1Department of Surgery and 2Department of Neuroscience, Brown University School of Medicine/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, Shinichiro Takeshita, James W. Hu, and David A. Bereiter. Cornea-Responsive Medullary Dorsal Horn Neurons: Modulation by Local Opioids and Projections to Thalamus and Brain Stem. J. Neurophysiol. 84: 1050-1061, 2000. Previously, it was determined that microinjection of morphine into the caudal portion of subnucleus caudalis mimicked the facilitatory effects of intravenous morphine on cornea-responsive neurons recorded at the subnucleus interpolaris/caudalis (Vi/Vc) transition region. The aim of the present study was to determine the opioid receptor subtype(s) that mediate modulation of corneal units and to determine whether opioid drugs affected unique classes of units. Pulses of CO2 gas applied to the cornea were used to excite neurons at the Vi/Vc ("rostral" neurons) and the caudalis/upper cervical spinal cord transition region (Vc/C1, "caudal" neurons) in barbiturate-anesthetized male rats. Microinjection of morphine sulfate (2.9-4.8 nmol) or the selective mu receptor agonist D-Ala, N-Me-Phe, Gly-ol-enkephalin (DAMGO; 1.8-15.0 pmol) into the caudal transition region enhanced the response in 7 of 27 (26%) rostral units to CO2 pulses and depressed that of 10 units (37%). Microinjection of a selective delta {[D-Pen2,5] (DPDPE); 24-30 pmol} or kappa receptor agonist (U50488; 1.8-30.0 pmol) into the caudal transition region did not affect the CO2-evoked responses of rostral units. Caudal units were inhibited by local DAMGO or DPDPE but were not affected by U50,488H. The effects of DAMGO and DPDPE were reversed by naloxone (0.2 mg/kg iv). Intravenous morphine altered the CO2-evoked activity in a direction opposite to that of local DAMGO in 3 of 15 units, in the same direction as local DAMGO but with greater magnitude in 4 units, and in the same direction with equal magnitude as local DAMGO in 8 units. CO2-responsive rostral and caudal units projected to either the thalamic posterior nucleus/zona incerta region (PO/ZI) or the superior salivatory/facial nucleus region (SSN/VII). However, rostral units not responsive to CO2 pulses projected only to SSN/VII and caudal units not responsive to CO2 projected only to PO/ZI. It was concluded that the circuitry for opioid analgesia in corneal pain involves multiple sites of action: inhibition of neurons at the caudal transition region, by intersubnuclear connections to modulate rostral units, and by supraspinal sites. Local administration of opioid agonists modulated all classes of corneal units. Corneal stimulus modality was predictive of efferent projection status for rostral and caudal units to sensory thalamus and reflex areas of the brain stem.
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INTRODUCTION |
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The trigeminal spinal nucleus (Vsp) is the
initial site of termination for trigeminal afferent fibers that encode
noxious mechanical, thermal, and chemical sensory information from the face as well as specialized structures such as the cornea, teeth, and
meninges (Dubner and Bennett 1983; Renehan and
Jacquin 1993
; Sessle 1987
). It has long been
appreciated that the most caudal portion of the Vsp, trigeminal
subnucleus caudalis (Vc), shares anatomical and physiological
properties with the spinal dorsal horn. However, unique organizational
features exist within the trigeminal brain stem complex that are not
seen at the spinal level and may contribute to trigeminal nociception.
First, craniofacial structures are represented at multiple levels of
the Vsp with an "onion skin-like" somatotopic organization
(Shigenaga et al. 1986a
,b
). Second, a dense fiber system
interconnects rostral and caudal portions of the Vsp (Ikeda et
al. 1984
; Jacquin et al. 1990
; Kruger et
al. 1977
; Panneton et al. 1994
). Third, the
magnitude and destination of efferent projections from second-order
neurons differ considerably for neurons located in different portions of the Vsp, especially to thalamus (Bruce et al. 1987
;
Fukushima and Kerr 1979
). The significance of multiple
representation, intersubnuclear communication, and efferent projection
status of neurons that receive input from a single craniofacial
structure, yet are located at different rostrocaudal levels of the Vsp,
in trigeminal pain is not certain. The cornea afferent system is well
suited to address these issues. The corneal epithelium is supplied
exclusively by small caliber afferents of the ophthalmic branch of the
trigeminal nerve, which terminate centrally in two spatially distinct
regions of the Vsp: a "rostral region," at the subnucleus
interpolaris/caudalis transition (Vi/Vc) and a "caudal region," at
the subnucleus caudalis/upper cervical cord transition (Vc/C1)
(Bereiter 1997
; Bereiter et al. 1996
;
Lu et al. 1993
; Marfurt 1981
;
Marfurt and Del Toro 1987
; Meng and Bereiter
1996
; Panneton and Burton 1981
; Strassman
and Vos 1993
).
