Separate populations of neurons in the rostral ventromedial medulla project to the spinal cord and to the dorsolateral pons in the rat

Transcription

1 Pacific University CommonKnowledge Faculty Scholarship (PHRM) School of Pharmacy 2004 Separate populations of neurons in the rostral ventromedial medulla project to the spinal cord and to the dorsolateral pons in the rat Amber V. Buhler Pacific University Herb K. Proudfit G. F. Gebhart Follow this and additional works at: Part of the Molecular and Cellular Neuroscience Commons, and the Pharmacy and Pharmaceutical Sciences Commons Recommended Citation Buhler, Amber V.; Proudfit, Herb K.; and Gebhart, G. F., "Separate populations of neurons in the rostral ventromedial medulla project to the spinal cord and to the dorsolateral pons in the rat" (2004). Faculty Scholarship (PHRM). Paper This Article is brought to you for free and open access by the School of Pharmacy at CommonKnowledge. It has been accepted for inclusion in Faculty Scholarship (PHRM) by an authorized administrator of CommonKnowledge. For more information, please contact

2 Separate populations of neurons in the rostral ventromedial medulla project to the spinal cord and to the dorsolateral pons in the rat Abstract Activation of neurons in the rostral ventromedial medulla (RVM) directly modulates spinal nociceptive transmission by projections to the spinal cord dorsal horn and indirectly by projections to neurons in the dorsolateral pons (DLP) that project to the spinal cord dorsal horn. However, it is not known whether the same neurons in the RVM produce both direct and indirect modulation of nociception. Deposits of the retrograde tracers Fluoro-Gold (FG) in the spinal cord dorsal horn and DiI in the DLP were used to determine whether the same RVM neurons project to both of these regions. Only 0.9+/-0.1% of RVM neurons retrogradely labeled with Fluoro-Gold from the spinal cord were also labeled with DiI placed in the DLP. In addition, spinally projecting RVM neurons were significantly larger than RVM neurons that project to the DLP. Finally, spinally projecting neurons were found predominantly on the midline and within the RVM; neurons that project to the DLP had a wider distribution and were present both within and outside of the RVM. Thus, separate and morphologically distinct populations of RVM neurons appear to modulate nociception by direct and indirect descending pathways. Keywords neurons, neural pathways, medulla oblongata, nociception, pons, rvm Disciplines Molecular and Cellular Neuroscience Pharmacy and Pharmaceutical Sciences This article is available at CommonKnowledge:

3 Brain Research 1016 (2004) Research report Separate populations of neurons in the rostral ventromedial medulla project to the spinal cord and to the dorsolateral pons in the rat A.V. Buhler*, H.K. Proudfit, G.F. Gebhart Department of Pharmacology, Carver College of Medicine, The University of Iowa, Bowen Science BLD 2-351, Iowa City, IA 52242, USA Accepted 13 April 2004 Available online Abstract Activation of neurons in the rostral ventromedial medulla (RVM) directly modulates spinal nociceptive transmission by projections to the spinal cord dorsal horn and indirectly by projections to neurons in the dorsolateral pons (DLP) that project to the spinal cord dorsal horn. However, it is not known whether the same neurons in the RVM produce both direct and indirect modulation of nociception. Deposits of the retrograde tracers Fluoro-Gold (FG) in the spinal cord dorsal horn and DiI in the DLP were used to determine whether the same RVM neurons project to both of these regions. Only 0.9 F 0.1% of RVM neurons retrogradely labeled with Fluoro-Gold from the spinal cord were also labeled with DiI placed in the DLP. In addition, spinally projecting RVM neurons were significantly larger than RVM neurons that project to the DLP. Finally, spinally projecting neurons were found predominately on the midline and within the RVM; neurons that project to the DLP had a wider distribution and were present both within and outside of the RVM. Thus, separate and morphologically distinct populations of RVM neurons appear to modulate nociception by direct and indirect descending pathways. D 2004 Elsevier B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation: anatomy and physiology Keywords: Retrograde tracer; DiI; Fluoro-Gold 1. Introduction Neurons of the rostral ventromedial medulla (RVM), including serotonergic neurons, project to the dorsal horn of the spinal cord [12] and produce both inhibition [12] and facilitation [16] of nociception. A population of RVM neurons also projects to the dorsolateral pons (DLP) [5,8], an area which includes a group of noradrenergic neurons, the A7 cell group, that innervates the spinal cord dorsal horn [6]. Electrical or chemical stimulation of noradrenergic A7 neurons also inhibits [19,20] and facilitates [14] nociception [9]. These projections mediate part of the antinociception produced by chemical stimulation of neurons in the RVM, as this effect is attenuated by microinjection of local anesthetic into the A7 region [15]. Further, the antinociception produced by chemical stimulation of neurons in the RVM is reduced by intrathecal administration of the a-adrenoceptor antagonists phentolamine or yohimbine [4,10] or by depletion of spinal NE [17]. These findings provide the anatomical basis for contributions of both serotonergic and noradrenergic systems to antinociception produced by RVM stimulation. The purpose of this study was to determine whether the RVM neurons that project to the dorsal horn of the spinal cord also project to the DLP. This study thus examines the anatomical basis of the functional interactions among neurons in the principal brainstem pain modulatory centers, the RVM and DLP. 2. Materials and methods 2.1. Retrograde tracer deposits * Corresponding author. Tel.: ; fax: address: amber-buhler@uiowa.edu (A.V. Buhler). Five male Sprague Dawley rats (Charles River Sasco, Portage, MI; g) were deeply anesthetized with /$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi: /j.brainres

