Brain Research, 547 (1991) 279-288 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 ADONIS 000689939116549B
279
BRES 16549
The projection of noradrenergic neurons in the A7 catecholamine cell group to the spinal cord in the rat demonstrated by anterograde tracing combined with immunocytochemistry Frank M. Clark and Herbert K. Proudfit The University of Illinois at Chicago, Department of Pharmacology, Chicago, IL 60680 (U. S. A.)
(Accepted 20 November 1990) Key words: Noradrenaline; A7 cell group; Phaseolus vulgaris leukoagglutinin; Fluoro-Gold; Antinociception; Dopamine-fl-hydroxylase; Tyrosine hydroxylase
Noradrenergic neurons located in the A5, A7 and locus coeruleus/subcoeruleus (LC/SC) catecholamine cell groups innervate all levels of the spinal cord. However, the specific spinal cord terminations of these neurons have not been clearly delineated. This study determined the spinal cord terminations of the A7 catecholamine cell group using the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in combination with dopamine-fl-hydroxylase (DBH) immunocytochemistry. In addition, the spinal cord projections of A7 neurons were examined by measuring the reduction in the density of DBH-immunoreactive axons in specific regions of the spinal cord after a unilateral electrolytic lesion of the A7 cell group. The results of these experiments indicate that noradrenergic neurons in the A7 cell group project primarily in the ipsilateral dorsolateral funiculus and terminate most heavily in the dorsal horn (laminae I-IV). 1NTRODUCTION The spinal cord is innervated by a dense plexus of norepinephrine-containing axon terminals 4'11"29. Although these axon terminals are present throughout the spinal cord, they are most concentrated in the superficial dorsal horn, the ventral horn motoneuron pool, lamina X, and are especially dense in the thoracic and sacral intermediolateral cell columns. Since there are only scattered norepinephrine-containing cell bodies in the upper cervical segments of the spinal cord 22"3°, it has been assumed that the origin of the noradrenergic innervation of the spinal cord arises mainly from one or more of the 7 noradrenergic cell groups ( A 1 - A 7 ) described by Dahlstr6m and Fuxe 8. There is now general agreement that only the pontine noradrenergic cell groups, A5, A6 (locus coeruleus, LC) and A7, project to the spinal cord 19'27'33-36. Although the location of the noradrenergic neurons that comprise the A5 and A6 cell groups have been clearly defined, the anatomical description of the A7 group is somewhat ambiguous and appears to differ among species. For example, in the cat the A7 cell group is considered to include the nucleus K611iker-Fuse (KF), the nucleus subcoeruleus (SC) and the medial and lateral
parabrachial nuclei 24. All 4 of these cell groups are closely associated and lie just lateral and ventral to the LC 24. However, the organization of the A7 and A6 cell groups in the rat is much different. In this species there are only a few widely distributed norepinephrine-containing neurons in the medial and lateral parabrachial nuclei. In addition, the SC is located immediately ventral to the LC and these two cell groups may be considered as one anatomical 7'25 and functional 16A7 unit: the LC/SC complex. However, the A7 cell group in the rat, originally described by Dahlstr6m and Fuxe s, is distinct from the LC/SC complex and forms a continuous band of neurons located ventral to the ventral part of the superior cerebellar peduncle and dorsal to the rubrospinal tract at the level of the caudal third of the pons 15A8'21. Although the noradrenergic neurons located in the A7 cell group have been shown to project to the spinal cord 19'33-36, the specific sites in the spinal cord where these noradrenergic neurons terminate have not been determined. The purpose of this study was to examine the spinal cord projections of noradrenergic neurons in the A7 cell group using anterograde tracing with Phaseolus vulgaris leucoagglutinin ( P H A - L ) . PHA-L-labeled axons and terminals that also contained norepinephrine were identified using dopamine-fl-hydroxylase ( D B H )
Correspondence: H.K. Proudfit, Department of Pharmacology (mc 868), University of Illinois at Chicago, P.O. Box 6998, Chicago, IL 60680, 1LS.A.
280
immunocytochemistry, a presumptive marker for noradrenergic somata and axon terminals ~3. In addition, the projections of A7 neurons to the spinal cord were examined by making unilateral electrolytic lesions of the A7 cell group and measuring the subsequent reductions in the density of DBH-immunoreactive (DBH-ir) axon terminals in different areas of the spinal cord. Preliminary results of these experiments have been reported previously 5.
MATERIALS AND METHODS
Animal preparation These experiments were done using adult female SpragueDawley derived rats (Sasco, Inc., Omaha, NE) that weighed between 300 and 410 g. Animals were anesthetized with ketamine (100 mg/kg) and immobilized in a stereotaxic frame. Stereotaxically guided micropipets or metal electrodes were used for PHA-L injections or electrolytic lesions, respectively.
