 |
INTRODUCTION |
The midbrain periaqueductal gray (PAG) is part of a modulatory circuit that controls spinal and trigeminal nociceptive dorsal horn neurons (Behbehani 1995
; Fields and Basbaum 1978
; Fields et al. 1991
; Gebhart 1986
; Stamford 1995
; Willis 1988
; Willis and Westlund 1997
). The PAG is divided into functional columns that are activated under different circumstances and elicit different patterns of behavior (Bandler et al. 1991
). One of these columns, the ventrolateral PAG, controls dorsal horn nociceptive neurons primarily through a relay in the rostral ventromedial medulla (RVM) (Aimone et al. 1987
; Dostrovsky et al. 1983
; Fields and Basbaum 1978
; Fields et al. 1995
). We recently demonstrated that the inhibition of dorsal horn nociceptive responses by activation of ventrolateral PAG neurons is mediated in part by endogenous opioids acting at the µ-opioid receptor (Budai and Fields 1998
). Because only partial reversal of PAG inhibition was achieved and because opioid antagonists only partially reverse the inhibition of spinal nocifensor reflexes by stimulation of the RVM (Hammond et al. 1997
; Jensen and Yaksh 1984
; Zorman et al. 1982
), it is likely that other neurotransmitters contribute significantly to the control of spinal nociceptive transmission by the PAG-RVM pathway. One likely candidate for such a role is norepinephrine acting through
2-adrenergic receptors.
There is a dense concentration of both norepinephrine (Dahlström and Fuxe 1965
) and
2-adrenergic receptors in the dorsal horn (Jones et al. 1982
; Nicholas et al. 1993a
; Roudet et al. 1993
; Unnerstal et al. 1984
; Young and Kuhar 1980
). Although there are no noradrenergic neurons in the RVM or PAG, there is evidence that brain stem noradrenergic neurons contribute to the PAG-RVM inhibition of spinal nociceptive transmission. The adrenergic innervation of the spinal cord dorsal horn arises from the A5, the locus coeruleus, and the A7 noradrenergic cell groups in the pons (Clark and Proudfit 1993
, 1988; Schroder and Skagerberg 1985
; Westlund et al. 1982
;). There is a projection from the rostral PAG to A5 (Kwiat and Basbaum 1990
), A7 and to locus coeruleus (Cameron et al. 1995
), and a dense projection from the RVM to the A7 noradrenergic cell group (Clark and Proudfit 1993
; Holden and Proudfit 1998
). Furthermore, the nucleus reticularis paragigantocellularis lateralis, which is part of the RVM, receives a major projection from the PAG and projects to the locus coeruleus (Van Bockstaele et al. 1991
).
There is behavioral and electrophysiological evidence indicating that brain stem noradrenergic neurons contribute to the pain-modulating action of the PAG-RVM-dorsal horn pathway. Direct spinal application of adrenergic agonists produces behavioral analgesia (Barbaro et al. 1985
; Reddy et al. 1980
) and inhibition of dorsal horn neurons through
2-adrenergic receptors (Calvillo and Ghignone 1986
; Jones and Gebhart 1986
; Peng et al. 1996
). Peng et al. (1996)
, using microdialysis to apply
2-adrenergic antagonists, recently provided evidence of a tonic and PAG-evoked
2-adrenoceptor-mediated inhibition of nociceptive dorsal horn neurons. However, in that study inhibition of dorsal horn nociceptive responses was elicited by electrical stimulation of the dorsal PAG, and reversal of this inhibition required doses of
2-receptor antagonists that significantly elevated background and evoked activity in the absence of PAG stimulation (Peng et al. 1996
). These studies provided suggestive evidence indicating that the PAG modulates nociceptive dorsal horn neurons in part via spinally projecting brain stem noradrenergic neurons.
