Receptor binding sites for cholecystokinin, galanin, somatostatin, substance P and vasoactive intestinal polypeptide in sympathetic ganglia

0306452?:92

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Pergamon Press plc ('1991IBRO

RECEPTOR BINDING SITES FOR CHOLECYSTOKININ, GALANIN, SOMATOSTATIN, SUBSTANCE P AND VASOACTIVE INTESTINAL POLYPEPTIDE IN SYMPATHETIC GANGLIA P. W. MANTYH,*?~ M. D. CATTON,? C. J. ALLEN,? M. E. LABENSKI,? J. E. MAGCZO$ and S. R. VIGNA/ SMolecular Neurobiology Lab. (1 Sl), Research and Psychiatry Service, VA Medical Center, Minneapolis. MN 55417, U.S.A.

$Department of Psychiatry, University of Minnesota, Minneapolis. MN 55455, U.S.A. SDepartment of Biological Chemistry and Molecular j/Department

of Cell Biology,

Duke

Pharmacology, U.S.A.

Harvard

MA 02115. University

Medical

Durham.

Center,

Medical

School.

NC 27710.

Boston,

U.S.A.

Abstract-Sympathetic ganglia are innervated by neurupeptide-containing fibers originating from pre- and postganglionic sympathetic neurons, dorsal root ganglion neurons, and in some cases, myenteric neurons. In the present report receptor autoradiography was used to determine whether sympathetic ganglia express receptor binding sites for several of these neuropeptides including bombesin, calcitonin gene-related peptide-a, cholecystokinin, galanin, ~eurokinin A, somatostatin, substance P, and vasoactive intestinal polypeptide. The sympathetic ganglia examined included the rat and rabbit superior cervical ganglia and the rabbit superior mesenteric ganglion. High levels of receptor binding sites for cholecystokinin, galanin, somatostatin, substance P, and vasoactive intestinal polypeptide were observed in all sympathetic ganglia examined, although only discrete neuronal populations within each ganglion appeared to express receptor binding sites for any particular neuropeptide. These data suggest that discrete populations of postganglionic sympathetic neurons may be regulated by neuropeptides released from pre- and postganglionic sympathetic neurons, dorsal root ganglion neurons, and myenteric neurons.

The three

major

types

of

neurons

comprising

systems may also play a direct role in regulating PGS neurons within the sympathetic ganglia. Electrophysiological studies have provided support for this model by showing that PGS neurons are responsive to several neuropeptides including enkephahn, somatostatin and substance P.7~9~‘8~20~4’~4x~s2 High affinity receptor binding sites for substance P have also been shown to be present on both a prevertebral and paravertebral ganglion of the rat” and activation of vasoactive intestinal polypeptide (VIP) receptors in the superior cervical ganglion (SCG) causes an activation of second messenger signaling systems,‘.21 suggesting that several functional neuropeptide receptors are present in at least some sympathetic ganglia. While it is clear that there may be an interaction between neurons outside the sympathetic nervous system and postganglionic sympathetic neurons, it remains to be defined which functions might be regulated by such an interaction. Probably the best studied interaction, and the one we will focus on in the present report, is the interaction between DRG neurons and PGS neurons. It has become increasingly evident in the last decade that the neuropeptidecontaining DRG neurons that convey afferent somatosensory information from peripheral tissues to the spinal cord are also involved in the efferent regulation of the peripheral tissues they innervate.

the

nervous system are the preganglionic sympathetic neurons, postganglionic sympathetic (PGS) neurons and interneurons such as the small intensely fluorescent cells. The classical studies of Langley led to an early view of the sympathetic ganglia as relay stations, with the postganglionic neurons transmitting essentially the same signal as they received from preganglionic fibers2” Later, with the demonstration of interneurons within the sympathetic ganglion and the observation that there is generally not a 1: 1 correspondence between the fibers entering and leaving the ganglion, it was proposed that integration and processing of sympathetic activity also occurred in the sympathetic ganglia. This concept was further expanded with the demonstration that dorsal root ganglia (DRG) neurons and myenteric neurons project to some sympathetic ganglia, 6 x~‘0~“~‘5.34~36~43 suggesting that other neuronal .~. sympathetic

*To whom correspondence should be addressed, at: Molecular Neurobiology Lab. (151). Research and Psychiatry Service, VA Medical Center, Minneapolis, MN 55417, U.S.A. Ahhre~:iutions: CGRPa, calcitonin gene-related peptide-cc; DRG, dorsal root ganglia; IML, intermediolateral cell column; PGS, postganglionic sympathetic; SCG, superior cervical ganglia; SMG, superior mesenteric ganglia; SP, substance P; VIP, vasoactive intestinal polypeptide. 739

740

I’. 8’

MANIYH cv d.

