Neurokinin A coexists with substance P and serotonin in ventral medullary spinally projecting neurons of the rat

Peptides, Vol. 15, No. 6, pp. 1003-1011, 1994 1994 ElsevierScienceLtd Printed in the USA.All rights reserved 0196-9781/94 $6.00 + .00

Pergamon 0196-9781(94)E0069-H

Neurokinin A Coexists With Substance P and Serotonin in Ventral Medullary Spinally Projecting Neurons of the Rat KRISTINA

NEVIN,

HUANG

ZHUO

AND

CINDA

J. H E L K E l

Department o f Pharmacology and Neuroscience Program, Uniformed Services University o f the Health Sciences, Bethesda, M D 20814 R e c e i v e d 18 J a n u a r y 1994 NEVIN, K., H. ZHUO AND C. J. HELKE. Neurokinin A coexists with substance P and serotonin in ventral medullary spinally projecting neurons of the rat. PEPTIDES 15(6) 1003-1011, 1994.--The coexistence of neurokinin A (NKA) with substance P (SP) and serotonin (5-HT) in ventral medullary neurons of the parapyramidal region and nucleus raphe pallidus of the rat was studied using multiple immunofluorescence labeling. Nearly all of the NKA-immunoreactive (IR) cells in the parapyramidal region and raphe pallidus were SP-IR and 5-HT-IR, whereas about 70% of the SP-IR neurons and about 60% of the 5-HT-IR neurons contained NKA-IR. There were no apparent differences in the patterns of coexistence between parapyramidal and raphe pallidus neurons. NKA-IR neurons, which colocalized SP-IR and 5-HT-IR, were studied for projections to the lumbar and thoracic spinal cord by use of retrograde transport of fluorescent tracer. Whereas about 50% of the retrogradely labeled neurons of the parapyramidal region and raphe pallidus contained NKA-IR, nearly all of the NKA-IR neurons projected to the thoracic and lumbar spinal cord. In addition, some NKA-IR neurons in the ventral medulla were retrogradely labeled with tracer from localized injections into the thoracic intermediolateral cell column. In summary, this study demonstrated that NKA-IR is colocalized with SP-IR in bulbospinal serotonergic neurons of the parapymmidal region and raphe pallidus, which are known to regulate sensory, motor, and autonomic activities of the spinal cord. Intermediolateral cell column Thoracic spinal cord

Lumbar spinal cord

Pampyramidal region

N E U R O K I N I N A ( N K A ) (substance K), substance P (SP), and neurokinin B (NKB) are e n d o g e n o u s tachykinins and putative neurotransmitters of the m a m m a l i a n nervous system (10,20,24,27,30). Substance P is present in neurons of the ventral medulla, which project to the spinal cord, and which colocalize serotonin (5-HT) and thyrotropin-releasing hormone (TRH) (57,11,15,36). These ventral medullary neurons, located in the raphe pallidus and the parapyramidal region, play an important role in modulating sensory afferent, m o t o r output, and autonomic activity (3,17,33,34). In contrast, N K B - I R (which is encoded by a separate gene) is not found in these ventral medullary neurons (42). The presence of N K A in these ventral medullary bulbospinal neurons has not been clearly defined. Alternate R N A splicing of the primary transcripts of the SP/NKA gene results in the production of three SP-encoding m R N A s ; two o f these (/3-, and 3,-preprotachykinin m R N A ) contain the sequences for both SP

Raphe pallidus

Tachykinins

and N K A , and the third (c~-preprotachykinin m R N A ) lacks the sequence for N K A (21,31). Thus, it is anticipated that certain SP-IR neurons would colocalize N K A - I R and that nearly all N K A - I R neurons would colocalize SP-IR. A preliminary report localized N K A - I R neurons in the parapyramidal region and the raphe pallidus of the ventral medulla, and detected N K A - I R fibers and terminals in the spinal cord (13). However, it has not been established whether these N K A - I R neurons also colocalize SP-IR and 5-HT-IR, and/or project to the spinal cord. The purpose of this study was to see the extent of N K A coexistence with SP and 5-HT in the ventral medulla neurons, and to determine their projections to the spinal cord. METHOD