The dorsal horn site of action of such descending adrenergic control is largely unknown. Iontophoretically applied norepinephrine produces a potent and selective inhibition of the nociceptive responses of dorsal horn cells (Headley et al. 1978
) with no significant change in the responses to innocuous brush or iontophoresed DL-homocysteic acid (Fleetwood-Walker et al. 1985
) suggesting a presynaptic mechanism. On the other hand, norepinephrine iontophoresis was reported to inhibit firing of dorsal horn neurons evoked either by iontophoresed glutamate (Howe and Zieglgansberger 1987
; Millar et al. 1993
; Willcockson et al. 1984
) or DL-homocysteic acid in nociceptive units (Belcher et al. 1978
), a result more consistent with a direct postsynaptic action. Iontophoretic application of the
2-adrenoceptor agonist clonidine was also reported to inhibit the responses to iontophoresed glutamate in nociceptive dorsal horn neurons (Millar et al. 1993
).
In the current studies we confirmed and extended these findings through the use of bicuculline (BIC) to stimulate neurons in the ventrolateral PAG while iontophoretically applying adrenergic antagonists at the level of the dorsal horn. We demonstrate that local release of norepinephrine mediates the inhibition by PAG neurons of dorsal horn neuronal responses to noxious heat via an
2 adrenoceptor. In addition, with iontophoretic application of excitatory amino acids (EAAs), we provide evidence that both presynaptic and postsynaptic
2 adrenoceptors can inhibit sacral nociceptive dorsal horn neurons. Finally, we demonstrate a direct
1-mediated excitatory action of norepinephrine in sacral nociceptive dorsal horn neurons.
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METHODS |
Anesthesia and surgery
Male Sprague-Dawley rats (300-450 g; Bantin and Kingman, Hayward, CA) were initially anesthetized with pentobarbital sodium (50 mg/kg ip). A catheter was inserted into an external jugular vein for supplementary anesthetic. For single-unit recordings, the sacral spinal cord was exposed by a laminectomy, and the rat was placed in a stereotaxic apparatus. Holes were drilled in the skull, and the dura was removed to allow microinjections into the PAG. The spinal cord was covered with a pool of warmed mineral oil. Body temperature was kept at 37°C by a water-heated blanket beneath the rat and an infrared heat lamp from above. Heart rate was monitored and maintained within normal limits for lightly anesthetized rats. Recordings were commenced
1 h after surgery. During the experiments, the animals were maintained in a lightly anesthetized state with a continuous intravenous infusion of methohexital sodium (0.3-0.6 ml/h of 20-mg/ml solution). The infusion rate was adjusted so that the rats showed no sign of discomfort, but the tail flick reflex could be evoked by application of noxious heat (43-45°C) to the tail.
Microinjections
Microinjections of BIC into the PAG were made through a 31-gauge stainless steel injection cannula that extended 5 mm below a 25-gauge guide cannula. BIC was freshly dissolved in physiological saline (100 ng/µl), and volumes of 0.10-0.30 µl were manually delivered from a 1.0-µl Hamilton syringe over a period of 2 min. PAG BIC was used because it does not activate fibers of passage but produces a robust, transient inhibition of the tail flick reflex via the RVM (Roychowdhury and Fields 1996
) and strongly inhibits dorsal horn nociceptive neurons (Budai and Fields 1998
; Sandkühler et al. 1989
).
Extracellular recording
Extracellular, single-unit recordings were made from neurons of the dorsal horn of the sacral spinal cord. Recording/iontophoresis electrodes were constructed from a seven-barreled array of thin wall borosilicate glass capillary tubings (1.5 mm OD, 1.12 mm ID, Frederick Haer, Bowdoinham, ME). The center barrel contained a 7-µm carbon fiber creating a low-impedance (0.4-0.8 M
at 1 kHz) recording electrode. Drugs were iontophoretically delivered from the surrounding six barrels. Action potentials were displayed on an oscilloscope, and activity of single units was isolated by using a window discriminator (BAK Electronics, Germantown, MD). Collection of experimental data as well as iontophoretic delivery of drugs were automated with a multifunction instrument control and data acquisition board (NB-MIO-16, National Instruments, Austin, TX) interfaced with a Power Macintosh 8100 computer programmed in LabVIEW (National Instruments, Austin, TX). Detailed description of data acquisition hardware and software is given elsewhere (Budai 1994
).