Thus neuropeptide-containing DRG neurons have been implicated in both the afferent central transmission of sensory information and in the efferent regulation of inflammatory. immune. and wound healing responses.‘7,2X.32A question which remains is whether sensory neurons directly regulate a specific set of target cells or whether the sensory neuron can also act indirectly via the sympathetic nervous system.24 One example of a sensory neuron apparently exerting an indirect action on a peripheral tissue, via the sympathetic nervous system, is in the rat cornea. After neonatal capsaicin treatment, which preferentially destroys the thin, unmyefinated neuropeptidecontaining DRG and trigeminaf ganglion neurons, treated animals often develop multiple cutaneous lesions, which in the cornea result in ulcerations.“5 Since several of the neuropeptides which are synthesized and released by DRG neurons have been shown to be mitogenic for several lines of cultured cells,3* it was thought that the capsaicin-sensitive DRG neurons play a direct trophic role by releasing neuro~ptides which stimulate epidermal mitogenesis. A somewhat unexpected finding was that animals which were neonataffy capsaicin-treated, and then chemically sympathectomized, did not develop the cornea1 fesions.46 While there are several ways to interpret these results, it is clear that there appears to be an interaction between the sensory and sympathetic neurons and that this interaction is important in maintaining the integrity of a peripheral tissue these two systems jointly innervate. In the present report we use receptor autoradiography to define further the neurochemicaf organization by which neuropeptides may exert receptor-mediated actions via PCS neurons. Using this approach we will define: (1) which of eight neuropeptides (all of which are known to be synthesized and released by DRG neurons and some of which are synthesized and released by myenteric neurons and PGS neurons themselves) have the proper neurochemi~af organization to produce functionaf receptor-mediated responses on PGS neurons in two different species; (2) whether this organization differs in a paravertebraf ganglion (superior cervi-

cal ganglion) and a prevertebraf ganglion (superior mesenteric ganglion); and (3) whether all sympathetic neurons within a sympathetic ganglion express a similar level of each neuropeptide receptor binding site. EXPERIMENTAL PROCEDURES

Tissue preparation

Five male adult New Zealand White rabbits (Universal Animals, Inglewood, CA) and tive male adult SpragueDawley rats (250 g, Simonsen, Gilroy, CA) were overdose-d with Nembutal and perfused transcardially with 250 ml of 0.1 M phosphate-buffered saline at 4°C pH 7.4. The animals were placed on ice and the superior cervical ganglia, superior mesenteric ganglia (rabbit only), dorsal root ganglia (T4) and spinal cord (T4) were rapidly dissected out,

The spinal cord was included as a positive conrrol smce receptor binding sites for all eight of the neuropeptides examined in the present study have been shown to he present in this tissue. The DRG was included since tibers aming from these neurons are known to innervate the sympathetic ganglia’*,” and binding sites on these fibers could be a potentially confusing source of receptor binding sites in the sympathetic ganglia. The tissues were blocked. frozen in Tissue Tek and mounted on a brass microtome chuck. Frozen sections (15pm) were cut on a cryostat microtome and thaw-mounted onto gefatin-coated slides and stored at -70°C in boxes containing desiccant for up to three months. RadioligandA

The radioligands used in the present study were all labeled with “‘1 by conventional methods and purified by reversephase high performance liquid chromatography to a specific activity of approximately 2000 Ci/mmol. Receptor binding protocols

Receptor autoradiography was performed as previously described.‘sm’2 The slide-mounted tissue sections were brought to room temperature and then placed consecutively in a preincubation medium, an incubation medium, a wash solution and distilled water. The preincubation and washes were performed by imme~ing the entire slide in the appropriate solution whereas the incubation with the radio&and was performed by placing the slides on a flat surface and covering the sections with 1.5ml of the incubation medium. To estimate the non-specific binding, paired serial sections were incubated as described above except that the appropriate unlabeled peptide was added to the incubation solution at a final concentration of 1 PM. Bombesin

