Cornea-Responsive Medullary Dorsal Horn Neurons: Modulation by Local Opioids and Projections to Thalamus and Brain Stem

Harumitsu Hirata,1 Shinichiro Takeshita,1 James W. Hu,3 and David A. Bereiter1,2

 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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 · kg-1 · 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 MOmega , 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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Fig. 1. Experimental setup for unit recording at the rostral or caudal transition region, delivery of CO2 pulses to the cornea, and microinjection of drugs into the caudal transition region.

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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Fig. 2. Example of microinjection site at the caudal caudalis/upper cervical spinal cord (Vc/C1) transition region. White arrows indicate the micropipette tract, and black arrows estimate the diffusion of dye restricted to the dorsal horn region. Injection volume, 300 nl delivered over 5 min. Calibration, 400 µm.

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 chi 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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 3. Example of a rostral unit with a cornea only receptive field (RF) that was enhanced by morphine. A: peristimulus time histograms (PSTHs) from a type I rostral unit displaying a progressive increase in firing rate with increasing concentrations of CO2 (top panel), greatly enhanced spontaneous activity and evoked responses to CO2 pulses after local microinjection of morphine (25 ng/nl; 300 nl) into caudal Vc/C1 region (middle panel), and reversal of enhanced activity after naloxone (0.2 mg/kg iv, bottom panel). B: summary of the response to CO2 pulses corrected for changes in background activity, i.e., RCO2. C: recording site located at the subnucleus interpolaris/caudalis (Vi/Vc) transition region and microinjection site centered near the lamina IV-V border at the caudal Vc/C1 transition region.

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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Table 1. Summary of effects of local microinjection of opioid receptor agonists into the caudal Vc/C1 region on rostral and caudal CO2-responsive corneal units



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Fig. 4. Recording sites for rostral units that displayed either facilitation (left side) or depression (right side) to CO2 pulses after microinjection of morphine or D-Ala, N-Me-Phe, Gly-ol-enkephalin (DAMGO) into the caudal Vc/C1 transition region were distributed similarly at the Vi/Vc transition region. Arrow indicates the location of unit shown in Fig. 3. Calibration, 1 mm.

To determine whether multiple pathways mediated the effects of mu receptor agonists on rostral units, 15 CO2-responsive cells were tested after local DAMGO and, subsequently, after systemic morphine (3 mg/kg iv). Morphine altered 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. Figure 5 shows an example of a type I unit in which the predrug responses to 80% CO2 (Fig. 5A) were depressed by local DAMGO injected into the caudal transition region and enhanced after intravenous morphine (Fig. 5B). Naloxone reversed the CO2 responsiveness of this unit to predrug levels as shown by the summary of late RCO2 values in Fig. 5C. Two additional cells were each enhanced by local DAMGO and inhibited by systemic morphine.



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Fig. 5. Example of a rostral unit that displayed opposite responses to mu opioid agonists microinjected into the caudal region vs. systemic administration. A: responses to 0 and 80% CO2 pulses. B: inhibition of CO2-evoked activity after local microinjection of DAMGO (2.4 pmol) into Vc/C1 transition region. C: 15 min after morphine (1.0 mg/kg iv) reversed the inhibition caused by local DAMGO and 15 min after naloxone (0.2 mg/kg iv) a large increase in spontaneous and CO2-evoked activity was seen. C: summary graph for this unit corrected for changes in spontaneous activity (RCO2). D: cutaneous RF for this unit was inside the tip of nose and displayed wide dynamic range (WDR)-like properties. Recording site was somewhat dorsal at the Vi/Vc transition compared with the location of most rostral corneal units.

Microinjection of the selective delta opioid agonist, DPDPE, into the caudal transition region had no effect on the spontaneous activity or the CO2-evoked responses of six rostral units tested (Table 1). In one case, a type I rostral unit, the response to 80% CO2 was enhanced after intravenous morphine while prior CO2 testing after local DPDPE had no effect. Similarly, local microinjection of the kappa opioid agonist, U50,488H, into the caudal transition region had no effect on the spontaneous activity or the CO2-evoked responses of the five rostral units tested (Table 1). In one case subsequent intravenous morphine depressed the response to 80% CO2 pulses (not shown).