Single unit extracellular recordings were made from selected dorsal horn neurons responding to noxious heat delivered by a projector lamp focused on the blackened ventral surface of the tail. A thermistor probe placed in contact with the heated area was used to provide feedback control of the heat stimulus. Temperature ramps were generated from a holding temperature of 35°C to a peak of 50°C at a rate of 2°C/s. Neurons were characterized as low threshold (LT), nociceptive specific (NS), or wide dynamic range (WDR) by their responses to mechanical stimuli of increasing strength. Both innocuous (brush and pressure) and noxious (pinch and squeeze that was felt as painful by the experimenter) stimuli were applied to the excitatory receptive fields of the tail.
Microiontophoresis and drugs
Microiontophoresis was performed with a five-channel Neurophore BH-2 controller with automatic current balancing (Medical Systems, Greenvale, NY). Drug barrels of the combined recording/iontophoresis electrode contained one of the following freshly made solutions: 100 mM N-methyl-D-aspartate Na (NMDA) in 100 mM NaCl (pH 8.0), 20 mM kainic acid (KA) in 180 mM NaCl (pH 8.0), 100 mM clonidine HCl in distilled water (pH 5.7), 50 mM methoxamine HCl in 150 mM NaCl (pH 4.5), 50 mM idazoxan HCl (RX 781094) in 50 mM NaCl (pH 4), 20 mM benoxathian HCl in 180 mM NaCl (pH 4.5), 10 mM yohimbine HCl in 160 mM NaCl (pH 5), 5 mM [D-Ala2,methyl-Phe4,Gly-ol5]enkephalin (DAMGO) in 160 mM NaCl (pH 5.5), and 2% Pontamine Sky Blue in 100 mM sodium acetate. NMDA and Pontamine Sky Blue were delivered by negative currents; all other compounds were ejected by positive current. All drugs were obtained from RBI (Natick, MA) except Pontamine Sky Blue, which was purchased from BDH Chemicals (Poole, England).
Histological verification
Recording sites were marked by ejection of Pontamine Sky Blue with 1 µA negative current for 20 min. Although not all marks were found, the depth of recording was noted in all cases so that cell locations could be approximated. Sites of microinjections into the PAG or RVM were labeled by microinjections of 0.25 µl Pontamine Sky Blue dye through the injection cannula. At the end of the experiment, the animal was euthanized with an overdose of methohexital and intracardially perfused with physiological saline followed by 10% formalin. Recording and injection cannulae locations were verified histologically in 50-µm thin sections counterstained with neutral red. Positions of the Pontamine Sky Blue marks were established with the stereotaxic atlas of Paxinos and Watson (1986)
. Histologically verified brain microinjection sites and spinal cord recording sites are shown in Fig. 1.

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| FIG. 1.
Histological verification of sites of bicuculline (BIC) microinjection into the midbrain periaqueductal gray (PAG) and sites of recordings taken from the dorsal horn of the rat caudosacral spinal cord. Experiments with dorsal horn idazoxan iontophoresis are shown. The rest of the PAG injection and spinal recording sites were all in the same general distribution but not shown for clarity. * and : wide dynamic range (WDR) and nociceptive specific (NS) cells, respectively. Sections were adapted from the atlas of Paxinos and Watson (1986) .
