Projections of neurons in the ventromedial medulla to pontine catecholamine cell groups involved in the modulation of nociception

Brain Research, 540 (1991) 105-115 Elsevier

105

BRES 16275

Projections of neurons in the ventromedial medulla to pontine catecholamine cell groups involved in the modulation of nociception Frank M. Clark and Herbert K. Proudfit The University of Illinois at Chicago, Department of Pharmacology, Chicago, IL 60612 (U.S.A.) (Accepted 21 August 1990)

Key words: Norepinephrine; Phaseolus vulgaris leucoagglutinin; Fluoro-Gold; A5 cell group; A7 cell group; Locus coeruleus; Nucleus raphe magnus; Gigantocellular nucleus pars alpha; Paragigantocellular nucleus

Stimulation of neurons in the nucleus raphe magnus (RMg) or the adjacent gigantoceilular nucleus pars alpha (Gia) and paragigantocellular nucleus (PGi) produces antinociception which is partially mediated by bulbospinal noradrenergic neurons. Since no norepinephrine-containing neurons are located in either the RMg or the Gia/PGi, it is likely that neurons located in these nuclei have axonal connections with the spinally-projecting catecholamine neurons located in the A5, A6 (locus coeruleus), or A7 catecholamine cell groups. To provide evidence for such connections, the anterograde tracer, Phaseolus vulgaris-leucoagglutinin (PHA-L), was injected into the RMg or Gia/PGi and labeled axons were identified near catecholamine-containing neurons labeled with dopamine-fl-hydroxylase-immunoreactivity(DflH-ir). A dense field of PHA-L-positive terminals was seen within the A7 cell group which was mainly ipsilateral to PHA-L injections made into either the RMg or the Gia/PGi. Many PHA-L-positive terminals were closely apposed to DflH-ir A7 perikarya or proximal dendrites. A modest number of terminals was seen within the A5 and LC cell groups. In the second experiment, a unilateral injection of the retrograde tracer, Fiuoro-Gold, was made into the A7 cell group and brainstem sections were processed for serotonin (5-HT) immunocytochemistry. Many neurons retrogradely labeled with Fluoro-Gold were seen in the RMg, but a much larger number were found in the Gia/PGi. Less than 5% of these Fluoro-Gold-labeled cells contained 5-HT-immunoreactivity. The results of these experiments indicate that the RMg and Gia/PGi have a substantial population of non-serotonergic neurons which project to the A7 noradrenergic cell group.

INTRODUCTION A great deal of convincing evidence indicates that n e u r o n s in the nucleus raphe magnus (RMg) and the a d j a c e n t gigantocellular nucleus pars alpha ( G i a ) and p a r a g i g a n t o c e l l u l a r nucleus (PGi) m o d u l a t e nociception. For e x a m p l e , electrical or chemical stimulation of either the R M g 1'4"16"18'29'31'32'34"36 or the G i a / P G i 4'36 produces p o t e n t antinociception. This antinociceptive effect appears to be m e d i a t e d by both spinally-projecting noradrenergic and serotonergic neurons since it is a t t e n u a t e d by intrathecal injections of either serotonergic or alpha n o r a d r e n e r g i c antagonists 4'1s'32"36 or by depletion of either spinal cord serotonin or n o r e p i n e p h r i n e 34. Since both the R M g and G i a / P G i contain serotonergic neurons 14 that project to the spinal cord 6, it is not surprising that serotonergic antagonists partially block the antinociception p r o d u c e d by R M g or G i a / P G i stimulation. H o w e v e r , because there are no catecholamine cell bodies located within either the R M g or the Gia/ PGi 14, it is likely that stimulation of neurons in the R M g and G i a / P G i activates spinally-projecting n o r e p i n e p h -

