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Descending modulation of dorsal horn biogenic amines as determined by in vivo dialysis
Neuroscience Letters, 108 (1990) 231-236
231
Elsevier Scientific Publishers Ireland Ltd. NSL 06542
Descending modulation of dorsal horn biogenic amines as determined by in vivo dialysis R a y m o n d H. Abhold 1 and Robert M. Bowker 2 ~Department of Biology, California State University at Fresno, Fresno, CA 93740-0073 (U.S.A.) and 2Department of Anatomy, Michigan State University, East Lansing, M148824 (U.S.A.)
(Received 20 April 1989;Revisedversion received 23 August 1989;Accepted23 August 1989) Key word~." Norepinephrine;Serotonin; Spinal cord; Nucleus raphe magnus; Nucleus gigantocellularis;
Microdialysis Bipolar concentric stimulating electrodes were placed medially within the nucleus raphe magnus and laterally within the nucleus gigantocellularis. The levels of norepinephrine (NE), serotonin (5-HT), and 5-hydroxyindolaceticacid (5-HIAA)within the dorsal horn of the spinal cord were measured before and after electrical stimulation of these brainstem nuclei using in vivodialysiscoupledwith high pressure liquid chromatography and electrochemical detection. Stimulation medially significantlyincreased the levels of all three amines. While simulation laterally also increased NE, both 5-HT and 5-HIAAwere significantly reduced. The relevance of these findingsto descendingmodulation of ascending nociceptiveneural activity within the dorsal horn is discussed.
Nociception appears to be consequent to communication between peripheral afferent fibers and ascending neuronal pathways within the spinal cord, such as the spinothalamic and spinoreticular tracts. Though this neural activity may be controlled at any of the synaptic junctions in the ascending pathways, studies on periaqueductal gray stimulation-induced analgesia have led to the conclusion that direct modulation of nociceptive information occurs within the dorsal horn of the spinal cord [2]. Two descending projections that appear to be particularly effective in modulating this activity arise from (1) the serotonergic neurons of the medullary raphe and adjacent ventral brainstem and (2) the noradrenergic neurons of the pons (A5-A7 cell group). Direct stimulation of the neurons within the nucleus raphe magnus (NRM) and adjacent nucleus gigantocellularis (NGC) can increase threshold to pain and induce apparent analgesia [7]. Anatomical studies have demonstrated that the efferent projections from these nuclei are principally serotonergic in nature, though peptidergic projections both with and without colocalized serotonin (5-HT) have been described [1]. Inhibition of pain transmission may also be achieved by activation of noradrenerCorrespondence." R.H. Abhold, Department of Biology,California State Universityat Fresno, Fresno, CA 93740-0073, U.S.A.
0304-3940/90/$ 03.50 © 1990Elsevier ScientificPublishers Ireland Ltd.
232
gic pathways as well as by iontophoretic application of norepinephrine (NE) into the dorsal horn [4]. That serotonergic and noradrenergic pathways function at the level of the spinal cord is substantiated by evidence that the analgesia induced by stimulation of the periaqueductal gray can be attenuated by spinal application of phentolamine and methylsergide [2]. However, complete attenuation of the antinociceptive response could only be obtained with the simultaneous application of both antagonists. The modulation of nociception by descending serotonergic and noradrenergic fibers may itself be indirectly mediated. Such an intermediary role has been proposed for enkephalinergic interneurons within the dorsal horn since the microinjection of morphine into the dorsal horn induces analgesia and naloxone blocks both morphine and stimulation-induced analgesia [9]. Several groups have reported measuring the release of 5-HT and NE into intrathecal superfusates of the spinal cord subarachnoid space in response to the stimulation of serotonergic and noradrenergic descending pathways [3, 10]. It is not known, however, whether the results of these studies accurately describe corresponding activity within the spinal cord. Though increased levels of 5-hydroxyindoleacetic acid (5-HIAA) within the dorsal horn have been measured by in vivo voltammetry in response to stimulation of the NRM [5], it is not clear whether the increase reflects the release and/or metabolism of 5-HT or the direct release of the 5-HT metabolite itself. In the present study, the stimulated release of 5-HT and NE directly into the dorsal horn was examined using in vivo dialysis coupled with high pressure liquid chromatographic separation and electrochemical detection (HPLC ECD).