Sections were first preincubated in 10 mM HEPES (pH 7.4) for 5 min followed by an incubation in 10 mM HEPES (pH 7.4), 4.7 mM KCI, 13OmM NaCI, 5 mM MgCf,, 1 mM EGTA. 0.1% bovine serum albumin. 100 me/ml bacitracin and IO0pM[‘251]-~yr4)-~m~in for 1h.49 xfter the incubation the sections were washed four times for 2min each in a solution of 10 mM HEPES (pH 7.4) and 0.01% bovine serum albumin, followed by two brief dips in ddH,O at 4°C. Calcironin gene-related peptide-a

Slide-mounted tissue sections were preincubated in 50mM Tris-HCI buffer (pH 7.4) for 5 min and subsequently incubated in 50mM Tris-HCI buffer (pH 7.4), 5 mM MgCI,, 2 mM EGTA and 100 pM [‘*sI]i~ohistidyl’~~ human calcitonin gene-related peptide-cc (CGRPa) (Amersham) for 2 h. Afterwards the slide-mounted tissue sections were washed (four times, 3 min each) in a solution of 50 mM Tris-HCl (pH 7.4), 0.1% bovine serum albumin, followed by two brief dips in ddH,O at 4”C.‘*

The radioligand used in the present study was “‘I-Bolton Hunter-labeled cholecystokinin-8 (sulfated; [i2JI]cholecystokinin, Amersham). Sections were preincubated for 30 min at room temperature in 50 mM Tris-HCl (pH 7.4) containing 130 mM NaCi, 4.7 mM KCl, 5 mM MgCl,, 1 mM EGTA (Tris saline buffer) and 0.5% bovine serum albumin.37 The sections were then incubated in Tris saline buffer containing 0.025% bacitracin, 1.0 mM dithiothreitol, 2 pg/ml chymostatin, 4 pg/ml leupeptin (pH 6.5) and 100 pM [iZsI]cholecystokinin-8 for 150min at room temperature. Afterwards the sections were washed six times, for 15 min each, in fresh incubation buffer, containing the 0.5% bovine serum albumin at 4°C. The sections were then dipped twice in ddH,O at 4°C.

Binding

sites for neuropeptides

Galanin

741

ganglia

KCI, 5 mM MnCl,, 1 mM EGTA, 1% bovine serum albumin, 1 mg/ml bacitracin and 100pM [“‘I]VIP (pH 7.4) at 20°C. Afterwards the sections were washed twice for 15 min at 4°C in the incubation solution without the radioactive ligand.i’ The sections were then dipped twice in ddHzO at 4’C.

Tissue sections were preincubated for 1Omin in IOmM HEPES buffer (pH 7.4, 20°C) followed by a 60 min incubation in the same buffer with 100 pM [‘2sI]galanin (monoiodinated porcine galanin labeled using chloramine-T). The sections were then washed (four times, 3min each) in the same buffer.“’

Autoradiography

Neurokinin A

After the final dip in ddH,O the slides were brought into the cold room (4°C) and allowed to air dry for 3 h. When completely dry the slide-mounted tissue sections were placed in apposition to LKB tritium-sensitive film alongside iodinated standards (Amersham). After one to three weeks the LKB film wa> developed in D-19 developer, fixed, and washed. In selected sections where we wished to obtain a higher degree of histological resolution, the radioligand was fixed to its binding site using paraformaldehyde vapors and then processed for standard emulsion-dipped autoradiogautoradiograms were raphy. ” Finally the emulsion-dipped developed, placed in Carnoy’s fixative for 3 h, Nissl stained, and mounted with Histoclad. Dark-field and bright-field photomicrographs were then taken of the silver grains and counterstained sections, respectively. Using this approach allowed us to generate three complementary images: the LKB autoradiograms which were analysed for quantitative densitometry. the autoradiograms of the emulsion-dipped slides which provided detailed histological resolution of the binding sites under the developed silver grains, and the counterstained section which allowed us to identify the cell type expressing the specific binding site. Controls for chemographic artifacts were made by performing the binding exactly as described except that the radioligand was omitted from the incubation medium. To estimate quantitatively the density of radiolabeled neuropeptide binding sites, microdensitometry was performed as previously described.?*

