Atkinson Pain Research Laboratory, Division of Neurosurgery, Barrow Neurological Institute, Phoenix, Arizona 85013
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ABSTRACT |
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Craig, A. D., K. Krout, and D. Andrew. Quantitative Response Characteristics of Thermoreceptive and Nociceptive Lamina I Spinothalamic Neurons in the Cat. J. Neurophysiol. 86: 1459-1480, 2001. The physiological characteristics of antidromically identified lamina I spinothalamic (STT) neurons in the lumbosacral spinal cord were examined using quantitative thermal and mechanical stimuli in barbiturate-anesthetized cats. Cells belonging to the three main recognized classes were included based on categorization with natural cutaneous stimulation of the hindpaw: nociceptive-specific (NS), polymodal nociceptive (HPC), or thermoreceptive-specific (COOL) cells. The mean central conduction latencies of these classes differed significantly; NS = 130.8 ± 55.5 (SD) ms (n = 100), HPC = 72.1 ± 28.0 ms (n = 128), and COOL = 58.6 ± 25.3 ms (n = 136), which correspond to conduction velocities of 2.5, 4.6, and 5.6 m/s. Based on recordings made prior to any noxious stimulation, the mean spontaneous discharge rates of these classes also differed: NS = 0.5 ± 0.7 imp/s (n = 47), HPC = 0.9 ± 0.7 imp/s (n = 59), and COOL = 3.3 ± 2.6 imp/s (n = 107). Standard, quantitative, thermal stimulus sequences applied with a Peltier thermode were used to characterize the stimulus-response functions of 76 COOL cells, 47 HPC cells, and 37 NS cells. The COOL cells showed a very linear output from 34°C down to ~15°C and a maintained plateau thereafter. The HPC cells showed a fairly linear but accelerating response to cold below a median threshold of ~24°C and down to 9°C (measured at the skin-thermode interface with a thermode temperature of 2°C). The HPC cells and the NS cells both showed rapidly increasing, sigmoidal response functions to noxious heat with a fairly linear response between 45 and 53°C, but they had significantly different thresholds; half of the HPC cells were activated at ~45.5°C and half of the NS cells at ~43°C. The 20 HPC lamina I STT cells and 10 NS cells tested with quantitative pinch stimuli showed fairly linear responses above a threshold of ~130 g/mm2 for HPC cells and a threshold of ~100 g/mm2 for NS cells. All of these response functions compare well (across species) with the available data on the characteristics of thermoreceptive and nociceptive primary afferent fibers and the appropriate psychophysics in humans. Together these results support the concept that these classes of lamina I STT cells provide discrete sensory channels for the sensations of temperature and pain.
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INTRODUCTION |
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Lamina I of the spinal dorsal
horn is an integral component of the central neural representation of
pain and temperature sensations (see reviews by Craig
2000a; Perl 1984
). Lamina I neurons receive monosynaptic, modality-selective input from A
and C primary afferent thermoreceptors and nociceptors. Accordingly, the original
physiological description of spinal lamina I neurons by
Christensen and Perl (1970)
reported cells responsive
selectively to noxious cutaneous stimulation (pinch and/or heat) as
well as cells responsive to innocuous thermal stimulation, some of
which were selectively sensitive to cooling and inhibited by warming
and some of which responded both to cold and to noxious stimuli (see
also Kumazawa et al. 1975
; Mosso and Kruger
1973
).
Lamina I is also the major source of ascending output from the
superficial dorsal horn. Lamina I axons in cats and primates ascend
contralaterally in the lateral spinothalamic tract (STT), which is the
critical pathway for the sensations of pain, temperature, itch, and
sensual touch in humans (Craig 2000b). Studies in the cat of antidromically identified lamina I trigeminothalamic cells by
Dostrovsky and Hellon (1978)
and lamina I spinothalamic
cells by Craig and Kniffki (1985)
described unit
responses that were consistent with the general pattern described by
Christensen and Perl (1970)
. We now recognize three main
categories of lamina I projection neurons: nociceptive-specific (NS)
cells, innocuous thermoreceptive-specific (COOL or WARM) cells, and
polymodal nociceptive (HPC) cells sensitive to noxious
heat, pinch, and noxious cold (Craig and Bushnell 1994
; Craig and Hunsley
1991
; Craig and Serrano 1994
). The available
evidence indicates that these three groups have different response
properties, different ascending conduction velocities, and different
susceptibility to descending or pharmacological modulation
(Craig and Serrano 1994
; Dostrovsky et al.
1983
). Recent findings, based on intracellular labeling,
indicate that these three categories are also distinct morphologically
and correspond to the three major anatomical types of lamina I neurons
that can be distinguished in horizontal sections (Han et al.
1998
). These three physiological and anatomical categories of
lamina I STT cells have also been recognized in the monkey
(Craig et al. 1999
; Dostrovsky and Craig
1996
; Kumazawa et al. 1975
; Zhang and
Craig 1997
).
Thus these categories of lamina I STT neurons appear to be robust and
biologically significant classes of neurons. Various aspects of the
physiological characteristics of these neurons have been examined in
prior studies (Craig and Bushnell 1994; Craig and
Hunsley 1991
; Craig and Kniffki 1985
;
Craig and Serrano 1994
). The present report provides a
quantitative description of the response properties of these three
classes of lamina I STT cells in the cat based on a large sample of
neurons examined in this laboratory with standardized stimuli across
several series of experiments. The present data quantitatively
differentiate these classes of neurons and enable a comparison of the
stimulus-response properties of these lamina I STT neurons with
available psychophysical results.
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METHODS |
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General procedures
All experiments were conducted in accordance with the guiding
principles for the care and use of animals of the American
Physiological Society, and the protocols were approved by the local
Institutional Animal Care and Use Committee. The data reported were
gathered in experiments performed on a total of 162 domestic cats
(2.0-6.5 kg) of either sex across several different series of
experiments (e.g., Andrew and Craig, 2001; Craig
and Andrew 1999
; Craig and Bushnell 1994
;
Craig and Hunsley 1991
; Craig and Serrano
1994
; Dostrovsky and Craig 1993
; Han and
Craig 1993
; M. Karpuchina and A. D. Craig, unpublished
results; B. Lumb and A. D. Craig, unpublished results). The
animals were anesthetized with pentobarbital sodium (40 mg/kg ip). A
catheter was placed in the right cephalic vein for administration of
intravenous anesthetic supplements to maintain areflexia. The head,
neck, back, and left hindlimb were shaved. Eye salve was applied, 0.5%
marcaine was injected subcutaneously at incision sites, and a local
anesthetic was sprayed in the ear canals. A single intravenous
injection of dexamethasone (10 mg) was administered to prevent edema. A
catheter placed in the left common carotid artery enabled measurement
of blood pressure with a transducer, and the trachea was cannulated.
Pancuronium bromide (~0.4 mg/h) was injected to induce paralysis, and
a positive pressure respirator was used. The animals were ventilated
with 75% air and 25% O2, and end-tidal
CO2 was maintained at 3.5-4.5%. Anesthetic depth (areflexia, stable blood pressure) was verified every hour before each injection of the paralytic agent. Core temperature was
maintained at 37.5°C with a heating pad and (if necessary) a
feedback-controlled infra-red heat lamp from a safe distance. The
ambient room temperature was maintained at 23-25°C. The animal's head was mounted in a stereotaxic frame, and a craniotomy was made to
allow access to the right thalamus. A laminectomy was performed to
expose the lumbosacral enlargement, and vertebral clamps were used to
suspend the animal. The spinal cord was covered at all times with
Tyrode's solution or artificial cerebrospinal fluid, which was
maintained at 38°C with a nichrome heating element. A pneumothorax
was performed to reduce cord pulsations as needed.
Recording sites were marked with lesions made by passing 20-30 µA (cathodal) for 30-40 s through the recording electrode. At the end of the experiments, the animals were perfused with buffered 10% formalin or the lumbosacral spinal cord was removed and immersed in fixative. Spinal and thalamic tissue blocks were 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.
Antidromic activation
To place the array of antidromic stimulating electrodes, single-
and multiunit recordings of somatosensory responses in the somatotopic
ventroposterior (VP) thalamus on the right side were made using large
tungsten-in-glass microelectrodes (~25- to 40-µm tip diam).