4 A.V. Buhler et al. / Brain Research 1016 (2004) ketamine (80 mg/kg) and xylazine (4 mg/kg, i.p.) and surgically prepared. The retrograde tracer Fluoro-Gold (FG, Fluorochrome, Englewood, CO; in saline) was deposited bilaterally on the surface of the L4 L6 spinal cord dorsal horn to retrogradely label spinally projecting neurons in the RVM. The FG deposit was made using 1 2 mm 2 Gel- Foam (Upjohn, Kalamazoo, MI) pledgets saturated with a 2% FG solution that were placed on the surface of the spinal cord directly over the dorsal root entry zone [7]. Silk sutures were used to close the muscle layers and the skin. A 0.5 Al volume of 0.05% DiI (Molecular Probes, Eugene, OR; in methanol) was also microinjected unilaterally into the DLP using a 33 gauge stainless steel injector inserted into a guide cannula placed immediately above the DLP using appropriate stereotaxic coordinates relative to bregma (AP 34.4 mm, ML 38.8 mm, DV 24.5 mm). Fig. 1. (A) FG deposits in transverse sections taken from the lumbar spinal cords of five rats (An1 An5). The irregular profiles indicate the extent of the FG diffusion at the site of application. (B) DiI deposits in transverse sections through the DLP of five rats (labeled An1 An5). Grey lines indicate the dense central core of injections, while black lines indicate the extent of diffusion from the site of DiI injection. The numbers under each section indicate the distance (mm) from Bregma. (C) Representative sections at and 9.8 mm from Bregma, indicating the location of several cell groups: seventh nerve (7n), dorsal cochlear nucleus (DC), gigantocellular reticular nucleus (Gi), gigantocellularis pars alpha (GiA), paragigantocellularis (LPGi), lateral superior olive (LSO), facial nucleus (7), pyramidal tract (py), nucleus raphe magnus (RMg), spinal trigeminal tract (sp5), superior paraolivary nucleus (SPO), trapezoid nucleus (Tz), anterior ventral cochlear nucleus (VCA) and posterior ventral cochlear nucleus (VCP). The RVM is defined as the triangular area within the bold lines.