fine-containing neurons. T h e r e is strong anatomical evidence that the only c a t e c h o l a m i n e - c o n t a i n i n g neurons which p r o j e c t to the spinal cord are located in the A5, locus coeruleus (LC), and A 7 cell groups 24'41-44. The p u r p o s e of the e x p e r i m e n t s described in this r e p o r t was to d e t e r m i n e if neurons located in the R M g or G i a / P G i have axonai projections to neurons located in the spinally-projecting catecholamine cell groups. These experiments were done using two e x p e r i m e n t a l approaches. The first involved injecting the a n t e r o g r a d e tracer, Phaseolus vulgaris leucoagglutinin ( P H A - L ) , ' into the R M g or the G i a / P G i and d e t e r m i n i n g the presence of P H A - L - l a b e l e d axons and axon terminals in the LC, A 5 and A 7 cell groups. The second a p p r o a c h involved injecting the r e t r o g r a d e tracer, F l u o r o - G o l d , into the A 7 cell group and examining the R M g and G i a / P G i for r e t r o g r a d e l y - l a b e l e d cells. The results of these experiments indicated that neurons located in the R M g or G i a / P G i have m a j o r axonal p r o j e c t i o n s to the A 7 cell group, but fewer p r o j e c t i o n s to the L C and A5 cell group.

Correspondence: H.K. Proudfit, Department of Pharmacology (M/C 868), University of Illinois at Chicago, P.O. Box 6998, Chicago, IL 60680, U.S.A. 0006-8993/91/$03.50 ~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)

106 MATERIALS AND METHODS

Animal preparation All experiments were performed using adult female SpragueDawley derived rats (Sasco, Inc.), weighing 270-450 g. Animals were anesthetized with ketamine (100 mg/kg) and immobilized in a stereotaxic frame. Injections of PHA-L into the RMg and Gia/PGi, and Fluoro-Gold injections into the A7 cell group were made using stereotaxically-guided micropipettes. The anatomical nomenclature for reticular formation nuclei that was used in this report was described by Andrezik and Beitz2 and that for the catecholamine cell groups was described by Dahlstr6m and Fuxe t4,

Anterograde tracing experiments Iontophoretic injections of PHA-L (Vector Laboratories) were made into the RMg of 17 rats using the following stereotaxic coordinates: AP, -2.5; V, -0.5 and L, +0.0 mm with respect to the interaural line. Injections were made in the Gict/PGi of 8 animals. Stereotaxic coordinates for these injections were: AP, -2.5; V, +0.0 and L, +0.7 mm. The incisor bar was set at -2.5 mm for all PHA-L injections. Iontophoretic current (5-10/~A) and pipette tip diameters (10-15/~m) used to deliver PHA-L were similar to those used by Gerfen and Sawchenko ~5. Animals were maintained for 10 days, at which time they were deeply anesthetized with pentobarbital and perfused intracardially with 200 ml cold saline, 300 ml of cold 4% phosphate-buffered paraformaldehyde (pH 7.4), followed by 200 ml of cold 10% sucrose in 0.1 M phosphate buffer (pH 7.4). Brains and spinal cords were removed and stored at 4 °C for at least 3 days in a solution containing 30% sucrose in 0.1 M phosphate buffer (pH 7.4). Brainstem tissue was blocked and 40 kem transverse sections were cut on a cryostat microtome and processed for PHA-L and dopamine-fl-hydroxylase (D/3H) immunocytochemistry. All immunocytochemistry was done on free-floating sections. Sections were washed twice in 187 mM phosphate-buffered saline (PBS; pH 7.4) between each antibody incubation unless otherwise stated. Sections were cut, washed with cold PBS, treated for 20 rain with 2.5% H202 in methanol to inactivate endogenous peroxidasesu, washed 4 times with PBS and incubated for 48 h at 4 °C in pooled goat anti-PHA-L (Vector) and rabbit anti-D/3H (Eugene Tech) antibody solutions which contained 0.5% Triton X-100. Both antibodies were diluted 1:1000. Following the 2-day incubation, the tissue was warmed to room temperature and washed in PBS. Sections were incubated for 30 min in a solution containing pooled donkey anti-goat immunoglobulins (1:80 dilution; Chemicon), swine anti-rabbit immunoglobulins (1:80 dilution; Accurate) and 0.5% Triton X-100. The sections were washed in PBS, then placed in a solution containing goat peroxidase-antiperoxidase (PAP) complex (Chemicon) diluted 1:180 for 30 min. Sections were washed with PBS again and incubated for 6 rain in a solution that contained 0.05% filtered diaminobenzidene (DAB) and 0.005% HzO z in PBS. Sections were then washed in PBS and incubated in rabbit PAP (Cappel) diluted 1:180 for 30 rain. Sections were then washed in PBS and incubated for 4 min in a 50-raM Tris-HC! buffer (pH 8) which contained filtered DAB (0.013%) and nickel ammonium sulfate (0.67%) to which 0.005% H20 2 was added. Sections were then washed, mounted on gelatin-coated slides and coverslipped with Permount.