Microelectrode penetrations were initiated anteriorly and laterally
(AP9.5, ML6.5) and then more posteriorly for the purpose of locating
the small (approximate diam: ~0.25 mm) region at the dorsomedial
aspect of the trigeminal representation in which units responsive
selectively to cooling the ipsilateral tongue are found (Auen et
al. 1980; Landgren 1960
) and also to identify
the ventral border of the ventral posterior thalamus at the external
medullary lamina. The array consisted of eight concentric bipolar
electrodes (Rhodes NE-100 or NEX-100) evenly spaced at 1-mm
mediolateral intervals. The electrodes were staggered vertically so
that the medial two electrodes were placed in the vicinity of nucleus
submedius, the third electrode was near the ventral aspect of the basal
part of the ventral medial nucleus, the fourth electrode was near the
dorsomedial aspect of the ventral posterior medial nucleus, the fifth
electrode was near the ventral posterior inferior nucleus, the sixth
electrode was near the posterior nucleus dorsal to the ventral
posterior lateral nucleus, and the seventh and eighth electrodes were
at the ventral margin of the ventral posterior lateral nucleus. Bipolar
stimuli applied between the inner and outer conductors of single
electrodes were delivered to each of the electrodes in the thalamic
array with the following parameters: 500-1,000 µA, 2-ms duration,
three or four pulses at 200-333 Hz, inner (or outer) contact negative.
Bipolar stimulation afforded discrete activation from individual
electrodes, generally with thresholds of 30-150 µA, though in some
cases, the array was apparently not well placed and it was necessary to
use stronger stimuli or monopolar stimulation (indifferent attached to
incised cranial skin or the tongue). The array was cleaned following
each experiment with an ultrasonic denture cleaner, and electrodes were
replaced as needed.
Lamina I unit characterization
Recordings were obtained from cells in the superficial dorsal horn at a depth of 200-600 µm near the dorsal root entry zone of the left lumbosacral spinal cord (L6-S1 segments) with platinum-plated tungsten-in-glass microelectrodes having tip sizes of ~15 µm. In general, the electrode first encountered large-diameter muscle spindle primary afferent fibers having regular ongoing discharge. Lamina I was recognized just ventral to these fibers by the presence of units with irregular ongoing activity. Such ongoing activity could often be inhibited by radiant warmth and excited by innocuous cooling. Lamina I was also recognizable by the presence of units antidromically activated from the thalamic electrode array. Below this, we find a 200-µm zone in the outer substantia gelatinosa where our electrodes rarely pick up unitary discharges, and this is followed by field potentials and multi-unit responses to low-threshold mechanical stimulation in inner lamina II and laminae III-V.
The microelectrode was moved so that a single unit was isolated on the
basis of spike amplitude, and antidromic activation from the thalamus
was verified. The antidromic nature of the activation was determined on
the basis of a constant latency response, a discrete all-or-none
threshold to stimulation, and the ability to follow a train (at
200-333 Hz) of at least three stimuli with constant latencies. All
units that fulfilled these criteria and showed collision (extinction)
with ongoing or evoked orthodromic spikes also demonstrated a
definitive critical collision interval (Lipski 1981;
Ranck 1975
) when tested.
Antidromically activated single units were tested with natural
cutaneous stimulation, including limb movement, tap, innocuous and
noxious mechanical (brush, cotton wisp, flat forceps), innocuous cooling (beaker filled with wet ice), innocuous warming (radiant heat
lamp), noxious heat (heat lamp), and noxious cold (ice-cold beaker for
20-30 s) stimuli (Craig and Hunsley 1991; Craig
and Serrano 1994
). All identified cells were tested with all
stimulus modalities. The cells were classified as
thermoreceptive-specific cells if they were sensitive only to cooling
(COOL); NS cells if they were sensitive only to heat, pinch, or both;
and polymodal nociceptive (HPC) cells if they were sensitive to noxious
heat, pinch, and noxious cold. Wide dynamic range cells that respond to
both innocuous and noxious mechanical stimuli also occur in lamina I,
but these rarely project to thalamus in the cat (Craig and
Kniffki 1985
; Craig and Serrano 1994
;
Hylden et al. 1986
).
The COOL cells (formerly called COLD cells) were recognized by their
irregular spontaneous activity, which could be inhibited by innocuous
radiant warming of the skin, by their vigorous and immediate 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. Some COOL units occasionally respond with a brief
phasic discharge to pinch or noxious ("paradoxical") heat, as
described before (Craig and Hunsley 1991;
Dostrovsky and Craig 1996
; Dostrovsky and Hellon
1978
; Kumazawa et al. 1975
; or for cold-specific
primary afferents: Dodt and Zotterman 1952
). 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, and their responses to noxious cold stimuli are occasionally delayed by several seconds (see Craig and Kniffki 1985
;
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 or both pinch and heat and
their insensitivity to cooling and noxious cold. Nociceptive cells
recorded late in a particular experiment occasionally displayed weak
low-threshold mechanical sensitivity, which was ascribed to
sensitization by prior noxious stimulation.
The present analysis includes only lamina I STT cells of these three
main types; other, less frequent cells that respond selectively to
deep, chemical, warm or slow mechanical stimuli are described separately (Andrew and Craig 2001; Craig and
Kniffki 1985
; Andrew and Craig, unpublished data). Spontaneous
discharge was measured over a 2-min interval prior to stimulation or
characterization. Receptive fields were determined by manual mapping
with threshold stimuli and indicated on figurines. The receptive fields
of the identified units in each class were consistent with the
descriptions in prior articles (e.g., Craig and Hunsley
1991
; Craig and Kniffki 1985
; Craig and
Serrano 1994
) and are not addressed in this report.
Quantitative stimulation
Quantitatively controlled thermal stimuli were applied with a
custom-built thermoelectric (Peltier) stimulator (20 × 20 mm) placed over a unit's receptive field. A thermocouple fixed to the
surface of the Peltier and covered with thermally conductive epoxy
(Marlow Industries, Dallas, TX) was used for feedback control of the
temperature of the thermode. However, the temperature used for analysis
was the mean of repeated measurements at the interface between the
thermode and the skin with a separate, calibrated thermocouple
(0.005-in Type T, Omega CL23A, Stamford, CT), which differed from that
of the thermocouple embedded on the face of the thermode and from the
temperature of the glabrous dermis; this measured temperature, which
provides a reliable standard for comparisons between laboratories,
varied less than ±0.4°C between sessions. The thermoelectric
stimulator we used could not deliver subzero temperature commands. The
stimulus sequences were designed to measure stimulus-response functions
across a broad range of temperatures with minimal habituation or
sensitization (Craig and Hunsley 1991; Craig and
Serrano 1994
).
The cooling stimulus command sequence consisted of a descending staircase series of 4°C cooling steps of 20- or 30-s duration beginning from a baseline temperature of 34°C and proceeding to a final command temperature of 6 or 2°C. The command temperatures were 34, 30, 26, 22, 18, 14, 10, and 6°C and in some cases also 2°C, which produced thermode-skin interface temperatures of 34.5, 32.1, 28.7, 25.5, 22.1, 18.7, 15.7, 12.5, and 9°C, respectively. Warming steps (10 s) were appended at the end of this sequence to command temperatures of 44, 48, and 52°C to test for heat sensitivity, although these were of little use for quantitative analysis (see RESULTS) due to the adaptation properties of COOL cells.
The heat stimulus sequence consisted of a series of 20-s heating steps
(at 9 or 15°C/s) from a baseline of 34°C to command plateau
temperatures 58 or 62°C in 4°C increments with a 1-min hold at
34°C between steps. The heat sequence thus included command steps of
42, 46, 50, 54, 58, and 62°C, which corresponded to skin-thermode interface temperatures of 38.5, 42.7, 45.5, 48.5, 53.0, and 57.7°C, respectively. These thermal stimulus sequences produce stable and
reproducible stimulus-response functions (see RESULTS).
For quantitative mechanical stimulation, we used a custom-built,
hand-held, force-monitored (strain gauge) pincher with a 3-mm2 circular contact surface that enabled
graded pressure/pinch stimuli to be applied at a variety of skin
surfaces. This device was based on the design described by
Burgess and Perl (1967) as stimulator 2 in their Fig. 2. We first put the pincher in contact with the skin for several seconds
to accommodate the thermal sensitivity of HPC cells (see
RESULTS), and then we applied an ascending staircase series
of steps of increasing forces (0-1,000 g at ~100
g increments, ~5 s per step) by monitoring the applied
force visually on an oscilloscope. This device produces a graded
sensation of pain in humans when applied to the skin between digits
beginning at a force of ~300 g.