Activation of a common population of corneal afferent nerves is
sufficient to evoke multiple aspects of corneal nociception such as
pain sensation (Beuerman and Tanelian 1979;
Kenshalo 1960
; Lele and Weddell 1959
),
motor reflexes (Evinger et al. 1993
), and
autonomic/endocrine reflexes (Bereiter et al. 1994
,
1996
). Thus one interpretation of a dual organization is
that second-order neurons at the rostral and caudal transition regions
mediate different aspects of corneal pain, and intersubnuclear
connections serve to recruit or coordinate the activity of these
cell groups. Specialization of function is supported by previous
reports that corneal units located at the rostral and caudal transition
regions display different receptive field (RF) properties (Meng
et al. 1997
, 1998
). A second distinctive feature
between rostral and caudal units was a dose-related facilitation of
corneal input to a significant percentage of rostral units after
systemic morphine, whereas caudal units only were inhibited
(Hirata et al. 1999
; Meng et al. 1998
).
Local administration of morphine at the caudal transition region
mimicked the facilitatory effect of systemic morphine on rostral
corneal units and suggested a role for intersubnuclear connections in
this response (Meng et al. 1998
). Since morphine binds
to more than one opioid receptor subtype (Yaksh 1997
),
one goal of the present study was to determine which opioid receptor
subtype(s) mediated the excitability changes in rostral and caudal
corneal units after local microinjection into the caudal transition
region. Also, since corneal units located at the rostral transition
region have heterogeneous RF properties, a second goal was to determine
whether different classes of corneal units responded similarly to
opioid receptor agonists. Corneal units were classified according to
corneal stimulus modality, convergent cutaneous RF properties, and
efferent projection status. Efferent projections to the thalamus were
tested by antidromic stimulation methods from electrodes placed in the
posterior nucleus, since we determined that corneal units did not
project to other thalamic regions (Hirata et al. 1999
).
Units also were tested for projections to the superior
salivatory/facial motor nucleus region, a brain stem region concerned
with trigeminal-evoked eyeblink reflexes (Pellegrini et al.
1995
) and lacrimation (Toth et al. 1999
).
Portions of these data have been presented previously in preliminary
form (Bereiter et al. 1999
).
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METHODS |
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Surgical preparation
Male rats (295-450 g, Sprague-Dawley, Harlan) were anesthetized
initially with pentobarbital sodium (70 mg/kg ip) prior to surgery. The
left femoral artery (blood pressure monitor) and jugular vein
(anesthesia and drug infusions) were catheterized, and after
tracheotomy, animals were artificially respired with oxygen-enriched
room air. Anesthesia was maintained by continuous infusion of
methohexital sodium (35-40 mg · kg1 · h
1) and 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 prior to the electrophysiological recording
session. The animal was placed in a stereotaxic frame, and a portion of
the occipital bone and C1 vertebra was 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. Expiratory end-tidal CO2
was monitored continuously and kept at 3.5-4.5% by adjusting tidal
volume. Mean arterial pressure (MAP) remained above 100 mmHg throughout
the experiment. Body temperature was maintained at 38°C with a
heating blanket and thermal probe.
Electrophysiology recording techniques
Brain stem neurons were recorded extracellularly using tungsten
electrodes (9 M, FHC, Bowdoinham, ME) as described previously (Hirata et al. 1999
; Meng et al. 1997
).
Neurons recorded at the rostral Vi/Vc transition were approached at an
angle of 28° off vertical and 45° off midline (see Fig.
1). Neurons recorded in laminae I-II at
the caudal Vc/C1 transition were approached at an angle of 43° off
vertical and 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. 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 (G3) through a DAQ interface
board using LabVIEW software (National Instruments, Austin, TX). These
data also were digitized (NeuroData) and stored on VCR tape as a backup
and for further off-line analyses.
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In each animal preparation a single cornea-responsive neuron was
isolated and several general response properties were determined as
reported previously (Hirata et al. 1999; Meng et
al. 1997
). Units were tested for A- and/or C-fiber type corneal
input (electrical stimuli) in which responses occurring at latencies of
>30 ms were assumed to indicate C-fiber input (Hu 1990
;
Meng et al. 1997
). Mechanical threshold for corneal
input was assessed with calibrated von Frey filaments. Convergent
cutaneous receptive fields (RF) were examined by applying innocuous
mechanical stimulation followed by noxious pinch and deep pressure to
the ipsilateral face. Corneal units with a convergent cutaneous RF were
classified as low threshold mechanoreceptive (LTM), wide dynamic range
(WDR), or nociceptive specific (NS) units as described previously
(Hirata et al. 1999
; Hu 1990
; Meng
et al. 1997
). Neurons with no apparent cutaneous RF were
classified as cornea only (CO) units. Each corneal unit was tested for
possible efferent projections to the contralateral thalamic posterior
nucleus/zona incerta region (PO/ZI) and the ipsilateral superior
salivatory/facial motor n. region (SSN/VII). The stereotaxic
coordinates (in mm) for PO/ZI were as follows: 3-5 caudal to bregma,
2.5 lateral, and 6-7 ventral to the brain surface (Paxinos and
Watson 1986
). To stimulate the SSN/VII while avoiding the
transverse sinus, the electrode was angled rostral 2° off vertical at
the coordinates: 4 mm caudal to lambda, 2 mm lateral, and 5-7 mm
ventral to the cerebellar surface. Stimulation of the SSN was confirmed
by the presence of "bloody tears," the porphyrin-rich mucous
secretion from the Harderian glands that are supplied by premotor
inputs from SSN (Beuregard and Smith 1994
). Antidromic
testing occurred at a fixed anterior-posterior/medial-lateral position
while moving the electrode array vertically along a 4-5 mm
dorsal-ventral tract that passed through the PO/ZI or SSN/VII region.
