Departments of 1Neuroscience and 2Surgery, Brown University/Rhode Island Hospital, Providence, Rhode Island 02903; and 3Faculty of Dentistry, University of Toronto, Toronto, Ontario M5G 1G6, Canada
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ABSTRACT |
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Hirata, Harumitsu, James W. Hu, and David A. Bereiter. Responses of Medullary Dorsal Horn Neurons to Corneal Stimulation by CO2 Pulses in the Rat. J. Neurophysiol. 82: 2092-2107, 1999. Corneal-responsive neurons were recorded extracellularly in two regions of the spinal trigeminal nucleus, subnucleus interpolaris/caudalis (Vi/Vc) and subnucleus caudalis/upper cervical cord (Vc/C1) transition regions, from methohexital-anesthetized male rats. Thirty-nine Vi/Vc and 26 Vc/C1 neurons that responded to mechanical and electrical stimulation of the cornea were examined for convergent cutaneous receptive fields, responses to natural stimulation of the corneal surface by CO2 pulses (0, 30, 60, 80, and 95%), effects of morphine, and projections to the contralateral thalamus. Forty-six percent of mechanically sensitive Vi/Vc neurons and 58% of Vc/C1 neurons were excited by CO2 stimulation. The evoked activity of most cells occurred at 60% CO2 after a delay of 7-22 s. At the Vi/Vc transition three response patterns were seen. Type I cells (n = 11) displayed an increase in activity with increasing CO2 concentration. Type II cells (n = 7) displayed a biphasic response, an initial inhibition followed by excitation in which the magnitude of the excitatory phase was dependent on CO2 concentration. A third category of Vi/Vc cells (type III, n = 3) responded to CO2 pulses only after morphine administration (>1.0 mg/kg). At the Vc/C1 transition, all CO2-responsive cells (n = 15) displayed an increase in firing rates with greater CO2 concentration, similar to the pattern of type I Vi/Vc cells. Comparisons of the effects of CO2 pulses on Vi/Vc type I units, Vi/Vc type II units, and Vc/C1 corneal units revealed no significant differences in threshold intensity, stimulus encoding, or latency to sustained firing. Morphine (0.5-3.5 mg/kg iv) enhanced the CO2-evoked activity of 50% of Vi/Vc neurons tested, whereas all Vc/C1 cells were inhibited in a dose-dependent, naloxone-reversible manner. Stimulation of the contralateral posterior thalamic nucleus antidromically activated 37% of Vc/C1 corneal units; however, no effective sites were found within the ventral posteromedial thalamic nucleus or nucleus submedius. None of the Vi/Vc corneal units tested were antidromically activated from sites within these thalamic regions. Corneal-responsive neurons in the Vi/Vc and Vc/C1 regions likely serve different functions in ocular nociception, a conclusion reflected more by the difference in sensitivity to analgesic drugs and efferent projection targets than by the CO2 stimulus intensity encoding functions. Collectively, the properties of Vc/C1 corneal neurons were consistent with a role in the sensory-discriminative aspects of ocular pain due to chemical irritation. The unique and heterogeneous properties of Vi/Vc corneal neurons suggested involvement in more specialized ocular functions such as reflex control of tear formation or eye blinks or recruitment of antinociceptive control pathways.
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INTRODUCTION |
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The cornea is a densely innervated organ (Rozsa and
Beuerman 1982), supplied exclusively by small myelinated and
unmyelinated sensory fibers that lack specialized endings
(Beuerman et al. 1983
; MacIver and Tanelian
1993
; Rozsa and Beuerman 1982
; Zander and
Weddell 1951
), that has long been associated with pain
sensation. In addition, direct activation of corneal nerve terminals
evokes protective reflexes such as eye blinks (Evinger et al.
1993
), tear formation (Stern et al. 1998
),
endocrine and cardiovascular responses (Bereiter et al.
1996
), as well as causing pain (Beuerman and Tanelian
1979
; Kenshalo 1960
; Lele and Weddell
1959
). Although the general classes of primary afferent fibers
that supply the cornea are well-established (see Belmonte and
Gallar 1996
), far less is known regarding the properties of the
second-order brain stem neurons that receive input from corneal
nociceptors. If the pathways for autonomic, motor and sensory responses
to corneal stimuli are activated from a common population of
nociceptors, then the initial relay in the trigeminal spinal nucleus
(Vsp) onto second-order trigeminal neurons must play a critical role in
mediating the various aspects of corneal nociception. As proposed for
the spinal dorsal horn (Laird and Cervero 1991
;
Price and Dubner 1977
), the contribution of second-order
neurons to the various aspects of nociception can be inferred on the
basis of adequate knowledge of critical features such as neuron
location, stimulus encoding properties, response to analgesics, and
efferent projections to higher brain centers associated with
established functions. The cornea afferent system provides a useful
model to test this hypothesis for trigeminal nociception.
The sensory innervation of the corneal epithelium is supplied by the
ophthalmic division of the trigeminal nerve (Marfurt et al.
1989; Zander and Weddell 1951
). Axonal
tract-tracing studies indicate that the central branches of corneal
nerves terminate in two regions of the Vsp: at the subnucleus
interpolaris/subnucleus caudalis (Vi/Vc) transition and in laminae
I-II at the subnucleus caudalis/upper cervical cord (Vc/C1)
(Marfurt 1981
; Marfurt and Del Toro 1987
;
Panneton and Burton 1981
). Also, c-fos gene expression, a marker for intense neural activation, is produced mainly at the Vi/Vc
and Vc/C1 transition regions after corneal stimulation, a finding
consistent with the conclusion that neurons in these regions receive
the majority of direct input from cornea nociceptors (Bereiter
1997
; Bereiter et al. 1996
; Lu et al.
1993
; Meng and Bereiter 1996
; Strassman
and Vos 1993
). However, the basis for a dual representation of
the cornea at the Vi/Vc and Vc/C1 transition regions and its importance
in mediating the various aspects of corneal nociception remains
uncertain. Recently, we determined that the general properties of
corneal-responsive neurons at the Vi/Vc and Vc/C1 transition regions
displayed substantial differences (Meng et al. 1997
,
1998
). For example, all Vc/C1 corneal units received
convergent cutaneous input that could be classified as nociceptive
(i.e., wide dynamic range or nociceptive specific) and the responses to
electrical test stimulation of the cornea were inhibited by morphine.