For most units, each stimulus sequence was repeated two or more times,
with a 3- to 5-min interval between trials. Unit responses to
successive thermal stimulus sequences applied at or near the same
stimulation site were averaged. 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. Analog microelectrode records, feedback control
signals, blood pressure, and voice records were stored on tape.
Voltage-discriminated unit discharges were used to generate
peri-stimulus time histograms using custom programs on a UNIX computer
(Masscomp 5400) or a Power1401 interface and the program Spike2
(Cambridge Electronic Design, Cambridge, UK) on a PC. Thresholds were
taken as the first temperature or pressure at which a response clearly
distinguishable from background activity was visibly apparent (i.e.,
where we judged an increment larger than the range of sampled ongoing
activity). Responses were measured from histograms (1-s bins) as
dynamic (first 5 s), static (remainder of stimulus period), and
total (entire stimulus period), beginning with the onset of each
stimulus step. The force applied during each manual pinch stimulus was
measured in the middle of each individual step; these records were not
averaged. Parametric (2, t-test,
ANOVA, General Linear Model, k-means cluster analysis) and
nonparametric statistics (Wilcoxon rank, Kruskal-Wallis, as required by
normality tests) were used with post hoc verification to compare the
responses of subclasses of cells, using the programs SigmaStat/SigmaPlot (SPSS, Chicago, IL) and CSS Statistica (Statsoft, Tulsa, OK). Stimulus-response curves were fitted with linear, power or
four-parameter sigmoidal functions. The linear model used was
y = ax + b, the power law model
used was y = y0 + axb, and the four-parameter sigmoidal
model used was y = y0 + a/(1 + e{exp[
(x
x0)/b]}).
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RESULTS |
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General characteristics of lamina I STT neurons
Lamina I STT neurons were recorded at depths of 200-600 µm
below the surface in segments
L6-S1. The identification
of antidromically activated STT projection neurons at these depths
provides assurance that the units were lamina I neurons because STT
neurons are only very rarely located in laminae II-III in the cat
(Carstens and Trevino 1978; Zhang et al.
1996
). In addition, lesions made at the sites of lamina I STT
units were identified in 37 cats, and these were all located
histologically in lamina I; they were concentrated in the dorsal cap at
the apex of the dorsal horn, such as that shown in Fig.
1, where retrograde labeling results
indicate that lamina I STT cells are concentrated, but recordings were
also made more medially.
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A total of 364 units were antidromically identified as lamina I STT neurons by the demonstration of a high-frequency, constant latency train response (Fig. 2A). This was accepted as a sufficient condition if collision (extinction) of the first antidromic action potential in the train response by a closely preceding (ongoing or evoked) action potential was observed. Definitive demonstration of a critical collision interval (2 × latency + ~1 ms for refractory period) was obtained for every cell tested (n = 65) that fulfilled these criteria (Fig. 2, B and C). The mean antidromic latency of this population was 83.2 ms, the mean conduction distance was 330 mm, and thus the overall mean conduction velocity was 5.0 m/s.
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Of the total sample, 136 cells were identified as COOL lamina I STT cells, 128 as HPC lamina I STT cells, and 100 as NS lamina I STT cells. (The infrequent lamina I STT cells of other types were not included in the present analysis.) The mean conduction latencies for these classes were COOL = 58.6 ± 25.3, HPC = 72.1 ± 28.0, and NS = 130.8 ± 55.5 (SD) ms, respectively, which correspond to mean conduction velocities of 5.6, 4.6, and 2.5 m/s. The box plots in Fig. 3A graphically compare the measured conduction latencies of these classes. The conduction latencies of these three classes were significantly different (ANOVA and post hoc t-tests, P < 0.001).
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Spontaneous (ongoing) discharge rates were measured for 107 COOL, 59 HPC, and 47 NS lamina I STT cells (total = 213) that were isolated
prior to any intentional noxious stimulation of the hindlimb. The mean
ongoing rates for these three cell types were COOL, 3.3 ± 2.6;
HPC, 0.9 ± 0.7; and NS, 0.5 ± 0.7 (SD) imp/s. As shown in
the box plots in Fig. 3B, the median rates were lower, being
2.7, 0.9, and 0.2 imp/s, respectively. The ongoing rate of the COOL
units was significantly different from that of the HPC and NS units
(P < 0.001). The difference between HPC and NS cells
did not achieve statistical significance, although in an independent
subset of these data (n = 97) (Andrew and Craig
2001), the difference between the ongoing rates of HPC and NS
cells approached significance (P < 0.08) in accordance
with our general impression from these recordings.
The thalamic electrodes from which the units were antidromically
activated were compared for these classes, but in contrast to a prior
comparison (Craig and Kniffki 1985), no significant correlation was observed. This must have resulted from the inherently variable placement of the fixed array in different experiments, and the
failure of particular electrodes in some experiments because detailed
antidromic mapping of single lamina I STT units using a mobile array
has shown clear distinctions between the projections of
thermoreceptive-specific and nociceptive lamina I STT cells (Craig and Dostrovsky 2001
; Dostrovsky and Craig
1993
).
The quantitative stimulus-response characteristics of samples of 76 COOL, 47 HPC, and 37 NS lamina I STT cells are described in the following text. The receptive field of nearly all of these units included a portion of or all of the glabrous ventral hindpaw, and the quantitative thermal and mechanical stimuli were applied to this region. Many units were tested with the standard stimulus sequences more than once: for example, 66% of the COOL cells were tested two or more times and 41% were tested three or more times. The responses to repeated standard test stimuli were in general quite reproducible. For example, Fig. 4 shows two original responses of a COOL unit to a standard cooling staircase stimulus along with the response curves for all four applications of the same stimulus, which are nearly identical. The responses of HPC cells to cold, heat and pinch stimuli and of NS cells to heat and pinch stimuli were similarly reproducible.
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Characteristics of COOL lamina I STT cells
The response characteristics of COOL lamina I STT units were examined with standard staircase stimuli that tested their sensitivity to successive 20- or 30-s temperature steps of ~4°C from a baseline of 34 or 38°C down to a thermode-skin interface temperature of 12.7° (n = 64; thermode command temperature of 6°C) or 9°C (n = 12; command temperature of 2°C). This sequence evoked reproducible cooling responses (Fig. 4) that provided a stimulus-response function for each unit based on the mean discharge rate over the entire response to each temperature step for all sequences applied.
Figure 5 shows the responses of eight
COOL lamina I STT units to the standard test stimulus that are
representative of the variety observed with respect to onset threshold,
sensitivity to decreasing temperature, and overall discharge rate.
These examples also show that the static responses of COOL cells showed
variable adaptation during the step stimuli, and further, they document the variable presence of a dynamic response component at the onset of
each temperature step. Dynamic responses in general began at warmer
temperatures than static responses and quickly saturated (and/or
decremented) at cooler temperature steps. However, there was otherwise
no correlation between the dynamic sensitivity and the overall response
patterns of these cells (cf. Hutchison et al. 1997).
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The warming steps that were included in the test sequence following the
cooling staircase (shown in Fig. 4) were much less effective at
demonstrating the warming inhibition that is uniquely characteristic of
COOL cells than radiant heat applied over a larger field. The warming
steps could not be used as a quantitative measure because the response
histograms to warming were also confounded by the increases in ongoing
discharge produced following each warming step (Fig. 4) that reflect
longer-term (minutes) adaptation processes that we did not study (cf.
Darian-Smith 1994; Dostrovsky and Hellon
1978
). The warming steps included noxious heat test stimuli
that produced interface temperatures of 45.5 and 48.5°C, and only 17 COOL units showed weak (paradoxical) responses to these stimuli; such
responses were not graded (confirmed by supplemental use of the
standard noxious heat sequence in 6 of these). One exceptional cell
that did show a graded heat response was nonetheless categorized as a
COOL cell (rather than an HPC cell) because it was clearly inhibited by
radiant warming.
The mean stimulus-response function of the entire population of 76 COOL
lamina I STT cells (Fig. 6A)
demonstrates the two most salient features of the COOL lamina I STT
population: the close relationship of the average discharge of the COOL
units with decreasing temperature and the saturation of this response at temperatures below ~15°C. The left-most data point on this graph
is based on the 12 units for which a thermode-skin interface temperature of 9°C was tested, and the standard error at this point
is accordingly larger. Over the range of 15-34°C, this correlation is remarkably linear (Fig. 6B); at every point, the mean and
standard error of the population response are within the 95%
confidence limits of the regression line. The regression line has a
correlation coefficient of r = 0.59 and a slope of
0.57 Hz/°C. The 95% confidence limits of the regression span a
width on the abscissa of 1.5°C, meaning that this is the temperature
difference at the skin-thermode interface that can be discriminated
reliably (P < 0.05) by this population of COOL lamina
I STT neurons. Plotting the mean population response on a normalized
scale (that is, with the maximal discharge of each cell set at 100%;
not shown), reduces the effect of the differences between cells in
absolute discharge rate and provides a higher correlation coefficient
(r = 0.80) with a slope of
3.91%/°C and a 95%
confidence limit width of 0.8°C.