Antidromic testing was done after encoding properties were assessed to
avoid possible persistent effects from high-intensity and
high-frequency stimulation. Stimuli were presented from two arrays of
two (1- or 2-mm separation) concentric bipolar stimulating electrodes
(SNE-100, Rhodes Medical Instruments). Antidromically evoked spikes
were defined by a constant latency (<0.1 ms jitter), high-frequency
following (0.1 ms pulse, 200-300 Hz, 20 ms train duration) and a
collision with orthodromically driven spikes occurring within a
critical time window (Lipski 1981
). A stimulus intensity of 500 µA was defined as the maximum allowable current for specific activation. The brain loci of lowest current for antidromic activation were marked electrolytically (30 µA, 30 s).
Corneal stimulation by carbon dioxide
The setup for delivery of CO2 pulses to
the corneal surface is shown in the schematic diagram of Fig. 1. Pulses
of CO2 gas of different concentrations were
obtained by mixing the outflow from tanks containing 100%
CO2 and air through a proportional gas mixer as
monitored from the bleeder valve output by an infrared detector
(CapStar 100, CWE). Humidified CO2 gas mixtures
were delivered at a constant flow to the left cornea through a short length of polyethylene tubing (~2 mm ID) positioned ~5 mm from the
corneal surface. The timing and duration of CO2
pulses (40 s duration, minimum of 4 min between pulses) were
computer-controlled by LabVIEW software. In most experiments, only the
responses to CO2 pulses of 0 and 80% were
compared. However, in initial experiments the responses to a full range
of CO2 concentrations (0, 30, 60, 80, and 95%)
were examined to confirm that the threshold and slope of the
stimulus-response curves were similar to those reported previously
(Hirata et al. 1999). Special care was taken to keep the
cornea moist during surgery and the recording period with normal saline.
Experimental design and opioid drug administration
The main protocol consisted of an initial test using 0%
CO2 to determine the flow rate that had a minimal
effect on neural activity. This was followed by two pulses of 80%
CO2 presented at 15-min intervals. At 6 min after
the second control 80% CO2 pulse, microinjection
of opioid receptor agonist into the caudal Vc/C1 region began and
continued over 5 min. Then 4 min later (i.e., 15 min after previous
80% CO2 pulse) another series of test pulses was
presented, followed by naloxone (0.2 mg/kg iv), and a final series of
CO2 test pulses. In a subset of experiments (n = 15), the responses to CO2
pulses were determined after local injection of opioid drugs and,
subsequently, after systemic morphine (0.5-3.0 mg/kg iv), then after
naloxone. Microinjections of opioid drugs were directed at the caudal
Vc/C1 transition region via a calibrated thick-walled pipette (40-80
µm OD, Fisher) delivered by a pneumatic device. The rostrocaudal
position of the pipette corresponded to the location where we have
recorded caudal corneal units previously (Hirata et al.
1999; Meng et al. 1997
, 1998
). The rostral edge of the C1 vertebrae served as a landmark at which the
pipette was inserted 500-800 µm medial to the lateral edge of the
brain stem and lowered ~500 µm below the dorsal surface of the
spinal cord. Microscopic observation of the meniscus movement monitored
the rate (60 nl/min) and volume (180-300 nl) of injection. As shown by
the example of Fig. 2, a 300-nl injection
volume spread spherically over ~1.0 mm to completely bathe the caudal
region without diffusing to the rostral Vi/Vc transition region 4-5 mm away. The drugs used for microinjections into the caudal Vc/C1 region
were as follows: morphine sulfate (25 µg/µl, 37 mM), selective mu
opioid receptor agonist, D-Ala, N-Me-Phe, Gly-ol-enkephalin (DAMGO; RBI; 10 or 20 µM), delta opioid agonist, enkephalin,
[D-Pen2,5] (DPDPE; RBI; 100 µM),
and selective kappa receptor agonist, trans-(±)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzene-acetamide methane sulfonate (U50488H; RBI; 50 or 100 µM). The mixed opioid receptor antagonist, naloxone hydrochloride (0.2 mg/kg iv), was used to
reverse the effects of mu and delta agonists. Because kappa agonist
injection did not affect corneal unit activity, no kappa-selective
antagonist was employed. All drugs used for local microinjection were
dissolved in artificial cerebrospinal fluids (150 mM NaCl, 2.6 mM KCl,
1.3 mM CaCl2, and 1.8 mM
MgCl2), respectively. Solutions of morphine
sulfate (0.5-3 mg/kg) and naloxone hydrochloride (0.2 mg/kg iv) that
were given systemically were diluted in physiological saline.