By contrast, many Vi/Vc units had receptive fields (RFs) restricted to
the corneal surface, and morphine enhanced the responses to corneal
electrical test stimuli. Although these results suggested that Vi/Vc
and Vc/C1 units process corneal input differently, it was important to
determine the encoding properties across a range of stimulus
intensities using a well-defined, natural-occurring test stimulus. In
the present study, pulses of CO2 gas of varying
concentrations were applied to the cornea, and the responses of Vi/Vc
and Vc/C1 units were recorded before and after morphine. Corneal
nociceptors respond reliably to CO2 pulses at
concentrations similar to that which evoke pain sensation in humans
(Chen et al. 1995
). Because the stimulus-response
function of corneal nociceptors to graded concentrations of
CO2 pulses has been described, comparison to the
pattern for second-order neurons would address the issue of corneal
sensory information transfer at multiple levels of the trigeminal
neural axis. Also, corneal-responsive neurons were tested for
projections to the contralateral thalamus, because it was predicted
that Vi/Vc and Vc/C1 corneal units that contribute to the
sensory-discriminative aspects of corneal nociception should encode
CO2 stimulus intensity and project to sensory
regions of the thalamus.
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METHODS |
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Animals and surgery
Male rats (270-446 g, Sprague-Dawley, Harlan) were anesthetized
initially with pentobarbital sodium (70 mg/kg ip) before surgery. The
left femoral artery (blood pressure monitor) and jugular vein (anesthesia and drug infusions) were catheterized, and after
tracheostomy, animals were artificially respired with oxygen-enriched
room air. Anesthesia was maintained by a continuous infusion of
methohexital sodium (~35 mg · kg1 · h
1) and later switched to a mixture of
methohexital sodium (26-40 mg · kg
1 · h
1) and the paralytic agent, gallamine
triethiodide (14-32 mg · kg
1 · h
1), after completion of all surgical
procedures and just before the electrophysiological recording session.
The animal was placed in a stereotaxic frame, and a portion of the
occipital bone and C1 vertebra were removed to expose the dorsal
surface of the medulla. The brain stem surface was bathed in warm
mineral oil. A dental drill was used to remove a small portion of bone
on the right side of the skull for placement of the array of antidromic
stimulating electrodes. Expired end-tidal CO2 was
monitored continuously and kept at 3.5-4.5% by adjusting volume or
rate of the respirator. Mean arterial pressure (MAP) remained above 100 mmHg throughout all experiments. Body temperature was maintained at
38°C with a heating blanket and thermal probe.
Electrophysiology recording techniques
Extracellular unit recordings were made using tungsten
electrodes (9 M, FHC, Brunswick, ME) as described previously
(Meng et al. 1997
). Neurons recorded at the Vi/Vc
transition were approached at an angle of 28° off vertical and 45°
off midline. Neurons recorded in laminae I-II at the Vc/C1 transition
were approached at an angle of 43° off vertical, 60° off midline
and were found just before exiting the lateral dorsal horn, 300-500
µm after surface penetration. Mechanical (von Frey filaments) and
electrical (0.1-1 ms duration, maximum of 1.0 mA, 0.2 Hz) stimulation
of the cornea was used to search for responsive neurons. Electrical
stimuli were applied from a bipolar electrode (2 mm separation, FHC)
mounted on the ear bar and placed lightly on the cornea.
Well-discriminated unit activity was amplified, displayed on a digital
oscilloscope to monitor spike shape and amplitude, and passed through a
window discriminator. Discriminated neural spikes, MAP, and a marker for CO2 stimulus pulses were acquired and
displayed on-line with an Apple computer (PowerMac 7100) through a
Lab-NB interface board using LabVIEW software (National Instruments).
Also, these data were digitized (NeuroData) and stored on VCR tape as a
backup and for further off-line analyses.
In each animal preparation a single neuron was isolated, and several
general response properties were determined initially: A- or C-fiber
type corneal input (electrical stimuli), mechanical thresholds for
corneal input (von Frey filaments), presence of a convergent cutaneous
RF, and tests for antidromic activation from the contralateral
thalamus. Responses to electrical stimulation of the cornea occurring
at latencies of >30 ms were assumed to indicate C-fiber input
(Hu 1990; Meng et al. 1997
). The
ipsilateral face was explored for possible cutaneous input to
corneal-responsive neurons, by first applying innocuous mechanical
stimulation and then noxious pinch and deep pressure.
Corneal-responsive neurons with a convergent cutaneous RF were
classified as low-threshold mechanoreceptive (LTM), wide dynamic range
(WDR), or nociceptive specific (NS) units (Hu 1990
;
Hu et al. 1981
). LTM units responded to hair movement or
light touch and did not increase in discharge rate with more intense
stimuli. WDR units were sensitive to both nonnoxious and noxious
mechanical stimuli and displayed an increase in firing rate with
increasing stimulus intensity. NS units were activated only by noxious
pinch applied to the cutaneous RF. Neurons with no apparent cutaneous
receptive field were classified as cornea only (CO) units.
Antidromic stimulation
An array of two or four (1 or 2 mm tip separation) concentric
bipolar stimulating electrodes (Rhodes Medical Instruments, SNE-100 or
SNE-300) was used to stimulate medial (nucleus submedius, SM) or
lateral (ventral posteromedial nucleus, VPM; and posterior nuclear
group, PO) regions of the thalamus. The stereotaxic coordinates (AP
from bregma, ML from midline, and DV from dorsal brain surface) were
(in mm: 3, 0.5,
7 mm for SM;
3.5, 2.5,
6 mm for VPM;
4.6, 3,
6 mm for PO as adapted from the atlas of Paxinos and Watson
(1986)
. Antidromic electrodes were lowered slowly while passing
current (center negative; 0.1 ms, 20-800 µA, 1 or 8 Hz). Antidromic
activation was defined by evoked activity at a constant latency (<0.1
ms jitter), ability to follow high-frequency stimulation (>200 Hz,
20-ms train duration), and collision with spikes evoked orthodromically
by corneal stimulation (see Lipski 1981
). The minimum
intensity for antidromic activation was obtained by varying the
electrode depth, and all putative antidromic spikes were evoked by
current intensities of <550 µA.
Corneal stimulation by carbon dioxide
Different concentrations of CO2 were
obtained by mixing the outflow from tanks containing 100%
CO2 and air through a proportional gas mixer
(MX18; BOC, Warwick, RI) after the method of Chen et al.