Animals and Surgical Procedures Experiments were conducted on 40 male Sprague-Dawley rats (350-450 g; Taconic Farms, Germantown, NY). Animals

The opinions or assertions herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD or the USUHS. The experiment reported herein was conducted according to the principles set forth in the Guidefor the Care and the Use of Laboratory Animals, Institute of Animal Resources, National Research Council, DHEW Pub. No. 74-23. Requests for reprints should be addressed to Dr. Cinda J. Helke, Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814.

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FIG. 1. Photomicrographs of the parapyramidal region (A,B) and raphe pallidus (C,D) showing adjacent sections immunostained for NKA (A,C) and SP (B,D). Arrows indicate neurons immunostained for NKA and SP. Calibration bar = 30 gm~

VENTRAL MEDULLARY NEUROKININ A NEURONS

lOO o

~

8o

~

~o

E .E_

40

~

2o 9 NKA with SP

NKA with 5-HT

NKA with SP & 5-HT

$P with NKA

5-HT with NKA

FIG. 2. Bar graph showing the percent (mean + SEM) of NKA-IR neurons that also contained SP-IR (NKA with SP), 5-HT-IR (NKA with 5-HT), or both SP- and 5-HT-IR (NKA with SP and 5-HT), SP-IR neurons that contained NKA (SP with NKA), and 5-HT neurons that contained NKA (5-HT with NKA) in the parapyramidal region (shaded bars) and raphe pallidus (hatched bars). N = 8.

were anesthetized with halothane (2% in oxygen) and treated ICV with colchicine (100/~g/10 ~tl of 0.9% NaCI). After a 24-h survival period, the rats were deeply anesthetized with chloral hydrate (400 mg/kg, IP) and perfused transcardially with phosphate-buffered saline (PBS) followed by Zamboni's fixative (pH 7.3). The brains were removed, postfixed overnight in Zamboni's fixative, and then placed in 30% sucrose in PBS overnight. Retrograde transport of fluorescent tracers was used to identify lumbar and thoracic spinal projecting neurons of the ventral medulla, as previously described (5,42). To do this, laminectomies of appropriate level of the thoracic or lumbar spinal cord were performed under halothane anesthesia. The exposed spinal cord was stabilized by clamping the spinous process to a rigid spinal frame of a Kopf stereotaxic apparatus. A total volume of 2/A of 2% FluoroGold (Fluorochrome Inc., Englewood, CO) in 0.9% NaC1 was injected bilaterally over four penetrations into the seventh-eighth segments of the thoracic spinal cord or the fourth-sixth lumbar segments of the spinal cord. The penetration was made approximately at the point of the dorsal root entrance and aimed to the ventral horn of the spinal cord. There were two injections for each penetration. After the first injection to the ventral horn, the needle was withdrawn about 0.5 mm to deposit the tracer to the dorsal portion of the spinal cord. To investigate IML-projecting neurons, 40-50 nl of undiluted rhodamine-labeled latex microspheres (Lumafluor, New City, NY) were unilaterally injected into the left IML of the third thoracic segment of the spinal cord, as previously described (36). Fortyeight hours after injection of a tracer, the rats were treated with colchicine (100 gg/10 /zl). Approximately 24 h later, the rats were perfused as described above, and brains and spinal cords were removed. The spinal cord segment injected with tracer was verified by counting the spinal cord rootlets. The location and spread of the tracers at the injection site was evaluated in multiple 30-#m spinal cord sections.