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Data analysis
Neural recording data were acquired at a resolution of 1-s bins
and displayed by LabVIEW as peristimulus time histograms (PSTHs), exported to a spreadsheet, and analyzed off-line. Since the background activity of most units fluctuated during the interstimulus period between CO2 pulses, a response magnitude
(RCO2) was calculated that subtracted the
background activity prior to each pulse. 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 response to a
CO2 pulse often was seen as an early (<5 s) and
late (7-22 s) component in which only the late component was shown to
be proportional to CO2 concentration (Hirata et al. 1999). The latency for the late
RCO2 (>7 s after stimulus onset) 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). Similarly, the
response duration was defined as the time from the onset of the late
RCO2 until the value of three consecutive bins no
longer exceeded the mean +2 SD of the background activity. Average
RCO2 values for each CO2
test series (i.e., 0 and 80% CO2) under
different conditions (i.e., predrug, postdrug, postdrug plus naloxone)
were analyzed statistically by ANOVA corrected for repeated measures
(Winer 1971
), and individual comparisons were made with
the Newman-Keuls test. The present data assessed only the late
component responses to CO2 pulses (late
RCO2). A change in late
RCO2 after opioid drug administration was
considered significant if it exceeded the preopioid value by >50%.
Comparison of the frequency of occurrence of
CO2-responsive and nonresponsive units projecting
to SSN/VII or PO/ZI was determined by
2 analysis.
Histology
At the end of the experiment the animal was deeply
anesthetized with an overdose of methohexital sodium (60 mg/kg iv) and perfused through the heart with saline followed by 10% Formalin containing potassium ferrocyanide. Blocks of medulla and thalamus were
frozen, sectioned at 40 or 80 µm, respectively, and stained with
cresyl violet. Recording sites at the rostral Vi/Vc and caudal Vc/C1
transition regions and antidromic stimulation sites in thalamus and
rostral brainstem were reconstructed and drawn on a standardized series
of brain outlines adapted from the atlas of Paxinos and Watson
(1986).
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RESULTS |
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General properties of corneal units
A total of 97 rostral and 32 caudal units were tested for
responses to CO2 pulses after identification by
mechanical and electrical stimulation of the cornea. Forty-four of 97 rostral units (45.4%) and 14 of 32 caudal units (43.8%) also
responded to 80% CO2 pulses. The general
properties and frequency of occurrence of different classes of
CO2-responsive units were similar to those
reported previously (Hirata et al. 1999). Rostral units
were further classified on the basis of the stimulus-response pattern
to CO2 pulses. Type I rostral units were
encountered most often (>70% of rostral
CO2-responsive units) and displayed a progressive
increase in firing rates as CO2 concentrations
were increased (Fig. 3A). Type
II rostral units displayed an early inhibitory phase prior to an
increase in firing rate as CO2 concentration
increased (see Hirata et al. 1999
) (Fig. 8A).
Unless otherwise noted, the effects of opioid drugs were not selective
for different classes of CO2-responsive rostral units. All caudal units that responded to CO2
pulses displayed S-R functions similar to type I rostral units.
Repeated presentation of 80% CO2 test pulses (40 s duration, at 15-min intervals) did not cause significant
tachyphylaxis as seen by changes in RCO2 (14 ± 16% and 17 ± 17%, mean ± SE, P > 0.05, ANOVA, response to 1st pulse vs. 2nd pulse, for rostral,
n = 17, and caudal units, n = 10, respectively).
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Effects of microinjections of opioid receptor agonists into the caudal transition region
ROSTRAL VI/VC UNITS. Microinjection of morphine or DAMGO into the caudal transition region had similar effects on CO2-evoked responses and were analyzed as one treatment group. Seven of 27 rostral units tested after local morphine or DAMGO were enhanced (>50% change in late RCO2; range, 55-850%, P < 0.01 ANOVA, vs. 80% CO2 pre-DAMGO), 10 units had a reduced response, and 10 units were not affected (Table 1). An example of a type I rostral unit that was enhanced by local morphine is shown in Fig. 3. Opioid-induced modulation of evoked responses appeared as a change in RCO2 magnitude with no effect on response duration or latency. In initial experiments, units were tested across the full range of CO2 concentrations (0-95%) after microinjection of morphine as shown in Fig. 3. However, in most experiments in which morphine or DAMGO was injected, and in all experiments after DPDPE or U50,488H, only the responses to 0 and 80% CO2 were examined. The effects of morphine and DAMGO were reversed by antagonist naloxone (Fig. 3A, bottom panel). Although Fig. 3A (middle panel) also revealed an increase in spontaneous activity after morphine, effects on spontaneous activity were seen in only 7 of the 17 rostral units in which morphine or DAMGO altered significantly (increase or decrease of >50% vs. predrug) the response to CO2 pulses. Mu opioid-induced changes in spontaneous activity were in the same direction as that for the evoked responses in only four of seven cases. The changes in spontaneous activity began during the 5-min injection period and persisted for at least 30 min or until naloxone was given. The duration of the effect of local DAMGO on CO2-evoked responses was examined in five rostral units and persisted for at least 35 min. For the seven units enhanced by local morphine or DAMGO, four units were cornea-only cells, two units had an LTM-like RF, and one had a WDR-like RF on the eyelid. Of the 10 units depressed by local morphine or DAMGO, 2 were cornea only cells, 4 had an LTM-like RF on the eyelid, 3 had a WDR-like RF on the eyelid, and 1 had an inhibitory RF inside the nostril. These results suggested that RF properties of rostral units alone did not predict the response to opioid drugs. The recording sites for rostral units whose evoked responses were enhanced (Fig. 4, left) or depressed (right) were distributed similarly at the rostral transition region. The recording locations for units not responsive to CO2 pulses were similar to those of responsive units.