(1995). A constant flow of mixed gas was humidified and, after activation of an electronic switch, diverted to the left cornea through
a short length of polyethylene tubing (~2 mm ID). The separation
between the tip of the tubing and the corneal surface was ~5 mm. To
estimate the force (in mg) caused by the flow of air, the tubing was
positioned 5 mm above and perpendicular to the surface of a precision
balance, and air pulses of varying flow rates were applied. At 260 ml/min the force displacement was ~1 mg, and at 515 ml/min the force
was 4 mg, values below or near the detection threshold for humans
(Belmonte et al. 1999
). The dead space from the
electronic valve to the tip of tubing was 1.6-1.7 ml and created a
time delay of ~1 s. The timing and duration of
CO2 pulses were computer controlled by the
LabVIEW program (designed originally by D. Budai, University
California, San Francisco and modified by H. Hirata, Rhode Island
Hospital). Concentrations of CO2 were 0, 30, 60, 80, and 95% as monitored from the bleeder valve output by an infrared
detector (CapStar 100, CWE). An ascending series of
CO2 pulses were used in most cases, and the
duration of each pulse was 30 or 40 s presented at an
interstimulus interval (ISI) of 4 min. The flow rate varied from 200 to
600 ml/min and was reduced to a minimum, depending on the cell's
sensitivity to 0% CO2. The flow of air alone
(0% CO2) onto the cornea often produced
transient initial (<5 s) discharges in neurons, but this activity was
rarely proportional to CO2 concentration and was
readily distinguishable from the late discharges (>10 s delay, Fig.
2). Special care was taken to keep the cornea moist throughout surgery
and the recording period with normal 0.9% saline. The integrity of the
corneal surface was confirmed by a small rise in MAP that occurred
after high concentrations (>80%) of CO2 in each
animal preparation.
Experimental design and drug administration
An initial control (no drug) series of CO2
test stimuli consisted of pulses (0, 30, 60, 80, and 95%) of 30 or
40 s in duration with 4 min between each pulse. Morphine sulfate
(MS; Marsam Pharmaceutical) was given (0.5-3.5 mg/kg iv) over 2-3
min, followed after 10 min by another series of
CO2 pulses. A single dose of morphine (1.5 or 3.0 mg/kg iv) was used for most Vc/C1 corneal units, because we determined
previously (Meng et al. 1998) that doses lower than 1.0 mg/kg, had no effect on activity evoked by electrical stimulation of
the cornea. For most Vi/Vc corneal units, morphine was given in
cumulative doses of 0.5, 1.0, and 2.0 mg/kg up to a maximum of 3.5 mg/kg. After CO2 testing in the presence of
morphine, naloxone hydrochloride (RBI) was given (0.2 or 0.4 mg/kg iv)
and after 10 min a final series of CO2 pulses was
presented. The response properties of a single neuron were studied in
each experiment.
Data analysis
Neural recording data were acquired and displayed by LabVIEW as
peristimulus time histograms (PSTHs) of spikes per 1-s bins. Together
with signals for MAP and CO2 stimulus onset and
offset, each data set was exported to a spreadsheet and analyzed
off-line. Spontaneous activity (see Table
1) was determined by averaging the spike
counts during the 2-min epoch preceding the first
CO2 test series. Because the background activity
of most units fluctuated during the 4-min period between
CO2 pulses, estimation of response magnitude
(RCO2) required that the background activity
before each CO2 pulse be accounted for
statistically. The RCO2 was defined as the spike
count per bin (1 s) that exceeded the mean plus 2 times the standard
deviation (SD) for the 2-min epoch of background activity sampled
immediately preceding each CO2 test pulse. The latency for RCO2 was defined as the earliest time
after stimulus onset for which three consecutive 1-s bins displayed a
spike count that exceeded the mean ± 2 SD of background activity
(i.e., the RCO2 value). Similar methods have been
used by us (Hirata and Aston-Jones 1994) and others
(Neugebauer et al. 1993
) to determine response magnitude
and latency in central neurons with substantial background activity.
Similarly, the onset of the inhibitory period observed in type II Vi/Vc
neurons after CO2 stimuli was defined as the
minimum time at which the discharge rate fell below the mean ± 2 SD of the background activity for at least three consecutive 1-s bins
(see Fig. 8). The average RCO2 values for each
CO2 concentration under multiple experimental
conditions (i.e., no drug, morphine, morphine plus naloxone) were
analyzed statistically by ANOVA, corrected for repeated measures
(Winer 1971
), and individual comparisons were made with
the Newman-Keuls test. Post hoc analyses revealed distinct classes of
CO2-responsive Vi/Vc corneal units. At the Vi/Vc
transition, CO2-responsive corneal units of
different types (I, II, or III) were analyzed separately. At the Vc/C1
transition, all CO2-responsive corneal units
displayed a similar stimulus-response function and were considered as a
single category. Additional groups of Vi/Vc and Vc/C1 corneal units did
not respond to CO2 pulses before or after
morphine.
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Histology
At the end of the experiment, the animal was deeply anesthetized
with an overdose of methohexital sodium (60 mg/kg iv) and perfused
transcardially with 10% Formalin containing potassium ferrocyanide.
Blocks of medulla and thalamus were frozen, sectioned at 40 or 80 µm,
respectively, and stained with cresyl violet. The recording sites in
Vi/Vc and Vc/C1 regions of medulla were reconstructed from the tissues
containing DC lesions. Antidromic stimulation sites in contralateral
thalamus were reconstructed from brain sections containing the Prussian
blue reaction product and drawn on a standard series of outlines
adapted from the atlas of Paxinos and Watson (1986).
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RESULTS |
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General properties
These results derive from 39 Vi/Vc and 26 Vc/C1 neurons that
responded to mechanical and electrical stimulation of the cornea and
subsequently were tested for sensitivity to CO2
pulses. As shown in Table 1, most Vi/Vc corneal units had low levels of spontaneous activity (<0.5 spikes/s), whereas Vc/C1 cornea units had
comparatively higher levels (>0.5 spikes/s; Vi/Vc vs. Vc/C1, P < 0.01) before CO2 testing.
Cornea RFs were determined by lightly rubbing the cornea with a von
Frey filament. At the Vi/Vc, 4 of 28 neurons that were classified as CO
units had a corneal RF that included only a portion of the cornea.
However, for all Vi/Vc units that had a convergent cutaneous RF
(n = 11), and for all 26 Vc/C1 units, the corneal RF
covered the entire surface. The average von Frey threshold for Vi/Vc
corneal units (111.9 ± 30.1 mg) was numerically lower than Vc/C1
units (139.0 ± 41.9 mg); however, this difference was not
significant statistically (P > 0.05, Mann-Whitney
U test). Based on the latency to electrically evoked corneal
activity 87.1% (27 of 31) of Vi/Vc neurons were classified as
receiving A-fiber only input and four cells as receiving A- plus
C-fiber volleys. Similarly, 86.7% (13 of 15) of Vc/C1 units received
A-fiber only input from the cornea. The recording sites for Vi/Vc
corneal units were located near the ventral edge of the Vsp within 0.5 mm of the obex, and Vc/C1 corneal units were recorded within
superficial laminae ~4 mm caudal to the obex (Fig.
1) consistent with previous reports
(Meng et al. 1997). The recording locations of
CO2-responsive neurons (Fig. 1, left side of each outline) and nonresponsive corneal neurons
(right side) were similar.