0.5
/
/
/
~j
0
mm
r '\
\
Fig. l. Diagramatic representation of dialysis probe placement and dimensions within the dorsal horn of the spinal cord. The stainless-steel body of the probe is colored dark gray whereas the light gray represents epoxy cement. That portion of the probe through which dialysis occurs is uncolored and is approximately 500 ,um in length• The tip of the outlet tubing is positioned medially within the clear area and above the cement that seals the end of the dialysis tubing•
233 The dialysis probes were constructed according to the methods outlined by Robinson and Wishaw [6] with the following modifications: (1) inlet tubing - fused silica capillary tubing (75 pm i.d., 150/lm o.d., Polymicro Technologies), (2) probe body - 29 gauge stainless-steel tubing (175/lm i.d., 330/lm o.d.), (3) dialysis tubing - regenerated cellulose hollow fiber dialysis tubing (9000 MWCO, 150/~m i.d., 175/~m o.d., Spectrum Medical Industries) with a functional length of 500 pm, (4) outlet tubing - Microbore polyimide tubing (75/2m i.d., 115/lm o.d., Polymicro Technologies), and (5) inlet and outlet tubing were joined to the body of the probe by means of Intramedic PE 20 (380/~m i.d., 1090/~m o.d.) polyethylene tubing. Fig. l shows a diagramatic representation of this probe and its dimensions relative to the dialysis site. The efficiency of amine diffusion through the dialysis fiber when expressed as mean _ S.E.M. percent recovery ofNE, 5-HIAA and 5-HT was 18.4_ 1.1, 15.4_+0.5, and 13.7_ 0.8, respectively. Male Sprague-Dawley rats (350-450 g) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a stereotaxic apparatus. Concentric bipolar stimulating electrodes (0.3 mm diameter) were stereotaxically placed within the NRM (A/P -3.4 mm, M/L 0.0 ram, D/V 0.0 mm, relative to the interaural line) or within the NGC (A/P - 2 . 4 mm, M/L 1.0 mm, D/V 0.0 mm). Noradrenergic fiber bundles descending through the NGC have been previously visualized by tyrosine-hydroxylase immunocytochemistry (Bowker, unpublished observations). On electrical stimulation, a Grass model $9 stimulator was used to generate 100/iA, 100 j~s pulses at a frequency of l0 Hz and for a total period of l0 min. After exposing the lumbar and sacral levels by means of a dorsal laminectomy, the spinal cord was rigidly fixed in an elevated position by clamps on the spinal processes. The dura mater was opened and functioning dialysis probes were inserted bilaterally into the dorsal horn to an approximate depth of 1.0 mm using a micromanipulator and microscopic visualization. The dialysis probes were connected to 2.5 ml gas-tight Hamilton syringes mounted on a Harvard infusion pump which delivered the microdialysis buffer to the probes at 2.13/~l/min. This buffer had an adjusted pH of approximately 6.0 at room temperature and consisted of 150 mM NaC1, 3 mM KCI, 2 mM CaCl2, and l mM MgCI2 dissolved in HPLC grade water. After 2 h equilibration, samples were collected at 20 min intervals into 0.5 ml polypropylene microfuge tubes containing 50/~l HPLC-ECD mobile phase (see below). Before and after each stimulation 2-3 samples were obtained in order to establish a baseline. Samples were analyzed by HPLC -ECD within 1 h of collection. On HPLC-ECD analysis, the entire sample was injected directly onto a 250 x 4.6 mm ODS reverse phase column (Cis, 5/1m). The mobile phase contained 100 mM citrate, 75 mM NaH2PO4, 0.88 mM heptanesulfonate, 0.1 mM EDTA, 12% MeOH, and sufficient 6 N NaOH to adjust the apparent pH to 4.2. This buffer was preoxidized just prior to sample injection (+0.4 V) and immediately after column separation (+ 0.01 V) in order to maximize electrochemical detection. Detection of biogenic amines within the column effluent was performed by working electrode (+0.30 V) in line with an ESA amperometric controller (ESA, Inc., Bedford, MA). Standard curves ranging from 0.01 to 1.0 pmol were used for peak identification and quantifi-
234 cation. Statistical significance was d e t e r m i n e d using paired t-tests a n d was accepted at P < 0 . 