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CAUDAL VC/C1 UNITS. Microinjection of DAMGO into the caudal transition region inhibited the CO2-evoked response of each of four caudal units tested (Table 1) and reduced the spontaneous activity of three of four cells. Similarly, local injection of the delta agonist, DPDPE, inhibited the CO2-evoked activity of each of four caudal cells tested and inhibited the spontaneous activity in three of four cells. Unlike some rostral units, no caudal unit showed enhanced spontaneous or evoked activity after mu or delta opioid agonists. Local injection of the kappa agonist, U50,488H, into the caudal transition region did not affect the evoked or spontaneous activity of four caudal units tested. Recording loci of all caudal units were within the superficial laminae (I-II) approximately 4-5 mm caudal to the obex (see example in Fig. 8E).
Efferent projections
Table 2 summarizes the status of
rostral and caudal units tested for efferent projections to the
ipsilateral SSN/VII region in the rostral medulla and/or the
contralateral PO/ZI region in the diencephalon in which units were
grouped according to CO2 responsiveness.
CO2-responsive rostral and caudal units projected with similar frequency to the SSN/VII or PO/ZI regions. Rarely, rostral
(1 of 21 tested) or caudal (1 of 8 tested)
CO2-responsive units sent collateral projections
to both the SSN/VII and PO/ZI regions. By contrast, rostral and caudal
units that did not respond to CO2 pulses
displayed distinctly different projection patterns. As summarized in
Table 2, rostral units not responsive to CO2 pulses projected only to SSN/VII, and caudal units projected only to
PO/ZI. No CO2-unresponsive units (19 units
tested) sent collateral projections to both SSN/VII and PO/ZI. Overall,
2 analyses revealed that the projection
patterns for rostral and caudal CO2-responsive
units were significantly different (P < 0.001) from
unresponsive units. Representative lesion sites for antidromic
activation of rostral and caudal CO2-responsive
units (+CO2, right side of each
outline) and CO2-nonresponsive units (
CO2, left side) are shown in Fig.
7. At the PO/ZI region the majority of
antidromic sites were located in the most caudal and ventral portions
of PO. The average conduction velocity for rostral units that projected
to PO/ZI was 3.9 ± 0.9 m/s (mean ± SE; range, 7.2-1.3 m/s;
n = 6) and was significantly greater (P < 0.05, ANOVA) than that seen for caudal units (1.6 ± 0.5 m/s;
range, 2.2-0.6 m/s; n = 3). This finding suggested
that the projection of CO2-responsive caudal
units to the PO/ZI was mediated by smaller and more slowly conducting
fibers than that of rostral CO2-responsive units.
Rostral units projecting to SSN/VII had an average conduction velocity of 2.0 ± 0.3 m/s (range, 2.6-0.7 m/s; n = 10)
and was not different (P > 0.05 ANOVA) from that of
caudal units (1.6 ± 0.5 m/s; range, 2.6-1.0 m/s;
n = 3). The range of currents that activated units projecting to PO/ZI was 16-180 µA and for units projecting to SSN/VII was 10-400 µA. The effect of local opioid agonists on neural
activity was not predictive of efferent projection status. For example,
type I rostral CO2-responsive units (see Fig. 3) that were enhanced by local morphine or DAMGO projected to either the
SSN/VII or PO/ZI regions. An exception to this was the projection status of type II rostral units. These cells displayed a distinctive early inhibitory phase to CO2 pulses followed by
excitation (see Hirata et al. 1999
) (Fig.
8A). Four of six type II
rostral units projected to SSN/VII and none to the PO/ZI region. Three
of the six type II units were inhibited by local DAMGO, and three were not affected.
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Antidromic stimulation methods were used to determine whether caudal corneal units (n = 3) projected directly to the rostral transition region. Two CO2-responsive caudal units, each with a WDR-like convergent cutaneous RF, were activated optimally from the caudal-most of a two-electrode array placed in the rostral transition region as shown by the example of Fig. 8. The estimated conduction velocity for each cell was ~6 m/s based on an inter-electrode distance of 5 mm. A third caudal corneal unit that did not respond to CO2 pulses was driven orthodromically from the rostral transition region.