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The individual stimulus-response functions of these 76 COOL lamina I
STT units are shown in Fig.
7, segregated
according to threshold for clarity. The majority (n = 52) responded to the initial cooling step (from 34 to 32.1°C), and
the mean response of this group clearly saturated at least by 15.7°C.
A few neurons had identified thresholds at the subsequent cooling steps
of 28.7 (n = 12) or 25.5°C (n = 10).
Two units with thresholds of 18.7°C were categorized as COOL cells
because they had saturating responses to deep cold, were inhibited by
warming, and had no sensitivity to heat or pinch. One exceptional COOL
unit with a unique, bell-shaped response curve was not included in the
present analyses (shown in Fig. 1 of Craig and Hunsley
1991).
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We compared the normalized response curves of the individual units
(Fig. 8), and it became visibly apparent
that two subpopulations could be distinguished on the basis of their
output at 28.7°C being greater or less than 50%. The mean of one
subpopulation showed a rapid rise in discharge with decreasing
temperature and then a decelerating response function
(n = 26) and the mean of the other subpopulation showed
a slower onset and then a steeper (sigmoidal) response function
(n = 51). The former subset achieved a mean of 68% of
maximal output at 28.5°C and the latter subset 26% of maximal
discharge at this temperature. These two subsets were significantly
different (ANOVA, P < 0.0001, with post hoc t-test verification). Independent verification of this
segregation was obtained with a k-means cluster analysis of
COOL cells that differentiated the same subsets on the basis of either
normalized response or absolute discharge frequency (P < 105; not shown).
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Characteristics of HPC lamina I STT cells
The responses of HPC lamina I STT units were examined to cold
(n = 47), heat (n = 45), and pinch
(n = 20). The responses of HPC cells were qualitatively
and quantitatively distinct from those of COOL cells and NS cells. The
responses of four HPC lamina I STT cells to both cold and heat stimuli
are shown in Fig. 9, and these are
representative of the most salient features of the responses of HPC
neurons to thermal stimulation. In general, HPC lamina I STT neurons
displayed graded static responses to both cold and heat. Their
responses to cold continued to increase at noxious temperatures. Their
responses to heat usually were greater in magnitude than their
responses to cold. None of the HPC cells were inhibited by radiant
warming. The responses shown in Fig. 9 are also representative of the
variety of individual characteristics observed. Individual HPC units
had differing thresholds to cold and heat. A few units responded with
greater discharges to cold than to heat. Most showed strongly graded
responses, whereas others had rather weakly graded responses, and some
showed dynamic as well as static responses. As shown in Fig. 9
(bottom 2 unit records), the earliest responses of HPC cells
to cooling generally consisted of a phasic discharge, whereas static
responses became evident at colder temperatures (18.7°C).
|
The same staircase cooling sequences used to characterize COOL cells
were used to examine HPC cells. The mean stimulus-response function of
the 47 HPC lamina I STT cells tested with cooling stimuli (Fig.
10A) shows moderate ongoing
discharge at neutral temperatures, a mean onset threshold of ~25°C,
and an accelerating response curve that continues to rise at the
coldest skin-thermode interface temperatures tested. The left-most data
point on this graph is based on the seven units for which a
thermode-skin interface temperature of 9°C was tested. Regression
analyses of the mean population response over its entire extent using
various quantitative models produced correlations with similar
coefficients: linear r = 0.64, power r = 0.62 (exponent of 1.4), and sigmoidal r = 0.66. The
linear regression against discharge rate (not shown) had a slope of
0.3 Hz/°C and a 95% confidence limit width of 1.7°C. A linear
regression on the normalized mean response function (maximal response
set at 100%; not shown) produced a higher correlation coefficient of
r = 0.82 with a slope of
3.6%/°C and a 95%
confidence limit width of 1.1°C.
|
The individual stimulus-response functions to cold of all 47 HPC units
are plotted together in Fig. 10B. This shows that the units
had a variety of different thresholds, different rates of increase with
decreasing temperature, and different absolute discharge levels. The
histogram in Fig. 10C shows that the distribution of the
cold thresholds of these units was unimodal but broad. Of this sample,
40% (n = 19) began to discharge to cooling steps of
28.7 or 25.5°C from the baseline of 34°C and 68%
(n = 32) of the population were activated by a 22.1°C
step; thus approximately half of the HPC cells were activated at
~24°C. As can be visually appreciated from the plot in Fig.
10B, two subpopulations could be distinguished on the basis
of their discharge rate at 18.7°C (P < 106, t-test). These subpopulations
had mean discharge rates of 8.1 (n = 24) and 2.1 (n = 23) imp/s at 18.7°C, and they essentially divided the HPC cells into high- and low-response categories having thresholds
25.5°C or thresholds
22.1°C. The mean response
functions of these two subpopulations were significantly different
(P < 0.0001, ANOVA), but nonetheless both had
ascending trajectories that paralleled the overall mean curve. Nearly
identical subsets were differentiated independently by
k-means cluster analysis of absolute discharge rate or
normalized responses (P < 10
5).
A standard heat stimulus sequence was used to test 45 HPC lamina I STT
cells. We tested 18 cells with heat pulses ranging to 53.0°C
(skin-thermode interface), and we tested 27 cells with heat pulses to
57.7°C because some units had thresholds of 53°C and because most
units tested showed response increments at this temperature. The
responses shown in Fig. 9 illustrate the variety observed in the
patterns of HPC heat responses. All HPC cells had responses that were
maintained throughout the 20-s heat stimuli, and many showed an
increasing discharge during the stimuli. Some cells evinced
afterdischarge subsequent to the most intense heat stimuli that lasted
several minutes. If the noxious heat stimuli were repeated several
times, the HPC units often became sensitized to low-threshold
stimulation (see Craig and Kniffki 1985), and accordingly, the heat test sequence was generally applied sparingly. The average number of heat trials per HPC cell was 1.5; 34% of the
cells were tested with the heat sequence two or three times.
The mean heat-evoked stimulus-response function of the 45 HPC lamina I STT cells (Fig. 11A) illustrates the direct relationship of average discharge rate to increasing temperature above threshold. The regression in Fig. 11B shows that the middle portion of this response curve can be modeled by a linear function (r = 0.64). The regression line against discharge rate has a slope of 1.55 Hz/°C, and the width of the 95% confidence limits at the middle of the curve (~48°C) is 1.3°C. Sigmoidal and power law models over this range had similar regression coefficients. The individual stimulus-response functions of all 45 cells are plotted in Fig. 11C; these curves were similar for most of the HPC cells in this sample, though thresholds varied. The unimodal distribution of thresholds is shown in Fig. 11D; 55% of the population was activated with the 45.5°C step, and 91% was activated with the 48.5°C step.
|
The uniformity in the rate of rise of the responses of HPC cells to heat is clearly evident in a plot (Fig. 12A) of the individual response functions on a scale normalized to the response of each unit at the highest common temperature used (53.0°C). Despite the differing thresholds, nearly all HPC units showed the same proportional increase between 45 and 50°C. The continued increment at higher stimulus temperatures is also clear in the tails of these curves at the top right of the graph. Figure 12B shows the mean population response function normalized to the relative maximum of each individual unit (i.e., including maxima at 57.7°C). A linear regression over the middle of this curve (not shown) has a high correlation coefficient (r = 0.82), equivalent to the linear correlations of the normalized response function of the COOL cell population and the normalized cold response of the HPC cells; it has a slope of 6.8%/°C and a 95% confidence limit width of 0.83°C. Including the points at the initial rise along with the middle of the curve, however, enables a power law or exponential model to yield a similarly high correlation (r = 0.84; power exponent = 1.5). Nevertheless, a sigmoidal function (4-parameter model, a = 91, b = 2.1, x0 = 49, y0 = 5.0) provides a superb fit (r = 0.91) for the entire curve, as shown in Fig. 12B.
|
Quantitative responses to pinch stimulation were obtained for 20 HPC
lamina I STT cells. The examples shown in Fig.