Sections were brought to room temperature and placed in a preincubation medium (ls”C for 10 min) consisting of 5OmM Tri-HCI (pH 7.4). Afterwards the sections were incubated at 19°C for 2 h in a solution of 50 mM Tris-HCl (pH 8.0) containing 3 mM MnCl,, 200 mg/l bovine serum albumin, 2 mg/l chymostatin, 4 mg/l leupeptin, 40 mg/l bacitracin, and 100 pM iZ5f-Bolton Hunter neurokinin A. Following this incubation, the sections were washed four times in 50 mM Tris--HCi, for 5 min at 4°C (pH 7.4).4.‘9.‘2

Sections were preincubated in 70 mM Tris-HCI for 5 min followed by a 2 h incubation in 170 mM Tris-HCl (pH 7.4), 5 mM MgCl,, 1% bovine serum albumin, 20 mg/l bacitracin, and 100 pM [‘*“I]-(Tyr3)-somatostatin cyclic analog (o-Phe -Cys-Tyr-n-TrpLys-Thr-Cys-Thr--NHJ (which is similar to Sandostatin@;). The sections were then washed in 170 mM Tris-HCl buffer ( pH 7.4) and 0.01% bovine serum albumin four times for 4 min each.‘,4’ The sections were then dipped twice in ddH,O at 4°C. Substance P Slide-mounted tissue sections were brought to room temperature and placed in a preincubation medium (2O’C for 10 min) consisting of 50 mM Tris-HCl (pH 7.4) containing 0.005% (v/v) polyethyl~nimine. The slide-mounted sections were then incubated at 20°C for 1 h in a solution of 50mM Tris-HCl (pH 7.4) containing 3 mM MnCl,, 200 mg/l bovine serum albumin, 2 mg/l chymostatin, 4 mg/l leupeptin, 40 mg/I bacitracin, and 100pM lz51Bolton-Hunter substance I’. Following this incubation sections were rinsed with four washes of 50 mM Tris-HCl (pH 7.4; 4’C, 2 min each).‘.a.l’ The sections were then dipped twice in ddH,O at 4 C.

RESULTS

For each radioligand described we examined at least five rabbit and rat SCG, five rabbit superior mesenteric ganglia (SMG), five rat and rabbit spinal cords (T4) and five rat and rabbit DRG (T4). In general the same density and pattern of binding was observed in comparing results obtained in different animals. We will therefore describe the pattern of binding from one representative animal for each ligand and will note any major differences among animals in the observed binding. The results from these experiments are presented in Table I.

Vasoucrh~e inresrinal poiypeptide The radioligand used in the present study was [3-iodotyrosyl-“51]-vasoactive intestinal peptide ([“‘JVIP; Amersham). Tissue sections were preincubated in 10 mM HEPES buffer (pH 7.4) for 5 min at 20°C followed by a 2 h incubation in IO mM HEPES buffer, 130 mM NaCl. 4.7 mM Table

in sympathetic

1. Distribution

of receptor

binding

sites

Rat

Rabbit __..~

Neuropeptide Bombesin CGRPx Cholecystokinin Galanin Neurokinin A Somatostatin SP VIP

DRG

IML

SCG

DRG

IML

SCG

_

_

_ -

_ _

-

-

+ _ _ _ _ _

++ + “I-+ +++ i-

+ + -I- f + + -t + + + -t + +++ +-l-i

++++ ++++ _ _ _ _

++ + ++ _ +++ +

SMG

+++-I+++-t -

+++-I++++ _

++++ ++ ii++

++++ ++++ ii-ii-

Location and density of receptor binding sites for sensory neuropeptides on the intermediolateral cell column @ML), dorsal root ganglia (DRG), superior cervical ganglion (SCG), and superior mesenteric ganglion (SMG) in the rabbit and rat. Serially adjacent frozen sections (15 pm) were labeled as described in the text and opposed to LKB Ultrofilm for 10 days, Densitometric readings for each region were taken as described from the five different animals, averaged and corrected for the non-linearity of the film as previously described** using autoradiographic standards (Amersham). Values are expressed as a percentage of laminae 1 and 2 binding in the rabbit thoracic spinal cord for each ligand: ( - ) undetectable; ( + ) O.l-25.0%; ( + + ) 25.1-50.0%; ( + + + ) 50.1-75.0%; ( + + + + ) 75.1.-100.0%.

742

1’. w.

,btAN?‘YHcl rd.