Previously we reported that some rostral units that were not responsive to initial CO2 testing became responsive after intravenous morphine (Hirata et al. 1999). An example of a rostral type I unit in which the response to 80% CO2 pulses was "unmasked" after microinjection of DAMGO into the caudal transition region is shown in Fig. 6. Note that intravenous morphine also enhanced the activity of this unit (Fig. 6C). A total of 17 rostral units that were not responsive to initial CO2 testing were retested after local DAMGO, and 2 units behaved in a manner similar to the example shown. Six of the remaining 15 units also were tested after intravenous morphine, but none became responsive to CO2 pulses. Two rostral units that were initially not responsive to CO2 pulses were tested after microinjection of DPDPE or U50,488H, and neither cell became responsive.



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Fig. 6. Example of a rostral unit (type III) that became responsive to CO2 pulses only after local DAMGO injection into the caudal transition region. A: PSTHs in response to 0 and 80% CO2 pulses (15-min interpulse intervals) before drug injection. B: local injection of DAMGO (2.4 pmol in 240 nl) into the caudal transition region unmasked the response to CO2 (1st 2 80% pulses). Note that DAMGO-induced facilitation lasted <45 min since no response was seen after the 3rd CO2 pulse. C: systemic morphine (3.0 mg/kg iv) also unmasked the response to CO2 pulses and was reversed by naloxone (0.2 mg/kg iv). Note there was little spontaneous activity prior to morphine. D: WDR-like convergent cutaneous RF that included all 3 divisions of trigeminal system (shaded region). E: recording site was located at the rostral Vi/Vc transition region. Approximate spread of DAMGO within the medullary dorsal horn at the caudal transition region (shaded region) and micropipette tract (black). Calibration, 1 mm.

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, chi 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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Table 2. Summary of efferent projection status for CO2-responsive and mechanical only rostral and caudal corneal units



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Fig. 7. Antidromic projection status of rostral and caudal units in the thalamus (left panels) or brain stem (right panels) was related to CO2-responsiveness. Note that rostral and caudal units that responded to CO2 pulses (+CO2, right half of each outline) projected with similar frequency to either the contralateral posterior nucleus/zona incerta region (PO/ZI) region or the ipsilateral superior salivatory/facial nucleus (SSN/VII) region. Among units that did not respond to CO2 pulses (-CO2, left half of each outline), rostral units projected only to SSN/VII, whereas caudal units projected only to PO/ZI. The number to the right of each outline indicates anterior-posterior distance from bregma in mm. Sites that activated rostral () and caudal units () that were responsive to CO2 pulses are shown on the right side of each outline, and sites that activated non-CO2-responsive units are shown on the left. APT, anterior pretectal n.; ISN, inferior salivatory n.; MG, medial geniculate n.; mlf, med. longitudinal fasciculus; NRM, n. raphe magnus; NTS, n. tractus solitarius; p, pyramidal tr.; PO, posterior thalamic n.; SSN, superior salivatory n.; SV, sup. vestibular n.; Vo, trigeminal subnucleus oralis; Vtr, trigeminal spinal tr.; VII, facial motor n.; VPM, ventroposteromedial n.; ZI, zona incerta; 7g, genu of VII n.; 7n, facial nerve.



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Fig. 8. Example of a caudal unit that was antidromically activated from the rostral Vi/Vc transition region. A: constant latency of antidromic spikes () evoked by 3 consecutive stimuli (- - - - -). Latency, 0.8 ms. B: collision test. Cornea-induced orthodromic spike (*) blocked the occurrence of antidromically evoked spike (). C: antidromic spikes () followed high-frequency stimulation (down-arrow , 300 Hz, 0.1 ms). D: site of lowest current for antidromic activation located at the Vi/Vc transition region. Numbers within the circles indicate threshold current (µA, 0.1 ms duration). E: recording site in lamina I. F: WDR-like convergent cutaneous RF on upper and lower eyelids.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society