This processing produced brown staining of PHA-IMabeled axons and blue-black staining of D/3H-immunoreactive (Drill-Jr) somata and terminals. The location of PHA-L injection sites was assessed using camera lucida drawings made of brainstem sections which included the injection site. The distribution of PHA-L-labelcd axons and terminals within or near catecholaminc cell groups was determined by making drawings of the location of all labeled axons which were present within oz near the catecholaminc cell group from a single section.

Retrograde tracing studies Ten rats were anesthetized with ketamine (100 mg/kg) and iontophoretic injections of 2% Fluoro-Gold (Fluorochrome, Inc.) solution were made into the left A7 cell group of each rat using a glass micropipetet with a tip diameter of 25-30 ,urn. Fluoro-Gold was ejected from the micropipettes using 10 ttA anodal current pulses of 1 s duration with 1 s between pulses. Stereotaxic coordinates for the A7 cell group were AP, +1).2; V, +2.3 to +2.7 and L, +1.9 mm with respect to the interaural line with the incisor bar set at -2.5 mm. Three injections of Fluoro-Gold were made by varying the vertical coordinate from +2.3 mm to +2.7 mm. Ten days after surgery rats were intracardially perfused as described above. Frozen sections were cut from brainstem blocks and washed in cold PBS, then incubated for 20 min in a solution of 2.5% H20 2 in methanol. Coronal sections that included the RMg and Gia/PGi were washed 4 times and then incubated in rabbit anti-serotonin (5-HT) antibody (1:1000, Incstar) overnight at 4 °C. Sections near the injection site were incubated in rabbit anti-DflH antibody (1:I000) overnight at 4 °C. Sections were then warmed to room temperature, washed twice in PBS, then incubated in goat antirabbit immunoglobulins conjugated with rhodamine (1:80, Cappel) for 30 min. Sections were then mounted on slides and coverslipped with DPX (BDH Chemicals). All fluorescence microscopy was done using a Zeiss epifluorescence microscope. Brainstem sections that contained the FluoroGold injection site were examined and drawn using an X-Y plofter coupled to the microscope stage. Sections that contained the RMg and Gia/PGi were examined and retrogradely-labeled neurons, 5-HT-immunoreactive neurons and double-labeled neurons that contained both Fluoro-Gold and 5-HT-immunoreactivity were plotted at the appropriate locations on the brainstem sections using an X-Y plotter.

RESULTS

Anterograde tracer experiments T h e p u r p o s e o f t h e s e e x p e r i m e n t s w a s t o d e t e r m i n e if n e u r o n s in t h e R M g o r G i a / P G i p r o j e c t t o t h e A 5 , A 7 o r L C c a t e c h o l a m i n e cell g r o u p s . T h e a n t e r o g r a d e t r a c e r , PHA-L, RMg

was i o n t o p h o r e t i c a l l y i n j e c t e d i n t o e i t h e r t h e

or G i a / P G i ,

a n d p o n t i n e b r a i n s t e m tissue w a s

processed for i m m u n o c y t o c h e m i c a l visualization of both DflH-ir

and

PHA-L.

These

procedures

allowed

the

Fig. 1. Camera lucida drawings of a representative PHA-L injection site in the RMg and the location of PHA-L-labeled axons with respect to DflH-ir neurons in the pontine catecholamine cell groups. The drawings to the right of each brainstem section are enlargements of the various catecholamine cell groups which are enclosed by the boxes in the drawings on the left side. A: the location of the PHA-L injection site. The solid black area represents the dense core of the injection site while the stippled region shows the extent to which the PHA-L diffused into to the surrounding tissue. B: a brainstem section through the LC at the level of the genu of the facial nerve. The DflH-ir cell bodies are represented by the stippled area while the PHA-L-labeled axons and terminal segments are illustrated by the thick lines. C: a section through the caudal A5 cell group at the level of the exit of the facial nerve. The DflH-ir cell bodies are represented by the stippled shapes while the PHA-L-labeled axons are represented by thick lines. D: a similar drawing for the rostral A5 at the level of the trigeminal motor nucleus. These A5 neurons are located medial and ventral to the sensory root of the trigeminal nerve. E: the labeling of the A7 cell group which is located at the level of the caudal third of the inferior colliculus.