Bombesin

In the rabbit and rat thoracic spinal cord binding sites for bombesin ([‘2SI]-Tyr~-bombesin) are present in high concentrations only over laminae 1 and 2 of the spinal cord as previously described.” In laminae 1 and 2 of the thoracic spinal cord the specific/ non-specific binding ratios were greater than 95:5. Receptor binding sites for bombesin were not detected in the SCG of the rat and rabbit, the SMG of the rabbit or the DRG of the rat or rabbit. Calcitonin gene-related peptide-a

In the rabbit and rat thoracic spinal cord, receptor binding sites for CGRPa were observed in low concentrations over laminae 1 and 2, moderate concentrations over laminae 3-9, and highest concentrations in lamina 10. This pattern is similar to that previously reported in the human4’ and rat.22 A moderate concentration of receptor binding sites is also present in the region of the intermediolateral cell column (IML) in both the rabbit and rat. In laminae 1 and 2 of the thoracic spinal cord the specific/non-specific binding ratios were greater than 90: 10. No specific CGRPa binding sites were detected in the SCG of the rat or rabbit, the SMG of the rabbit or the DRG of the rat or rabbit.

In the rabbit and rat thoracic spinal cord, receptor binding sites for cholecystokinin are present in high con~ntrations over laminae 1 and 2, in low density in laminae 3-9, and in moderate concentrations in lamina 10. In laminae 1 and 2 of the spinal cord the specific/non-specific binding ratios are greater than 90 : 10. A high density of cholecystokinin binding sites was also observed in the rabbit DRG, whereas a low density of cholecystokinin binding sites was observed in the rat DRG. In the rat and rabbit SCG and in the rabbit SMG there is a high density of cholecystokinin binding sites. In the rat and to a greater extent in the rabbit SCG and SMG (Fig. I), different areas of the same ganglion have varying densities of cholecystokinin binding sites. Thus there appears to be a heterogeneous distribution of receptors in the ganglia, suggesting that certain groups of cells within one ganglion express a higher

--

- ---

.-.--.-

~___._ .__. .._..~~.

density ofcholecystokinin receptor binding sues than immediately adjacent groups of cells (Fig. I J. Gafanin

In the rabbit and rat, thoracic spinal cord receptor binding sites for galanin are present in high concentrations over laminae 1 and 2 and in low to moderate concentrations in laminae 3-10. In laminae I and 2 of the spinal cord the specific/non-specific binding ratios are greater than 95 : 5. A high density of galanin binding sites is also observed in the rabbit but not the rat DRG. A high density of galanin binding sites is observed in the rat and rabbit SCG and in the rabbit SMG. In the rat SCG and more conspicuously in the rabbit SCG and SMG (Figs 2 and 3, respectively), there does not appear to be a strict correlation between the area with the highest density of neuronal cell bodies (Figs 2c and 3c) and the area with the highest concentration of galanin binding sites (Figs 2d and 3d). Instead, there appears to be a highly heterogeneous distribution of galanin binding sites such that certain cell groups express extremely high concentrations of galanin binding sites, whereas immediately adjacent cell groups have only background levels of galanin binding. Neurokinin A

In the rabbit and rat thoracic spinal cord, receptor binding sites for neurokinin A are present in high concentrations over laminae 1 and 2 of the spinal cord as previously described.29 In these areas of the spinal cord the s~i~c/non-speci~c binding ratios are greater than 80:20. No receptor binding sites for neurokinin A were detected in the DRG of the rat or rabbit, the IML column of the spinal cord of the rat or rabbit, in the SCG of the rat or rabbit, or in the SMG of the rabbit. Somatostatin In the rat and rabbit thoracic spinal cord, receptor binding sites for somatostatin are present in high concentrations over laminae 1 and 2 and are not detectable in laminae 3-10. In laminae I and 2 the specific/non-specific binding ratios are greater than 95: 5. Binding sites for somatostatin are not detectable in the rat or rabbit DRG. A high concentration of somatostatin binding sites was observed

_.-._

Fig. I. Autoradiographic localization of [“51]cholecystokinin (‘251-CCK) binding sites in rabbit superior mesenteric ganglion (SMG). (a) Dark-field photomicrograph of LKB tritium-sensitive film which has been apposed to a section of the rabbit SMG which had been incubated with the ‘251-CCKligand. The white silver grains in a and d represent high concentrations of ‘2s1-CCK binding sites. The control section (b), serially adjacent to a, was treated identically, except that 1 HM of cold chol~ystokin~n was added to the incubation medium. In order to obtain the specific binding, the binding in b was subtracted from the binding in a. (c) Light-field photomicrograph of the same section seen in a which has been Nissl stained to visualize the neurons contained within the ganglion. (d) Dark-field photomicrograph of the same section seen in a and c which has been dipped in nuclear emulsion and then developed to allow a higher resolution of the i2%CCK binding sites. Note that in all the figures, c is the Nissl-stained section which generated the autoradiogram seen in d. Note that there is some regional variation in the density of “‘1-CCK binding sites visible in both a and d, whieh appears to be independent of the underlying cell density (c). Scale bars = 1.Omm (b); 0.2 mm (c).