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Cutaneous RF properties
Vi/Vc. As shown in Table 1, 72% (28 of 39) Vi/Vc corneal units were classified as CO, and of those that had a convergent cutaneous RF, 7 of 11 were classified as LTM. One LTM unit had a complex cutaneous RF that consisted of an excitatory field on upper eyelid and a low-threshold inhibitory RF on the nose. Two Vi/Vc units (multi-RF, Table 1) were excited by corneal stimulation but also had a low-threshold inhibitory cutaneous RF surrounding the cornea. Nine of 11 Vi/Vc corneal units had a cutaneous RF restricted to the ophthalmic division, one included ophthalmic and maxillary divisions, and one multi-RF Vi/Vc cell had a RF that included all three trigeminal divisions. The cutaneous RF of 9 of 11 Vi/Vc units was contiguous with the corneal surface. Five Vi/Vc units had a RF on the conjunctiva in addition to the skin; however, the conjunctiva RF properties were not studied further.
Vc/C1. As shown in Table 1, all Vc/C1 corneal units had a convergent cutaneous RF that could be classified as either WDR (21/26) or NS (5/26). No Vc/C1 units were classified as LTM or CO. The cutaneous RF for 24 of 26 Vc/C1 units was contiguous with the corneal surface; however, one unit had an NS field on the pinna, and another cell had a WDR-like field on the nose. One Vc/C1 neuron had a complex RF that included excitatory fields on cornea plus skin (ophthalmic and maxillary divisions) and a high-threshold inhibitory field on the nose. For 23 of 26 Vc/C1 corneal units, the convergent cutaneous RF was restricted to the ophthalmic division. Although the cutaneous RF area was not quantified, there did not appear to be overt differences in either RF size or location on the facial skin for Vi/Vc and Vc/C1 corneal units.
Responses to CO2 pulses applied to the cornea
Vi/Vc. Forty-six percent (18 of 39) of Vi/Vc neurons were excited by the initial series of CO2 pulses applied to the cornea. Two main patterns of response to graded concentrations of CO2 were seen for Vi/Vc neurons. Type I neurons were encountered most frequently (n = 11) and displayed a progressive increase in discharge rate to increasing concentrations of CO2 as shown by the example in Fig. 2A. Consistent evoked discharges to CO2 pulses began after a delay (late responses) in all 11 cells (range, 7-22 s after 95% CO2) and often outlasted the CO2 stimulus, especially at higher CO2 concentrations (80-95% CO2). Note in Fig. 2A that a transient (<5-s delay) variable increase in firing was seen at stimulus onset (early RCO2) that was not dependent on CO2 concentration. A positive relationship between the early RCO2 (where RCO2 = spike count >mean background + 2 SD) and CO2 concentration was seen in only 3 of 11 type I Vi/Vc neurons as defined by a significant late RCO2. By contrast, the late RCO2 was proportional to CO2 concentration (Fig. 2B) and the average late RCO2 for all type I Vi/Vc neurons to 80% CO2 pulses was significantly greater than to 60% CO2 pulses (P < 0.01, ANOVA, see Fig. 6A). The derived population response for type I Vi/Vc units was described by a positively accelerating power function with an exponent of 1.81 (r = 0.934, n = 11). In initial experiments CO2 pulses of 30 s duration were used; however, as seen by the PSTHs in Fig. 3, the late increase in firing pattern of type I cells often appeared after a 15- to 20-s delay and continued beyond the end of the stimulus. To address the concern that a consistent maximum stimulus-response function to graded concentrations of CO2 pulses of 30 s duration may have been obscured by a variable and incomplete "ON response," the CO2 pulse duration was increased to 40 s in subsequent experiments. As seen in Fig. 3B, the variability of the late RCO2 to 30-s pulses at high CO2 intensities (80 and 95%) was reduced by using 40-s pulses. The threshold concentration for the late RCO2 (defined as >20% increase in RCO2, sustained for 3 consecutive bins, above the RCO2 to 0% CO2) was 30% CO2 in five units, 60% in four units, and 80% in two type I Vi/Vc units. The average latency of the late RCO2 decreased significantly with an increase in CO2 concentrations (60-95%) for type I Vi/Vc corneal units (see Fig. 6B). Eight of 11 type I units were classified as CO, 1 as WDR, 1 as LTM, and 1 as a multi-RF cell.
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Vc/C1.
Fifty-eight percent (15 of 26) of Vc/C1 corneal units responded to
CO2 pulses. Compared with Vi/Vc corneal units,
the stimulus-response patterns of Vc/C1 units to
CO2 pulses (30-95%) were homogeneous. All Vc/C1
units displayed a progressive increase in discharge rate with
increasing CO2 intensity. A majority of Vc/C1
units (10 of 15) showed a progressive increase in firing rate
throughout the entire range of CO2 concentrations
(Fig. 4A, left column) and the
remainder displayed an apparent saturation to 95%
CO2 pulses (Fig. 4B, right column).
Two subclasses of Vc/C1 units with linear and saturated response
patterns also were apparent if a less conservative method of
subtracting only the mean background (versus mean ± 2 SD for
RCO2) was used to calculate the
RCO2. This suggested that the level of
spontaneous activity before CO2 testing did not
influence the classification (bottom panels of Fig. 4, ).
As shown in Fig. 5A, Vc/C1
units often responded to CO2 pulses (30 or
40 s duration) with a brief transient (<5 s delay) increase in
firing rate that was not related to CO2
concentration and, followed by a delay (range of latency, 8-29 s at
95% CO2), a late RCO2 that
was proportional to CO2 concentration
(P < 0.01, ANOVA). The late RCO2
often persisted for several seconds after stimulus offset, similar to
the response seen for type I Vi/Vc units (Fig. 3). Comparison of 30- and 40-s CO2 pulses for the average early and
late RCO2 values (calculated as spikes per
stimulus) shown in Fig. 5B were not different statistically
(P > 0.05, ANOVA). The average late
RCO2 stimulus-response function for all Vc/C1 units shown in Fig. 6A
revealed a progressive increase with greater CO2
intensity similar to that seen for Vi/Vc units. The derived population
response for all Vc/C1 corneal units was described by a positively
accelerating power function with an exponent of 1.41 (r = 0.889, n = 15). The latency for late
RCO2 onset also decreased significantly with
increasing CO2 intensity similar to that of Vi/Vc
units (Fig. 6B). The threshold CO2
concentration was 60% for 7 of 15 Vc/C1 units, 80% for 4 units, and
95% for 4 units. Although the results suggested that Vc/C1 units
required higher minimum CO2 concentration than
Vi/Vc units for activation, this was not significant (P > 0.05,
2 analysis). Thirteen of 15 Vc/C1
corneal units were classified as WDR on the basis of the properties of
the convergent cutaneous RF, and two cells were classified as NS
neurons.