Anterograde tracing experiments The anterograde tracer PHA-L was used to determine the spinal cord distribution of axons arising from neurons in the A7 cell group. Unilateral iontophoretic injections of PHA-L (Vector Laboratories) were made into the left A7 cell group. Coordinates were AP +0.15; V +2.5 and L +2.1 mm with respect to the interaural line with the incisor bar set at -2.5 mm. Iontophoretic current (5-!0 #A) and pipette tip diameters (10--15 /am) used to deliver PHA-L were similar to those used by Gerfen and Sawchenkoa2. Thirty days after the PHA-L injection, animals were perfused intracardiatly with 200 ml cold saline, 300 ml of cold 4% phosphate buffered paraformaldehyde (pH 7.4), followed by 200 ml of 10% sucrose in 0.1 M phosphate buffer. Brains and spinal cords were removed and stored at 4 °C in a solution containing 30% sucrose in 0.1 M phosphate buffer for at least 3 days. Forty/~m sections of brainstem and spinal cord were cut on a cryostat and free-floating sections were processed for PHA-L and DBH immunocytochemistry. Sections were washed twice in 187 mM phosphate-buffered saline (PBS; pH 7.4) between each antibody incubation unless otherwise stated. All antibody solutions contained 0.5% Triton X-100 in PBS. Sections were cut, washed with cold PBS, treated with 2.5% HzOz in methanol to remove endogenous peroxidase3, washed 4 times with PBS and incubated for 48 h at 4 °C in pooled goat anti-PHA-L (l:1000; Vector) and rabbit anti-DBH (1:1000; Eugene Tech) antibody solutions. Following the 2 day incubation, the sections were incubated for 30 min in pooled donkey anti-goat immunoglobulins (1:80; Chemicon) and swine anti-rabbit (1:80; Accurate) immunoglobulins. The sections were then placed in goat peroxidase anti-peroxidase (PAP; 1:180; Chemicon) for 30 min. Sections were incubated for 6 min in a solution which contained 0.05% filtered diaminobenzidene (DAB) and 0.005% H20 z in PBS. Tissue sections were then incubated in rabbit PAP (1:180; Cappel) for 30 min followed by a 4 minute incubation in a 50 mM Tris HC1 buffer (pH 8) which contained 0.013% filtered DAB, 0.005% H202 and 0.67% nickel ammonium sulfate. Finally, the tissue sections were mounted on gelatin-coated slides and coverslipped with Permount. This processing resulted in blue-black DBH-positive staining and brown PHA-L-labeled somata and terminals. Some of the spinal cord sections were processed using secondary antisera conjugated with fluorophores to allow visualization of axons that contained both PHA-L- and DBH-ir. These sections were incubated as above in pooled primary antibody for 48 h at 4 °C, then incubated for 30 min in donkey anti-goat immunoglobulins conjugated with fluorescein isothiocyanate (FITC; 1:80; Chemicon). Tissue was then incubated in sheep anti-rabbit immunoglobulins conjugated with rhodamine (1:80; Cappel), washed twice, mounted on gelatin-coated slides and
coverslipped with DPX (BDH Chemicals) The distribution of PHA-L-labeled axons z, ld terminals ill the spinal cord was mapped by making composite drawings of :dt PHA-L-labeled axons which appeared in 12 consecutive spinal cold sections. Composite drawings were made of sections from cervical. thoracic and lumbar segments of the spinal cord. The laminar distribution of PHA-L-labeled axons was estimated fr~m~ plates found in the atlas of Paxinos and Watson 23. Finally, estimates were made of the number of PHA-L-labeled axons that also contained DBH-ir. These estimates were made by systematically examining ten randomly selected sections from lumbar, thoracic and cervical spinal cord segments using an FITC filter ~,~ visualize PI-IAL-labeled axons. When PHA-L-labeled axons were identified, the rhodamine filter was used to determine if the same axon also contained DBH-ir. These double-labeled axons were assumed to have their origin in the A7 cell group,
Retrograde tracing studies It is possible that PHA-L injections in the A7 cell group may have labeled some non-catecholamine-containing neurons that project to the spinal cord. The number and location of non-catecholaminecontaining neurons in the area of the A7 cell group was estimated by examining brainstem sections for retrogradely labeled neurons following large bilateral injections of the retrograde tracer FluoroGold into the lumbar segments of the spinal cord. Spinally projecting catecholaminergic neurons were identified by the presence of both Fluoro-Goid and tyrosine hydroxy!ase-immunoreactivity (TH-ir). Tyrosine hydroxylase antiserum (Incstar) was used to label A7 catecholamine neurons because, in our experience, it produces a more intense staining than dopamine-fl-hydroxylase antiserum (Eugene Tech). Rats were anesthetized, a laminectomy was performed to expose the dorsal aspect of the lumbar spinal cord and 3-5 injections of 2% Fluoro-Gold (Fluorochrome, Inc) in a volume of 0.5 pl were made at different depths of the spinal cord. Ten days after surgery, the injected rats were intracardialiy perfused as described above. Brainstem and spinal cord sections were cut, washed in cold PBS and incubated for twenty min in 2.5% H20 2 in methanol. Spinal cord sections were washed 4 times in PBS. then mounted on slides and coverslipped with DPX. Brainstem sections were washed 4 times and then incubated overnight at 4 °C in a PBS solution that contained mouse anti-tyrosine hydroxylase antibody (1:1000; Incstar) and 0.5% Triton X-100. Sections were washed in PBS, incubated in goat anti-mouse immunoglobulins conjugated with rhodamine (1:80; Cappel) for 30 min and then mounted on slides and coverslipped with DPX,