Binding

sites for neuropeptides

Fig.

in sympathetic

1.

ganglia

743

744

P. W. MANTYH

er rif

Fig. 2. Autora~ographic localization of [‘2’I]gaimin (‘ZSI-GAL) binding sites in rabbit SCG. Note that as with the ‘2SI-CCK binding sites in Fig. I, the density of lZsI-GAL binding sites is not directly correlated with underlying density of the stained neurons, but rather varies over different regions of the ganglion. Scale bars = 1. I mm (b): 0.2 mm (c). See Fig. 1 for explanation.

Binding sites for ncuropeptides

in sympathetic ganglia

Fig. 3. Autorad~ograpbic ~ocaiizat~on of [‘~5~~galanin(‘“‘I-GAL) binding sites in rabbit superior mesenteric ganglion (SMG). Note that although the density of “5t-GAL binding sites is fan-homogeneous throughout the ganglion, the ‘Z51-GALbinding sites do appear to be associated with discrete neuronal pools as seen in the Nissl counterstained section (c). See Fig. 1 for explanation. Scale bars = I .2 mm (b): 0.2 mm (c).

745

746

P.

w. kfANTYH

et (II.

Fig. 4. Autoradiographic localization of [‘*‘I]somatostatin (‘251-SOM) in the rabbit superior mesenteric ganglion (SMG). Note the extremely heterogeneous distribution of ‘251-SOM binding sites (a and c). For explanation see Fig. 1. Scale bars = 1.2mm (b); 0.2 mm (c).

Binding

sites for neuropeptides

in the rat and rabbit SCG and in the rabbit SMG (Fig. 4). As with the other radioligands where specific binding was present in sympathetic ganglia, both the rat and more obviously in the rabbit SCG and SMG, there did not appear to be a strict correlation between the area with the highest density of cell bodies and the area with the highest concentration of somatostatin binding sites. As seen in Fig. 4a, the silver grains representing somatostatin binding sites are not uniformly distributed throughout the ganglion but rather are only present over specific areas of the ganglion, suggesting that some but not other groups of cells express the somatostatin binding sites. Substance

P

In the rabbit and rat thoracic spinal cord, receptor binding sites for substance P (SP) are present in high concentrations in laminae 1 and 2, in low concentrations in laminae 3 and 4, and in moderate concentrations in laminae 5-9. A very high concentration of receptors was observed in both lamina 10 and in the IML, as has been previously reported.’ In laminae 1 and 2 the s~cific/non-s~cific binding ratios are greater than 90: 10. No SP binding sites are detectable in either the rat or rabbit DRG. In contrast, a high density of SP binding sites was observed in the rat and rabbit SCG and in the rabbit SMG (Figs 5 and 6). In the rabbit SCG and SMG there did not appear to be a strict correlation between areas with the highest density of cell bodies and areas with the highest concentration of SP binding sites, again suggesting that certain cell groups express a higher concentration of SP binding sites than neighboring cell groups. Vasoactive

intestinal poI~peptide

In the rabbit and rat thoracic spinal cord, high densities of VIP binding sites are present over laminae 1 and 2, low densities over laminae 3-9, and high densities over lamina 10. No VIP binding sites were detectable in the rat or rabbit DRGs. In laminae 1 and 2 the speci~c/non-s~cific binding ratios were greater than 90: 10. A high density of VIP binding sites is present in the rat SCG and a very high density is present in the rabbit SCG and SMG (Figs 7 and 8). The distribution of VIP binding sites was highly heterogeneous in that while some cell groups had very high densities, neighboring cell groups exhibited only background densities.