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Effects of intravenous morphine
Vi/Vc. Four of six type I Vi/Vc units showed an increase in spontaneous activity (>50% change) after morphine (0.5-3.5 mg/kg iv), and two units were not affected. Of the type I units tested for CO2 responses before morphine, after morphine, and after naloxone, two units were markedly enhanced by morphine (Fig. 8A), one unit was inhibited and two were not affected. Both the excitatory and inhibitory effects of morphine on type I Vi/Vc units were reversed by naloxone. Morphine did not affect the early transient responses (<5-s delay) to CO2 pulses in type I units. There was no apparent difference in the recording location for Vi/Vc corneal units that were excited, inhibited, or unaffected by morphine.
Six type II Vi/Vc units were tested for CO2 responses before and after morphine. Morphine (cumulative dose of 0.5-3.5 mg/kg iv) caused an increase in background activity of one unit, decrease in four units, and no effect in one neuron. By contrast, morphine enhanced (>50% above premorphine value) the late RCO2 after high concentrations of CO2 (80 or 95%) in three units (see example in Fig. 8) and decreased the late RCO2 in two units. Morphine did not affect the duration of the inhibitory phase of type II units evoked by 30-95% CO2 pulses, although the example of Fig. 8 suggested that it may have been reduced in some cases. Twenty-one of 39 Vi/Vc corneal units did not respond to the initial series of CO2 pulses before morphine. However, after morphine a third category (type III, n = 3) of Vi/Vc neuron was uncovered that displayed a vigorous response to CO2 in naloxone-reversible manner. Eleven cells that initially were unresponsive to CO2 pulses were retested after morphine, and 3 of these 11 units displayed an increase in late RCO2 as shown in Fig. 7B. The dose of morphine required for uncovering CO2-evoked activity was >1 mg/kg in each case. The magnitude of the late RCO2 after 95% CO2 was enhanced significantly after morphine (P < 0.025, ANOVA, vs. premorphine or postnaloxone). The ability of naloxone to return type III units to a state of low CO2 responsiveness was not due to a general loss of unit amplitude or activity, because rubbing the cornea evoked a prompt and significant increase in firing rate (R, in Fig. 7B). Type III Vi/Vc units displayed low levels of background activity (<0.2 spikes/s) and morphine caused only small additional increases. Two of three type III Vi/Vc units were classified as CO, and one unit had an LTM-like RF on the upper and lower eyelids. All three type III units received A-fiber only input from the cornea. It was possible that the frequency of occurrence of type III cells (3 of 11) was underestimated, because seven additional corneal units that did not respond to the initial series of CO2 pulses were not retested after morphine. It was possible that the occurrence of CO2-unresponsive Vi/Vc neurons (21 of 39) may have been overestimated by desensitization caused by prior CO2 testing of cells that were not held long enough to be included in the data analyses.
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Vc/C1. Morphine inhibited the CO2 responsiveness of all Vc/C1 corneal units tested. A total of six Vc/C1 units were tested before morphine, after morphine (1.5 or 3.0 mg/kg iv), and after naloxone. As summarized in Fig. 9, the late RCO2 of Vc/C1 units, expressed as percent of control, was inhibited by morphine and reversed, at least partially, by naloxone. Because desensitization of Vc/C1 units was common, it was important to compare the percentage decrease due to repeated presentation of CO2 pulses alone (n = 5). Figure 9 indicates that repeat presentation of CO2 pulses alone (C1 vs. C2) caused numerically smaller reductions in the late RCO2 than was seen after 1.5 or 3 mg/kg morphine. Morphine never enhanced the late response of a Vc/C1 corneal unit and did not affect the early transient response to CO2 pulses. In contrast to type III Vi/Vc corneal units, no Vc/C1 unit that was unresponsive to the initial series of CO2 pulses became responsive after morphine (n = 5). Several additional Vc/C1 units were examined after morphine, but were lost before CO2 testing was completed; however, background activity was generally reduced by morphine (11 of 17 units) with no effect in six units.
Antidromic responses from contralateral thalamus
Vi/Vc. None of 18 Vi/Vc corneal units tested were activated antidromically from sites in the contralateral nucleus submedius (SM) or ventral posteromedial thalamic nucleus (VPM). Three Vi/Vc neurons also were tested for activation from sites in the caudal aspect of contralateral posterior thalamic nucleus (POc), and none were driven antidromically (bipolar concentric electrode, center negative, 0.2 ms pulse duration; up to 2.0 mA).
Vc/C1. None of 30 Vc/C1 corneal units tested were antidromically activated from sites within the SM or VPM. However, 7 of 19 Vc/C1 units were driven from POc. An example of a Vc/C1 unit antidromically activated from the POc is shown in Fig. 10. Two of seven Vc/C1 corneal units driven from sites in the contralateral POc were responsive to CO2 pulses, and five units were not. The average current required for antidromic activation was 329 µA (range, 84-540 µA; n = 7), and the latency to antidromic activation ranged from 2.8 to 8.0 ms. Calculated conduction velocities ranged from 1.8 to 5.4 m/s, suggesting that mainly small-diameter myelinated fibers mediated the projection from Vc/C1 neurons to the contralateral POc.
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DISCUSSION |
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The present study used a chemical stimulus of
CO2 pulses to determine the stimulus encoding
properties of Vi/Vc and Vc/C1 corneal units. It was predicted that
neurons that mediate the sensory-discriminative aspects of corneal pain
should encode the concentration of CO2, be
inhibited by systemic morphine, and project to the contralateral
thalamus. The results indicated that Vc/C1 corneal units satisfied each
of these criteria. Although Vi/Vc units also displayed a positive
stimulus-response function to increasing concentrations of
CO2, morphine often enhanced neural responsiveness to CO2, and Vi/Vc corneal units
were not activated antidromically from stimulation sites in the
contralateral thalamus. These results confirm and extend previous
findings (Meng et al. 1997, 1998
) that
Vi/Vc and Vc/C1 neurons likely contribute to different aspects of
corneal pain.
Dual representation of the cornea at the Vi/Vc and Vc/C1 transition regions
The caudal portion of the trigeminal spinal nucleus (Vsp),
subnucleus caudalis (Vc), shares anatomic and physiological
similarities with the spinal dorsal horn (Dubner and Bennett
1983; Sessle 1987
). However, the Vsp also
displays distinctive features suggesting that the initial integration
of nociceptive input from craniofacial structures may be processed
differently from that of other body tissues. One unique organizational
feature of craniofacial input to the Vsp not seen at the spinal level
is a multiple somatotopic representation within the Vsp. Multiple
representation of the cornea is suggested by transganglionic
tract-tracing studies in which the central projections of corneal
sensory nerves are seen to terminate mainly at the ventrolateral pole
of the Vi/Vc transition and in the lateral superficial laminae at the
Vc/C1 transition region (Marfurt 1981
; Marfurt
and Del Toro 1987
; Panneton and Burton 1981
).