Lesion experiments The purpose of these experiments was to use a different approach to determine the spinal cord distribution of axons arising from noradrenergic neurons in the A7 cell groupl These experiments determined the areas in the spinal cord where the density of noradrenergic axons was reduced following unilateral lesions of A7 neurons. Animals were anesthetized, mounted in a stereotaxie headholder and unilateral A7 lesions were made by passing a current of 2 mA for 5 s. Fourteen days after surgery, animals were intracardially perfused as described above. Brainstem and spinal cord sections were cut, washed in cold PBS, incubated in 2.5% H20 z in methanol for 20 min, washed 4 times, and then incubated at 4 °C for 48 h in a solution which contained rabbit anti-DBH antibody (1:1000; Eugene Tech). Sections were then incubated in goat anti-rabbit immunoglobulins (l:80; Atlantic), then incubated in rabbit PAP (1:180; Cappel) for 30 min. Sections were then incubated for 6 min in a solution which contained 0,05% filtered DAB and 0.005% HzO 2 in PBS for 6 min. Finally, the sections were mounted on gelatin-coated slides and coverslipped with Permount, The effect of A7 lesions on the number of DBH-ir axons in the lumbar segments of the spinal cord was assessed by quantifying the density of these axons on the ipsilateral and contralateral sides of each section using an image analysis system (JAVA, Jandc! Scientific) as described previously7. Briefly. DBH-ir axons were
281 visualized in spinal cord sections under darkfield illumination, the image was digitized and the density of DBH-ir axons in either the dorsal or the ventral horn was determined. The approximate area of the dorsal horn that was analyzed included laminae I through IV, while the approximate area of the ventral horn included laminae VIII and IX. The analysis included 20 consecutive sections from each of 4 rats with lesions that produced total unilateral destruction of the noradrenergic neurons in the A7 cell group. The effects of A7 lesions on the density of DBH-ir axons in the spinal cord was determined by comparing the density in the dorsal or ventral horn on the ipsilateral side with that on the contralateral side. The statistical significance of these comparisons was assessed using the Student's paired t-test. The destruction of noradrenergic neurons in the A7 cell group was verified by examining the lesioned area in sections that were stained using DBH immunocytochemistry.
RESULTS
Anterograde tracer experiments The purpose of these experiments was to determine the d i s t r i b u t i o n o f a x o n t e r m i n a l s in t h e s p i n a l c o r d t h a t arise f r o m n o r a d r e n e r g i c n e u r o n s l o c a t e d in t h e A 7 cell g r o u p . To t h i s e n d , t h e a n t e r o g r a d e t r a c e r P H A - L
was ionto-
p h o r e t i c a l l y e j e c t e d i n t o t h e r e g i o n o f t h e A 7 cell g r o u p and the location of PHA-L-labeled
a x o n t e r m i n a l s in t h e
spinal cord was assessed. An example of a representative P H A - L i n j e c t i o n in t h e A 7 cell g r o u p is s h o w n in Fig. 1. Three PHA-L injections that encompassed a majority of t h e D B H - i r n e u r o n s in t h e A 7 cell g r o u p a r e s h o w n in
A
PHA-L
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TH
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DBH
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Fig. 1. The location of a PHA-L injection that encompassed some of the spinally projecting noradrenergic neurons in the A7 cell group. Panel A shows the location of the PHA-L deposit in the A7 cell group. Panel B shows the location of DBH-ir neurons in the contralateral A7 cell group in the same transverse section. Panels C and D illustrate the location of spinally projecting A7 neurons in a different animal following a bilateral injection of Fluoro-Gold in the lumbar spinal cord. Panel C shows the noradrenergic neurons in the A7 cell group stained for TH-ir. Panel D shows the location of neurons in the A7 that were retrogradely labeled by Fluoro-Gold injected into the spinal cord. Note that every spinally projecting neuron seen in Panel D also contains TH-ir as shown in Panel C. The calibration bar in Panel A is 200/~m and represents the scale for Panels A and B. The calibration bar in Panel C is 100/~m and represents the scale for Panels C and D.
282
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Fig~ 2, PHA-L injection sites in the dorsolateral pontine tegmentum and the location of PHA-L-labeled axons and t e ~ a l s in the spinal cord. Sections A, B and C are :drawings made with a projection microscope which illustrate the location Of PHA-L injection Sites in transverse brainstem sections taken from 3 different rats. An
enlargement of the injection sites is shown tO the right of each section: The extent of PHA,L deposits is outlined by solid lines and DBH-ir neurons in the A7 are reprvsented by solid circles. The brainstem sections illustrated are those in which the maximum deposit of PHA-L was seen. The size of the injection site was taken to be the size of the dense core of PHA-L-Iabel, although some diffuse PHA-L labeling was seen outside the central core of the PHA-L deposit. Drawings of the distribution of PHA-L-labeled axons in lumbar segments of the spinal cord are shown on the far right. The drawing of each spinal cord section is a composite of 12 adjacent sections.