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Data analysis
Statistical evaluations were made using the total number of spikes evoked during each epoch of excitation by heat stimuli or iontophoretic application of an excitatory compound. The background neuronal discharge was calculated by averaging a 15-s period of ongoing activity preceding each epoch of excitation, and this value was subtracted from all evoked responses. Differences in magnitude between different response epochs of a single cell were confirmed by one-factor analysis of variance (ANOVA, with Student-Newman-Keuls test for posthoc analysis) by comparing the total number of spikes per excitation period. To make data from different experiments more comparable, analysis of pooled data was done after normalizing the baseline heat-evoked response to 100%. Means ± SD of a number (n) of observations are given throughout. A P value of <0.05 was considered significant in all cases. All statistical calculations were performed with GBStat software for the Macintosh (Dynamic Microsystems, Silver Spring, MD).
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RESULTS |
Experiments were carried out on a total of 89 noxious heat-responsive spinal dorsal horn neurons in 66 lightly anesthetized rats. Neurons were located between 100 and 600 µm from the surface of the dorsal horn, as estimated by microdrive readings. The dorsal horn neurons with histologically verified locations and the locations of the BIC microinjections in the PAG are illustrated in Fig. 1. All cells had excitatory receptive fields located on the tail. On the basis of their responses to mechanical stimuli of increasing intensity, including innocuous brush, pressure, noxious pinch, and squeeze, 72 of 89 cells were characterized as wide dynamic range (WDR; responding to both innocuous and noxious peripheral stimuli) neurons, whereas the remaining 17 cells were identified as nociceptive specific (NS; activated by noxious peripheral stimulation) neurons. By using a temperature ramp from 35 to 50°C at a rate of 2°C/s to stimulate their cutaneous receptive fields in the tail, these neurons produced a stable baseline response to heat stimuli repeated at 4-min intervals as shown in Fig. 2A.

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| FIG. 2.
Ratemeter recordings. A: effects of BIC microinjection into the PAG on the responses of a sacral spinal dorsal horn neuron to noxious heat delivered to the cutaneous receptive field on the tail. B-D: inhibition by PAG-BIC was reversed by iontophoretic application of 2-adrenoceptor antagonists idazoxan (ejected at 30 nA for 30 s) or yohimbine (50 nA for 60 s) but not by the 1-adrenoceptor antagonist benoxathian (30 nA, 60 s). Experiments were carried out in 4 separate animals.
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Reversal of PAG-evoked spinal inhibition by
2-adrenoceptor antagonists
Fifteen nanograms of BIC was microinjected into the midbrain PAG in 41 lightly anesthetized animals while extracellular recordings were taken from nociceptive dorsal horn neurons in the caudosacral region of the spinal cord. PAG-BIC injections caused a significant and long-lasting inhibition of responses to noxious heat in the majority (38/41) of the recorded dorsal horn neurons (Fig. 2A). Two cells showed an increased response to heat after PAG-BIC, whereas the responses in one neuron remained unaffected by PAG-BIC. Significant inhibition was taken as a minimum 25% decrease in the total number of spikes per heat stimulation epoch compared with the mean of three pre-BIC injection control responses. This minimum effect size was chosen arbitrarily to have a magnitude of inhibition sufficient to allow significant reversal with iontophoresis of adrenergic antagonists. The inhibition reached its maximum at 15-20 min and lasted for 30-60 min. Occasionally, neurons did not recover from the PAG-BIC-evoked inhibition during the entire length of the recording session. Drug effects were evaluated with the fourth and fifth responses after BIC administration, which occur at the approximate peak of the inhibitory effect. Within this time window the PAG-BIC inhibition of dorsal horn responses was to a mean of 37 ± 19% (n = 8) of the pre-BIC control (Fig. 3). Local iontophoresis of the selective
2-adrenoceptor antagonist idazoxan near the recorded dorsal horn cells partially reversed PAG-BIC-evoked spinal inhibition (Fig. 2B). Because greater amounts of the iontophoresed idazoxan by itself increased the heat-evoked responses of the dorsal horn cells (Fig. 5B), iontophoresis parameters were selected in a preliminary experiment so that the ejected idazoxan did not have a facilitatory effect in the absence of PAG-BIC. Thus, with ejection currents ranging from 20 to 30 nA for 30 s, idazoxan significantly reduced the PAG-BIC-induced spinal inhibition as illustrated in Fig. 2B. On the basis of the data pooled from 15 experiments, after PAG-BIC application and in the presence of idazoxan, a mean 82 ± 22% of the pre-BIC response was recorded (Fig. 3). Histological verification of the PAG-BIC injection sites as well as recording sites in the dorsal horn for the idazoxan experiments are shown in Fig. 1.