DISCUSSION

Data suggesting that these neurapeptjde binding sites correspond to ~u~ct~ona~ receptors on postgang~i~)n~c synpatheric neurons Previous reports have established that there are several potential sources of the neuropeptides that might occupy the neuropeptide receptors expressed by the PGS neurons. A potential source for somato-

747

in sympathetic ganglia

statin and VIP is a subpopulation of the PGS neurons themselves which has been shown to contain and presumably synthesize these peptides.25.” A second source for bombesin, cholecystokinin~ CGRPa, galanin, neurokinin A, SP, somatostatin and VIP in PGS ganglia is the DRG neurons which have been shown to provide a substantial innervation to prevertebral ganglia and to a lesser extent to paravertebral ganglia.‘O~” A third possible source of bombesin, cholecystokinin, and VIP in sympathetic ganglia which innervate the lower gastrointestina1 tract is myenteric neurons, which have been shown to innervate the coeliac-superior mesenteric gang1ia.W In the present study we have shown that both prevertebral and paravertebral sympathetic ganglia express receptor binding sites for several neuropeptides. Questions which the present study has not definitively addressed are whether these receptor binding sites correspond to functional receptors and whether it is the PGS neurons themselves or their supporting cells which are expressing these receptor binding sites. Previous studies, however, suggest that sympathetic ganglia do express several functio~l neuropeptide receptors. For example, VIP has been shown to increase inositol phosphohpid breakdown in the rat SCG, suggesting that the VIP binding sites present in this ganglion are coupled to a functional second messenger system and thus correspond to functional VIP receptors.‘,” Electrophysiological studies have shown that iontophoretic application of SP in the guinea-pig SMG’.” and chick SCG4’ produces a slow membrane depolarization which lasts for minutes, similar to the described actions of SP on CNS neurons. In the present study, while we have not been able to obtain unequivocal, single-cell resolution of the binding sites on PGS neurons (probably because when the binding sites are expressed by PGS neurons they are present on the intermingled dendrites, cell bodies and axons of the expressing cells), it is apparent in several figures presented here (Figs la, 2d, 3d, 4a, 5b, 7d and 8d) that the receptor binding sites present in sympathetic ganglia are more dense over clusters of PCS neurons than in the intervening space, which is composed primarily of the supporting cells. Neuronal organization s,vmpathetic ganglia

and compartmentalization

of

In the present report we have demonstrated that postganglionic sympathetic neurons express receptor binding sites for five of the eight neuropeptides examined. In addition, although clear differences in the synaptic organization and neuropeptide levels in paravertebral vs prevertebral ganglia have been reported,“,15*“5 with the neuropeptide receptor binding sites we examined there were no absolute differences in the expression of these binding sites between prevertebral and paravertebral ganglia. The distribution of several of the neuropeptide binding sites in the rat, and even more noticeably in the rabbit. do

748

P. W.

MANTYH et d.

Fig. 5. Autoradiographic localization of [“‘I]substance P (‘*‘I-SP) binding sites in the rat superior cervical ganglion (SCG). Note that the density of ‘251-SPbinding sites is not uniform (a and d) throughout the ganglion. See Fig. 1 for explanation. Scale bars = 1.2 mm (b); 0.2 mm (c).

Binding sites for neuropeptides in sympathetic

ganglia

749

Nissl

Fig. 6. Autoradiographic localization of [Tlsubstance P (‘2sI-SP) binding sites in the rabbit superior mesenteric ganglion (SMG). In this figure, a is a photomicrograph of the Nissl-stained section, b is a dark-field autoradiogram showing the distribution of “51-SP binding sites in section a, and c is the serially adjacent control section. See Fig. I for explanation. Scale bar = 1.2 mm.

not always appear to be directly correlated with cell density. Rather, some clusters of PGS neurons express a higher density of receptor binding sites than other adjacent cell groups within the same ganglion, suggesting that the neuropeptide receptor binding sites are expressed by discrete clusters of PGS neurons. What makes this observation of possible functional interest is that some topographical organization has been described in sympathetic ganglia26 and there appears to be a correlation between the neuropeptides expressed by a PGS neuron and the

particular type of tissue which the PCS neuron innervates; i.e. target specificity.“~” For example, neuropeptide Y is expressed by a subset of postganglionic sympathetic neurons and these neuropeptide Y-containing neurons appear to innervate specific target tissues.25 Whether there is also a correlation between the neuropeptide receptors that are expressed by PGS neurons and the target which they innervate is as yet unknown, but the neurochemical organization observed in the present study would be compatible with such an organization.