5 14 A.V. Buhler et al. / Brain Research 1016 (2004) Tissue preparation A period of 7 12 days was allowed for tracer transport and rats were then deeply anaesthetized with pentobarbital (50 mg/kg) and transcardially perfused with a 4% paraformaldehyde solution. Brainstem blocks were frozen and 30 Am transverse sections were cut on a cryostat microtome. Brainstem sections were mounted on Superfrost/plus slides (Fisher Scientific, Pittsburgh, PA) and coverslipped with Fluoromount G (EMS, Ft. Washington, PA) Fluorescence microscopy and data analysis Labeled neurons were visualized using epifluorescence microscopy. The distribution of neurons retrogradely labeled from the spinal cord, from the DLP, and neurons containing both labels were determined from detailed drawings made using digital camera lucida software (Neurolucida Microbrightfield, Colchester, VT). The analysis of these labeled neurons did not use stereological methods because the aim of this study was to determine the relative numbers of RVM neurons that project to the spinal cord, to the DLP and to both regions. Twelve non-adjacent sections from each of the five brains were drawn and the neurons in each section were plotted and counted. The sections were located at the following levels relative to Bregma (mm): 11.4, 11.2, 11.0, 10.9, 10.7, 10.5, 10.3, 10.2, 10.0, 9.8, 9.6 and 9.4. Estimates of the total numbers of double-labeled neurons, DiI-labeled neurons and FG-labeled neurons were determined by plotting the locations of these neurons and counting all the labeled neurons that were present in each section within the boundaries of the RVM. The RVM was defined as a roughly triangular area that comprises the nucleus raphe magnus, nucleus raphe pallidus and medial gigantocellularis reticular nucleus, pars alpha (see Fig. 1C). The total number of single and double-labeled neurons in the 12 sections was averaged and data from all 5 animals were combined. This analysis provides a quantitative estimate of the number of retrogradely labeled and double-labeled neurons that project to the DLP or the spinal cord from several regions at different levels in the brainstem. Estimates of the size of DiI- and FG-labeled neurons were determined from randomly selected brainstem sections from one animal under 40 magnification. The length and width of each neuron was measured by drawing a calibrated line across the long and short axis of each neuron using the Fig. 2. Representative fluorescence micrographs of fusiform RVM neurons labeled by a DiI deposit in the DLP (top panels) and FG deposit in the spinal cord (bottom panels). The left and right micrographs represent two different areas within the RVM. Arrows indicate several relatively rare double-labeled neurons. Scale bar = 60 Am.

6 A.V. Buhler et al. / Brain Research 1016 (2004) Neurolucida program. Finally, the spinal cord and the dorsolateral pons were sectioned and the extent of the FG and DiI deposits were mapped on camera lucida drawings made using Neurolucida tracing software. Statistical analyses of differences in size between neuronal populations were performed using Student s t-test. Differences were deemed significant if P < Animal care and use The experimental protocols were approved by The University of Iowa Animal Care and Use Committee and were in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals. Fig. 3. The distribution of DiI, FG and double-labeled neurons in 12 transverse sections from 12 different rostro-caudal levels of the RVM in a representative animal. Neurons located within the RVM, the area enclosed by the dashed lines, were counted. Symbols: DiI-labeled = red, FG-labeled = green, doublelabeled = purple. The sections are arranged from caudal ( 11.4 mm) to rostral ( 9.4 mm) and the numbers at the lower left of each section represent the distance in millimeters from Bregma.