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| FIG. 3.
Pooled response data illustrating the magnitude of the reversal of PAG-BIC-evoked inhibition by spinal iontophoresis of idazoxan, yohimbine, or benoxathian. Adrenergic compounds were applied iontophoretically as shown in Fig. 2. The total numbers of spikes per heat stimulation period are shown as mean percent of pre-BIC control in the absence (open columns) and presence of 2- or 1-adrenoceptor antagonists (shaded columns). Data represent the average of n experiments ± SD. Asterisks indicate significant difference from the BIC only experiment by analysis of variance (ANOVA); **P < 0.01.
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| FIG. 5.
A: antagonism by idazoxan (iontophoresed at 30 nA for 30 s) of the inhibitory effects of clonidine (20 nA, 30 s) on heat-related responses. B: dose-related effects of iontophoretically applied idazoxan on responses of a dorsal horn neuron activated by repetitive noxious heating of the cutaneous receptive field. Idazoxan was ejected at 90 nA for 5, 15, and 30 s as shown.
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Because idazoxan can interact with the nonadrenergic imidazoline I2 receptors, another nonimidazoline
2-adrenoceptor antagonist, yohimbine, was also tested. A partial to full reversal of PAG-BIC inhibition was observed after iontophoresis of yohimbine (Fig. 2C). Restoration of the heat response to a mean 86 ± 23% of the pre-BIC control (n = 4) was produced by yohimbine (Fig. 3). In contrast to the effects of
2-adrenoceptor antagonists, iontophoretic application of the selective
1-adrenoceptor antagonist benoxathian had no effect on the PAG-BIC inhibition (Figs. 2D and 3). Current and duration for benoxathian iontophoresis were selected to be sufficient to prevent methoxamine's facilitatory effects, as shown in Fig. 6C. These results strongly support the conclusion that the
2 adrenoceptor is necessary for the PAG-BIC-evoked inhibition.

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| FIG. 6.
Ratemeter recordings illustrating experiments with the 1-adrenoceptor agonist methoxamine and antagonist benoxathian, respectively, on the responses of dorsal horn neurons to EAAs and noxious peripheral heat. A: responses to NMDA (iontophoresed at 55 nA for 5 s) or kainic acid (KA; 28 nA, 5 s) were increased in approximately one-half of the neurons, whereas the heat-evoked responses remained unchanged in more than one-half of the neurons after methoxamine iontophoresis (30 nA, 15 s). B: selective 1-adrenoceptor antagonist benoxathian (60 nA, 30 s) had no significant effect on cell excitation by any stimulus modality. C: effects of methoxamine (30 nA, 15 s) were antagonized by benoxathian (60 nA, 30 s).
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Opposite effects of
2-adrenoceptor antagonist idazoxan and agonist clonidine on responses of dorsal horn neurons
In an effort to gain further insight into the mechanism of
-adrenergic control of dorsal horn pain transmission, we examined the effects of iontophoretically applied
-adrenergic agents on the responses of nociceptive dorsal horn neurons to the EAAs, NMDA, or kainic acid and to peripheral noxious heat. Iontophoresis of idazoxan at relatively high currents (60-100 nA for 30 s) led to an increase in the baseline heat as well as NMDA and kainic acid-evoked responses in the same neurons (Fig. 4A). In contrast, the
2-adrenoceptor agonist clonidine, when applied at relatively low iontophoretic currents (15-30 nA), produced a pronounced inhibition of the heat-evoked responses with only marginal effects on EAA-evoked activity (Fig. 4B). When applied at higher currents (50-80 nA), clonidine inhibited both EAA- and heat-evoked responses (Figs. 5A and 7C). These effects of clonidine were selective for
2-adrenergic receptors as they were completely antagonized by idazoxan in all neurons tested (Fig. 5A). Statistical analysis of pooled data from several experiments revealed a significant and selective inhibition of noxious heat-related responses at low clonidine ejection currents, whereas iontophoresed idazoxan at relatively high ejection currents caused a significant increase in both EAA- and heat-evoked responses (Fig. 4C).