0 1 . After receiving an overdose of anesthesia, each a n i m a l was sequentially perfused with physiologic saline a n d 2 4 % glutaraldehyde in 100 m M s o d i u m phosphate, pH 7.4. After isolation, the spinal cord was sectioned o n a freezing m i c r o t o m e at 90 ltm a n d then c o u n t e r stained with t h i o n i n for histologic verification of p r o b e placement. The effects of s t i m u l a t i o n medially within the N R M a n d laterally within the N G C are s h o w n in Table I. W h e n c o m p a r i n g the basal level o f 5 - H T as a percent of the total i n d o l a m i n e m e a s u r e d (5-HT + 5 - H I A A ) , 6 a n i m a l s exhibited very low levels of 5-HT whereas a n o t h e r 6 exhibited very high levels. Despite this difference, however, the responses of both groups were qualitatively the same. U n d e r c o n d i t i o n s when basal 5-HT levels were low (3.3 + 0.3% of total indolamine), s t i m u l a t i o n medially produced significant increases in N E (18.1 +2.3%), 5 - H I A A (21.1 _2.1%), a n d 5-HT (103.0 + 16.0%). T h o u g h s t i m u l a t i o n laterally also p r o d u c e d a significant increase in N E (91.2+6.3%), decreases in 5 - H I A A (11.0+2.4%) a n d 5-HT (43.4_+3.1%) were measured. Similar changes to these were observed when basal levels of 5-HT were high (96.5+0.2% of total indolamine). T h a t is, s t i m u l a t i o n medially significantly increased N E (16.9+1.4%), 5 - H I A A (38.9_+3.6%), a n d 5-HT (28.0_+4.6%), while
TABLE I BASAL AND STIMULATED LEVELS OF NE, 5-HIAA AND 5-HT WITHIN THE DORSAL HORN AS MEASURED BY IN VIVO DIALYSIS COUPLED WITH HPLC ECD % 5-HT=percent contribution of serotonin (5-HT) to total indolamine measured. Midline-stimulating electrode placement into the nucleus raphe magnus. Lateral = stimulating electrode placement into the nucleus gigantocellularis. Baseline(Base) and stimulated (St±m) levelsof norephinephrine (NE), 5-HIAA and 5-HT were measured and are expressed as pmol/20 min. %= Stim represented as a percent of Base. Each value repesents the mean ±S.E.M. of 6 independent determinations. %5-HT
3.3 +0.3
Amine
NE 5-HIAA 5-HT
96.5 +0.2
NE 5-HIAA 5-HT
Midline
Lateral
Base
Stim
%
Base
Stim
%
101.3 ± 8.1 1098.9 +83.5 32.9 ± 3.0
119.6" _+ 9.8 1327.5" _+62.3 65.7* i 5.9
118.1 ± 2.3 121.1 ± 2.1 203.0 ±16.0
92.0 + 3.9 1132.7 -+8Y4 42.7 _ 6.9
175.3" ± 6.4 1002.3" +93.7 24.3* ± 4.1
191.2 +_6.3 89.0 _+2.4 56.6 _+3.1
56.9 ± 2.9 13.3 + 0.8 356.0 ±24.5
66.5* i 3.2 18.5* _+ 1.2 451.4* ±20.4
116.9 -+ 1.4 [38.9 _+ 3.6 128.0 ± 4.6
59.8 +_ 2.7 15.5 ± 0.9 446.6 +23.2
119.0" i 8.7 12.2* i 0.7 309.6* -+22.8
198.7 __+9.9 79.0 ± 1.0 69.1 -+2.3
*Significantlydifferent from Base, P< 0.01.
235 stimulation laterally increased NE (98.7+9.9%) and decreased both 5-HIAA (21.0__ 1.0%) and 5-HT (30.9+2.3%). In the present study, basal levels of NE, 5-HT and 5-HIAA were measured in the dorsal horn of the spinal cord using in vivo dialysis coupled with HPLC-ECD. These levels were significantly increased by electrical stimulation of the NRM. Though stimulation of the NGC also increased NE, both 5-HT and 5-HIAA were significantly reduced. The amines measured by these methods were ultimately determined to be authentic by comparison of samples, standards, and samples spiked with standards. Finding relatively low basal levels of 5-HT in proportion to the total indolamine measured is consistent with previous studies using similar methodologies [3, 8]. The mechanism by which half of the experimental trials resulted in a higher proportion of 5-HT is not clear. While destruction of terminal fields by movement of the spinal cord relative to the dialysis probe may release 5-HT, such movement was mimimal even when examined under high magnification with a surgical microscope. Furthermore, the total amount of indolamine as well as the levels of both NE and 5-HIAA were significantly reduced rather than elevated when basal 5-HT levels were high. These latter findings would also argue against high basal levels of 5-HT resulting from higher neuronal activity. Neither could we establish a correlation between the basal level of 5-HT and the placement of the dialysis probe within the dorsal horn, i.e. superficial versus deep laminar placement. This conclusion is supported by the absence of trials measuring intermediate or equivalent levels of 5-HT and 5-HIAA. Finally, both 5-HT and 5-HIAA have been previously measured in superfusates of the spinal cord subarachnoid space [3, 10]. It has been shown that the addition of a selective inhibitor of 5-HT reuptake (fluoxetine) into the superfusate reduces the basal level of 5-HIAA and increases that of 5-HT [3]. This has led to the suggestion that the observed 5-HIAA results from the reuptake and metabolism of synaptically released 5-HT. In the present study, therefore, it is possible that high levels of 5-HT accompanied by low levels of 5-HIAA may result from the reduced reuptake and/or intracellular metabolism of 5-HT. Whatever the mechanism, it is clear that differences in the basal levels of 5-HT and 5-HIAA did not appreciably influence the response to electrical stimulation. Demonstrating that stimulation of the NRM increases 5-HT and 5-HIAA is consistent with anatomical evidence