Fig. 2. Brainstem sections were processed for both DBHand PHA-L-immunocytochemistry using the standard PAP method and the nickel,enhanced PAP method in order to identify the injection site. Although it was not possible to determine if a particular :neuron was immunoreactive for both D B H and PHA-L using the PAP method, neurons were observed within the injection site that were PHA-L-labeled (stained brown) and morphologically similar to the large multipolar blue-black DBHir neurons that were located in the A7 cell group, but outside the injection site. It is likely that these PHAL,labeled neurons also contained DBH-ir. Fig. 2 shows that axons labeled by a PHA-L injection that encompassed the A7 cell group were mainly located in the ipsilateral dorsolateral funiculus. By contrast, only a few labeled axons were seen in the ventral or
Fig. 3. The location of PHA:L injection sites-that did: not: include DBH-ir neurons in the A7 cell group. A: PHA-L injection located "dorsal to the DBH-ir neurons in the A7 cell group. B: PHA:L injection located rostral to DBH-ir neurons in t h e A 7 eetl group. C: PHA-L injection located ventral to DBH,ir neurons in the A7 cell group. The features of this figure are Similar to those described in the legend for Fig. 2.
A
B
C
D
Fig. 4. The distribution of PHA-L-laheled axons and terminals ~at different levels of the spinal cord following an injection of PHA-L in the A7 cell group. A: location of the PHA-L injection site in a transverse section through the dorsolateral pontine tegmentum: B: distribution of PHA-L-laheling in lumbar spinal cord s ~ e n t s . C: distribution of PHA-L-labeling in thoracic spinal cord segments. D: distribution of PHA-L-labeling in cervical spinal cord segments. Each drawing (B, C and D) is a composite of I2 contiguous transverse sections. This is the same case as illustrated in Figl 2A.
283
A
B
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PHA-L
B
DBH
Fig. 6. An example of a DBH-ir axon in the dorsal horn that was also anterogradely labeled by a PHA-L injection in the A7. Panel A illustrates two axons that were anterogradely labeled by PHA-L injected into the A7 cell group. Panel B shows that one of these axons (arrow 1) also contains DBH-ir, while the other (arrow 2) does not contain DBH-ir. Panel B also illustrates a DBH-ir axon that was not anterogradely labeled from the A7 cell group (arrow 3). The calibration bar is 25/zm.
missed the A7 cell group dorsally (Fig. 3A), rostrally (Fig. 3B) or ventrally (Fig. 3C). The n u m b e r of P H A L-labeled axons in l u m b a r spinal cord segments from these 3 animals was very sparse. The location of P H A - L - l a b e l e d axons in different levels of the spinal cord is shown in Fig. 4. In general the P H A - L labeling was similar in all spinal cord segments, although some minor differences were apparent. For example, the projection to the superficial dorsal horn in lumbar segments was greater than that in cervical and thoracic spinal cord segments. However, the projections
Fig. 5. Brightfield photomicrographs of transverse brainstem sections that illustrate the morphology of PHA-L-labeled axons and terminal segments in the spinal cord dorsal horn following a PHA-L injection in the A7. The axons and terminal segments shown in Panel A are located within about 500 ~m from the dorsal surface of the spinal cord which can be seen in the upper part of the panel. Panel B shows a high power photomicrograph of axons and varicosities in the dorsal horn. The calibration bar in Panel A is 100 /tm and that in Panel B is 40 ~tm.
ventrolateral funiculi on the ipsilateral side and the labeling in the contralateral white matter was very sparse. All 3 cases in which P H A - L was injected in the A7 cell group exhibited substantial labeling in the ipsilateral dorsal horn and was concentrated in the area that corresponds to laminae I - I V . Two of the A7 injections (Fig. 2B,C) produced dense labeling in the lateral part of the dorsal horn that approximately corresponds to laminae III and IV. Injections of P H A - L that did not include D B H - i r neurons in the A7 cell group produced only sparse labeling in the spinal cord. Fig. 3 shows the distribution ~f labeled axons produced by P H A - L injections that
to lamina X near the central canal and to the medial aspects of laminae VI-VII in the intermediate zone were more dense in the cervical and thoracic segments than those in the l u m b a r spinal cord segments. In addition, the descending A7 axons coursed through the lateral funiculus in cervical segments, while those in the l u m b a r or thoracic segments were located in the dorsolateral funiculus. Finally, there was only a minimal projection to the ipsilateral ventral horn or the contralateral spinal cord in all spinal cord segments. P H A - L - l a b e l e d axons and terminal segments in the spinal cord were short (usually less than 100/~m segments were seen in 40/~m sections), of fine diameter and seldom branched (Fig. 5). Estimates of the n u m b e r of doublelabeled axons revealed that nearly all of the axons and axon terminal segments which were anterogradely labeled with P H A - L also contained D B H - i r (Fig. 6). Only a few P H A - L - l a b e l e d axons did not contain DBH-ir.