1005 The coexistence of NKA and SP in the neurons of the ventral medulla was demonstrated by using the indirect immunofluorescence method on adjacent sections. Sections were preincubated in 3% normal goat serum in PBS containing 0.3% Triton X-100 for 30 rain. One set of sections was incubated in rabbit polyclonal anti-SP serum (l:1000; lncstar Corp., Stillwater, MN) for 24 h, and incubated in lissamine rhodamine (LRSC)-conjugated goat anti-rabbit IgG (1:50; Jackson ImmunoResearch Labs, West Grove, PA) for 1 h. The adjacent sections were incubated in rabbit polyclonal anti-NKA serum (1:250; SK-6 from Dr. Susan Leeman, Boston University; preabsorbed with 100 ~tg/ml SP peptide, see below) for 24 h, then incubated in fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (l: 100; Cappel, Downington, PA) for l h. The coexistence of SP, NKA, and 5-HT in the neurons of the parapyramidai region and raphe pallidus was determined using a combination of dual-color immunohistochemistry to identify NKA-IR and 5-HT-IR cells in one section, and singlecolor immunohistochemistry on an adjacent section to identify SP-IR cells. To do this, one set of sections was preincubated in 3% normal goat serum in PBS containing 0.3% Triton X-100 for 30 min, and incubated in a cocktail of rabbit anti-NKA serum (preabsorbed with 100 #g/ml SP) and guinea pig anti-5HT serum (l:1000; Arnel, New York, NY) for 24 h. Subsequently, sections were incubated in a cocktail of FITC-conjugated goat anti-guinea pig IgG (i:100; Jackson ImmunoResearch Labs, West Grove, PA) and LRSC-conjugated donkey anti-rabbit IgG (1:50; Jackson ImmunoResearch Labs) for 1 h. A set of adjacent sections was stained for SP-IR as described above. Sections were coverslipped with PermaFluor (Immunon/ Lipshaw, Detroit, MI) and viewed with a Leitz Diaplan microscope equipped for epifluorescent illumination. FITC was visualized with the Leitz I2 filter system, the LRSC- or rhodaminelabeled latex microspheres with the N2 filter system, and FluoroGold with the A filter system. For the dual-color fluorescence, 570-nm short-pass filter was used to completely prevent visualization of the LRSC under the I2 filter system and a 495nm short-pass filter was used to prevent visualization of the FITC under the A filter system.

Data Analysis Coexistence of SP and NKA in the parapyramidal region and raphe pallidus was determined by comparing immunoreactive cells in adjacent sections. Photomicrographs of entire regions of the left and right parapyramidal region and the raphe pallidus were taken from adjacent sections. Twenty to 30 morphologically intact sections from the rostral-caudal extent of the medulla (10.8-13.3 mm posterior to bregma) of each rat were used for cell counts. Tissue landmarks were used to verify cell location. Only positively stained cells with distinct nuclei were counted. The percentages of NKA-IR cells that had SP-IR, and SP-IR ceils that contained NKA-IR were made. To determine the percentage of NKA-IR cells that had SPIR and 5-HT-IR, pictures of5-HT-IR, NKA-IR, and SP-IR cells were taken and compared, and a count of multiply immunoreactive cells was made. The percentage of NKA-IR cells that had SP/5-HT-IR and the percentage of 5-HT-IR cells that had SP/NKA-IR was determined.

Immunohistochemistry The medulla was serially sectioned at 6 #m. Tissue sections were mounted on gelatin-coated slides and were processed for fluorescence immunohistochemistry. The region of the medulla that was studied extended 10.8-13.3 mm posterior to bregma (32).

Specificity Controls The ability of our staining procedures to distinctly label NKA and SP was verified by Larsson's immunoblotting procedure (22). Nitrocellulose paper was spotted with 2 t~l of varying concentrations (1-500 ng/#l) of NKA and SP, fixed with 4% para-

O

FIG. 3. Photomicrographs of neurons in the parapyramidal region (A-D) and the mphe pallidus (E-H) retrogradely labeled with FluoroGold from the lumbar spinal cord (A,E) and immunostained for NKA (B,F). 5-HT (C,G), and SP (D,H). (A,B,C) and (E,F,G) are views of the same section, and D and H are views of the respective adjacent section. Arrows indicate FluoroGold-labeled neurons containing NKA-, SP-, and 5-HT-IR. Calibration bar = 30 t~m.