that serotonergic neurons of this nucleus project to the spinal cord. These results also corroborate previous reports of stimulationinduced 5-HT and 5-HIAA release as measured in intrathecal superfusates [3]. Such demonstrations of 5-HT release into the dorsal horn during NRM stimulation are significant in that the 5-HT released is believed to mediate antinociception. Support for this idea is provided by studies in which iontophoresis of 5-HT into the dorsal horn inhibited neuronal activation by painful stimuli [4]. Furthermore, these effects of 5-HT were inhibited by blocking the 5-HT receptor with methylsergide [2] and potentiated by inhibiting 5-HT reuptake with fluoxetine [3]. The actions of 5-HT may itself be mediated, in part, by interneurons within the dorsal horn. Recent attention has focused on enkephalinergic interneurons since the application of morphine in the vicinity of dorsal horn neurons induces analgesia and since naloxone inhibits both
236
NRM stimulation-induced analgesia as well as the analgesia resulting from the spinal application of morphine [10]. However, naloxone does not completely block stimulation-induced analgesia. Increased NE release in response to NRM stimulation has been previously reported [3]. This increase in NE levels does not apparently result from activation of noradrenergic fibers within this nucleus nor from activation of fibers of passage. These authors have suggested that stimulation of the NRM activates projections to noradrenergic perikarya which, in turn, send projections to the spinal cord. While the exact location of these noradrenergic neurons remains to be established, those from the pons (AS-AT cell group) appear to be particularly effective in modulating pain transmission within the dorsal horn. That complete attenuation of the antinociceptive effects of periaqueductal gray stimulation could only be obtained with the simultaneous application of both phentolamine and methylsergide [2] suggests that noradrenergic pathways may induce analgesic effects independent of those produced by 5-HT. In the present study, stimulation in the region of noradrenergic fibers within the NGC significantly increased NE above baseline values and simultaneously decreased the levels of both 5-HT and 5-HIAA. The increase in NE supports anatomical evidence indicating that the noradrenergic fibers passing through the NGC terminate in the dorsal horn of the spinal cord. While the decrease in indolamines is not entirely understood, such results may reflect NE-mediated presynaptic inhibition of serotonergic terminals within the dorsal horn. I Bowker, R.M., Westlund, K.L., Sullivan, M.C., Wilber, J.F. and Coulter, J.D., Descending serotonergic, peptidergic and cholinergic pathways from the raphe nuclei: a multiple transmitter complex, Brain Res., 288 (1983) 33 48. 2 Hammond, D.L. and Yaksh, T.L., Antagonism of stimulation-produced antinociception by intrathecal administration of methylsergide or phentolamine, Brain Res., 298 (1984) 329 337. 3 Hammond, D.L., Tyce, G.M. and Yaksh, T.L., Eltlux of 5-hydroxytryptamine and noradrenaline into spinal cord superfusates during stimulation of the rat medulla, J. Physiol. (Lond.), 359 (1985) 151 162. 4 Headley, P.M., Duggan, A.W. and Griersmith, B.T., Selective reduction by noradrenaline and 5-hydroxytryptamine of nociceptive responses of cat dorsal horn neurons, Brain Res., 145 (1978) 185 189. 5 Rivot, J.P., Chiang, C.Y. and Besson, J.M., Increase of serotonin metabolism within the dorsal horn of the spinal cord during nucleus raphe magnus stimulation, as revealed by in vivo electrochemical detection, Brain Res., 238 (1982) 117 126. 6 Robinson, T.E. and Wishaw, I.Q., Normalization of extracellular dopamine in striatum following recovery from a partial unilateral 6-OHDA lesion of the substantia nigra: a microdialysis study in freely moving rats, Brain Res., 450 (1988) 209 224. 7 Satoh, M., Akaike, A., Nakazawa, T. and Takagi, H., Evidence for involvement of separate mechanisms in the production of analgesia by electrical stimulation of the nucleus reticularis paragigantocellularis and nucleus raphe magnus in the rat, Brain Res., 194 (1980) 525 529. 8 Sorkin, L.S., Steinman, J.L., Hughes, M.G., Willis, W.D. and McAdoo, D.J., Microdialysis recovery of serotonin released in spinal cord dorsal horn, J. Neurosci. Methods, 23 (1988) 131 138. 9 Yaksh, T.L., Spinal opiate analgesia: characteristics and principles of action, Pain, 1l (1981) 293 346. 10 Yaksh, T.L. and Tyce, G.M., Resting and K +-evoked release of serotonin and norepinephrine in vivo from the rat and cat spinal cord, Brain Res., 192 (1980) 133- 146.