107

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A

B

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da

E

Fig. 2. Illustration of a representative P H A - L injection site in the Gia/PGi and the anterograde labeling of axons within or near pontine catecholamine groups. See the legend that accompanies Fig. 1 for a more detailed description.

109

,6,

B

C

D

l::

F

Fig. 3. Detailed illustration of the PHA-L injection site in the RMg and the relationship between PHA-L-labeled axon terminals and DflH-ir neurons in the A7 cell group. A: a photomicrograph of the PHA-L injection site in the RMg where the largest deposit of PHA-L was seen. This section was taken from the level of the ascending seventh cranial nerve in the rostral medulla. B: a low power photomicrograph that shows DflH-ir neurons located in the caudal aspect of the A7 cell group. C,D: high power photomicrographs of the two DflH-ir A7 neurons that are labeled C and D in panel B. These photomicrographs illustrate the spatial relationship between PHA-Lqabeled axon terminals and DflH-ir neurons in the A7 cell group. E,F: camera lucida drawings of the photomicrographs in C and D. The DflH-ir A7 neurons are outlined by the thin lines while the PHA-L-labeled axon terminals are represented by the thick lines. A,B: bar = 500/am; C,D: bar = 10/am.

110 spatial r e l a t i o n s h i p s of P H A - L - l a b e l e d axons and termi-

A P H A - L i n j e c t i o n site which was located n e a r the

nal s e g m e n t s to D B H - i r n e u r o n s in the A 5 , A 7 and L C

rostral p o l e of the R M g at the level of the a s c e n d i n g

to be d e t e r m i n e d .

s e v e n t h cranial n e r v e (Fig. 1A) l a b e l e d m a n y axons and t e r m i n a l s e g m e n t s which w e r e l o c a t e d a m o n g the D f l H - i r cell bodies (Fig. 1 B - E ) . T h e A 7 cell g r o u p was densely labeled (Fig. 1E), while the caudal (Fig. I C ) and rostral (Fig. 1D) A 5 cell g r o u p s w e r e m o d e r a t e l y labeled. In contrast, the L C was v e r y sparsely l a b e l e d and only a few l a b e l e d axons w e r e s e e n in the v e n t r a l part of this nucleus (Fig. 1B). P H A - L was also i o n t o p h o r e t i c a i l y i n j e c t e d into the G i a / P G i in the a r e a i m m e d i a t e l y dorsal to the p y r a m i d at the level of the a s c e n d i n g s e v e n t h cranial n e r v e (Fig.

a A

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.

B

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Fig. 4. Illustration of a representative Fluoro-Gold injection site in the A7 cell group and the location of retrogradely-labeled neurons in sections through the rostral medulla. A: the Fluoro-Gold injection site. DflH-ir cell bodies are represented by filled circles. The dense core of the Fluoro-Gold injection site, depicted by the heavier stippled region, encompassed some of the A7 cell bodies. The more lightly stippled region, illustrating the extent to which Fluoro-Gold diffused from the injection site, encompassed most of the remaining A7 cell bodies in this section. Although not depicted in this figure, the Fluoro-Gold injection site included a portion of the motor nucleus of the fifth cranial nerve. B: the distribution of Fluoro-Gold-filled neurons in the rostral medulla. C,D: the distribution of Fluoro-Gold-filled cells at more caudal levels. The numbers to the right of the brainstem sections represent the approximate distance (mm) of each brainstem,section from the interaural line. E: the location of reticular formation nuclei in the rostral medulla. RMg, nucleus raphe magnus; Gia, gigantocellular nucleus pars alpha; PGi, paragigantocellular nucleus; Gi, gigantocellular reticular nucleus; PCRt, parvocellular reticular nucleus.