Retrograde tracer experiments It is possible that some of the P H A - L - l a b e l e d terminals seen in the spinal cord following a P H A - L injection into the A7 cell group originate from spinally projecting
284
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Fig. 7. The location of neurons in the pontine tegmentum that were retrogradely labeled following bilateral injections of Fluoro-Gold into the lumbar segments of the spinal cord. The location of retrogradely labeled neurons and those that exhibited TH-ir are plotted on transverse sections from 3 different levels of the A7 cell group (Sections A, B and C). The injection site, represented by the solid area, is shown in Section D. Neurons labeled with Fluoro-Gold are represented by crosses, TH-ir somata are represented by open circles and double-labeled somata that contained both TH-ir and Fluoro-Goid are represented by filled circles. All of the labeled neurons in each section have been plotted. The numbers to the right of each section represent the distance (mm) rostral to the interaural line. n o n - c a t e c h o l a m i n e neurons in or near the A 7 cell group. To d e t e r m i n e the n u m b e r and distribution of spinally p r o j e c t i n g non-catecholamine neurons in the vicinity of the A 7 cell group, large bilateral injections of the r e t r o g r a d e tracer, F l u o r o - G o l d , were m a d e into the l u m b a r spinal cord and the distribution of neurons i n t h e pontine t e g m e n t u m that contained both retrogradely t r a n s p o r t e d F l u o r o - G o l d and tyrosine hydroxylase-immunoreactivity (TH-ir) was d e t e r m i n e d (Fig. 7). The m a j o r i t y ( > 8 0 % ) o f the T H - i r neurons in the A 7 cell group were labeled with retrogradely t r a n s p o r t e d Fluoro-
Fig. 8. Photomicrograph of a transverse bramstem section that shows a representative unilateral electrolytic lesion that included the A7 cell group. The calibration bar is t.0 mm G o l d (Figs. 1C,D and 7). Within the area in which the TH-ir neurons of the A 7 cell group are distributed, the vast majority ( > 9 0 % ) of r e t r o g r a d e l y filled neurons were also labeled with T H - i r (Figs. 1C,D and 7). These results suggest that most of the spinally projecting neurons within the i m m e d i a t e vicinity of the A 7 cell group contain catecholamines. H o w e v e r , there was a large cluster of spinally projecting non-catecholamine-containing neurons located just medial to the A 7 cell group that e x t e n d e d in a ventrolateral direction from the ventrolateral margin of the cerebral aqueduct to just dorsal to the superior olivary nucleus (Fig, 7). This cluster of neurons appears to correspond to the dorsal and ventral subcoeruleus nuclei ~.
Lesion experiments A n additional e x p e r i m e n t a l a p p r o a c h was used to examine the projections of n o r a d r e n e r g i c A 7 neurons to the spinal cord. The p u r p o s e of these experiments was to m a k e unilateral electrolytic lesions of the A 7 cell group and m e a s u r e the subsequent reduction in the density of catecholamine-containing axon terminals in the spinal cord. A representative A 7 lesion is shown in Fig. 8 and Fig. 9 shows the effects of 4 such lesions on the density of D B H - i r axons in the spinal cord. Each lesion p r o d u c e d a statistically significant (P < 0.05) reduction in the density of D B H - i r axon terminals in the dorsal horn, but only one in 4 animals d e m o n s t r a t e d a statistically significant density reduction in the ventral horn. The reduction in terminal density in the dorsal horn varied from 13,8% to 29.0% and was not c o r r e l a t e d with the size of the lesion. For e x a m p l e , Fig. 9 A shows that a relatively
285 to the spinal cord 20'27'31'32-36. H o w e v e r , the funicular 50 40 30
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279
BRES 16549
The projection of noradrenergic neurons in the A7 catecholamine cell group to the spinal cord in the rat demonstrated by anterograde tracing combined with immunocytochemistry Frank M. Clark and Herbert K. Proudfit The University of Illinois at Chicago, Department of Pharmacology, Chicago, IL 60680 (U. S. A.)
(Accepted 20 November 1990) Key words: Noradrenaline; A7 cell group; Phaseolus vulgaris leukoagglutinin; Fluoro-Gold; Antinociception; Dopamine-fl-hydroxylase; Tyrosine hydroxylase
Noradrenergic neurons located in the A5, A7 and locus coeruleus/subcoeruleus (LC/SC) catecholamine cell groups innervate all levels of the spinal cord. However, the specific spinal cord terminations of these neurons have not been clearly delineated. This study determined the spinal cord terminations of the A7 catecholamine cell group using the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in combination with dopamine-fl-hydroxylase (DBH) immunocytochemistry. In addition, the spinal cord projections of A7 neurons were examined by measuring the reduction in the density of DBH-immunoreactive axons in specific regions of the spinal cord after a unilateral electrolytic lesion of the A7 cell group. The results of these experiments indicate that noradrenergic neurons in the A7 cell group project primarily in the ipsilateral dorsolateral funiculus and terminate most heavily in the dorsal horn (laminae I-IV). 1NTRODUCTION The spinal cord is innervated by a dense plexus of norepinephrine-containing axon terminals 4'11"29. Although these axon terminals are present throughout the spinal cord, they are most concentrated in the superficial dorsal horn, the ventral horn motoneuron pool, lamina X, and are especially dense in the thoracic and sacral intermediolateral cell columns. Since there are only scattered norepinephrine-containing cell bodies in the upper cervical segments of the spinal cord 22"3°, it has been assumed that the origin of the noradrenergic innervation of the spinal cord arises mainly from one or more of the 7 noradrenergic cell groups ( A 1 - A 7 ) described by Dahlstr6m and Fuxe 8. There is now general agreement that only the pontine noradrenergic cell groups, A5, A6 (locus coeruleus, LC) and A7, project to the spinal cord 19'27'33-36. Although the location of the noradrenergic neurons that comprise the A5 and A6 cell groups have been clearly defined, the anatomical description of the A7 group is somewhat ambiguous and appears to differ among species. For example, in the cat the A7 cell group is considered to include the nucleus K611iker-Fuse (KF), the nucleus subcoeruleus (SC) and the medial and lateral
parabrachial nuclei 24. All 4 of these cell groups are closely associated and lie just lateral and ventral to the LC 24. However, the organization of the A7 and A6 cell groups in the rat is much different. In this species there are only a few widely distributed norepinephrine-containing neurons in the medial and lateral parabrachial nuclei. In addition, the SC is located immediately ventral to the LC and these two cell groups may be considered as one anatomical 7'25 and functional 16A7 unit: the LC/SC complex. However, the A7 cell group in the rat, originally described by Dahlstr6m and Fuxe s, is distinct from the LC/SC complex and forms a continuous band of neurons located ventral to the ventral part of the superior cerebellar peduncle and dorsal to the rubrospinal tract at the level of the caudal third of the pons 15A8'21. Although the noradrenergic neurons located in the A7 cell group have been shown to project to the spinal cord 19'33-36, the specific sites in the spinal cord where these noradrenergic neurons terminate have not been determined. The purpose of this study was to examine the spinal cord projections of noradrenergic neurons in the A7 cell group using anterograde tracing with Phaseolus vulgaris leucoagglutinin ( P H A - L ) . PHA-L-labeled axons and terminals that also contained norepinephrine were identified using dopamine-fl-hydroxylase ( D B H )
Correspondence: H.K. Proudfit, Department of Pharmacology (mc 868), University of Illinois at Chicago, P.O. Box 6998, Chicago, IL 60680, 1LS.A.