231
Elsevier Scientific Publishers Ireland Ltd. NSL 06542
Descending modulation of dorsal horn biogenic amines as determined by in vivo dialysis R a y m o n d H. Abhold 1 and Robert M. Bowker 2 ~Department of Biology, California State University at Fresno, Fresno, CA 93740-0073 (U.S.A.) and 2Department of Anatomy, Michigan State University, East Lansing, M148824 (U.S.A.)
(Received 20 April 1989;Revisedversion received 23 August 1989;Accepted23 August 1989) Key word~." Norepinephrine;Serotonin; Spinal cord; Nucleus raphe magnus; Nucleus gigantocellularis;
Microdialysis Bipolar concentric stimulating electrodes were placed medially within the nucleus raphe magnus and laterally within the nucleus gigantocellularis. The levels of norepinephrine (NE), serotonin (5-HT), and 5-hydroxyindolaceticacid (5-HIAA)within the dorsal horn of the spinal cord were measured before and after electrical stimulation of these brainstem nuclei using in vivodialysiscoupledwith high pressure liquid chromatography and electrochemical detection. Stimulation medially significantlyincreased the levels of all three amines. While simulation laterally also increased NE, both 5-HT and 5-HIAAwere significantly reduced. The relevance of these findingsto descendingmodulation of ascending nociceptiveneural activity within the dorsal horn is discussed.
Nociception appears to be consequent to communication between peripheral afferent fibers and ascending neuronal pathways within the spinal cord, such as the spinothalamic and spinoreticular tracts. Though this neural activity may be controlled at any of the synaptic junctions in the ascending pathways, studies on periaqueductal gray stimulation-induced analgesia have led to the conclusion that direct modulation of nociceptive information occurs within the dorsal horn of the spinal cord [2]. Two descending projections that appear to be particularly effective in modulating this activity arise from (1) the serotonergic neurons of the medullary raphe and adjacent ventral brainstem and (2) the noradrenergic neurons of the pons (A5-A7 cell group). Direct stimulation of the neurons within the nucleus raphe magnus (NRM) and adjacent nucleus gigantocellularis (NGC) can increase threshold to pain and induce apparent analgesia [7]. Anatomical studies have demonstrated that the efferent projections from these nuclei are principally serotonergic in nature, though peptidergic projections both with and without colocalized serotonin (5-HT) have been described [1]. Inhibition of pain transmission may also be achieved by activation of noradrenerCorrespondence." R.H. Abhold, Department of Biology,California State Universityat Fresno, Fresno, CA 93740-0073, U.S.A.
0304-3940/90/$ 03.50 © 1990Elsevier ScientificPublishers Ireland Ltd.
232
gic pathways as well as by iontophoretic application of norepinephrine (NE) into the dorsal horn [4]. That serotonergic and noradrenergic pathways function at the level of the spinal cord is substantiated by evidence that the analgesia induced by stimulation of the periaqueductal gray can be attenuated by spinal application of phentolamine and methylsergide [2]. However, complete attenuation of the antinociceptive response could only be obtained with the simultaneous application of both antagonists. The modulation of nociception by descending serotonergic and noradrenergic fibers may itself be indirectly mediated. Such an intermediary role has been proposed for enkephalinergic interneurons within the dorsal horn since the microinjection of morphine into the dorsal horn induces analgesia and naloxone blocks both morphine and stimulation-induced analgesia [9]. Several groups have reported measuring the release of 5-HT and NE into intrathecal superfusates of the spinal cord subarachnoid space in response to the stimulation of serotonergic and noradrenergic descending pathways [3, 10]. It is not known, however, whether the results of these studies accurately describe corresponding activity within the spinal cord. Though increased levels of 5-hydroxyindoleacetic acid (5-HIAA) within the dorsal horn have been measured by in vivo voltammetry in response to stimulation of the NRM [5], it is not clear whether the increase reflects the release and/or metabolism of 5-HT or the direct release of the 5-HT metabolite itself. In the present study, the stimulated release of 5-HT and NE directly into the dorsal horn was examined using in vivo dialysis coupled with high pressure liquid chromatographic separation and electrochemical detection (HPLC ECD).