Fig. 7. Autoradiographic localization of [‘251]vasoactive intestinal polypeptide (~ZSI-VJP)binding sites in the rat superior cervical ganglion (SCG). See Fig. 1 for explanation. Scale bars = I. 1 mm (bf; 0.2 mm (cl.

Binding

Nissl

sites for neuropeptides

in sympathetic

751

ganglia

sbbit SMG

Fig. 8. Autoradiographic localization of [“‘Ijvasoactive intestinal polypeptide (‘zsI-VIP) binding sites in the rabbit superior mesenteric ganglion (SMG). This is the clearest demonstration of the often highly uneven distribution of neuropeptide binding sites in this sympathetic ganglion (a), and clearly demonstrates that while some cell groups have an extremely dense concentration of binding sites, neighboring cell groups in the same ganglion have receptor binding densities that are barely above background levels (c and d). See Fig. 1 for explanation. Scale bars = I .O mm (b); 0.2 mm (c).

752

1’. W.

Possible functions

of the receptor

binding

MANTYH

sites

One interesting aspect of the efferent regulation of peripheral target tissues by PGS neurons and DRG neurons is their regulation of normal growth and the wound healing response. While it was previously thought that these two peripheral neuronal systems had mutually exclusive functions, recent data suggest that these two neuronal systems interact to regulate the peripheral tissues they jointly innervate. Originally it was shown that neonatally capsaicin-treated rats had a significantly higher number of cornea1 ulcerations than controls,4s suggesting that the release of neuropeptides by sensory neurons might provide a tonic mitogenic stimulus to the cornea1 epithelial cells and without this tonic stimulus, cornea1 lesions would develop. However, in a second report it was noted that this cornea1 ulceration could be blocked by sympathectomy,46 suggesting that the interaction between the DRG neurons and PGS neurons is crucial in attempting to understand how these two peripheral neuronal systems regulate the normal growth and wound healing of the peripheral tissues they jointly innervate. Another example of the interaction between DRG neurons and PGS neurons has been noted clinically in the chronic pain syndrome known as reflex sympathetic dystrophy. In this syndrome, damage to PGS neurons results in a chronic pain state. Since a secondary pathology appears to be an alteration in the response properties of DRG neurons, these data suggest that PGS neurons normally play a role in regulating the response properties of DRG neurons. This concept is further supported by reports that DRG neurons express adrenergic binding sitesa and that an effective analgesic treatment is administration of adrenergic antagonists’4,‘h and/or chemical sympathectomy.14 Since the present study suggests that neuropeptides released from DRG neurons have the proper neurochemical organization to regulate PCS neurons and PGS neurons express receptor binding sites for neurotransmitters and neuropeptides released by DRG neurons, these

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data suggest a neurochemical basis by which DRG neurons could regulate PGS neurons and vice versa. While we have focused on the potential interactions between DRG neurons and PGS neurons, it is clear that there are at least two other possible sources of neuropeptides which may occupy the neuropeptide receptor binding sites expressed by PGS neurons. The first possible source is the PGS neurons themselves, which have been shown to synthesize somatostatin,*’ VIP25 and in certain circumstances SP.” Since PGS neurons are known to interact with other PGS neurons, both within the same and different ganglia, the present data suggest that if somatostatin, SP and/or VIP are released in a sympathetic ganglion, there are receptor binding sites which could interact with these released neuropeptides. Currently, however, we do not know if a single PGS neuron both synthesizes the neuropeptide and expresses the receptor binding site for that neuropeptide (an autoreceptor). A second possible source for the neuropeptide is in the peripheral target tissue itself, which may also send a neuropeptide-containing collateral fiber back to the PGS ganglia. This arrangement, where the target tissue contains neurons which themselves project back to the innervating sympathetic ganglia, has been described for bombesin, cholecystokinin, and VIP between myenteric neurons in the colon and in the guinea-pig coeliac-superior mesenteric ganglion complex.” It should also be pointed out, however. that an appropriate neuropeptide, released in the target tissue innervated by PGS neurons, may also be able to interact with the binding sites on the terminals of the PCS neuron in the target tissue itself, since it is likely that receptors which can be visualized in the cell body are also found in other parts of the PGS neuron.

Acknowledgements-This work was supported by the American Paralysis Association, Southern California Arthritis Foundation, Sloan Foundation and NIH grants NS-23970, NS-22961 and DK-40260.

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17 July 1991)

tract: localization by auto-