C ~

-2.3

D ~

-2.6

Gia

Fig. 5. Illustration of a Fluoro-Gold injection site in the A7 cell group that did not include the motor nucleus of the fifth cranial nerve. A: the injection site which encompassed the lateral part of the A7 cell group, but did not include the motor nucleus of the fifth cranial nerve. See the legend that accompanies Fig. 4 for a more detailed description.

111

A

0

D

Fig. 6. Illustration of Fluoro-Gold injection sites that were located rostral to the A7 cell group. DflH-ir neurons are plotted as filled circles.

2A). Although the relative distribution of PHA-Llabeled axons and terminals produced by injections in both the Gia/PGi and the RMg was similar, the P H A - L injection in the Gia/PGi appeared to label more axons than the injection in the RMg (Fig. 1). The most dense P H A - L labeling was seen within or near the A7 cell group (Fig. 2E). However, far fewer labeled axons or terminal segments were seen near catecholamine somata that were located in the caudal and rostral parts of the A5 cell group (Fig. 2C,D). The labeling in the LC was very sparse, but there was moderate labeling in the area just ventral to the LC (Fig. 2B). PHA-L-labeled axons and terminal segments were found in greater numbers on the side of the injection site following injections into either the RMg or the Gia/PGi. For example, the P H A - L injection site in the RMg (Fig. 3A) was located slightly lateral to the midline and the PHA-L-labeling in the brainstem in this animal was more dense on the side that contained the bulk of the P H A - L injection. Similarly, the PHA-L-labeling in the catecholamine cell groups produced by PHA-L injections in the Gia/PGi was much more dense on the side ipsilateral to the P H A - L injection site. Approximately 90% of the PHA-L-labeling in the brainstem produced by the PHAL injection illustrated in Fig. 2 was located on the same side as the injection site. Fig. 3 illustrates the location of PHA-L-labeled axons

and terminal segments near catecholamine neurons in greater detail. Although it is not possible to demonstrate the existence of synaptic contacts at the light microscopic level, Figs. 3C and 3D show high power photomicrographs of PHA-L-labeled axon terminals that are in close apposition to DflH-ir cell bodies in the A7 cell group. In both Figs. 3C and 3D, the PHA-L-labeled terminals are enlarged near the surface of the DflH-ir cell body which suggests the existence of a terminal bouton and a synaptic contact. The DflH-ir axon terminals can be distinguished from PHA-L-labeled axon terminals since the DflH-ir is blue-black whereas the PHA-L-label is brown. The PHA-L-labeled axons in the region of pontine catecholamine cell groups exhibited widespread branching (Figs. 1E and 2E) and axon terminals with numerous varicosities were also common (Figs. 3C and 3E). Some of the axons appeared to continue in a rostral direction past the A7 cell group which indicates that neurons in both the RMg and the Gia/PGi have projections which course through the A7 region. However, there were more axons in the A7 cell group than in the area rostral to the A7, which indicates that most of the PHAL-labeled axons terminate in the region of the A7 cell group. Retrograde tracer studies The results of the anterograde tracing experiments