0.5
/
/
/
~j
0
mm
r '\
\
Fig. l. Diagramatic representation of dialysis probe placement and dimensions within the dorsal horn of the spinal cord. The stainless-steel body of the probe is colored dark gray whereas the light gray represents epoxy cement. That portion of the probe through which dialysis occurs is uncolored and is approximately 500 ,um in length• The tip of the outlet tubing is positioned medially within the clear area and above the cement that seals the end of the dialysis tubing•
233 The dialysis probes were constructed according to the methods outlined by Robinson and Wishaw [6] with the following modifications: (1) inlet tubing - fused silica capillary tubing (75 pm i.d., 150/lm o.d., Polymicro Technologies), (2) probe body - 29 gauge stainless-steel tubing (175/lm i.d., 330/lm o.d.), (3) dialysis tubing - regenerated cellulose hollow fiber dialysis tubing (9000 MWCO, 150/~m i.d., 175/~m o.d., Spectrum Medical Industries) with a functional length of 500 pm, (4) outlet tubing - Microbore polyimide tubing (75/2m i.d., 115/lm o.d., Polymicro Technologies), and (5) inlet and outlet tubing were joined to the body of the probe by means of Intramedic PE 20 (380/~m i.d., 1090/~m o.d.) polyethylene tubing. Fig. l shows a diagramatic representation of this probe and its dimensions relative to the dialysis site. The efficiency of amine diffusion through the dialysis fiber when expressed as mean _ S.E.M. percent recovery ofNE, 5-HIAA and 5-HT was 18.4_ 1.1, 15.4_+0.5, and 13.7_ 0.8, respectively. Male Sprague-Dawley rats (350-450 g) were anesthetized with chloral hydrate (400 mg/kg, i.p.) and mounted in a stereotaxic apparatus. Concentric bipolar stimulating electrodes (0.3 mm diameter) were stereotaxically placed within the NRM (A/P -3.4 mm, M/L 0.0 ram, D/V 0.0 mm, relative to the interaural line) or within the NGC (A/P - 2 . 4 mm, M/L 1.0 mm, D/V 0.0 mm). Noradrenergic fiber bundles descending through the NGC have been previously visualized by tyrosine-hydroxylase immunocytochemistry (Bowker, unpublished observations). On electrical stimulation, a Grass model $9 stimulator was used to generate 100/iA, 100 j~s pulses at a frequency of l0 Hz and for a total period of l0 min. After exposing the lumbar and sacral levels by means of a dorsal laminectomy, the spinal cord was rigidly fixed in an elevated position by clamps on the spinal processes. The dura mater was opened and functioning dialysis probes were inserted bilaterally into the dorsal horn to an approximate depth of 1.0 mm using a micromanipulator and microscopic visualization. The dialysis probes were connected to 2.5 ml gas-tight Hamilton syringes mounted on a Harvard infusion pump which delivered the microdialysis buffer to the probes at 2.13/~l/min. This buffer had an adjusted pH of approximately 6.0 at room temperature and consisted of 150 mM NaC1, 3 mM KCI, 2 mM CaCl2, and l mM MgCI2 dissolved in HPLC grade water. After 2 h equilibration, samples were collected at 20 min intervals into 0.5 ml polypropylene microfuge tubes containing 50/~l HPLC-ECD mobile phase (see below). Before and after each stimulation 2-3 samples were obtained in order to establish a baseline. Samples were analyzed by HPLC -ECD within 1 h of collection. On HPLC-ECD analysis, the entire sample was injected directly onto a 250 x 4.6 mm ODS reverse phase column (Cis, 5/1m). The mobile phase contained 100 mM citrate, 75 mM NaH2PO4, 0.88 mM heptanesulfonate, 0.1 mM EDTA, 12% MeOH, and sufficient 6 N NaOH to adjust the apparent pH to 4.2. This buffer was preoxidized just prior to sample injection (+0.4 V) and immediately after column separation (+ 0.01 V) in order to maximize electrochemical detection. Detection of biogenic amines within the column effluent was performed by working electrode (+0.30 V) in line with an ESA amperometric controller (ESA, Inc., Bedford, MA). Standard curves ranging from 0.01 to 1.0 pmol were used for peak identification and quantifi-
234 cation. Statistical significance was d e t e r m i n e d using paired t-tests a n d was accepted at P < 0 . 