VENTRAL MEDULLARY NEUROKININ A NEURONS formaldehyde, and incubated with the primary antibodies for 24 h. The primary antibodies included polyclonal rabbit antiSP serum, polyclonal rabbit anti-NKA serum, and polyclonal rabbit anti-NKA serum preabsorbed with 100 ug/ml SP peptide (Peninsula, Belmont, CA). Immunoreactivities were visualized with the avidin-biotin-peroxidase procedure (18) using a Vectastain ABC kit (Vector Labs, Budingame, CA). The results were consistent with our previous study (12) (i.e., the rabbit anti-SP serum recognized SP, but not NKA). The rabbit anti-NKA serum recognized NKA (at 1 ng/~l) and SP (>-10 ng/#l). However, when preabsorbed with 100 #g/ml SP peptide, the SP immunorecognition of the NKA antibody was abolished whereas the NKA immunorecognition was unaffected. The anti-NKA serum was therefore routinely preabsorbed with 100 #g/ml SP peptide. The lack of nonspecific binding, the lack of inappropriate interactions in double immunofluorescence procedure, and the specificities of the SP and 5-HT primary antibodies and the secondary antibodies have been previously tested in this laboratory (12,13,35,36,38). RESULTS

Coexistence of NKA and SP Numerous NKA-IR and SP-IR neurons were found in both the parapyramidal region and raphe pallidus (Figs. 1, 3, 4). There was no preferential distribution of NKA-IR and SP-IR cells in the long axis of the parapyramidal region and raphe pallidus. In both regions, NKA-IR and SP-IR neurons were similarly distributed, although there appeared to be a greater number of SPIR cells than NKA-IR cells. In general, NKA-IR cells colocalized SP-IR, whereas many, but not all, SP-IR cells colocalized NKAIR. To investigate the extent of coexistence of NKA and SP in the neurons of the parapyramidal region and raphe pallidus, a total of 1386 and 1416 immunoreactive cells were counted in the parapyramidal region and raphe pallidus, respectively, from eight rats. In the parapyramidal region, 95% of NKA-IR neurons contained SP-IR and 74% of the SP-IR cells contained NKAIR [Figs. I(A,B), 2]. Most ofimmunostained cells were pyramidal in shape and were approximately 20 ttm in diameter. Similar results were found in the raphe pallidus, where 89% of the NKAIR cells colocalized SP-IR and 70% of the SP-IR cells colocalized NKA-IR [Figs. I(C,D), 2]. Raphe pallidus immunoreactive cells were generally pyramidal in shape, approximately 18 ttm in size, and were more frequently found at the rostral levels of the raphe pallidus.

Coexistence of NKA With 5-HT and SP In neurons of the parapyramidal region and raphe pallidus, all (99-100%) NKA-IR neurons contained 5-HT-IR. However, only about 60% of 5-HT-IR neurons contained NKA-IR [Figs. 2, 3(B,C,F,G), 4(B,C,F,G)]. There were no apparent differences in these patterns of colocalization between the parapyramidal region and the raphe pallidus. To examine the triple coexistence ofNKA-IR, SP-IR, and 5-HT-IR, 892 and 792 immunopositive cells were counted in the parapyramidal region and raphe pallidus, respectively, from eight rats. Ninety percent (or more) of the NKA-IR neurons in the parapyramidal region and raphe pallidus colocalized both SP-IR and 5-HT-IR (Fig. 2). On the other hand, only 54% of the 5-HT-IR cells in both the parapyramidal region and raphe pallidus had both SP-IR and NKA-IR (Fig. 2).

