data provide strong corroboration
for the behavioral anatomic findings. Thermoreceptive neurons have not
yet been recorded in this part of the thalamus in anesthetized cats,
but the present findings support the view that this region is a
primordial homologue of the much larger VMpo nucleus in primates and
especially humans (Blomqvist et al. 2000
), where
thermoreceptive-specific neurons have been identified (Craig et
al. 1994
, 1999
; Davis et al. 1999
).
This homology is supported too by the present evidence indicating that
nociceptive NS and HPC lamina I TSTT neurons from the trigeminal and
the spinal dorsal horn also terminate in ventral VMb. In the cat,
nociceptive-specific units have been identified within ventral VMb, and
such units include cells with input from the face and the paws
(Vahle-Hinz et al. 1987). Similarly, in primates and
humans, VMpo also contains many nociceptive-specific neurons
(Blomqvist et al. 2000
; Craig et al.
1994
; Lenz et al. 1993b
) that seem to underlie
the activation of insular cortex by noxious stimuli in human imaging
studies (see Craig and Dostrovsky 1999
; Moulton
et al. 2000
), and stimulation in this region in awake humans
evokes discrete, well-localized sensations of pain or cold
(Davis et al. 1996
, 1999
; Lenz et al. 1993a
,
1997
). The present findings do not differentiate the
projections of NS and HPC lamina I TSTT cells and indicate that both
project to the ventral VMb region, but other physiological evidence
nonetheless indicates that these two classes of nociceptive neurons
have differentiable roles in pain sensation (Andrew and Craig
1999
; Craig and Andrew 1999
; Craig and
Bushnell 1994
).
The present data show that virtually all thermoreceptive lamina I TSTT
neurons project to dmVPM in the cat, whereas nociceptive lamina I cells
do not. Curiously, the prior behavioral anatomic findings did not
reveal a role for this region in thermal sensation; a lesion that
damaged this region but not VMb had no measurable effect on a cat's
thermosensory behavior (Norrsell and Craig 1999). Thermoreceptive-specific neurons have been recorded in dmVPM
(Auen et al. 1980
; Landgren 1960
; present
study), although only orofacial RFs have been reported and most of
these were ipsilateral rather than contralateral in the anesthetized
cat. The role of dmVPM remains at present undetermined.
The present findings corroborate prior anatomical evidence that the
ventral aspect of VPL receives direct lamina I TSTT input from the
spinal cord (Craig 1987, 1991
; Craig and Burton
1985
) and indicate that this is a selectively nociceptive
input. Both specific and nonspecific nociceptive neurons have
repeatedly been recorded within the ventral periphery of VPL
(Bruggemann et al. 1994
; Gordon and Manson
1967
; Honda et al. 1983
; Horn et al.
1999
; Hutchison et al. 1992
; Kniffki and
Mizumura 1983
; Vahle-Hinz et al. 1987
;
Yokota et al. 1988
). This region seems to project to the
anterior cingulate, area 3a, and SII cortices (Craig and Kniffki 1985b
; Musil and Olson 1988
), which
differentiates it from the ventral aspect of VMb. This differs too from
the overall thalamocortical organization of the monkey (Craig
and Dostrovsky 1999
). Unlike the monkey, where nociceptive
neurons have been recorded in VP and in cortical areas 1-2 (reviewed
by Kenshalo et al. 2000
; Treede et al.
1999
), nociceptive neurons have rarely been recorded within the
core of the ventrobasal complex in the cat (Davis and Dostrovsky 1988
; Golovchinsky et al. 1981
; Honda et
al. 1983
; Kniffki and Mizumura 1983
;
Rydenhag and Roos 1986
). Nonetheless, area 3a is activated by noxious stimulation both in the cat (Iwata et al. 1986
) and in the primate (Tommerdahl et al.
1998
), where the source of this activation may be a collateral
input from VMpo (see Craig and Dostrovsky 1999
).
Clinical evidence also suggests a role for area 3a in pain sensation
(Perl 1984
).
Nociceptive neurons have been recorded in Po dorsal to VPM and VPL,
especially along the border between these nuclei (Bruggemann et
al. 1994; Davis and Dostrovsky 1988
; Horn
et al. 1999
; Hutchison et al. 1992
;
Vahle-Hinz et al. 1987
). The present findings provide evidence of lamina I TSTT terminations in Po that are generally quite
dorsal to this border. However, other nociceptive inputs to the dorsal
and ventral borders of VP could originate from deeper TSTT cells in
laminae V-VII (Yen et al. 1991
). In contrast, the present findings are certainly consistent with the original description of nociceptive-specific units in caudal Po in the cat (Poggio and Mountcastle 1960
), where many of the studies cited in the preceding text reported similar neurons. Recent evidence suggests that
this region may project to the most posterior portion of insular cortex
in the rat (Shi and Davis 1999
), yet the relationship of
caudal Po to vVMb remains to be determined.
The present findings corroborate prior work indicating a dense
projection of nociceptive lamina I TSTT neurons to Sm (Craig 1987, 1991
), and they extend our previous observations
(Craig and Dostrovsky 1991
) by showing that many
thermoreceptive-specific lamina I neurons project to Sm from the spinal
cord as well as from the trigeminal dorsal horn. This nucleus in cats
and rats contains nociceptive-specific units (Craig
1991
; Dostrovsky and Guilbaud 1990
;
Kawakita et al. 1993
; Miletic and Coffield
1989
), and it projects to ventrolateral orbital cortex, where
similar recordings have been obtained (Backonja and Miletic
1991
; Snow et al. 1992
). In contrast, the
analogous lamina I TSTT projection to medial thalamus in the monkey (to
the ventral caudal aspect of the medial dorsal nucleus) seems instead
to provide the source of thermosensory-modulated nociceptive activation
of the anterior cingulate cortex (see Craig and Dostrovsky
1999
); this constitutes another profound phylogenetic
difference between primates and nonprimates. Nonetheless, the role of
convergent thermoreceptive-specific input to medial thalamus may be
similar across these species in providing a substrate for the
inhibition of nociceptive processing by innocuous thermosensory
activity (Craig 1991
; Ericson et al. 1996
). This is consistent with the observation that lesions of Sm did not affect the thermosensory behavior of cats (Norrsell and Craig 1999
).
Conclusion
In summary, the present findings demonstrate with antidromic mapping of single, identified units that the thalamic projections of nociceptive (NS and HPC) and thermoreceptive-specific (COOL and WARM) lamina I TSTT cells differ, which is consistent with the many other features that distinguish these cell classes. These observations corroborate anatomical observations of the projection targets of lamina I TSTT neurons in the cat, they fit with behavioral observations on the role of the ventral aspect of VMb in thermal sensation in cats, and they provide further evidence supporting the view that ventral VMb in cats is a primordial homologue of the VMpo nucleus that is well developed only in primates and especially in humans.
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ACKNOWLEDGMENTS |
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The authors thank M. Tatum and M. Teofilo for technical assistance.
This work was supported by National Institutes of Health Grants NS-25616 (to A. D. Craig) and DE-05404 and NS-36824 (to J. O. Dostrovsky) and by the Atkinson Pain Research Fund administered by the Barrow Neurological Foundation.
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FOOTNOTES |
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Address for reprint requests: A. D. Craig, Div. of Neurosurgery, Barrow Neurological Institute, 350 W. Thomas Rd., Phoenix, AZ 85013 (E-mail: bcraig{at}chw.edu).
Received 29 November 2000; accepted in final form 17 April 2001.
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REFERENCES |
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