Similarly, c-fos immunocytochemical studies reveal that noxious thermal
(Lu et al. 1993
; Meng and Bereiter 1996
), mechanical (Strassman and Vos 1993
), or chemical
(Bereiter 1997
; Bereiter et al. 1996
;
Meng and Bereiter 1996
) stimulation of the corneal
surface produces a high-density of Fos-positive neurons in the Vi/Vc
and Vc/C1 transition regions. The basis for a dual representation of
the cornea in two spatially distinct portions of the Vsp remains
uncertain and two general organizational schemes can be considered.
First, it is possible that subpopulations of corneal primary afferent
neurons may project preferentially to either the Vi/Vc or Vc/C1
transition region. Such a segregation of corneal afferents could be
based on fiber type and/or modality. Partial support for fiber type
segregation derives from studies in which the marker for unmyelinated
fibers, isolectin B4, was injected into the trigeminal ganglion to
reveal a dense termination of C-fibers in the superficial laminae of Vc
but not at the Vi/Vc transition region (Sugimoto et al.
1997a
). By contrast, both substance P-like and
calcitonin gene-related peptide (CGRP)-like immunoreactivity (Meng and Bereiter 1996
; Strassman and Vos
1993
; Sugimoto et al. 1997b
), neuropeptide
markers for small diameter fibers, overlap substantially with the
location of Fos-positive neurons produced at the Vi/Vc and Vc/C1
transition regions by corneal stimulation. Also, we have determined
previously (Meng et al. 1997
), and confirmed in the
present study, that the activity evoked at A-fiber or A- plus C-fiber
latencies after electrical stimulation of the cornea occurred in a
similar proportion for Vi/Vc and Vc/C1 units. Thus these results
suggest that Vi/Vc and Vc/C1 corneal units receive a similar proportion
of afferent input on the basis of fiber diameter. Alternatively, it is
possible that corneal afferents distribute preferentially to the Vi/Vc
or Vc/C1 transition regions based on the stimulus modality encoded by
the primary afferent neuron. Recording studies, mainly from cat and
rabbit, indicate that the majority of corneal afferent fibers can be
classified as polymodal nociceptors (>70%), mechanoreceptive, or cold
receptors in descending frequency of occurrence (see Belmonte
and Gallar 1996
). In the first systematic examination of
corneal-responsive units within the caudal Vsp (Meng et al.
1997
), we determined that all Vc/C1 corneal units responded to
mustard oil stimulation of the cornea regardless if electrical stimuli
evoked activity at A-fiber alone or A- plus C-fiber latencies. By
contrast, Vi/Vc units typically responded to mustard oil only if
electrical stimulation of the cornea evoked activity at C-fiber-like
latency. A second line of evidence to suggest that Vi/Vc and Vc/C1
corneal units receive different forms of orofacial input concerns
convergent cutaneous RFs. At the Vc/C1 transition, all corneal units
received a convergent cutaneous RF that could be classified as
nociceptive, i.e., WDR- or NS-like. However, at the Vi/Vc transition,
>50% of corneal units had no convergent cutaneous RF, and of those
that received cutaneous input, most were classified as LTM confirming
previous findings (Meng et al. 1997
). Also, the number
of Fos-positive neurons produced at the Vi/Vc transition increased
similarly over a broad range of thermal stimuli applied to the cornea,
whereas at the Vc/C1 transition only noxious thermal intensity levels enhanced c-fos expression (Meng and Bereiter 1996
).
Despite the limitations of c-fos expression as a marker for central
pain pathways (see Bullitt 1990
; Strassman and
Vos 1993
), these results were consistent with the notion that
noxious thermal input from corneal nociceptors was processed
differently by neurons at the Vi/Vc and Vc/C1 transition regions.
Although the available evidence suggested that Vi/Vc and Vc/C1 corneal
units receive a different complement of sensory input, it was important
to examine this possibility more closely by determining the
stimulus-response function of corneal units to a naturally occurring
chemical stimulus.
Encoding of corneal stimuli by Vi/Vc and Vc/C1 neurons and primary afferent fibers
It has been proposed that a unique feature of nociceptive
processing compared with other sensory systems is a relative constancy of the stimulus-response function for nociceptive neurons across different levels of the neuroaxis (McHaffie et al.
1994). Comparison of the response properties of second-order
corneal units at the Vi/Vc and Vc/C1 transition regions to those of
corneal primary afferents permits inferences to be made regarding the
transfer of sensory information at the initial sites of integration of presumptive corneal pain pathways. First, the corneal RF area for
single primary afferent fibers from cat (Belmonte and Giraldez 1981
; Gallar et al. 1993
; Lele and
Weddell 1959
), rabbit (Tanelian and Beuerman
1984
) and rat (Mark and Maurice 1977
) comprise
only a fraction of the corneal surface as determined by mechanical stimulation. Although some polymodal afferent fibers supply up to 70%
of the corneal surface area (Belmonte et al. 1991
;
Gallar et al. 1993
), the present study indicated that
most brain stem units displayed whole corneal RFs, indicating
considerable spatial convergence onto second-order trigeminal neurons
at both the Vi/Vc and Vc/C1 transition regions. Second, the percentage
of neurons responsive to mechanical and chemical stimulation of the
cornea was similar for primary afferents and second-order neurons. In the cat, ~60% of mechanically sensitive corneal primary afferent fibers also responded to acetic acid (Belmonte et al.
1991
; Gallar et al. 1993
). Assuming that most
pH-sensitive corneal fibers also respond to CO2, as was
found in the rat skin in vitro preparation (Steen et al.
1992
), the present finding that 46% of Vi/Vc and 58% of Vc/C1
corneal units were excited by the initial series of CO2
pulses agreed well with the percentages reported for primary afferents.