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DISCUSSION |
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Opioid effects on trigeminal neurons
Local administration of morphine or DAMGO into the caudal Vc/C1
transition region altered the excitability of 63% of rostral units
responsive to CO2 stimulation of cornea. These
responses were selective for the mu opioid receptor subtype since
neither delta nor kappa agonist injection into the caudal transition
region altered the spontaneous or CO2-evoked
activity of rostral units. While rostral units either were enhanced or
inhibited by mu agonists, the CO2 responses of
all caudal units were inhibited by either mu or delta agonists, and
none were affected by kappa agonists. This finding suggested the
existence of two populations of opioid-sensitive corneal units in
laminae I-II at the caudal transition region. One population of caudal
units was inhibited by a mu opioid receptor mechanism and mediated the
changes in rostral unit excitability and a second population that was
inhibited by a delta receptor mechanism and did not contribute to
changes in rostral unit excitability. These results confirm and extend
our previous reports in which intravenous morphine enhanced the
responses of many rostral units to CO2 pulses
(Hirata et al. 1999) or to electrical or noxious thermal
stimulation of the cornea (Meng et al. 1998
) while
inhibiting all caudal units.
Although it is well accepted that spinal and supraspinal mechanisms
mediate opiate analgesia (see Yaksh 1997), the mechanism for opiate analgesia in trigeminal pain is less certain. The basis for
opiate action within the trigeminal system has emphasized the caudal
portions of the Vsp, trigeminal subnucleus caudalis (Vc). Anatomical
studies have confirmed that, similar to spinal dorsal horn, laminae
I-II of Vc contains dense immunoreactivity for mu (Ding et al.
1996
), delta (Arvidsson et al. 1995
), and kappa
opioid receptor subtypes (Mansour et al. 1996
) with
weaker staining in deeper laminae and throughout the more rostral
subnuclei. Behavioral studies have shown that direct microinjection of
morphine into Vc elevated facial heat pain thresholds in awake monkeys (Oliveras et al. 1986
) and attenuated heat-
(Rosenfeld et al. 1983
) or formalin-induced face rubbing
in rats (Duale et al. 1996
). Despite compelling evidence
for Vc involvement in opiate analgesia for trigeminal pain, few studies
have examined the response of second-order trigeminal neurons to
nociceptive input after local administration of opioid drugs. In
recordings from dorso-rostral Vc (0.5-2 mm caudal to obex),
iontophoresis of DAMGO or DPDPE caused only inhibition of
N-methyl-D-aspartate (NMDA)-evoked activity of
cells with cutaneous facial RFs classified as NS, WDR, and LTM and
located within superficial laminae (Zhang et al. 1996
). Some recorded cells likely were located in lamina I, and not substantia gelatinosa (SG), since inhibition of NMDA-evoked activity by mu or
delta agonists also was seen among identified trigeminothalamic projection neurons (Wang and Mokha 1996
). Thus when
compared with the present results, nociceptive units located in
superficial laminae of rostral Vc and at the caudal Vc/C1 transition
region appear to share common response properties to opioid drugs.
In contrast, studies using in vitro slice preparations of Vc to assess
the influence of opioid receptor-selective drugs have reported
significant differences compared with those seen in the present study.
For example, among SG cells recorded from horizontal slices of Vc in
guinea pig or rat, methionine enkephalin or DAMGO hyperpolarized the
majority of neurons while DPDPE had no effect (Grudt and
Williams 1994), whereas both DAMGO and DPDPE inhibited all
caudal units tested in the present study. The basis for differences in
opioid responsiveness of Vc neurons recorded in vivo compared with
those seen in vitro are not certain. However, as discussed by
Kohno et al. (1999)
, differences in the effects of
opioid drugs on SG cells obtained from spinal cord (inhibition by mu
and delta agonists) and Vc (inhibition by mu, but not by delta
agonists) slice preparations may be due to slice orientation (e.g.,
transverse vs. horizontal). In the one study that reported mainly
excitation of SG lumbar spinal neurons by morphine, a sagittal slice
preparation was used (Magnuson and Dickenson 1991
),
suggesting that specific dendritic orientation or longitudinal
circuitry within the slice may be necessary to observe some effects of
opioid drugs. Kappa opioid receptor involvement in Vc neural activity
has not received adequate attention. The kappa agonist U69593
hyperpolarized 35% of SG cells from horizontal slices of Vc in guinea
pig (Grudt and Williams 1993
), whereas local
microinjection of the kappa opioid agonist U50,488H did not affect
rostral or caudal corneal units. Although this is the first report that
local administration of a kappa opioid agonist did not modulate natural
nociceptive input to Vc neurons, it was possible that higher doses may
have had an effect. Hylden et al. (1991)
found either
facilitation or inhibition of lamina I spinal neurons after topical
application of micromole doses of U50,488H, whereas a maximum
of 30 pmol was microinjected in the present study.
Intersubnuclear connections and opioid analgesia
Unlike in spinal cord where nociceptive fibers supply a
given body locus and terminate around a single segmental level,
trigeminal nerves supply craniofacial structures such as cornea
(Bereiter 1997; Bereiter et al. 1996
;
Lu et al. 1993
; Marfurt and Del Toro 1987
; Strassman and Vos 1993
), temporomandibular
joint region (Hathaway et al. 1995
), and transverse
sinus (Strassman et al. 1994
) and terminate at multiple
levels of the Vsp. The role of multiple representation of orofacial
structures in trigeminal pain processing is not well defined.