7 16 A.V. Buhler et al. / Brain Research 1016 (2004) Results 3.1. Location of tracer deposits Labeling of bulbospinal neurons was achieved by retrograde transport of FG from the dorsal horn of the spinal cord. The location of FG deposits in the spinal cord was determined using fluorescence microscopy. The extent of local FG diffusion is plotted for all five animals and is predominately within the dorsal half of the spinal cord (Fig. 1A). Animals with more ventral spread of FG (An2 and An4) exhibited approximately the same number of FGlabeled neurons in the RVM as those with smaller, more dorsal tracer deposits. This suggests that the FG-labeled neurons in the RVM project predominately to the dorsal horn in all animals. Although dye uptake was not bilateral in An5, the number of labeled neurons was similar to that seen in the other animals. The labeling of RVM neurons that project to the DLP was achieved by retrograde transport of DiI from the DLP. The location and extent of DiI deposits was similarly determined using fluorescence microscopy and injection sites were mapped on a representative section through the DLP (Fig. 1B). There is substantial overlap between the location of DiI deposits and spinally projecting FG-labeled neurons in the DLP; however, the majority of FG-labeled neurons are found in areas just medial to the injection sites. Representative sections at the level of the RVM are included for reference (Fig. 1C) and dye deposits were not found to passively extend to this region Distribution of RVM neurons that project to the spinal cord and the DLP The locations of FG and of DiI labeled neurons (Fig. 2) were plotted for all animals at 12 different levels of the RVM to determine the distribution of projection neurons in the rostral/caudal axis. Most of the retrogradely labeled neurons were fusiform in shape (Fig. 2). The distribution of labeled neurons from one representative animal is shown in Fig. 3 and the average number of labeled neurons from all animals is presented in Fig. 4. While spinally projecting neurons were found almost exclusively on the midline and within the RVM, DLP-projecting neurons were present both within and outside of the RVM. DLP-projecting neurons located within the RVM had a fairly homogeneous distribution. A small cluster of strongly labeled cells was also seen in all animals in the ventral cochlear nucleus contralateral to the DLP injection site. Spinally projecting neurons were most numerous at the caudal levels of the RVM, with fewer labeled neurons at more rostral levels. In contrast, DLP-projecting neurons were most numerous at rostral levels. Double-labeled neurons were rare (0.9 F 0.1% of FG-labeled neurons in the RVM) at all rostro-caudal levels, but were most frequent between 11.1 and 10.3 mm posterior to Bregma (1.1 F 0.1% of FG-labeled neurons in the RVM). The number of retrogradely labeled neurons was similar regardless of whether DiI or FG was placed in the spinal cord Size differences between spinally projecting and DLPprojecting RVM neurons Because the size of spinally projecting and DLP-projecting RVM neurons appeared to be different in all animals studied, a quantitative determination of differences in the long (length) and short (width) axes of labeled fusiform neurons was undertaken in one animal. No difference in the size (length F S.E.M. width F S.E.M., Am) of spinally projecting (26.5 F F 0.5 Am, n = 45) and DLPprojecting RVM neurons (25.0 F F 0.9 Am, n = 12) was found in two sections just caudal to the RVM ( 12.6 to 12.8 mm from Bregma; Fig. 5A). In contrast, in two sections from the caudal RVM ( 11.0 to 11.2 mm from Bregma), spinally projecting RVM neurons (34.9 F F 0.7 Am, n = 110) were significantly larger (Student s t-test, P < 0.005) than DLP-projecting RVM neurons Fig. 4. Graphical representation of the average number ( F S.E.M.) of DiI-labeled, FG-labeled and double-labeled neurons at 12 levels of the RVM from 5 animals. Because of the relatively low number of double-labeled cells, the graph represents these at 10 the actual cell counts to allow visibility of the bars.