280
immunocytochemistry, a presumptive marker for noradrenergic somata and axon terminals ~3. In addition, the projections of A7 neurons to the spinal cord were examined by making unilateral electrolytic lesions of the A7 cell group and measuring the subsequent reductions in the density of DBH-immunoreactive (DBH-ir) axon terminals in different areas of the spinal cord. Preliminary results of these experiments have been reported previously 5.
MATERIALS AND METHODS
Animal preparation These experiments were done using adult female SpragueDawley derived rats (Sasco, Inc., Omaha, NE) that weighed between 300 and 410 g. Animals were anesthetized with ketamine (100 mg/kg) and immobilized in a stereotaxic frame. Stereotaxically guided micropipets or metal electrodes were used for PHA-L injections or electrolytic lesions, respectively.
Anterograde tracing experiments The anterograde tracer PHA-L was used to determine the spinal cord distribution of axons arising from neurons in the A7 cell group. Unilateral iontophoretic injections of PHA-L (Vector Laboratories) were made into the left A7 cell group. Coordinates were AP +0.15; V +2.5 and L +2.1 mm with respect to the interaural line with the incisor bar set at -2.5 mm. Iontophoretic current (5-!0 #A) and pipette tip diameters (10--15 /am) used to deliver PHA-L were similar to those used by Gerfen and Sawchenkoa2. Thirty days after the PHA-L injection, animals were perfused intracardiatly with 200 ml cold saline, 300 ml of cold 4% phosphate buffered paraformaldehyde (pH 7.4), followed by 200 ml of 10% sucrose in 0.1 M phosphate buffer. Brains and spinal cords were removed and stored at 4 °C in a solution containing 30% sucrose in 0.1 M phosphate buffer for at least 3 days. Forty/~m sections of brainstem and spinal cord were cut on a cryostat and free-floating sections were processed for PHA-L and DBH immunocytochemistry. Sections were washed twice in 187 mM phosphate-buffered saline (PBS; pH 7.4) between each antibody incubation unless otherwise stated. All antibody solutions contained 0.5% Triton X-100 in PBS. Sections were cut, washed with cold PBS, treated with 2.5% HzOz in methanol to remove endogenous peroxidase3, washed 4 times with PBS and incubated for 48 h at 4 °C in pooled goat anti-PHA-L (l:1000; Vector) and rabbit anti-DBH (1:1000; Eugene Tech) antibody solutions. Following the 2 day incubation, the sections were incubated for 30 min in pooled donkey anti-goat immunoglobulins (1:80; Chemicon) and swine anti-rabbit (1:80; Accurate) immunoglobulins. The sections were then placed in goat peroxidase anti-peroxidase (PAP; 1:180; Chemicon) for 30 min. Sections were incubated for 6 min in a solution which contained 0.05% filtered diaminobenzidene (DAB) and 0.005% H20 z in PBS. Tissue sections were then incubated in rabbit PAP (1:180; Cappel) for 30 min followed by a 4 minute incubation in a 50 mM Tris HC1 buffer (pH 8) which contained 0.013% filtered DAB, 0.005% H202 and 0.67% nickel ammonium sulfate. Finally, the tissue sections were mounted on gelatin-coated slides and coverslipped with Permount. This processing resulted in blue-black DBH-positive staining and brown PHA-L-labeled somata and terminals. Some of the spinal cord sections were processed using secondary antisera conjugated with fluorophores to allow visualization of axons that contained both PHA-L- and DBH-ir. These sections were incubated as above in pooled primary antibody for 48 h at 4 °C, then incubated for 30 min in donkey anti-goat immunoglobulins conjugated with fluorescein isothiocyanate (FITC; 1:80; Chemicon). Tissue was then incubated in sheep anti-rabbit immunoglobulins conjugated with rhodamine (1:80; Cappel), washed twice, mounted on gelatin-coated slides and
coverslipped with DPX (BDH Chemicals) The distribution of PHA-L-labeled axons z, ld terminals ill the spinal cord was mapped by making composite drawings of :dt PHA-L-labeled axons which appeared in 12 consecutive spinal cold sections. Composite drawings were made of sections from cervical. thoracic and lumbar segments of the spinal cord. The laminar distribution of PHA-L-labeled axons was estimated fr~m~ plates found in the atlas of Paxinos and Watson 23. Finally, estimates were made of the number of PHA-L-labeled axons that also contained DBH-ir. These estimates were made by systematically examining ten randomly selected sections from lumbar, thoracic and cervical spinal cord segments using an FITC filter ~,~ visualize PI-IAL-labeled axons. When PHA-L-labeled axons were identified, the rhodamine filter was used to determine if the same axon also contained DBH-ir. These double-labeled axons were assumed to have their origin in the A7 cell group,