Interruption of connections between Vc and more rostral subnuclei of
Vsp significantly affected nociceptive activity of rostral units,
mainly in Vo (Davis and Dostrovsky 1988
;
Dostrovsky et al. 1981
; Greenwood and Sessle 1976
; Scibetta and King 1969
). Convergent lines
of evidence also suggest that longitudinal connections between the
caudal and rostral transition regions contribute to the mechanisms
underlying opioid analgesia in corneal pain. Microinjection of mu
opioid agonists into the caudal transition region modulated the
activity of 63% of CO2-responsive rostral units.
This extended initial results in which topical application of morphine
to the dorsal surface of the caudal transition region enhanced the
response of rostral corneal units to electrical or thermal stimulation
(Meng et al. 1998
). Other studies have assessed Vc
connections with subnucleus oralis (Vo) in mediating the effects of
opioid drugs in the rat. Ujihara et al. (1987)
reported
that tooth pulp input to Vo cells was inhibited by conditioning stimuli
applied to the dorsal surface of rostral Vc, an effect that was blocked
by iontophoresis of naloxone onto the Vo unit. Furthermore, tooth pulp
input to Vo was inhibited by iontophoresis of enkephalin, suggesting
that enkephalinergic neurons from Vc modulated the activity of tooth pulp-responsive Vo cells. More recently, electrical stimulation of
facial skin at C-fiber strength evoked activity in Vo units that was
reduced by systemic morphine (Dallel et al. 1996
).
Microinjection of morphine into rostral Vc, near the Vi/Vc transition
region, also prevented C-fiber input to Vo convergent units, whereas
local injection into Vo had no effect (Dallel et al.
1998
). These results support the hypothesis that
intersubnuclear connections play a critical role in opioid analgesia
for other trigeminal pain conditions as well as for corneal pain. The
exact pathway from the caudal transition region necessary for opioid
modulation of rostral unit activity is not known. A direct connection
from caudal units to the rostral transition region was suggested by
evidence that some caudal units were driven antidromically (see Fig.
8); however, the involvement of a supraspinal loop remains possible.
Indeed, some rostral units were modulated by local injection of mu
agonists into the caudal region and displayed changes in corneal
responsiveness in the opposite direction after intravenous morphine,
suggesting that multiple pathways contribute to the opioid-mediated
effects on rostral cell activity.
The basis for opioid analgesia at spinal levels has emphasized a
linkage between neurons in superficial and deep laminae. Systemic or
local administration of mu agonists enhanced the activity of neurons in
superficial laminae in spinal dorsal horn (Duggan and North
1984; Jones et al. 1990
; Sastry and Goh
1983
; Woolf and Fitzgerald 1981
). However, in
contrast to the present results, when tested over a range of doses,
enhancement of spinal laminae I-II neurons by low doses of morphine
generally gave rise to depression after higher doses (Craig and
Serrano 1994
; Dickenson and Sullivan 1986
). Thus
the progressive increase in firing rate that occurred with increasing
doses of morphine among 25-40% rostral corneal units (Hirata
et al. 1999
; Meng et al. 1998
) appeared to be a unique property of rostral corneal units not commonly seen in other
neurons of the medullary or spinal dorsal horn. A second related
observation suggesting differences in the organization of opioid
mechanisms at trigeminal versus spinal levels was the finding that
caudal cornea units located in laminae I-II were never enhanced by
morphine or DAMGO (present study; Hirata et al. 1999
;
Meng et al. 1998
). In contrast, one proposed mechanism for opioid analgesia at the spinal cord level occurs through laminae I-II neurons in which enhancement (or disinhibition) of SG cells inhibit spinothalamic tract cells in deep dorsal horn (Duggan and North 1984
; Jones et al. 1990
; Sastry
and Goh 1983
; Woolf and Fitzgerald 1981
). While
it was possible that trigeminal SG cells (i.e., small lamina II
neurons) were excluded from our analyses due to electrode sampling
bias, it remains possible that the microcircuitry connecting
superficial with deep dorsal horn neurons may be different for
trigeminal and spinal systems. On anatomical grounds it has been
suggested that the rostral transition region contain displaced SG cells
(Phelan and Falls 1989
). Thus one interpretation of the present results is that some rostral units that were enhanced by
morphine function as displaced SG cells projected back to deeper laminae of Vc to inhibit trigeminothalamic projection cells. This may
also explain why some in vitro preparations of trigeminal brain stem
have failed to find neurons with enhanced responses after DAMGO
(Grudt and Williams 1994
), since the tissue slice did
not include the ventral area containing enhanced rostral units at the
rostral transition region. Although the present results confirmed a
significant effect of delta agonists on Vc neurons (Zhang et al.
1996
), it was unlikely that delta-responsive caudal corneal
units modulate rostral unit activity and, correspondingly, unlikely
that delta opioid-mediated analgesia depends on intersubnuclear connections for expression.