8 A.V. Buhler et al. / Brain Research 1016 (2004) Fig. 5. Graphical representations of the size of RVM neurons that project to the spinal cord (FG-labeled) and DLP (DiI-labeled). Neuron width is plotted on the ordinate and length is plotted on the abscissa. Samples of labeled neurons in the RVM in two sections were taken from each of three different rostro-caudal levels: (A) inferior olivary nuclei ( 12.6 to 12.8 mm from Bregma), (B) caudal RVM ( 11.0 to 11.2 mm from Bregma) and (C) rostral RVM ( 9.6 to 9.8 mm from Bregma). In (C), DiIlabeled neurons have been subdivided into two groups based on their location within the trapezoid nucleus (ntz)/superior olivary nucleus (SPO) or within the remainder of the RVM. (23.7 F F 0.6 Am, n = 56; Fig. 5B). The single double-labeled neuron in these sections was Am. In two sections from the rostral RVM ( 9.6 to 9.8 mm from Bregma), spinally projecting RVM neurons (30.1 F F 0.8 Am, n = 38) were also significantly larger (Student s t-test, P < 0.005) than DLP-projecting RVM neurons (17.4 F F 0.2 Am, n = 210; Fig. 5C). These DLP-projecting RVM neurons were comprised of two distinct populations, both significantly smaller than spinally projecting RVM neurons. One population of these smaller cells was found within the region of the nucleus of the trapezoid body/superior paraolivary nucleus (15.6 F F 0.2 Am, n = 163) and the other population was located within the remainder of the RVM (23.8 F F 0.5 Am, n = 44). Both of these populations of DLP-projecting RVM neurons were significantly smaller (Student s t-test, P < 0.005) than spinally projecting RVM neurons. Although there were no double-labeled neurons within the RVM in these two sections, there were two located outside the boundaries of the RVM (34.2 F F 0.6 Am, n = 2) that are included for comparison. The differences in size between spinally projecting and DLP-projecting RVM neurons may be due solely to differences in neuron orientation to the plane of transverse section. That is, spinally projecting RVM neurons may be oriented parallel and DLP-projecting RVM neurons may be oriented perpendicular to the plane of section. This possibility was tested by measuring a sub-group of DLP-projecting cells in the caudal RVM that appeared to be oriented parallel to the plane of section. Although the average size of this sub-group of neurons was predictably larger than that of the total population of DiI-labeled cells in the RVM of these sections, these neurons (27.0 F F 0.6 Am, n = 30) were still significantly smaller (Student s t-test, P < 0.005) than similarly located FG-labeled neurons (34.9 F F 0.7 Am, n = 110). Double-labeled neurons had the same dimensions when visualized under the UV or rhodamine filters, suggesting that the difference in size between the DiI- and FG-labeled populations is not due to differences in size discrimination between the two fluorophores. To further rule out this possibility, one animal was labeled with DiI from the spinal cord and retrogradely labeled cells in the RVM were measured from one section at 11.0 mm from Bregma. DiI-labeled spinally projecting RVM neurons (31.0 F F 0.5 Am, n = 81) were not significantly different in size (Student s t-test, P>0.05) from FG-labeled spinally projecting RVM neurons from the same level in a different animal (34.9 F F 0.7 Am, n = 110) and were significantly larger (Student s t-test, P < 0.005) than DiI-labeled DLP-projecting neurons (23.7 F F 0.6 Am, n = 56). 4. Discussion Both electrical and chemical activation of neurons in the RVM modulate spinal nociceptive transmission by activation of neurons with direct projections to the spinal cord dorsal horn [12]. Activation of neurons in the RVM also produces antinociception mediated by spinally projecting noradrenergic neurons [1,2,10,11] that are located in the A7 cell group of the DLP [15]. These results indicate that