This also suggested that select classes of CO2-sensitive neurons were not excluded from the present study due to microelectrode sampling bias. In fact, it was more likely that the percentage of
CO2-responsive Vsp neurons was underestimated because
repeated presentation of high concentrations of CO2
desensitized most Vc/C1 units and some Vi/Vc units. Also, some Vi/Vc
corneal units may be tonically inhibited, because morphine caused an
apparent disinhibition of the responses to CO2 pulses (type
III units). It is not known whether corneal primary afferent fibers
exist in the rat that respond to chemical irritants only and not to
mechanical stimuli as reported for the rabbit (Tanelian
1991
). At the Vi/Vc transition, two types of cells responded to
the initial series of CO2 pulses. Type I Vi/Vc units
displayed a progressive increase in discharge rate with increasing
CO2 concentration and type II Vi/Vc units showed a reduced
discharge rate at 30-60% CO2 concentrations and a
progressive increase in firing at higher concentrations. At the caudal
Vc/C1 transition, most units displayed a progressive increase in firing
rate with increasing CO2 concentration. Third, despite some
units in each category that displayed a saturation-like response to
95% CO2, it was unexpected to find similar CO2
thresholds and an average derived population response for type I, type
II, and Vc/C1 units that was described by a positively accelerating power function with an exponent of >1.0. This suggested that a majority of CO2-responsive neurons at the Vi/Vc and Vc/C1
transition regions received similar chemosensory information from
corneal primary afferents. In the cat A
and C-fiber corneal
afferents responded to CO2 pulses with an increasing firing
rate to CO2 concentration and an average power exponent of
1.12 (Chen et al. 1995
). In the rabbit the
stimulus-response pattern of corneal primary afferent fibers to a
variety of irritant chemicals also was fit by an exponential function
(Beuerman et al. 1992
). It is not yet known whether
corneal afferent fibers in the rat display a similar stimulus-response
pattern to graded concentrations of CO2 or acid. Most Vi/Vc
and Vc/C1 corneal units responded to CO2 pulses with long
latencies (10-20 s), whereas Chen et al. (1995)
found
most primary afferent fibers in the cat responded within 5 s. The
basis for this difference is not certain; however, the evoked pressor
responses to CO2 pulses also occurred after a 10- to 20-s
delay (see Fig. 8). The integration of
corneal sensory information by second-order neurons may require spatial
and/or temporal summation. Alternatively, it is possible that
CO2-responsive nerve endings are situated deeper within the
corneal epithelium of rat compared with cat. Small differences in the
barrier thickness or the temperature of the gas delivered to the cornea
could affect the diffusion of CO2 and/or the enzymatic
conversion to protons by carbonic anhydrase, which, in turn, activate
nerve endings (Steen et al. 1992
). Fourth,
desensitization of Vi/Vc and Vc/C1 units often was seen after repeated
stimulation with pulses at high CO2 concentrations
(80-95%). This result differed from that seen after repeated noxious
thermal stimulation of the cornea in which Vc/C1 units became
sensitized and Vi/Vc units generally had reduced responses (Meng
et al. 1997
). Interestingly, in the cat the activity of corneal
primary afferents also was enhanced by repeated noxious thermal
stimulation (Belmonte and Giraldez 1981
) and reduced by
repeated CO2 pulses (Chen et al. 1995
).
Carstens et al. (1998)
have noted that repeated
application of irritant chemicals such as nicotine or capsaicin to the
corneal surface caused pronounced tachyphylaxis in rostral Vc neurons.
Collectively, these results indicated that type I Vi/Vc and Vc/C1
corneal units displayed similar stimulus-response patterns to
CO2 pulses consistent with that expected to be transmitted
from corneal primary afferent fibers, and sufficient to encode
CO2 concentration. Type II Vi/Vc units had more complex
responses to CO2 pulses that must derive from central
processing.
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Effects of morphine
Despite similar responses to the
initial series of CO2 pulses, Vi/Vc and Vc/C1
corneal units had markedly different
responses to CO2
after morphine. Morphine enhanced the responses to
CO2 pulses of ~50% of type I and type II Vi/Vc
corneal units, whereas all Vc/C1 units were inhibited. The enhancement
of Vi/Vc units after morphine often appeared as an increase in
spontaneous activity accompanied by an increase in the
CO2 stimulus-response pattern to the previous
series of CO2 pulses. A third category, so-called type III Vi/Vc corneal units, became "unmasked" by morphine in that
they were initially unresponsive to CO2 pulses
and became responsive after morphine. However, it was possible that
type III units represented cells that had become desensitized by prior corneal testing. The effects of morphine on Vi/Vc and Vc/C1 units were
reversed by naloxone, indicating an opioid receptor-selective action.
These results confirmed and extended previous findings in which
morphine enhanced the activity of Vi/Vc units and inhibited that of
Vc/C1 corneal units to electrical or noxious thermal stimulation of the
corneal surface in a naloxone-reversible manner (Meng et al.
1998). Although less well-described than the inhibitory
effects, morphine or mu opioid receptor agonists have been reported to enhance the activity of nociceptive neurons in the spinal dorsal horn
after systemic (Craig and Serrano 1994
), intrathecal
(Dickenson and Sullivan 1986
), or iontophoretic
(Jones et al. 1990
) routes of administration. Among
medullary dorsal horn neurons, morphine increased the activity of some
cold-responsive (Mokha 1993
) and nociceptive
trigeminothalamic projection neurons in the rat (Wang and Mokha
1996
). Also, morphine did not attenuate c-fos gene expression produced by corneal stimulation at the Vi/Vc transition but did cause a
significant reduction at the Vc/C1 transition (Bereiter 1997
). In contrast to the progressive facilitative effects of increasing doses of intravenous morphine on Vi/Vc corneal units, the
majority of studies in which low doses of opioids were shown to enhance
neural activity also concluded that higher doses were inhibitory
(however, see Craig and Hunsley 1991
; Grudt and
Williams 1994
). For example, Craig and Serrano
(1994)
found that low doses of morphine (0.125 mg/kg iv)
increased the activity in 9 of 13 lamina I neurons in cat spinal cord
to noxious pinch or heat stimulation, whereas higher doses inhibited
most neurons. In rat spinal dorsal horn, intrathecal doses of <5 µg
morphine enhanced the evoked activity at C-fiber intensity, but higher
doses were inhibitory (Dickenson and Sullivan 1986
).
Similarly, low doses of opioids enhanced the activity of some dorsal
root ganglion cells in culture (Crain and Shen 1990
);
however, when tested on peripheral A
fibers, morphine caused only
inhibition of spontaneous activity (Russell et al.
1987
). Although lower doses of morphine than used here (0.5 mg/kg iv) may have enhanced the responses of caudal Vc/C1 corneal units
to CO2 pulses, it seemed unlikely that a common mechanism could explain the enhanced activity of both Vi/Vc corneal units and spinothalamic tract lamina I nociceptive neurons. Unlike the
effects on spinal dorsal horn neurons, higher doses of morphine (cumulative maximum dose, 3.5 mg/kg iv) only rarely inhibited Vi/Vc
corneal units in this or a previous study (Meng et al.
1998
). The exact mechanisms that underlie morphine enhancement
of Vi/Vc corneal units remain uncertain. Microinjection of morphine
into the Vc/C1 transition region could mimick the effect of systemic morphine on some Vi/Vc units, suggesting a possible action via intersubnuclear projections in Vsp (Meng et al. 1998
).