0 1 . After receiving an overdose of anesthesia, each a n i m a l was sequentially perfused with physiologic saline a n d 2 4 % glutaraldehyde in 100 m M s o d i u m phosphate, pH 7.4. After isolation, the spinal cord was sectioned o n a freezing m i c r o t o m e at 90 ltm a n d then c o u n t e r stained with t h i o n i n for histologic verification of p r o b e placement. The effects of s t i m u l a t i o n medially within the N R M a n d laterally within the N G C are s h o w n in Table I. W h e n c o m p a r i n g the basal level o f 5 - H T as a percent of the total i n d o l a m i n e m e a s u r e d (5-HT + 5 - H I A A ) , 6 a n i m a l s exhibited very low levels of 5-HT whereas a n o t h e r 6 exhibited very high levels. Despite this difference, however, the responses of both groups were qualitatively the same. U n d e r c o n d i t i o n s when basal 5-HT levels were low (3.3 + 0.3% of total indolamine), s t i m u l a t i o n medially produced significant increases in N E (18.1 +2.3%), 5 - H I A A (21.1 _2.1%), a n d 5-HT (103.0 + 16.0%). T h o u g h s t i m u l a t i o n laterally also p r o d u c e d a significant increase in N E (91.2+6.3%), decreases in 5 - H I A A (11.0+2.4%) a n d 5-HT (43.4_+3.1%) were measured. Similar changes to these were observed when basal levels of 5-HT were high (96.5+0.2% of total indolamine). T h a t is, s t i m u l a t i o n medially significantly increased N E (16.9+1.4%), 5 - H I A A (38.9_+3.6%), a n d 5-HT (28.0_+4.6%), while
TABLE I BASAL AND STIMULATED LEVELS OF NE, 5-HIAA AND 5-HT WITHIN THE DORSAL HORN AS MEASURED BY IN VIVO DIALYSIS COUPLED WITH HPLC ECD % 5-HT=percent contribution of serotonin (5-HT) to total indolamine measured. Midline-stimulating electrode placement into the nucleus raphe magnus. Lateral = stimulating electrode placement into the nucleus gigantocellularis. Baseline(Base) and stimulated (St±m) levelsof norephinephrine (NE), 5-HIAA and 5-HT were measured and are expressed as pmol/20 min. %= Stim represented as a percent of Base. Each value repesents the mean ±S.E.M. of 6 independent determinations. %5-HT
3.3 +0.3
Amine
NE 5-HIAA 5-HT
96.5 +0.2
NE 5-HIAA 5-HT
Midline
Lateral
Base
Stim
%
Base
Stim
%
101.3 ± 8.1 1098.9 +83.5 32.9 ± 3.0
119.6" _+ 9.8 1327.5" _+62.3 65.7* i 5.9
118.1 ± 2.3 121.1 ± 2.1 203.0 ±16.0
92.0 + 3.9 1132.7 -+8Y4 42.7 _ 6.9
175.3" ± 6.4 1002.3" +93.7 24.3* ± 4.1
191.2 +_6.3 89.0 _+2.4 56.6 _+3.1
56.9 ± 2.9 13.3 + 0.8 356.0 ±24.5
66.5* i 3.2 18.5* _+ 1.2 451.4* ±20.4
116.9 -+ 1.4 [38.9 _+ 3.6 128.0 ± 4.6
59.8 +_ 2.7 15.5 ± 0.9 446.6 +23.2
119.0" i 8.7 12.2* i 0.7 309.6* -+22.8
198.7 __+9.9 79.0 ± 1.0 69.1 -+2.3
*Significantlydifferent from Base, P< 0.01.
235 stimulation laterally increased NE (98.7+9.9%) and decreased both 5-HIAA (21.0__ 1.0%) and 5-HT (30.9+2.3%). In the present study, basal levels of NE, 5-HT and 5-HIAA were measured in the dorsal horn of the spinal cord using in vivo dialysis coupled with HPLC-ECD. These levels were significantly increased by electrical stimulation of the NRM. Though stimulation of the NGC also increased NE, both 5-HT and 5-HIAA were significantly reduced. The amines measured by these methods were ultimately determined to be authentic by comparison of samples, standards, and samples spiked with standards. Finding relatively low basal levels of 5-HT in proportion to the total indolamine measured is consistent with previous studies using similar methodologies [3, 8]. The mechanism by which half of the experimental trials resulted in a higher proportion of 5-HT is not clear. While destruction of terminal fields by movement of the spinal cord relative to the dialysis probe may release 5-HT, such movement was mimimal even when examined under high magnification with a surgical microscope. Furthermore, the total amount of indolamine as well as the levels of both NE and 5-HIAA were significantly reduced rather than elevated when basal 5-HT levels were high. These latter findings would also argue against high basal levels of 5-HT resulting from higher neuronal activity. Neither could we establish a correlation between the basal level of 5-HT and the placement of the dialysis probe within the dorsal horn, i.e. superficial versus deep laminar placement. This conclusion is supported by the absence of trials measuring intermediate or equivalent levels of 5-HT and 5-HIAA. Finally, both 5-HT and 5-HIAA have been previously measured in superfusates of the spinal cord subarachnoid space [3, 10]. It has been shown that the addition of a selective inhibitor of 5-HT reuptake (fluoxetine) into the superfusate reduces the basal level of 5-HIAA and increases that of 5-HT [3]. This has led to the suggestion that the observed 5-HIAA results from the reuptake and metabolism of synaptically released 5-HT. In the present study, therefore, it is possible that high levels of 5-HT accompanied by low levels of 5-HIAA may result from the reduced reuptake and/or intracellular metabolism of 5-HT. Whatever the mechanism, it is clear that differences in the basal levels of 5-HT and 5-HIAA did not appreciably influence the response to electrical stimulation. Demonstrating that stimulation of the NRM increases 5-HT and 5-HIAA is consistent with anatomical evidence that serotonergic neurons