13 illustrate graded responses above
the indicated thresholds using the manually applied staircase sequence
of increasing forces. Thresholds varied across units and generally were
between 200 and 600 g (equivalent to pressures of 67-233
g/mm2). Pinches that produced visible skin damage
resulted in prolonged afterdischarge (Fig. 13C). The forceps
were normally placed in position on the skin for 5-10 s before
pressure was applied to accommodate the thermal sensitivity of the HPC
cells. Included in Fig. 13D are two examples where initial
responses were obtained on contact prior to pinching (right)
or during the initial stages of the pinch stimulus (left)
because of adequate stimulation by the cool (room temperature) forceps.
Such responses were eliminated by prewarming the forceps to neutral
skin temperature, in clear contrast to the graded low threshold
responses that were obtained in these experiments from non-STT wide
dynamic range lamina I cells (see Craig and Serrano
1994).
|
A plot of the individual forces applied across all tests on these 20 HPC lamina I STT cells (Fig. 14A) shows that the sampling of force was nearly continuous although the regularity of the applied force steps is clearly visible. The plot of the individual stimulus-response functions of all 20 cells (Fig. 14B) illustrates the variety of ongoing rates, thresholds, and discharge ranges and reveals that the measured response curves of some units were not smooth. The mean frequency response of this sample of HPC units, plotted with bidirectional error bars to indicate the range of measured forces at each point (Fig. 14C), shows clearly that the average threshold was almost 400 g and that the population response increased monotonically above threshold. The regression line on absolute discharge rates above threshold (not shown) has a correlation coefficient of r = 0.54, a slope of 1.7 Hz/100 g, and a 95% confidence limit width at the midpoint of 150 g. The linear regression of the normalized data above threshold (Fig. 14D), which accommodates the wide range of discharge rates in this limited sample, has a higher correlation coefficient (r = 0.79), equivalent to the correlations for the normalized responses of HPC cells to their other two stimulus modalities, cold and heat, and to that of COOL cells. The width of the 95% confidence limits at the center of the normalized regression is 58 g.
|
Characteristics of NS lamina I STT cells
Of the total of 100 NS cells recorded, 61 were sensitive to both
heat and pinch, 19 only to heat, and 17 only to pinch (and also
included 3 sensitive only to deep noxious mechanical stimulation). None
of the NS cells showed any sensitivity to cooling or to noxious cold.
The standard heat stimulus sequence was used to characterize 37 NS
lamina I STT cells. Of these, 25 were sensitive to both noxious heat
and pinch, and 12 were sensitive only to noxious heat. We tested all 37 cells with heat pulses 48.5°C (skin-thermode interface), 35 cells
with heat pulses
53.0°C, and 24 cells with heat pulses
57.7°C.
The individual responses shown in Fig.
15 are representative of the variety
observed among NS cells with respect to ongoing discharge, threshold,
evoked discharge rate, and the rate of response increment to graded
heat. All NS cells had responses that were maintained throughout the
20-s heat stimuli, and many showed an increasing discharge during the
stimuli. Some cells evinced afterdischarge lasting several minutes.
Repetition of the noxious heat stimuli could sensitize NS units to
low-threshold stimulation (see Craig and Kniffki 1985
),
and accordingly, the heat sequence was applied sparingly. The average
number of heat trials per NS cell was 1.8; 44% were tested two or
three times.
|
The mean heat stimulus-response function of these 37 NS lamina I STT cells (Fig. 16A) summarizes the direct relationship of average discharge rate to increasing temperature. The plot of the individual stimulus-response functions of all 37 cells (Fig. 16B) shows that these curves were similar across NS cells although their thresholds varied. Figure 16C shows that the middle portion of this response curve is linear (r = 0.60); the regression line has a slope of 1.0 Hz/°C, and the width of the 95% confidence limits at the center of the curve (~48°C) is 1.7°C. Sigmoidal and power law models had similar regression coefficients.
|
The distribution of heat thresholds for NS cells (Fig. 17) is lower and broader than that of HPC cells, and this difference is significant (P < 0.002, General Linear Model). For NS cells, 38% of the population was activated with at least the 42.7°C step, and 76% was activated with the 45.5°C step. The mean response functions of each separate threshold group nonetheless have a trajectory similar to the overall mean function, as shown in Fig. 17. The thresholds of NS cells sensitive to both pinch and heat did not differ from those sensitive only to heat.
|
The plot of the individual NS heat-response functions normalized to the response of each unit at 53.0°C is shown in Fig. 18A. Nearly all NS units showed a similar proportional increase between 45 and 50°C and continued to increase at higher stimulus temperatures. Figure 18B shows the mean population response function normalized to the relative maximum of each individual unit (i.e., including maxima at 57.7°C). This strongly resembles the corresponding curve for HPC cells (cf. Fig. 12B). The heat-evoked stimulus-response functions for NS and HPC neurons were not significantly different if compared across all temperatures or only above threshold (P > 0.7, 2-factor repeated-measures ANOVA). A linear regression at the middle of this normalized curve (not shown) has a high correlation coefficient (r = 0.75), equivalent to the that of the normalized heat-response function of HPC cells, and it has a similar slope of 6.8%/°C; however, the width of the 95% confidence limit (1.9°C) is double that of the HPC normalized regression. A sigmoidal function (a = 93, b = 2.7, x0 = 48, y0 = 4.2) again provides a superb fit (r = 0.88) for the entire curve, as shown in Fig. 18B. Nonetheless comparison of this graph with the corresponding graph for HPC cells (Fig. 12B) clearly reveals the significantly lower threshold of the mean NS heat-response function.
|
Quantitative responses to pinch stimuli were obtained for 10 NS lamina
I STT cells. All of these were responsive to both pinch and heat;
quantitative results were not obtained for any cells responsive only to
pinch in the present sample (see Craig and Kniffki
1985). The forces applied in studying these cells matched those
applied to HPC cells (cf. Fig. 14A). The plot of the
individual stimulus-response functions of all 10 NS cells (Fig.
19A) illustrates the variety
of discharge rates and thresholds observed (ranging from 200 to 600 g). Nearly all cells showed a direct, monotonic relationship
to force above threshold. The mean frequency response function, plotted
with bidirectional error bars to indicate the range of forces at each
point (Fig. 19B), reflects the large variability in absolute
discharge rates in this relatively limited sample. This mean curve is
not distinct from that of the HPC cells (cf. Fig. 14C;
P > 0.7, ANOVA), but it shows a threshold of ~300
g (i.e., ~100 g/mm2), which is
noticeably lower than that of HPC cells. The regression line of the
mean frequency response of these units over the entire range of forces
(not shown) has a correlation coefficient of r = 0.59, a slope of 1.4 Hz/100 g and a 95% confidence limit width at the
midpoint of 174 g. Linear regression of the normalized data above
threshold (Fig. 19C), which accommodates the variability in
discharge rates in this limited sample, has a high correlation coefficient (r = 0.74), equivalent to the other
normalized response functions described above, with a slope of
10.6%/100 g, and with a 95% confidence limit width of 74 g.
|
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DISCUSSION |
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Technical considerations
The present data were necessarily obtained in anesthetized cats.
The anesthetic used, intravenous pentobarbital sodium, interacts with
GABA receptors and naturally causes some depression of responsiveness (Belelli et al. 1999). However, like other observers
(e.g., Hori et al. 1984
), we find that second-order
spinal neurons are nonetheless quite responsive. When stable vital
signs were uniformly maintained, the lamina I STT neurons we recorded
showed similar responsiveness across different experiments, and the
responses of individual cells were reproducible, in some cases over
observation periods of up to several hours. We have obtained similar
recordings using several different anesthetic regimens in this
laboratory (e.g., chloralose, alphaxalone/alphadalone, or 1.2%
halothane), and similar responses were also reported by
Christensen and Perl (1970)
and Kumazawa et al.
(1975)
in decerebrate cats without anesthesia. Higher
response frequencies were reported in lamina I spino-parabrachial neurons recently by Bester et al. (2000)
in the rat
(under 0.5% halothane and 67% nitrous oxide) and lamina I
trigeminothalamic neurons may be even more sensitive in the awake,
behaving monkey (cf. Maixner et al. 1989
). Nonetheless,
quantitative characterization of a sample of antidromically activated
lamina I STT cells of this size could practicably be obtained only in
this preparation in the cat.
Relatively few standard stimuli were used for characterization. The
graded, quantitative stimulus sequences we used adequately covered the
response ranges of the units to the modalities tested with a discrete
number of test intervals, and they were designed to minimize the
possibilities of fatigue, habituation, or sensitization of responses.