Spinally Projecting NKA-IR Neurons Bilateral injections of FluoroGold into the thoracic or lumbar spinal cords deposited the fluorescent tracer throughout the gray

1007 matter, including the dorsal and ventral horns, the area surrounding the central canal, the IML, and the adjacent white matter of the spinal cord. Injections into T7-8 and into L4-6 were restricted to the thoracic and lumbar segments, respectively. Retrogradely labeled cells were found throughout the medulla including the parapyramidal region, raphe pallidus, raphe obscurus, raphe magnus, paragigantocellular reticular nucleus, and nucleus reticularis magnocellularis pars alpha. Because the distribution of retrogradely labeled bulbospinal neurons has previously been extensively described (4,5,25,26,37), these data will not be described here in detail. From four rats, 339 and 307 immunostained and FluoroGold-labeled cells of the parapyramidal region and raphe pallidus were counted, respectively, to study NKA-IR, SP-IR, and 5-HT-IR neurons that project to the lumbar spinal cord. In both the parapyramidal region and raphe pallidus, all of the NKA/SP/5-HT-IR neurons were labeled by FluoroGold, whereas approximately 48% of the FluoroGoldlabeled cells in both parapyramidal region and raphe pallidus were NKA/SP/5-HT-IR (Fig. 3). Similarly, NKA/SP/5-HT-IR neurons in the parapyramidal region and raphe pallidus were retrogradely labeled from the thoracic spinal cord. Among the 553 and 485 immunostained and FluoroGoid-labeled cells, respectively, all of the NKA/SP/ 5-HT-IR cells in both the parapyramidal region and raphe pallidus were labeled by FluoroGold. Of the FluoroGold-labeled cells, 52% in both parapyramidal region and raphe pallidus were NKA/SP/5-HT-IR (Fig. 4). Restricted injections of rhodamine-labeled latex microspheres into the T3 IML resulted in retrogradely labeled NKA-IR neurons in the parapyramidal region and raphe pallidus (Fig. 5). The distribution of retrogradely labeled neurons was similar to that previously reported (14,35,36). Although the number of cells retrogradely labeled from the IML was much smaller than the number labeled by the larger injections of FluoroGold, the distribution of NKA-IR cells retrogradely labeled from restricted IML injections was similar to that noted with the larger spinal cord injections of FluoroGold. In addition, some of the IMLprojecting NKA-IR neurons colocalized 5-HT-IR. DISCUSSION The data presented here show that NKA-IR is colocalized with SP-IR in a subset of serotonergic neurons of the ventral medulla that projects to the thoracic and lumbar spinal cord.

Verification of Labeling Procedures Because of the similar carboxyl-terminals (Phe-X-Gly-LeuMet-NH2) of NKA and SP, the primary antibody raised against one tachykinin may be immunoreactive to the other. Present results from the immunoblot testing confirmed our previous results that the rabbit polyclonal antibody for SP (Incstar Corp.) recognized SP but not NKA. The cross-reaction of the Leeman SK-6 NKA antibody to SP can be abolished by preabsorption with 100/~g/ml of SP (12). However, it is not known whether this NKA antibody immunocytochemically detects the N-terminally extended (e.g., neuropeptide K and neuropeptide 3") or truncated forms of NKA [e.g., NKA(3-10)] (23).

Precursors for Ventral Medullary SP and NKA The present study demonstrated that almost all ventral medullary NKA-IR neurons had SP-IR. These data are consistent with the finding that/3- and "r-preprotachykinin mRNA, which encode both SP and NKA, were abundant in the ventral medulla

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FIG. 5. Photomicrographsofa NKA-IRneuron (A) in the parapyramidalregionthat was retrogradelylabeledwith rhodamine-labeledlatex microspheres (B) from the IML of the thoracic spinal cord. The asterisk in inset at (A) shows the locationof the NKA-IRcell, and the shaded area in the inset at (B) demonstrates the size of rhodamine-labeledmicrospheresdeposited at the third thoracic segmentof the spinal cord. Calibrationbar = 30/~m.