112 indicated that neurons in the RMg and Gic~/PGi project to the region of the A7 cell group. The purpose of the second series of experiments was to further investigate this apparent monosynaptic connection and to determine the location of neurons in the RMg or Gia/PGi that project to the A7 cell group. The retrograde tracer, Fluoro-Gold, was iontophoretically injected into the region of the A7 cell group and the location of retrogradely-labeled cell bodies in brainstem sections containing the RMg and Gia/PGi was determined. Fig. 4 illustrates the results for one of the 6 animals examined in this experiment. Fig. 4A shows that the Fluoro-Gold injection site was centered on the A7 cell group and included most of the A7 cell bodies. Fig. 4 B - D illustrates the location of retrogradely-labeled cell bodies at 3 levels of the rostral medulla. Both the RMg and Gia/PGi contained retrogradely-labeled cell bodies, although the number of labeled cell bodies was far greater in the Gia/PGi. Fluoro-Gold-filled neurons were found in the Gia/PGi bilaterally although there was a strong ipsilateral predominance. The largest number of retrogradelylabeled Fluoro-Gold neurons was found in the parvocellular reticular nucleus (PCRt) located lateral and dorsal to the Gia/PGi. Unlike the Gia/PGi, this nucleus exhibited a large bilateral population of Fluoro-Goldlabeled neurons. The large number of labeled neurons in the PCRt is probably due to the inclusion of the motor nucleus of the fifth cranial nerve (MoV) in the FluoroGold injection site since neurons in the PCRt have been shown to project to the MoV 20'39"40. To determine whether the PCRt neurons project to both the A7 cell group and MoV nucleus, Fluoro-Gold injections were made that included the A7 neurons, but not MoV neurons. Fig. 5A shows the location of a Fluoro-Gold injection that is centered on the ventral lateral border of the A7 cell group, but did not include any MoV cell bodies. The injection of Fluoro-Gold at this site produced retrograde labeling of many RMg and Gia/PGi neurons, but only a small number of PCRt neurons were labeled. In addition, this injection produced a dense cluster of Fluoro-Gold-labeled neurons in the contralateral dorsal cochlear nucleus. The injection of PHA-L into the RMg labeled many axons that appeared to pass through the A7 cell group in a caudal to rostrai trajectory. It is possible that the axons that pass through the A7 region are responsible for the retrograde labeling seen in the RMg and Gia/PGi following the Fluoro-Goid injections illustrated in Figs. 4 and 5. To examine this possibility, Fluoro-Gold injections were made into the area rostral to the A7 and the distribution of labeled neurons in the rostral medulla was determined. The injection of Fluoro-Gold into 4 sites rostral to the A7 cell group produced retrograde labeling

in only a few neurons located in the rostral medulla (Fig. 6). Since there is a substantial population of 5-HTcontaining neurons in the RMg and Gi~,/PGi. it is possible that some of these neurons may project to the A7 cell group. To explore this possibility, brainstem sections were processed for 5-HT immunocytochemistry and examined for double-labeled neurons that contained both 5-HT-immunoreactivity (5-HT-ir) and retrogradelytransported Fluoro-Gold. However, very few FluoroGold-filled neurons within the RMg or Gia/PGi contained 5-HT-ir. Thus, only one to two double-labeled neurons were found in 15 brainstem sections from each of 3 animals. DISCUSSION There is substantial evidence which suggests that the antinociception induced by stimulating neurons in the RMg or Gia/PGi is mediated by both raphe-spinal serotonergic neurons and bulbospinal noradrenergic neurons 3°. Since neither the RMg nor the Gia/PGi contain noradrenergic neurons, this antinociceptive effect is presumably mediated by spinally-projecting noradrenergic neurons which are located in the A5, A6 (LC) or A7 catecholamine cell groups 24"41 44. The purpose of the present experiments was to determine if neurons in either the RMg or Gia/PGi project to the A5, A7 or the LC. To this end, iontophoretic injections of the anterograde tracer, PHA-L, were made into the RMg or Gia/PGi and the location of PHA-L-labeled axons and axon terminals within the A5, A7, or LC was determined. The results indicated that the A7 cell group receives a dense projection from both the RMg and the Gia/PGi, while the A5 cell group receives a substantially smaller projection from these two nuclei. In contrast, the RMg and Gia/PGi have negligible projections to the LC. This latter observation is consistent with previous reports which indicate that neurons in the RMg have either a very sparse projection to the LC 12 or no discernible projection to this cell group 3. While these observations do not provide any direct evidence that axonal projections from either the RMg or Gia/PGi make synaptic contacts on catecholamine neurons, there is suggestive evidence that such synaptic contacts may exist. For example, many of the PHAL-labeled axons within the catecholamine cell groups branched extensively which suggests that these axons probably terminate within a few hundred microns. In addition, most of the branching axons contained numerous varicosities which have been shown to be associated with synaptic contacts made by PHA-L-labeled axons 45. Furthermore, some PHA-L-labeled axons appeared to be