Lamina I nociceptive neurons are readily sensitized by repeated noxious
stimuli (Christensen and Perl 1970; Craig and
Kniffki 1985
; Hylden et al. 1989
; Woolf
et al. 1994
), and nociceptive activity is reduced if short
interstimulus intervals are used (Christensen and Perl
1970
; LaMotte and Campbell 1978
). However, the
stimuli used certainly did not characterize all aspects of the
responses of these neurons. For example, our paradigm avoided the
long-term adaptation and contrast effects in COOL lamina I cells
associated with maintained temperatures, which produce significant hysteresis when descending/ascending sequences are used (e.g., Dostrovsky and Hellon 1978
) although this prevented
analysis of warm responses in COOL cells. Also we did not use brief
thermal stimulus pulses, which have enabled demonstrations that dynamic cooling or heating responses provide behaviorally significant information (Bushnell et al. 1983
; Darian-Smith
1984
; Robinson et al. 1983
), and we did
not use subzero, freezing temperatures (cf. Beise et al.
1998
; Simone and Kajander 1996
). As noted
further in the following text, the use of a broad range of differently sized probes and a force-controlled stimulator will enable a more thorough characterization of noxious mechanical sensitivity (e.g., Andrew and Craig 1999
; Garell et al.
1996
). Nonetheless the present data provide quantitative
descriptions that differentiate these three categories of lamina I STT
neurons and provide the basis for a comparison of their functional
properties with psychophysical findings.
Last, these data should represent a relatively unbiased sample of the
three main types of lamina I STT neurons. We recorded most units in the
dorsal cap at the apex of the dorsal horn (or more medially), where
lamina I STT neurons of all three morphological types are concentrated
(Zhang et al. 1996). The thalamic antidromic electrode array was intended to activate terminal axons at most of the
anatomically recognized lamina I STT projection sites in the thalamus
(Craig 1987
), and in many experiments, all of the electrodes at these sites were effective in activating lamina I neurons
antidromically. However, the relative proportions of COOL, HPC, and NS
cells sampled are not representative (cf. Zhang et al.
1994
) because we sought particular types of cells in particular experiments. In addition, it is much more difficult to maintain the
stable, long-term recordings necessary for quantitative
characterization from NS cells, especially those with very long
conduction latencies, than from COOL and HPC cells. Although
quantitative characterizations of NS cells selective for pinch are not
included in this sample, we have observed graded mechanical responses
both informally and systematically (Andrew and Craig
1999
) in such cells that were similar to those reported in the
present study.
Comparison of the distinct classes of lamina I STT cells
The three categories of cells we have characterized constitute the
vast majority of lumbosacral lamina I STT cells in the cat. These cell
classes are distinguished by a variety of characteristics. The
available evidence indicates that they differ physiologically in their
response characteristics, their afferent fiber inputs, and their
susceptibility to descending and pharmacological modulation (for
references, see Craig 2000a; Han et al.
1998
). Evidence has also been reported indicating that they
differ anatomically in the distribution of their terminations in the
thalamus, in their somato-dendritic morphology, and in their labeling
for the NK-1 receptor (Dostrovsky and Craig 1993
;
Han et al. 1998
; Yu et al. 1999
).
The COOL, HPC, and NS lamina I STT cells have distinctly 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. The present data
quantify these different response characteristics and clearly
distinguish the cold sensitivity of HPC lamina I STT cells from that of
COOL cells by demonstrating their significantly lower (colder)
thresholds and their accelerating activity to noxious cold, which
contrasts with the saturation of COOL cell activity below ~15°C
(Fig. 20). The present data also
quantitatively distinguish HPC cells from NS cells because the
population of HPC cells has significantly higher heat thresholds and a
steeper mean response function. Similarly, the mean response of HPC
cells to pinch seems to have a higher threshold than that of NS cells.
|
These selective response properties probably reflect afferent input
from selective categories of primary afferent fibers as discussed in
detail in the following text. As shown previously using electrical
stimulation of the skin and hindlimb peripheral nerves in the cat
(Craig and Kniffki 1985; and confirmed during the course
of the present experiments), NS cells receive predominantly A
-fiber
input, while HPC cells are dominated by monosynaptic C-fiber input, and
COOL cells respond to both A
- and C-fibers. [Cervero et al.
(1979)
similarly divided lamina I nociceptive cells into class
3a (with A
input) and class 3b (with A
and C input), but they
could not distinguish HPC cells because they did not utilize cold
stimuli.] Selective primary afferent convergence onto lamina I STT
cells is convincingly highlighted by our recent demonstration of
histamine-selective cells, which seem to receive monosynaptic input
exclusively from a distinct subset of C-fibers (Andrew and Craig
2001
). Such neurons are also distinguished by their unique
central conduction velocities, and as confirmed in the present
experiments, the central conduction velocities of COOL, HPC, and NS
cells are also significantly different (see Craig and Kniffki
1985
; Craig and Serrano 1994
; Dostrovsky
and Craig 1996
): 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. The
statistical significance of the pairwise differences in conduction latencies among COOL, HPC, and NS lumbosacral lamina I TSTT cells, even
within small subsets of these data (e.g., Andrew and Craig 2001
; Craig and Dostrovsky 2001
;
Craig and Serrano 1994
), indicates anatomical
differences between these cell classes.
Anatomical differences are directly revealed by the demonstration that
COOL cells have significantly different thalamic projections from
nociceptive (HPC and NS) cells in the cat (Craig and Dostrovsky 2001; Dostrovsky and Craig 1993
) and by recent
observations, based on intracellular labeling in the cat, indicating
that the somata of these physiological classes of lamina I cells are
morphologically distinct: 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
). This anatomical and
physiological correspondence is supported as well by evidence in the
primate (Craig et al. 1999
; Yu et al. 1999
). The present physiological observations on the conduction velocities of these cell classes directly support these anatomical correspondences because Golgi studies reported that fusiform lamina I
cells have unmyelinated axons, whereas pyramidal and multipolar cells
have myelinated axons (Gobel 1978
; Lima and
Coimbra 1986
).
Furthermore the present data confirm earlier reports that these cell
classes have different spontaneous discharge rates (see Andrew
and Craig 2001; Craig and Kniffki 1985
), which
probably also reflects their different afferent inputs. In addition,
these cell classes respond differently to descending and
pharmacological modulation: COOL cells are not inhibited by brain stem
stimulation, whereas NS cells are (Dawson et al.
1981
; Dostrovsky et al. 1983
); COOL
lamina I STT cells are not inhibited by morphine, whereas both NS and
HPC cells are (Craig and Hunsley 1991
; Craig and
Serrano 1994
); and, most fusiform and multipolar lamina I STT
cells bear NK-1 receptors, whereas few pyramidal cells do (Yu et
al. 1999
).
These three classes do not include all lamina I TSTT cells. The lamina
I STT projection seems to constitute an interoceptive (or homeostatic)
afferent pathway comprising several distinct modality-selective sensory
channels that carry activity representing different aspects of the
physiological condition of all tissues of the body (Craig
1996; Craig and Dostrovsky 1999
; Craig et
al. 2000
). Additional classes of lamina I STT cells that are
selectively chemo-sensitive (Andrew and Craig 2001
) or
responsive to warmth (Craig and Dostrovsky 2001
;
Dostrovsky and Hellon 1978
) or to deep (muscle, joint)
input (Craig and Kniffki 1985
) have been documented,
whereas others may be viscero-receptive, metabo-receptive or responsive
to C-fiber mechanoreceptors, although these have yet to be studied in
sufficient detail (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
).
Together all of these differences substantiate the conclusion that the
three categories of COOL, HPC, and NS lamina I STT cells form robust
and biologically distinct classes of ascending sensory neurons that
receive input from specific classes of modality-selective afferent
fibers. This conclusion is consistent with the concept that these
classes constitute "labeled lines" whose activity may correspond
directly to psychophysically distinct sensations. As discussed in
the following text, the COOL cells are clearly a unique component of
the STT that provide a substrate for discriminative thermal sensibility
(Craig et al. 2000); the HPC cells provide a reasonable
and quantitative explanation for the thermal grill illusion of pain,
which produces a sensation that mimics the burning sensation of noxious
cold (Craig and Bushnell 1994
; Craig et al. 1996
); and the NS cells are uniquely selective for noxious
mechanical stimuli and/or cutaneous heat. The specificity inherent in
the lamina I STT projection is underscored by the histamine-selective lamina I STT cells we have recently documented, which clearly have the
distinguishing characteristics of a labeled line for itch
(Andrew and Craig 2001
). Furthermore preliminary
evidence with novel, sophisticated paradigms suggests that NS cells can be associated with the psychophysically distinguishable sensation of
"first" (sharp) pain and HPC cells with "second" (burning) pain
(see following text) (Andrew and Craig 1999
;
Craig and Andrew 1999
).