9 18 A.V. Buhler et al. / Brain Research 1016 (2004) stimulation of RVM neurons co-activates spinally projecting neurons in the RVM and DLP that together constitute a parallel descending modulatory system. Such co-activation may be produced by spinally projecting RVM neurons that also project to, and activate, spinally projecting noradrenergic A7 neurons. This possibility is supported by reports which indicate that substance P-containing RVM neurons project to the spinal cord [3] and substance P- containing RVM neurons project to the DLP [18]. RVM enkephalin-containing neurons also project to the spinal cord [3] and the DLP [8]. However, the very small number of RVM neurons found in the present study that project to both the dorsal horn and the DLP argues against this possibility and indicates that spinally projecting RVM and noradrenergic neurons are independently controlled. This conclusion is supported by evidence which indicates that microinjection of the muscarinic cholinergic agonist carbachol in the RVM activates spinally projecting A7 neurons [15], but not raphe-spinal serotonin-containing neurons [4]. The present experiments also found that spinally projecting RVM neurons were significantly larger than RVM neurons that project to the DLP. This finding further reinforces the conclusion that RVM neurons that project to the spinal cord and those that project to the DLP are two distinct populations. It is unlikely that the difference in neuron size is due to neurochemical content because, as discussed above, many of the RVM neurons that project to the spinal cord contain the same transmitters as those that project to the DLP. Another possible explanation for the difference in size is the difference in projection distance of the two populations. However, this explanation is not supported by the finding that the spinally projecting and DLP-projecting RVM neurons located just caudal to the RVM were the same size. Furthermore, previous studies have found no relationship between neuron size and axonal length in chick propriospinal neurons [13]. These experiments demonstrate that there are separate populations of projection neurons in the RVM. One population projects directly to the spinal cord dorsal horn. Another population of RVM neurons projects to the DLP and by this indirect route similarly modulates spinal nociceptive transmission. These findings suggest that the modulation of spinal nociceptive transmission produced by activation of neurons in the RVM is mediated by at least two relatively independent projections to the spinal cord: RVM neurons projecting directly to the dorsal horn, and a separate pathway from another population of RVM neurons that projects to the DLP. Acknowledgements This work was supported by NIH grants DA (GFG), DA (HKP) and T32 DK (AVB). We thank Angela Borton, Judy Choi, Matt Severidt and Cecilia Sayago for their assistance with animal surgeries and cell counting and Mike Burcham for producing the graphics. References [1] L.D. Aimone, S.L. Jones, G.F. Gebhart, Stimulation-produced descending inhibition from the periaqueductal gray and nucleus raphe magnus in the rat: mediation by spinal monoamines but not opioids, Pain 31 (1987) [2] N.M. Barbaro, D.L. Hammond, H.L. Fields, Effects of intrathecally administered methysergide and yohimbine on microstimulation-produced antinociception in the rat, Brain Research 343 (1985) [3] R.M. Bowker, K.N. Westlund, M.C. Sullivan, J.F. Wilber, J.D. Coulter, Descending serotonergic, peptidergic and cholinergic pathways from the raphe nuclei: a multiple transmitter complex, Brain Research 288 (1983) [4] M.S. Brodie, H.K. Proudfit, Antinociception induced by local injections of carbachol into the nucleus raphe magnus in rats: alteration by intrathecal injection of monoaminergic antagonists, Brain Research 371 (1986) [5] F.M. Clark, H.K. Proudfit, Projections of neurons in the ventromedial medulla to pontine catecholamine cell groups involved in the modulation of nociception, Brain Research 540 (1991) [6] F.M. Clark, H.K. Proudfit, The projection of noradrenergic neurons in the A7 catecholamine cell group to the spinal cord in the rat demonstrated by anterograde tracing combined with immunocytochemistry, Brain Research 547 (1991) [7] A.T. Hama, J.M. Fritschy, D.L. Hammond, Differential distribution of GABA A receptor subunits on bulbospinal serotonergic and nonserotonergic neurons of the ventromedial medulla of the rat, Journal of Comparative Neurology 384 (1997) [8] J.E. Holden, H.K. Proudfit, Enkephalin neurons that project to the A7 catecholamine cell group are located in nuclei that modulate nociception: ventromedial medulla, Neuroscience 83 (1998) [9] J.E. Holden, E.J. Schwartz, H.K. Proudfit, Microinjection of morphine in the A7 catecholamine cell group produces opposing effects on nociception that are mediated by alpha1- and alpha2-adrenoceptors, Neuroscience 91 (1999) [10] T.S. Jensen, T.L. Yaksh, Spinal monoamine and opiate systems partly mediate the antinociceptive effects produced by glutamate at brainstem sites, Brain Research 321 (1984) [11] T.S. Jensen, T.L. Yaksh, Comparison of antinociceptive action of morphine in the periaqueductal gray, medial and paramedial medulla in rat, Brain Research 363 (1986) [12] M.J. Millan, Descending control of pain, Progress in Neurobiology 66 (2002) [13] T. Nakasone, Relationship between neuronal size and axonal length in chick propriospinal neurons, No to Shinkei-Brain and Nerve 39 (1987) [14] K. Nuseir, H.K. Proudfit, Bidirectional modulation of nociception by GABA neurons in the dorsolateral pontine tegmentum that tonically inhibit spinally projecting noradrenergic A7 neurons, Neuroscience 96 (2000) [15] K. Nuseir, B.A. Heidenreich, H.K. Proudfit, The antinociception produced by microinjection of a cholinergic agonist in the ventromedial medulla is mediated by noradrenergic neurons in the A7 catecholamine cell group, Brain Research 822 (1999) 1 7. [16] F. Porreca, M.H. Ossipov, G.F. Gebhart, Chronic pain and medullary descending facilitation, Trends in Neurosciences 25 (2002) [17] D.J. Smith, A.A. Hawranko, P.J. Monroe, D. Gully, M.O. Urban, C.R. Craig, J.P. Smith, D.L. Smith, Dose-dependent pain-facilitatory and inhibitory actions of neurotensin are revealed by SR 48692, a nonpeptide neurotensin antagonist: influence on the antinociceptive effect of morphine, Journal of Pharmacology and Experimental Therapeutics 282 (1997)

10 A.V. Buhler et al. / Brain Research 1016 (2004) [18] D.C. Yeomans, H.K. Proudfit, Projections of substance P-immunoreactive neurons located in the ventromedial medulla to the A7 noradrenergic nucleus of the rat demonstrated using retrograde tracing combined with immunocytochemistry, Brain Research 532 (1990) [19] D.C. Yeomans, H.K. Proudfit, Antinociception induced by microinjection of substance P into the A7 catecholamine cell group in the rat, Neuroscience 49 (1992) [20] D.C. Yeomans, F.M. Clark, J.A. Paice, H.K. Proudfit, Antinociception induced by electrical stimulation of spinally projecting noradrenergic neurons in the A7 catecholamine cell group of the rat, Pain 48 (1992)