Retrograde tracing studies It is possible that PHA-L injections in the A7 cell group may have labeled some non-catecholamine-containing neurons that project to the spinal cord. The number and location of non-catecholaminecontaining neurons in the area of the A7 cell group was estimated by examining brainstem sections for retrogradely labeled neurons following large bilateral injections of the retrograde tracer FluoroGold into the lumbar segments of the spinal cord. Spinally projecting catecholaminergic neurons were identified by the presence of both Fluoro-Goid and tyrosine hydroxy!ase-immunoreactivity (TH-ir). Tyrosine hydroxylase antiserum (Incstar) was used to label A7 catecholamine neurons because, in our experience, it produces a more intense staining than dopamine-fl-hydroxylase antiserum (Eugene Tech). Rats were anesthetized, a laminectomy was performed to expose the dorsal aspect of the lumbar spinal cord and 3-5 injections of 2% Fluoro-Gold (Fluorochrome, Inc) in a volume of 0.5 pl were made at different depths of the spinal cord. Ten days after surgery, the injected rats were intracardialiy perfused as described above. Brainstem and spinal cord sections were cut, washed in cold PBS and incubated for twenty min in 2.5% H20 2 in methanol. Spinal cord sections were washed 4 times in PBS. then mounted on slides and coverslipped with DPX. Brainstem sections were washed 4 times and then incubated overnight at 4 °C in a PBS solution that contained mouse anti-tyrosine hydroxylase antibody (1:1000; Incstar) and 0.5% Triton X-100. Sections were washed in PBS, incubated in goat anti-mouse immunoglobulins conjugated with rhodamine (1:80; Cappel) for 30 min and then mounted on slides and coverslipped with DPX,
Lesion experiments The purpose of these experiments was to use a different approach to determine the spinal cord distribution of axons arising from noradrenergic neurons in the A7 cell groupl These experiments determined the areas in the spinal cord where the density of noradrenergic axons was reduced following unilateral lesions of A7 neurons. Animals were anesthetized, mounted in a stereotaxie headholder and unilateral A7 lesions were made by passing a current of 2 mA for 5 s. Fourteen days after surgery, animals were intracardially perfused as described above. Brainstem and spinal cord sections were cut, washed in cold PBS, incubated in 2.5% H20 z in methanol for 20 min, washed 4 times, and then incubated at 4 °C for 48 h in a solution which contained rabbit anti-DBH antibody (1:1000; Eugene Tech). Sections were then incubated in goat anti-rabbit immunoglobulins (l:80; Atlantic), then incubated in rabbit PAP (1:180; Cappel) for 30 min. Sections were then incubated for 6 min in a solution which contained 0,05% filtered DAB and 0.005% HzO 2 in PBS for 6 min. Finally, the sections were mounted on gelatin-coated slides and coverslipped with Permount, The effect of A7 lesions on the number of DBH-ir axons in the lumbar segments of the spinal cord was assessed by quantifying the density of these axons on the ipsilateral and contralateral sides of each section using an image analysis system (JAVA, Jandc! Scientific) as described previously7. Briefly. DBH-ir axons were
281 visualized in spinal cord sections under darkfield illumination, the image was digitized and the density of DBH-ir axons in either the dorsal or the ventral horn was determined. The approximate area of the dorsal horn that was analyzed included laminae I through IV, while the approximate area of the ventral horn included laminae VIII and IX. The analysis included 20 consecutive sections from each of 4 rats with lesions that produced total unilateral destruction of the noradrenergic neurons in the A7 cell group. The effects of A7 lesions on the density of DBH-ir axons in the spinal cord was determined by comparing the density in the dorsal or ventral horn on the ipsilateral side with that on the contralateral side. The statistical significance of these comparisons was assessed using the Student's paired t-test. The destruction of noradrenergic neurons in the A7 cell group was verified by examining the lesioned area in sections that were stained using DBH immunocytochemistry.