(9,21). Likewise, the presence of a small percentage of SP-IR neurons that do not colocalize NKA-IR is consistent with RIA data (2,27) and may indicate the presence of a population ofc~preprotachykinin mRNA-containing (encodes for SP but not NKA) cells. However, an alternate explanation is that the sensitivity of the immunofluorescence procedure for NKA may be less than that for SP-IR.

NKA and SP in Serotonergic Neurons The presence of SP-IR in serotonergic neurons of the ventral medulla is well established (5,6,15,36). Our current results indicate that NKA-IR is also present in these serotonergic neurons of the parapyramidal region and raphe pallidus. Moreover, because most of the SP-IR neurons of the ventral medulla contained TRH-IR and 5-HT-tR (36), by inference, many of the NKA/ SP/5-HT-IR neurons are likely to additionally colocalize TRHIR. Moreover, the presence of a population ofserotonergic neurons that does not contain SP-IR (or NKA-IR) is consistent with previous findings and is consistent with the idea of at least two populations of ventral medullary serotonergic neurons (36,40,41 ).

Spinal Projections of NKA Neurons Retrograde transport studies with the fluorescent tracer, FluoroGold, showed that virtually all of the NKA/SP]5-HT-IR neurons of the parapyramidal region and the raphe pallidus projected to the thoracic and lumbar spinal cord. NKA-IR, SP-IR, and 5-HT-IR are present in nerve terminals of the lumbar and thoracic cord (16,29,39), and the coexistence of SP-IR with 5HT-IR in these terminals is well documented (39-41). Although demonstration of the coexistence of NKA-IR in these terminals has not been directly studied, it is likely that these nerve terminals arise from ventral medullary neurons (11,13) and contain NKAIR colocalized with SP-IR in serotonergic nerve terminals. For both lumbar and thoracic spinal projecting neurons, approximately 50% of the FluoroGold-labeled neurons in the parapyramidal region and raphe pallidus contained NKA/SP/5-HT-IR. These data indicate that there are multiple neurocliemical populations of parapyramidal region and raphe pallidus spinally projecting neurons. In addition to studying spinal projecting neurons of the thoracic cord using FluoroGold, IML-projecting neurons were specifically investigated by injection of rhodamine-labeled latex microspheres into the IML. Rhodamine-labeled microspheres

FIG. 4. Photomicrographs of neurons in the parapyramidal region (A-D) and raphe pallidus (E-H) retrogradely labeled with FluoroGold from thoracic spinal cord (A,E) and immunostained for NKA (B,F), 5-HT (C,G), and SP (D,H). (A,B,C) and (E,F,G) are views of the same section, and D and H are views of the respective adjacent section. Arrows indicate FluoroGold-labeledneurons containing NKA-, SP-, and 5-HT-IR. Calibration bar = 30/zm.

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were used because they allow a discrete injection with very little diffusion, as occurs with other retrograde tracers, including FluoroGold. In concerning a potential underestimate on these medullospinal IML-projecting neurons as most of rhodaminelabeled neurons were lightly labeled, a quantitative study of IMLprojecting ventral medullary neurons was not performed; however, we visualized ventral medullary N K A - I R cells that were labeled by rhodamine-labeled microspheres. Because N K A almost completely coexists with SP in bulbospinal parapyramidal region and raphe pallidus neurons, it is likely that N K A - I R coexists with SP-IR in neurons of the parapyramidal region and raphe pallidus that project to the IML.

The physiological importance of these coexisting neurochemicals is complex and not well understood. They may interact with one another to either evoke or inhibit the release of the others. For example, at the level of the spinal cord, SP has been shown to inhibit the release of 5-HT (28) whereas 5-HT has been shown to evoke the release of SP (19). Together, colocalized neurochemicals may affect receptor binding (1) and neuronal excitability (8). ACKNOWLEDGEMENT This work was supported by NIH grant RO1 NS24876.

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A NEURONS

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