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| FIG. 4.
Ratemeter recordings showing the opposite effects of 2-adrenoceptor antagonist idazoxan (A) vs. the agonist clonidine (B) on the responses of a dorsal horn neuron to iontophoresed excitatory amino acids (EAAs), N-methyl-D-aspartate (NMDA, ejected at 33 nA for 5 s), and kainic acid (KA, 16 nA for 5 s) and noxious heat to the cutaneous receptive field. Note the general potentiation by idazoxan (iontophoresed at 70 nA for 60 s) using longer duration application than required to block PAG-BIC-evoked inhibition. Clonidine (20 nA, 10 s) had a more potent inhibitory effect on the heat-evoked responses than EAA-evoked responses. Pooled response data (C) show the effects of iontophoresed idazoxan or clonidine on the responses of dorsal horn neurons to application of NMDA or KA by iontophoresis or to noxious heating of the cutaneous receptive field. Calculations were made on the basis of the total number of spikes per excitation period and shown as mean percent of preadrenergic manipulation. Data represent the average of n experiments ± SD *P < 0.05, **P < 0.01 denotes significant differences from control (100%), P < 0.05 indicates significantly decreased heat responses as compared with NMDA and KA responses by ANOVA.
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| FIG. 7.
Representative experiments showing the effects of iontophoretically applied -adrenergic drugs on a dorsal horn neuron that was excited by iontophoresed NMDA (ejected at 45 nA for 5 s) and kainic acid (KA, 22 nA, 5 s) and noxious stimulation of the receptive field. A: general excitatory effect of the activation of 1-adrenoceptors by methoxamine (40 nA, 20 s). B: blockade of the 2-adrenoceptors with idazoxan (30 nA, 30 s) also resulted in an enhancement of the evoked neuronal firing, whereas (C) activation of the 2-adrenoceptors by clonidine (25 nA for 15 s) profoundly inhibited the responses to noxious heat. Recordings A-C were taken from the same neuron at ~30-min intervals.
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Facilitation of dorsal horn neurons by
1 adrenoceptors
The effects of
1-adrenoceptor activation were tested by iontophoresis of methoxamine, a selective
1-adrenoceptor agonist (Trendelenberg 1972
) near dorsal horn neurons that were repetitively excited with iontophoretic application of EAAs (NMDA and kainic acid) and noxious heating of their cutaneous receptive field. Methoxamine caused an increase in the kainic acid-evoked responses in 11 of 15 neurons tested (Fig. 6A). In three of these neurons methoxamine increased kainate responses only, whereas in three other neurons enhanced kainate responses were accompanied by significantly elevated NMDA and heat responses as shown in Fig. 6A. Responses to NMDA were increased or decreased in roughly equal numbers of neurons (Table 1). Noxious heat-evoked cell firing remained at control level in 8 of 15 neurons, whereas a significant increase was observed in 5 units in the presence of methoxamine (Table 1). Although pooled data suggest a link between the effect of methoxamine application on NMDA and heat response, we found no consistent correlation. The
1-adrenoceptor antagonist benoxathian had no effects by itself but antagonized the facilitatory effects of methoxamine (Fig. 6, B and C). Enhancement of EAA and heat-evoked neuronal firing by methoxamine and idazoxan and inhibition by clonidine of noxious heat responses were observed in those neurons (n = 3) for which a complete data set could be obtained (Fig. 7). In these neurons, activation of
1 adrenoceptors by methoxamine increased both heat and EAA responses, whereas activation of
2 adrenoceptors by clonidine, when applied at low dose, inhibited responses to noxious heat significantly more than responses to EAAs.