However, the association of these cell classes with distinct sensations
cannot be made without first comparing the quantitative response
characteristics of these cells with the physiological characteristics
of primary afferents and with the psychophysical characteristics of
pain and temperature sensations. Furthermore these putative
associations are necessarily qualified by the recognition that the
activity of these cells must be integrated in the forebrain with the
concomitant activity of other types of cells that project rostrally in
the STT and in other pathways (Craig and Dostrovsky 1999; Price 1988
; Willis
1985
).
Comparison with prior lamina I cell and primary afferent recordings
COOL CELLS.
Quantitative data on trigeminal thermoreceptive-specific COOL lamina I
neurons were obtained in several prior studies (Davies et al.
1985; Dickenson et al. 1979
;
Hutchison et al. 1997
; Poulos 1981
),
including lamina I trigemino-thalamic cells (Dostrovsky and
Hellon 1978
). Although many authors have taught that peripheral cold-specific afferent fibers have bell-shaped response functions with
reduced activity at low temperatures (e.g., Darian-Smith 1984
; Iggo 1969
), work in the laboratories of
both Hellon and Poulos first revealed that many trigeminal lamina I
COOL cells have maintained responses at cold temperatures. As these
investigators suggested, this probably reflects convergent input from
cold-specific primary afferent fibers having a continuous range of
thresholds and response maxima, that is, ranging from the most
sensitive fibers that have high response maxima and reduced responses
at lower temperatures (Iggo 1969
) to the least sensitive
fibers that only respond at low (cold) temperatures (Duclaux et
al. 1980
; LaMotte and Thalhammer 1982
). The
convergent input to COOL cells also produces receptive fields that are
considerably larger than those of cold-specific primary afferent
fibers, and it obscures the pronounced bursting activity often
described in cold-specific afferents. On the other hand, lamina I COOL
cells (with unidentified projections) having bell-shaped response
curves have been repeatedly observed in the trigeminal nucleus caudalis
(e.g., Davies et al. 1985
; Hutchison et al.
1997
; Poulos 1981
); although our paradigm did
not incorporate the long adaptation times (>3 min) used in many prior
studies, we encountered only one such lamina I STT cell in the cat's
lumbosacral cord. Hutchison et al. (1997)
separated trigeminal COOL cells in the rat into three types, distinguishing those
with bell-shaped response curves from others having weaker or stronger
dynamic responses; however, neither of these characteristics distinguished subsets of COOL lamina I STT cells in the present study.
An unexpected statistical distinction was found between cells that were
sensitive to the first cooling step but had a decelerating response
curve and cells that had colder thresholds and a steeper response
function; these differences could be explained by the selective
convergence of cold-specific afferents that innervate the epidermis or
the dermis, respectively (Ivanov et al. 1982
; see also
Hensel 1981
), but the biological significance of this distinction depends on its thalamo-cortical processing. Nonetheless the
present data demonstrate clearly for the first time that the mean
population stimulus-response function of COOL lamina I STT cells shows
a very linear output over the entire range of cooling sensation and
that the output is maintained at a near constant level below this
range. These characteristics strongly parallel the available
psychophysical evidence, as described in the following text.
HPC CELLS.
Although the original reports of lamina I units described cells
responsive both to cooling and to noxious stimuli, most intervening studies did not utilize cold stimuli. As a result, polymodal
nociceptive lamina I STT HPC cells were not distinguished in nearly all
prior studies, and there is almost no prior quantitative data
(Craig and Bushnell 1994; Craig and Serrano
1994
). We have identified HPC lamina I STT units also in the
monkey (Dostrovsky and Craig 1996
), whereas cells having
similar polymodal sensitivity were classified in prior studies as
"high-threshold" (Willis et al. 1974
; however, see
Discussion in Kumazawa et al. 1975
) or as "wide dynamic range" (Ferrington et al. 1987
; Price
et al. 1976
). Lamina I spino-parabrachial neurons with similar
polymodal responses were also described in the rat by Bester et
al. (2000)
, though they were classified as nociceptive-specific cells.
NS CELLS.
Graded responses to noxious heat have been described for NS lamina I
projection cells in several prior studies in the monkey. Price
et al. (1976) described NS lamina I trigeminothalamic cells that encoded graded noxious heat, albeit less well than wide dynamic range cells in the deep dorsal horn. Bushnell et al.
(1984)
, Ferrington et al. (1987)
, and
Maixner et al. (1989)
reported similar findings for
samples based primarily on trigeminothalamic or STT neurons in lamina
I, and they also concluded that NS cells encode noxious heat intensity,
albeit less well than wide dynamic range cells. On the other hand,
Price et al. (1978)
and Kenshalo et al.
(1979)
found similar responses to heat across categories and
across laminae (see also McHaffie et al. 1994
). In
contrast, Coghill et al. (1993)
concluded that
unidentified NS superficial dorsal horn cells in the rat do not encode
long-term, repeated heat stimulation. Nevertheless, all of these
studies reported NS thresholds of ~43-47°C and stimulus-response curves for identified NS lamina I projection neurons using brief stimuli (5-30 s) that were positively accelerating near threshold and
more or less linear thereafter. However, cold stimuli were not used to
categorize lamina I cells in these prior studies, so NS, HPC, and wide
dynamic range cells may have been intermixed (see Han et al.
1998
). The present data show clearly that NS lamina I STT cells
in the cat have a sigmoid response function to noxious heat, with a
range of thresholds significantly lower than those of HPC cells, a
median threshold of ~43°C, and a fairly linear output between 43 and 53°C. The recent quantitative data of Bester et al.
(2000)
on NS lamina I spino-parabrachial neurons in the rat are
nearly identical (reported mean threshold, 43.6°C).
Comparison with human psychophysical characteristics
The stimulus-response functions of each of these classes of lamina
I STT cells for each modality showed a remarkably linear response
region. All of the linear regressions had a correlation coefficient
near r 0.6 based on absolute discharge frequency, which improved to r
0.8 if based on a normalized
response function. Although we prefer the absolute rate measure a
priori, a choice between these two response functions can only be based
on the properties of the thalamic and cortical cells that are activated by these STT cells, along with appropriate behavioral comparisons. We
briefly compare these data with human psychophysical results in the
absence of comparable physiological data on lamina I STT cells in the primate.
COOL CELLS AND THE SENSE OF TEMPERATURE.
The COOL cell response function was linear between 34 and 15°C and
nearly flat below that. This is directly comparable to the linear
magnitude estimation function of humans over this range (see Fig. 5 in
Darian-Smith 1984) and also to the relatively flat, maintained sensation of cold in the noxious cold range below ~15°C (Chen et al. 1996
; Chery-Croze 1983
).
Similarly, a linear increase in regional cerebral blood flow between 34 and 20°C has been measured with positron emission tomography only in
the cortical terminus of the lamina I STT pathway in humans
(Craig et al. 2000
). Although different cooling
thresholds are obtained using different methods (e.g., Yarnitsky
and Sprecher 1994
), psychophysical data obtained with stimulus
parameters similar to those we used indicate coolness thresholds of
~1.2-2.0°C on the human hand (Greenspan et al.
1993
; Hilz et al. 1995
), which is equivalent to
the ~1.5°C temperature difference that could be differentiated
reliably by the mean response function of COOL cells (~0.8°C for
the normalized mean). Paired, forced-choice tests with brief stimulus
pulses produce detection thresholds ~0.1°C (Chen et al.
1996
; Darian-Smith 1984
), and comparable sensitivity has been noted for trigeminal COOL cells (Craig et al. 1999
; Dostrovsky and Hellon 1978
), although
a direct quantitative comparison would require equivalent tests on COOL
lamina I STT cells. The sensitivity to the rate of cooling changes in
lamina I COOL cells was also found to parallel human psychophysical
reports (Davies et al. 1983
). Thus the response
characteristics of COOL lamina I STT cells support the conclusion that
they constitute a discrete sensory channel for innocuous thermal
(cooling) sensation. There seem to be no other ascending neurons that
show comparable sensitivity to innocuous temperatures, with the
exception of WARM lamina I STT cells, characterization of which is
needed yet to complement this analysis (Andrew and Craig, unpublished results).