of this nucleus project to the spinal cord. These results also corroborate previous reports of stimulationinduced 5-HT and 5-HIAA release as measured in intrathecal superfusates [3]. Such demonstrations of 5-HT release into the dorsal horn during NRM stimulation are significant in that the 5-HT released is believed to mediate antinociception. Support for this idea is provided by studies in which iontophoresis of 5-HT into the dorsal horn inhibited neuronal activation by painful stimuli [4]. Furthermore, these effects of 5-HT were inhibited by blocking the 5-HT receptor with methylsergide [2] and potentiated by inhibiting 5-HT reuptake with fluoxetine [3]. The actions of 5-HT may itself be mediated, in part, by interneurons within the dorsal horn. Recent attention has focused on enkephalinergic interneurons since the application of morphine in the vicinity of dorsal horn neurons induces analgesia and since naloxone inhibits both
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NRM stimulation-induced analgesia as well as the analgesia resulting from the spinal application of morphine [10]. However, naloxone does not completely block stimulation-induced analgesia. Increased NE release in response to NRM stimulation has been previously reported [3]. This increase in NE levels does not apparently result from activation of noradrenergic fibers within this nucleus nor from activation of fibers of passage. These authors have suggested that stimulation of the NRM activates projections to noradrenergic perikarya which, in turn, send projections to the spinal cord. While the exact location of these noradrenergic neurons remains to be established, those from the pons (AS-AT cell group) appear to be particularly effective in modulating pain transmission within the dorsal horn. That complete attenuation of the antinociceptive effects of periaqueductal gray stimulation could only be obtained with the simultaneous application of both phentolamine and methylsergide [2] suggests that noradrenergic pathways may induce analgesic effects independent of those produced by 5-HT. In the present study, stimulation in the region of noradrenergic fibers within the NGC significantly increased NE above baseline values and simultaneously decreased the levels of both 5-HT and 5-HIAA. The increase in NE supports anatomical evidence indicating that the noradrenergic fibers passing through the NGC terminate in the dorsal horn of the spinal cord. While the decrease in indolamines is not entirely understood, such results may reflect NE-mediated presynaptic inhibition of serotonergic terminals within the dorsal horn. I Bowker, R.M., Westlund, K.L., Sullivan, M.C., Wilber, J.F. and Coulter, J.D., Descending serotonergic, peptidergic and cholinergic pathways from the raphe nuclei: a multiple transmitter complex, Brain Res., 288 (1983) 33 48. 2 Hammond, D.L. and Yaksh, T.L., Antagonism of stimulation-produced antinociception by intrathecal administration of methylsergide or phentolamine, Brain Res., 298 (1984) 329 337. 3 Hammond, D.L., Tyce, G.M. and Yaksh, T.L., Eltlux of 5-hydroxytryptamine and noradrenaline into spinal cord superfusates during stimulation of the rat medulla, J. Physiol. (Lond.), 359 (1985) 151 162. 4 Headley, P.M., Duggan, A.W. and Griersmith, B.T., Selective reduction by noradrenaline and 5-hydroxytryptamine of nociceptive responses of cat dorsal horn neurons, Brain Res., 145 (1978) 185 189. 5 Rivot, J.P., Chiang, C.Y. and Besson, J.M., Increase of serotonin metabolism within the dorsal horn of the spinal cord during nucleus raphe magnus stimulation, as revealed by in vivo electrochemical detection, Brain Res., 238 (1982) 117 126. 6 Robinson, T.E. and Wishaw, I.Q., Normalization of extracellular dopamine in striatum following recovery from a partial unilateral 6-OHDA lesion of the substantia nigra: a microdialysis study in freely moving rats, Brain Res., 450 (1988) 209 224. 7 Satoh, M., Akaike, A., Nakazawa, T. and Takagi, H., Evidence for involvement of separate mechanisms in the production of analgesia by electrical stimulation of the nucleus reticularis paragigantocellularis and nucleus raphe magnus in the rat, Brain Res., 194 (1980) 525 529. 8 Sorkin, L.S., Steinman, J.L., Hughes, M.G., Willis, W.D. and McAdoo, D.J., Microdialysis recovery of serotonin released in spinal cord dorsal horn, J. Neurosci. Methods, 23 (1988) 131 138. 9 Yaksh, T.L., Spinal opiate analgesia: characteristics and principles of action, Pain, 1l (1981) 293 346. 10 Yaksh, T.L. and Tyce, G.M., Resting and K +-evoked release of serotonin and norepinephrine in vivo from the rat and cat spinal cord, Brain Res., 192 (1980) 133- 146.