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TABLE 1.
Effects of the 1-adrenoceptor agonist methoxamine on the responses of dorsal horn neurons to iontophoresed excitatory amino acids or to peripheral noxious heat
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DISCUSSION |
This study confirms and extends in several ways previous work indicating that norepinephrine acting through the
2 adrenoceptor contributes to the descending inhibitory control by neurons in the PAG of nociceptive transmission in the spinal cord dorsal horn (Cahusac et al. 1995
; Calvillo and Ghignone 1986
; Reddy et al. 1980
). By using BIC to activate neurons in the ventrolateral PAG, our data provide direct evidence that PAG neurons, as opposed to fibers of passage, engage noradrenergic projections to the dorsal horn to produce an
2-mediated inhibitory effect. Our experiments also provide definitive data implicating the
2 adrenoceptor in the PAG-evoked inhibition. First, both idazoxan and yohimbine markedly reduce the PAG-BIC inhibitory effect. This is critical because yohimbine, which shares idazoxan's
2-antagonist action, has no imidazoline antagonism. Second, ejection currents for the
2 antagonists used to reduce the PAG-BIC inhibition were just sufficient to block clonidine's inhibitory effect but had no effect on equivalent inhibition produced by the µ-opioid receptor agonist DAMGO, thus demonstrating the specificity of the antagonism. Third,
2-antagonist ejection currents were used that did not produce a change in baseline or heat-evoked activity in the absence of PAG-BIC; thus the reversal of inhibition cannot be attributed to a subtractive effect of baseline enhancement.
The current studies also provide new information on the dorsal horn site of action of the PAG-evoked,
2-mediated inhibition. Because PAG-BIC produced no consistent changes in EAA-evoked activity while markedly decreasing noxious heat-evoked responses (Budai and Fields 1998
), our data indicate that the
2-adrenoceptor-mediated descending inhibitory effect is directed primarily to the inputs to the nociceptive neurons that we have recorded.
One possibility is that descending inhibitory control by norepinephrine targets the terminals of primary afferent nociceptors. Several lines of evidence are consistent with this view. The monoamine transmitter norepinephrine is found in high concentration in the superficial laminae of the spinal cord (Dahlström and Fuxe 1965
; Kuraishi et al. 1983
). This distribution parallels that of adrenoceptors in the spinal gray matter, as demonstrated by autoradiography (Roudet et al. 1993
; Young and Kuhar 1980
), ligand-binding techniques (Jones et al. 1982
; Unnerstal et al. 1984
), and recently by in situ hybridization (Nicholas et al. 1993b
). Neurons in dorsal root ganglia that give rise to primary afferent fibers contain all three
2-adrenergic receptor subtypes (
2a,
2b, and
2c) mRNAs (Gold et al. 1997
; Nicholas et al. 1993b
, 1996
). The superficial layers of the spinal dorsal horn, where nociceptive primary afferent fibers terminate most densely, contain dense
2-adrenoceptor binding, whereas only a small number of dorsal horn cells contain
2-adrenoceptor mRNA. An alternative possibility is that the detection of
2-adrenergic receptors in some dorsal horn neurons may require more sensitive techniques such as those used by Gold et al. (1977). Norepinephrine or clonidine significantly reduce the evoked release of glutamate from spinal cord synaptosomes (Kamisaki et al. 1993
) and the release of substance P-like material and calcitonin gene-related peptide (CGRP) from spinal cord slices (Bourgoin et al. 1993
). Because CGRP is restricted to the terminals of primary afferents in the spinal cord, this result directly supports the idea that norepinephrine targets this site. Iontophoretically applied norepinephrine (Belcher et al. 1978
; Fleetwood-Walker et al. 1985
; Headley et al. 1978
) produces a potent and selective inhibition of the nociceptive responses of dorsal horn cells with no statistically significant change in the responses to innocuous brush or iontophoresed DL-homocysteic acid. This selectivity is consistent with a presynaptic site of action. The
2-selective agonist clonidine mimicked these effects, whereas the
1 agonist phenylephrine and the
agonist isoprenaline did not. Antagonism of the norepinephrine effect by yohimbine and idazoxan but not by the potent
1-selective antagonists WB4101 and prazosin also supports the involvement of an
2 receptor (Fleetwood-Walker et al. 1985
).