Efferent projections of corneal units
The efferent projection status of rostral and caudal units was
dependent on corneal stimulus modality (i.e.,
CO2-responsive vs. unresponsive units). Rostral
or caudal units that were responsive to CO2
pulses projected with similar frequency to the PO/ZI, a region presumed
to mediate sensory-discrimination of corneal pain, or the SSN/VII, a
brain stem region necessary for somato-autonomic reflex responses to
corneal stimulation. However, among units not responsive to
CO2 pulses, rostral units projected only to SSN/VII and caudal units projected only to PO/ZI. By contrast, responsiveness to mu agonists did not predict the efferent projection status of CO2-responsive rostral units. Rostral
cells that either were enhanced or inhibited by local injection of mu
agonists into the caudal transition region projected with similar
frequency to PO/ZI or to SSN/VII. These results agree generally with a
previous report in which 37% of CO2-responsive
caudal units, although no rostral units, projected to PO/ZI
(Hirata et al. 1999). However, in that study, emphasis
was directed at determining whether rostral or caudal units projected
to ventroposteromedial n. (VPM) or nucleus submedius of thalamus, and
only three rostral units were tested for projections to PO/ZI. Rostral
and caudal units responsive to thermal and electrical stimulation also
have been tested for projections to the parabrachial/Kolliker-Fuse
nuclear complex in which no rostral units but >80% of caudal units
were antidromically driven (Meng et al. 1997
). The
finding that rostral and caudal units not responsive to
CO2 pulses (i.e., mechanical only) units have
distinctly different projection targets was not expected. The
significance of this may relate to the specialized homeostatic functions necessary to maintain visual acuity. Thus activation of
mechanical only rostral units with corneal only RFs may be sufficient
for spontaneous lacrimation and eyeblinks evoked by inputs not
necessarily perceived as painful. While
CO2-responsive cells also would evoke reflex
tearing and eyeblinks, these cells would be expected to encode noxious
inputs of other modalities such as thermal and chemical. It is
interesting to note that the two caudal units antidromically activated
from the rostral transition region were
CO2-responsive, whereas a third unit was
classified as mechanical only and was driven orthodromically from the
rostral region. This suggests that CO2-responsive
units may rely more heavily on intersubnuclear connections for full
expression of function.
Significance of rostral and caudal units for corneal pain processing
Two general concepts relevant for sensory processing may be
considered to account for second-order corneal units being clustered into a rostral and caudal cell group. First, such a dual organization may provide for parallel processing of sensory input and a level of
redundancy to ensure adequate protective reflex activity for visual
function under painful conditions. Parallel processing is supported by
the observation that corneal primary afferents that encode mechanical
only and polymodal or noxious chemical stimulus modalities terminate in
the rostral and caudal transition regions. Among cells excited by
polymodal input, e.g., CO2-responsive units, both
rostral and caudal units displayed similar encoding properties to
CO2 pulses (Hirata et al. 1999)
and projected to similar supraspinal brain regions (present study).
Alternatively, a dual organization may provide for specialization of
function. Indeed, some properties of rostral units were never seen in
caudal corneal units. For example, nearly 50% of rostral units
displayed a "cornea only" RF with no apparent convergent input from
other facial regions, whereas all caudal units were classified as WDR or NS on the basis of a cutaneous RF (Meng et al. 1997
).
A significant percentage of rostral units, but no caudal units, were
enhanced by mu opioid agonists (Hirata et al. 1999
;
Meng et al. 1998
), a finding that suggested a
specialized role in opioid analgesia and in recruitment of descending
controls. Finally, when corneal stimulus modality was taken into
account, among rostral and caudal units not activated by
CO2 pulses (i.e., mechanical only cells), each
group projected to separate supraspinal targets (SSN/VII and PO/ZI,
respectively). Specialization of function by rostral and caudal units
also may underlie recruitment of activity during different levels of
stimulus intensities. Thus as noted above, activity in mechanical only
rostral units may be sufficient to maintain spontaneous lacrimation and
eyeblinks under nonpainful conditions, whereas mechanical only caudal
units project to the thalamus to mediate a sense of corneal itch or
irritation. Early clinical reports support the notion of specialization
since after trigeminal tractotomy at the level of the obex for facial
pain, some patients reported loss of pain sensation to corneal
stimulation while retaining a sense of touch (Sjoqvist
1938
). The role of intersubnuclear connections in trigeminal
sensation may be most important during painful conditions. Such
connections may serve to recruit and organize output functions from
neurons located at different rostrocaudal levels of Vsp.
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ACKNOWLEDGMENTS |
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The authors thank A. Benetti for excellent technical assistance.
This study was supported in part by National Institute of Neurological Disorders and Stroke Grant NS-26137 to D. A. Bereiter.
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FOOTNOTES |
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Address for reprint requests: D. A. Bereiter, Brown University School of Medicine/Rhode Island Hospital, Depts. of Surgery and Neuroscience, 222 Nursing Arts Bldg., Providence, RI 02903-4970 (E-mail: DBereiter{at}lifespan.org).
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 4 February 2000; accepted in final form 11 May 2000.
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REFERENCES |
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