Systemic morphine also could act locally to disinhibit interneurons at the Vi/Vc transition region or to activate opioid-dependent descending control pathways from higher brain areas. Systematic testing of possible peripheral effects of morphine on corneal afferent fibers has
not been reported; however, in humans topical morphine reduced the pain
associated with corneal injury without affecting corneal sensitivity in
uninjured patients (Peyman et al. 1994
). Recent anatomic
evidence indicates some opioid receptor activity in corneal nerves
(Wenk and Honda 1999
). Thus it cannot be excluded that a
portion of the morphine-induced inhibition of Vc/C1 units could occur
through action at peripheral sites, whereas enhancement of Vi/Vc unit
activity by morphine likely occurs via central mechanisms.
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Projections of Vi/Vc and Vc/C1 corneal units
If the contribution of second-order neurons to the various
aspects of nociception (e.g., sensory-discrimination, motor reflexes, autonomic reflexes) is determined in part by the strength of
projections to specialized brain areas (see Laird and Cervero
1991; Price and Dubner 1977
), then
identification of the efferent projection targets of Vi/Vc and Vc/C1
corneal units may aid in defining their roles in corneal nociception.
The efferent projections of Vi/Vc and Vc/C1 corneal units were markedly
different as determined by antidromic activation methods. With an
electrode array implanted in the contralateral thalamus directed at
sites within the PO group, VPM and SM, it was determined that 7 of 19 Vc/C1 corneal units projected to the PO group and none of 30 units
projected to the VPM or SM. By contrast, none of 21 Vi/Vc corneal units were antidromically activated from sites in the contralateral thalamus.
These results were similar to those of a previous study in which >80%
of Vc/C1 corneal units, but none of the Vi/Vc units, were
antidromically activated from sites in the parabrachial complex and
Kolliker-Fuse nucleus (Meng et al. 1997
). Recently, we
have combined c-fos immunocytochemistry and retrograde tracing methods to determine whether Vi/Vc and Vc/C1 corneal units project to the
contralateral thalamus (Bereiter et al. 1999
). Numerous
Fos-positive/FluoroGold double-labeled neurons were found in lamina I
at the Vc/C1 transition after FluoroGold injection into the caudal PO
group confirming the results of antidromic activation; however, some
double-labeled cells also were seen at the Vi/Vc transition. Numerous
double-labeled cells were seen at the Vi/Vc, but not at the Vc/C1
transition after FluoroGold injections into SM. No Fos-positive neurons
were seen to project to VPM from the Vi/Vc or Vc/C1 transition regions. The two main conclusions drawn from this study were that Vc/C1 corneal
units project selectively to the PO group and, second, neither Vi/Vc or
Vc/C1 corneal units project significantly to the contralateral VPM.
This finding differs from results seen in the cat in which corneal
units were recorded throughout the VPM (Hayashi 1995
).
Anterograde tracer injections into superficial laminae of Vc in the
rat were reported to label terminals in PO and VPM of contralateral
thalamus (Iwata et al. 1992
). However, others have
reported that injection of FluoroGold into VPM of the rat did not
retrogradely fill neurons in those regions of the Vsp that corresponded
to the location of Vi/Vc or Vc/C1 corneal units (Dado and
Giesler 1990
). It is not certain why Vi/Vc corneal units could
not be antidromically driven from the PO group or SM despite anatomic
evidence of such efferent projections (Bereiter et al.
1999
; Dado and Giesler 1990
; Yoshida et
al. 1991
). Possibly, these projections involve sparse or very
fine terminal regions in the thalamus. Also, some corneal-responsive
neurons recorded at the Vi/Vc transition may be local circuit neurons.
In recent preliminary studies, we have found several Vi/Vc corneal
units that could be activated antidromically from sites in or near the superior salivatory nucleus/facial motor nucleus region (Bereiter et
al. 1999
). This is consistent with results from Pelligrini et
al. (1995)
in the guinea pig indicating direct projections from
Vi/Vc units to the facial motor nucleus in control of eye blinks. The
function of the POc in sensory processing remains uncertain. The POc
has been referred to as a phylogenetically primitive thalamic
processing area because it is not somatotopically organized (see
Diamond 1995
). Many POc neurons have large cutaneous RFs
(Perl and Whitlock 1961
) and receive convergent input
from multiple sensory systems (LeDoux et al. 1987
).
Because stimulation of sites in the POc antidromically activated mainly
nociceptive neurons in the cervical dorsal horn (Dado et al.
1994
), this suggested a significant role for the POc in pain processing.
Functional significance
The results of the present study confirm and extend previous
findings (Meng et al. 1997, 1998
) to
conclude that corneal-responsive neurons at the Vi/Vc and Vc/C1
transition regions serve different functions in corneal nociception.
Corneal units at the Vc/C1 transition behaved, in most respects,
similar to spinal dorsal horn nociceptive neurons. Vc/C1 corneal units
received convergent cutaneous input that could be classified as
nociceptive (i.e., WDR or NS), were always inhibited by morphine, and
often displayed efferent projections to supraspinal regions associated
with higher-order processing of nociceptive input (parabrachial complex
or thalamic PO group). These features were consistent with the
hypothesis that Vc/C1 corneal units mediated the sensory-discriminative
aspects of corneal pain as well as the autonomic reflex adjustments
that accompany pain sensation. Despite similar stimulus-response
functions to CO2 pulses, Vi/Vc corneal units
displayed unique properties not seen at the Vc/C1 transition or the
spinal dorsal horn. Vi/Vc corneal units often had RFs restricted to the
corneal surface, were facilitated by a broad range of morphine doses,
and were rarely activated antidromically from sites within the medial
or lateral sensory thalamus. The function of Vi/Vc units in corneal nociception remains uncertain. The available evidence suggests that
Vi/Vc corneal units contribute to ocular-specific functions such as eye
blink reflexes and lacrimation. However, the enhanced responsiveness of
Vi/Vc units after morphine and the anatomic evidence of projections to
the SM (Bereiter et al. 1999
; Dado and Giesler
1990
; Yoshida et al. 1991
) is consistent with a
role in recruitment of endogenous antinociceptive controls. These
results support the notion that corneal chemosensory information is
relayed without significant transformation to different levels of the trigeminal system. Last, evidence for differential roles of Vi/Vc and
Vc/C1 neurons in corneal nociception is better supported by the
response to morphine and efferent projections than by the encoding
properties to chemical irritant stimuli.
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ACKNOWLEDGMENTS |
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We thank D. Bereiter and A. Benetti for excellent technical assistance and Dr. Ian Meng of the Department of Neurology, University of California, San Francisco, for valuable suggestions during the early stages of these experiments.
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FOOTNOTES |
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Address for reprint requests: D. A. Bereiter, Rhode Island Hospital/Brown University, Depts. of Surgery and Neuroscience, 222 Nursing Arts Bldg., Providence, RI 02903.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 April 1999; accepted in final form 1 July 1999.
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REFERENCES |
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