It is important to point out that our results do not conclusively implicate the primary afferent terminal as the site of the observed descending
2-mediated inhibition. Other possible targets for this inhibitory effect include the somata or terminals of relay interneurons in the superficial dorsal horn. In fact, in in vitro studies, North and Yoshimura (1984)
demonstrated that neurons in the substantia gelatinosa of the adult rat are hyperpolarized by norepinephrine acting through an
2 receptor. Substantia gelatinosa neurons are directly contacted by primary afferent nociceptors and serve as excitatory relays to neurons in other laminae (Light and Kavookjian 1988
; Light and Perl 1979a
,b
; Willis and Coggeshall 1991
; Willis and Westlund 1997
).
One of the most intriguing findings in this study is the apparently direct
1-adrenoceptor-mediated excitatory effect. Although there are other reports of noradrenergic excitatory effects on dorsal horn neurons (Howe and Zieglgansberger 1987
; Jones 1992
; Millar and Williams 1989
; North and Yoshimura 1984
; Peng et al. 1996
; Todd and Millar 1983
), the current study provides the first evidence that the effect is a direct
1-mediated postsynaptic action and that it is exerted on the same neurons whose response to noxious stimulation is inhibited through an
2-adrenoceptor mechanism. We found no evidence for a tonic or PAG-elicited
1 adrenergic effect because the
1-antagonist benoxathian did not alter baseline firing, heat-evoked excitation, or PAG-BIC-evoked inhibition. However, a descending
1-mediated excitatory effect was recently reported after opioid injection into the A7 region (Holden and Proudfit 1997
). Neurons in the A7 region could be excited by PAG-BIC via the RVM (Holden and Proudfit 1998
).
Electron microscopic studies revealed that axonal boutons directly contact the somata and/or dendrites of lamina I, IV, and V spinothalamic tract neurons. Catecholaminergic boutons appose the somata and dendrites of intracellularly filled spinothalamic tract cells characterized as high-threshold and wide dynamic range neurons (Westlund et al. 1990
). These observations clearly indicate a direct innervation of spinothalamic tract neurons by catecholaminergic neurons, providing an anatomic substrate for a postsynaptic adrenergic excitatory effect on nociceptive dorsal horn neurons. However, amines were also reported to inhibit spinothalamic tract neurons and reduce their responses to glutamate (Willcockson et al. 1984
).
It is puzzling that the same neurotransmitter can potentially have opposing actions on the firing of a neuron. One interesting possibility is that different populations of brain stem noradrenergic neurons target the
1 and
2 receptors in the dorsal horn. In fact, Proudfit and colleagues have come to the same conclusion based on anatomic (Holden and Proudfit 1998
) and behavioral studies (Holden and Proudfit 1997
). They demonstrated that different pharmacological manipulations in the A7 region of the brain stem can have either an
1-mediated excitatory effect or an
2-mediated inhibitory effect on spinally mediated behavioral responses to noxious stimulation.
In summary, the present experiments established that PAG neurons inhibit nociceptive dorsal horn neurons through an
2-adrenoceptor mechanism. Our evidence indicates that the location of the receptor is presynaptic to the recorded neurons. We also demonstrated that norepinephrine can directly enhance EAA-evoked excitation of the same nociceptive dorsal horn neurons whose inputs are inhibited by an
2-adrenoceptor mechanism.