113 closely apposed to DflH-ir somata (Fig. 3C-F). It is possible that some of the axons passing through the P H A - L injection sites in the RMg or Gia/PGi were labeled by the injection. However, the results of a systematic study of P H A - L uptake into axons of passage ~3 has shown that while axons of passage are capable of incorporating and transporting PHA-L, they do so to a far lesser extent than neuronal cell bodies. It is unlikely, therefore, that fibers of passage in the RMg or Gia/PGi could account for more than a small percentage of the PHA-L-labeled axons that were seen in the A7 region. To provide additional evidence for projections from neurons in the RMg and Gia/PGi to the A7 cell group, iontophoretic injections of the retrograde tracer, FluoroGold, were made into the region of the A7 cell group and brainstem sections were examined for the presence of retrogradely-labeled neurons in the RMg and Gia/PGi. The results indicated that both the RMg and Gia/PGi contained neurons which project to the A7 region. However, it appeared that the Gia/PGi contained many more A7-projecting neurons than did the RMg. This finding is consistent with the observation that P H A - L injections in the Gia/PGi labeled a much greater number of axons in the A7 cell group than similar injections in the RMg. Similar experiments were done to determine whether serotonergic neurons located in the RMg or Gia/PGi project to the A7 cell group. Brainstem sections processed for 5-HT immunocytochemistry exhibited only a small number of double-labeled neurons in the RMg or Gia/PGi that contained both Fluoro-Gold and 5-HT-ir. Thus, serotonergic neurons located in the RMg and Gia/PGi do not appear to have a significant projection to the A7 cell group. It is conceivable that the retrogradely-filled neurons seen in the RMg or Gia/PGi were not labeled by Fluoro-Gold transported into axon terminals within the A7, but instead were labeled by uptake of Fluoro-Gold into damaged axons passing through the A7 region. This possibility is supported by the observations of Schmued and Fallon 37 who demonstrated that Fluoro-Gold can be taken up and retrogradely transported by cut or damaged axons, but not by intact axons. However, there are several factors that argue against this possibility and support the assumption that the observed retrograde labeling resulted from the uptake of Fluoro-Gold into axon terminals in the A7 cell group. First, the amount of damage caused by the Fluoro-Goid injections was minimized by the use of iontophoretic ejection from glass micropipettes with small tip diameters. Second, a dense terminal field of PHA-L-iabeled axons and axon terminals was demonstrated in the A7 region following either RMg or Gia/PGi PHA-L injections, but the density of

axons was far less in the area rostral to the A7. This observation suggests that many RMg and Gia/PGi neurons terminate in the A7 region. In addition, the PHA-L-labeled axons in the A7 region exhibited extensive branching and varicosities which indicates the existence of synaptic terminals. The injection of Fluoro-Gold in the A7 region illustrated in Fig. 4 included the rostral portion of the trigeminal motor nucleus which may account for the heavy labeling seen in the parvocellular reticular nucleus (PCRt). Moreover, Fluoro-Gold injections that encompassed DflH-ir neurons in the A7 cell group, but not neurons in the trigeminal motor nucleus, did not produce retrograde labeling of neurons in the PCRt (Fig. 5). These observations are consistent with those of others 2°' 39,40 who demonstrated a major projection to the trigeminal motor nucleus from neurons located in both the medial and lateral aspects of the PCRt. The results of the experiments described in this report demonstrate that neurons in the RMg have axonal projections to the A7 catecholamine cell group. Although there is suggestive evidence that RMg neurons project to the A7 region 5, no definitive evidence for such a projection has been previously reported. These results provide an anatomical basis for the observations that the antinociception produced by stimulation of neurons in the ventromedial medulla (RMg and Gia/PGi) is media t e d ' b y both bulbospinal serotonergic neurons and spinally-projecting noradrenergic neurons. It has been well-documented that electrical or chemical stimulation of the RMg or Gia/PGi produces potent antinociception 1' 4,10,18,29,31-36. This antinociception is apparently due to activation of neurons located within these nuclei and not fibers of passage since stimulation induced by microinjection of glutamate, known to selectively activate cell bodies, produces a similar antinociceptive effect 22"36. Several lines of evidence indicate that the antinociception produced by RMg or Gia/PGi stimulation is mediated by serotonergic raphe-spinal neurons and by bulbospinal noradrenergic neurons. For example, the antinociception induced by electrical or chemical stimulation of the RMg or Gia/PGi is attenuated by intrathecal administration of either serotonergic or noradrenergic antagonists 4'18'22"32 or by depletion of spinal cord serotonin or norepinephrine content 34. Similarly, stimulation of the RMg or the Gia/PGi releases both 5-HT and NE into spinal cord superfusates of anesthetized rats 17'33. The location of the noradrenergic neurons activated by stimulation of neurons located in the RMg or Gia/PGi can be assumed to be one or more of the pontine catecholamine cell groups that contain spinally-projecting noradrenergic neurons: the A5, A7 or L C 24'41-44. There is evidence that stimulation of the A5 cell group 25-2s, the L C 21"23"38 and