HPC CELLS AND THE SENSATION OF COLD PAIN.
The stimulus-response function of HPC cells to cold had a median
threshold of ~24°C and showed a fairly linear increase at colder
temperatures. Prior analyses using the thermal grill illusion of pain
provided evidence indicating that the burning sensation of noxious cold
results from HPC lamina I STT activity that is normally inhibited in
the forebrain by COOL lamina I STT activity (Craig and Bushnell
1994; Craig et al. 1996
). Human psychophysical studies (Chen et al. 1996
; Chery-Croze
1983
; Wolf and Hardy 1941
) clearly document that
the perceptual attributes of cold pain are distinct from those of
innocuous cooling and heat pain, and that, while thresholds for cold
pain vary over a wide range, the median threshold appears to be
~15°C, at which temperature HPC cell activity is accelerating and
COOL cell activity becomes saturated. At temperatures below (colder
than) the threshold for cold pain, human perception of cold shows
little change, but the sensation of pain increases linearly. In
addition, studies in humans and monkeys have recently shown that the
slope of the psychophysical response function for cold pain is
significantly lower (i.e., has a larger detection limen) than that of
the sensations of innocuous cooling or heat pain, and also that the
pain intensity or unpleasantness of a noxious cold stimulus of
~10°C is similar to that of a noxious heat stimulus of ~47°C
(Morin and Bushnell 1998
; Rainville et al.
1999
). The present quantitative data on the thresholds, slopes, and intensities of the responses of HPC cells parallel these findings in all respects. Thus our findings support the concept that the burning
sensation of cold pain is engendered by HPC lamina I STT activity,
integrated at thalamo-cortical levels with COOL lamina I STT activity.
This is also consistent with the parallel inhibition of cold pain and
of HPC lamina I STT cells by systemic and topical morphine
(Craig and Serrano 1994
; Han and Craig
1993
). Note that this concept can also explain why ambient
temperatures below ~24°C generate an increasing sense of thermal
(or homeostatic) discomfort (see Craig 1996
).
Nevertheless, the unexplored forebrain integration of HPC activity with
the activity of lamina V STT cells that can also respond to noxious
cold (below ~15°C), noxious heat and pinch in the monkey
(Kenshalo et al. 1982
) at present qualifies this conclusion.
HPC AND NS CELLS AND THE SENSATION OF HEAT PAIN.
The stimulus-response functions to heat of both HPC and NS lamina
I STT cells were sigmoidal curves with steep, linear increases between
45 and 53°C. Based on the measured 95% confidence limits of the
linear regressions over the working range of these stimulus-response functions, the HPC population could reliably (P < 0.05) distinguish steps of ~1.3°C (~0.8°C for the normalized
mean), whereas the confidence limits for the NS population were larger
at ~1.7°C (and ~1.9°C for the normalized mean). The human pain
report is described as linear above threshold by many investigators
(Handwerker and Kobal 1993; Hardy et al.
1952
; LaMotte and Campbell 1978
; Robinson
et al. 1983
), although others who include the threshold region
describe heat pain sensation as an accelerating power function (see
Price 1988
). With regard to heat pain discrimination,
Hardy et al. (1952)
described a "dol" scale for
measuring heat pain with steps of ~0.8°C, and Taylor et al.
(1993)
found that the difference between "slight pain" and
"moderate pain" was reproducibly reported at steps of ~1.1°C.
[In addition, paired forced-choice tests in humans and monkeys
indicate detection thresholds for heat pain of ~0.1-0.4°C
(Bushnell et al. 1983
; Robinson et al. 1983
), but further experiments are needed to compare the
activity of HPC and NS cells with such results.] Although the NS and
HPC heat-response curves could not be statistically distinguished, the
median threshold of HPC cells was ~45.5°C, whereas that of NS cells
was significantly lower at ~43°C, and the inflection points in the
respective sigmoidal models (Figs. 12 and 18) are also offset.
Similarly, humans describe heat at temperatures slightly below pain
threshold as "pricking," as "heat" or as "prepain" but
describe temperatures above a threshold of ~45.5°C as "burning" pain (Boring 1942
; Handwerker and Kobal
1993
; Hardy et al. 1952
; LaMotte and
Campbell 1978
). These parallel characteristics suggest that the
C-fiber-dominated HPC lamina I STT cells may be associated with
"second" pain, that is, the burning sensation above heat pain
threshold, and that the A
-dominated NS lamina I STT cells may be
associated with "first" pain, that is, the pricking heat sensation
at temperatures below and near pain threshold. To address this
question, we suggest that these associations be tested with the
repeated brief contact heat paradigm recently refined by Vierck et al. (1997)
, who showed that, at sufficiently short
interstimulus intervals and sufficiently high temperatures, repeated
heat stimuli cause a selective increase in second pain reports
accompanied by a reduction in first pain sensation (see also
Price et al. 1978
), and that this summation can be
"re-set" at short intertrial intervals. Indeed, preliminary
evidence using this paradigm suggests that HPC, but not NS, lamina I
STT cells show responses that parallel this phenomenon (Craig
and Andrew 1999
).
HPC AND NS CELLS AND THE SENSATION OF MECHANICAL PAIN.
The sensation of mechanical pain (pinch) is certainly distinguishable
from thermal pain. However, as noted in the preceding text, there is
little evidence available on the stimulus-response functions of primary
nociceptors, central neurons or human perception to graded mechanical
stimulation, and the present limited data set is insufficient to
distinguish the role of NS neurons from that of HPC neurons in
mechanical pain sensation. Recently, Greenspan and colleagues initiated
use of a refined set of stimuli (Andrew and Greenspan
1999; Garell et al. 1996
; Greenspan and
McGillis 1991
, 1994
) and reported that mean pain
threshold in humans is ~100 g/mm2 with
probes tips having area on the order of 1 mm2 and
that pain sensation increases fairly linearly with increasing stimulus
intensity. Notably, a differentiable sensation of "sharpness" was
reported with thin probes below pain threshold. With the 3 mm2 pincher used in the present study on glabrous
skin, the stimulus-response functions for mechanical stimulation of NS
and HPC units increased linearly above thresholds of ~300 g (NS) or
~400 g (HPC) or ~100-125 g/mm2. Thus the
present data are consistent with the view that the response properties
of NS and HPC lamina I STT cells parallel human mechanical pain
perception. This comparison again recommends use of the more refined
stimulus protocol of Greenspan and colleagues to extend the present
characterization of lamina I STT neurons, which should enable
differentiation of the functional roles of different classes of
nociceptive spinal neurons in mechanical nociception. Preliminary
evidence using their protocols suggests that NS, but not HPC, lamina I
STT cells show responses that parallel A
nociceptor activity and the
sensation of first pain (Andrew and Craig 1999
). In
addition, such experiments could elucidate prior evidence
(Cervero et al. 1988
) that implicated NS lamina I cells
in the temporal augmentation of maintained noxious mechanical stimuli,
an as yet unexplained characteristic of human pain sensation (Adriaensen et al. 1984
; Andrew and Greenspan
1999
; see also Schmidt et al. 2000
).
Conclusions
The present results quantitatively differentiate the characteristics of the three main classes of lamina I STT cells in the cat (COOL, HPC, and NS), thereby validating the qualitative distinctions in their modality selectivity and corroborating the many other features that distinguish these cell classes. The response characteristics of these neurons parallel available psychophysical data on temperature and pain sensations, and thus our findings provide strong evidence consistent with the conclusion that these neurons constitute biologically distinct classes that receive input specifically from subsets of modality-selective afferent fibers and whose activity corresponds directly with psychophysically distinct sensations. These results support the view that lamina I STT neurons provide discrete ascending sensory channels, or "labeled lines," for pain, temperature and itch sensations. Additional experiments are in progress, using more refined stimulus paradigms, that can significantly extend these results, further differentiate these neurons from other types of ascending cells, and directly test this hypothesis in cats and monkeys.
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ACKNOWLEDGMENTS |
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We thank colleagues who participated in these experiments over the past several years: M. C. Bushnell, J. O. Dostrovsky, Z.-S. Han, S. Hunsley, M. Karpuchina, B. Lumb, and L. Serrano.
Support for this laboratory was provided by National Institutes of Health Grants NS-25616 and DA-07402 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, Atkinson Pain Research Laboratory, Div. of Neurosurgery, Barrow Neurological Institute, 350 W. Thomas Rd., Phoenix, AZ 85013 (E-mail: bcraig{at}chw.edu).
Received 26 December 2000; accepted in final form 20 April 2001.
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REFERENCES |
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