114 the A7 region 19"46 produces antinociception. H o w e v e r , since the present anatomical results indicate that the A7 cell group receives the strongest projection from both the R M g and G i a / P G i , it is likely that spinally-projecting neurons in the A 7 cell group mediate, at least in part, the antinociception induced by stimulation of neurons in the R M g and G i a / P G i . Since the R M g and G i a / P G i both contain a substantial p o p u l a t i o n of spinally-projecting serotonergic neurons 69, it is possible that these serotonergic neurons may also project to the A7 cell group. H o w e v e r , the failure of F l u o r o - G o l d injections in the A 7 cell group to retrogradely label 5-HT-ir neurons in the ventromedial medulla argues against this possibility. These results suggest that non-serotonergic neurons in the R M g and G i a / P G i that project to A7 neurons may be involved in modulating nociception. A d d i t i o n a l evidence for this conclusion is the observation that microinjection of cholinergic agonists into the R M g can p r o d u c e antinociception by selective activation of spinally-projecting noradrenergic neurons without activating raphe-spinal serotonergic neurons. Thus, microinjection of the cholinergic agonist carbachol p r o d u c e s antinociception that is blocked by REFERENCES 1 Akaike, A., Shibata, T., Satoh, M. and Takagi, H., Analgesia induced by microinjection of morphine into, and electrical stimulation of, the nucleus reticularis paragigantoceUularis"of rat medulla oblongata, Neuropharmacology, 17 (1978) 775-778. 2 Andrezik, J.A. and Beitz, A.J., Reticular formation, central gray and related tegmental nuclei. In G. Paxinos (Ed.), The Rat Nervous System, Vol. 2, Hindbrain and Spinal Cord, Academic Press, Orlando, 1985, pp. 1-28. 3 Aston-Jones, G., Ennis, M., Pieribone, V.A., Nickell, W.T. and Shipley, M.T., The brain nucleus locus coeruleus: restricted afferent control of a broad efferent network, Science, 234 (1986) 734-737. 4 Barbaro, N.M., Hammond, D.L. and Fields, H.L., Effects of intrathecally administered methysergide and yohimbine on microstimulation-produced antinociception in the rat, Brain Research, 343 (1985) 223-229. 5 Bobillier, P., Seguin, S., Petitjean, F., Salvert, D., Touret, M. and Jouvet, M., The raphe nuclei of the cat brainstem: a topographical atlas of their efferent projections as revealed by autoradiography, Brain Research, 113 (1976) 449-486. 6 Bowker, R.M., Steinbusch, H.W.M. and Coulter, J.D., Serotonergic and peptidergic projections to the spinal cord demonstrated by a combined retrograde HRP histochemical and immunocytochemical staining method, Brain Research, 211 (1981) 412-417. 7 Bowker, R.M., Westlund, K.N. and Coulter, J.D., Origins of serotonergic projections to spinal cord in rat: an immunocytochemical-retrograde transport study, Brain Research, 226 (1981) 187-199. 8 Bowker, R.M., Westlund, K.N., Sullivan, M.C., Wilber, J.F. and Coulter, J.D., Transmitters of the raphe-spinal complex: immunocytochemical studies, Peptides, 3 (1982) 291-298. 9 Bowker, R.M., Westlund, K.N., 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 Research, 288 (1983) 33-48.

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Acknowledgements. This work was supported by USPHS Grant DA 03980 from the National Institute on Drug Abuse. We would like to thank Nick Kriho for his expert help with the photomicrography.

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