RESULTS
Anterograde tracer experiments The purpose of these experiments was to determine the d i s t r i b u t i o n o f a x o n t e r m i n a l s in t h e s p i n a l c o r d t h a t arise f r o m n o r a d r e n e r g i c n e u r o n s l o c a t e d in t h e A 7 cell g r o u p . To t h i s e n d , t h e a n t e r o g r a d e t r a c e r P H A - L
was ionto-
p h o r e t i c a l l y e j e c t e d i n t o t h e r e g i o n o f t h e A 7 cell g r o u p and the location of PHA-L-labeled
a x o n t e r m i n a l s in t h e
spinal cord was assessed. An example of a representative P H A - L i n j e c t i o n in t h e A 7 cell g r o u p is s h o w n in Fig. 1. Three PHA-L injections that encompassed a majority of t h e D B H - i r n e u r o n s in t h e A 7 cell g r o u p a r e s h o w n in
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Fig. 1. The location of a PHA-L injection that encompassed some of the spinally projecting noradrenergic neurons in the A7 cell group. Panel A shows the location of the PHA-L deposit in the A7 cell group. Panel B shows the location of DBH-ir neurons in the contralateral A7 cell group in the same transverse section. Panels C and D illustrate the location of spinally projecting A7 neurons in a different animal following a bilateral injection of Fluoro-Gold in the lumbar spinal cord. Panel C shows the noradrenergic neurons in the A7 cell group stained for TH-ir. Panel D shows the location of neurons in the A7 that were retrogradely labeled by Fluoro-Gold injected into the spinal cord. Note that every spinally projecting neuron seen in Panel D also contains TH-ir as shown in Panel C. The calibration bar in Panel A is 200/~m and represents the scale for Panels A and B. The calibration bar in Panel C is 100/~m and represents the scale for Panels C and D.
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Fig~ 2, PHA-L injection sites in the dorsolateral pontine tegmentum and the location of PHA-L-labeled axons and t e ~ a l s in the spinal cord. Sections A, B and C are :drawings made with a projection microscope which illustrate the location Of PHA-L injection Sites in transverse brainstem sections taken from 3 different rats. An
enlargement of the injection sites is shown tO the right of each section: The extent of PHA,L deposits is outlined by solid lines and DBH-ir neurons in the A7 are reprvsented by solid circles. The brainstem sections illustrated are those in which the maximum deposit of PHA-L was seen. The size of the injection site was taken to be the size of the dense core of PHA-L-Iabel, although some diffuse PHA-L labeling was seen outside the central core of the PHA-L deposit. Drawings of the distribution of PHA-L-labeled axons in lumbar segments of the spinal cord are shown on the far right. The drawing of each spinal cord section is a composite of 12 adjacent sections.
Fig. 2. Brainstem sections were processed for both DBHand PHA-L-immunocytochemistry using the standard PAP method and the nickel,enhanced PAP method in order to identify the injection site. Although it was not possible to determine if a particular :neuron was immunoreactive for both D B H and PHA-L using the PAP method, neurons were observed within the injection site that were PHA-L-labeled (stained brown) and morphologically similar to the large multipolar blue-black DBHir neurons that were located in the A7 cell group, but outside the injection site. It is likely that these PHAL,labeled neurons also contained DBH-ir. Fig. 2 shows that axons labeled by a PHA-L injection that encompassed the A7 cell group were mainly located in the ipsilateral dorsolateral funiculus. By contrast, only a few labeled axons were seen in the ventral or
Fig. 3. The location of PHA:L injection sites-that did: not: include DBH-ir neurons in the A7 cell group. A: PHA-L injection located "dorsal to the DBH-ir neurons in the A7 cell group. B: PHA:L injection located rostral to DBH-ir neurons in t h e A 7 eetl group. C: PHA-L injection located ventral to DBH,ir neurons in the A7 cell group. The features of this figure are Similar to those described in the legend for Fig. 2.
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C
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Fig. 4. The distribution of PHA-L-laheled axons and terminals ~at different levels of the spinal cord following an injection of PHA-L in the A7 cell group. A: location of the PHA-L injection site in a transverse section through the dorsolateral pontine tegmentum: B: distribution of PHA-L-laheling in lumbar spinal cord s ~ e n t s . C: distribution of PHA-L-labeling in thoracic spinal cord segments. D: distribution of PHA-L-labeling in cervical spinal cord segments. Each drawing (B, C and D) is a composite of I2 contiguous transverse sections. This is the same case as illustrated in Figl 2A.
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Fig. 6. An example of a DBH-ir axon in the dorsal horn that was also anterogradely labeled by a PHA-L injection in the A7. Panel A illustrates two axons that were anterogradely labeled by PHA-L injected into the A7 cell group. Panel B shows that one of these axons (arrow 1) also contains DBH-ir, while the other (arrow 2) does not contain DBH-ir. Panel B also illustrates a DBH-ir axon that was not anterogradely labeled from the A7 cell group (arrow 3). The calibration bar is 25/zm.
missed the A7 cell group dorsally (Fig. 3A), rostrally (Fig. 3B) or ventrally (Fig. 3C). The n u m b e r of P H A L-labeled axons in l u m b a r spinal cord segments from these 3 animals was very sparse. The location of P H A - L - l a b e l e d axons in different levels of the spinal cord is shown in Fig. 4. In general the P H A - L labeling was similar in all spinal cord segments, although some minor differences were apparent. For example, the projection to the superficial dorsal horn in lumbar segments was greater than that in cervical and thoracic spinal cord segments. However, the projections
Fig. 5. Brightfield photomicrographs of transverse brainstem sections that illustrate the morphology of PHA-L-labeled axons and terminal segments in the spinal cord dorsal horn following a PHA-L injection in the A7. The axons and terminal segments shown in Panel A are located within about 500 ~m from the dorsal surface of the spinal cord which can be seen in the upper part of the panel. Panel B shows a high power photomicrograph of axons and varicosities in the dorsal horn. The calibration bar in Panel A is 100 /tm and that in Panel B is 40 ~tm.
ventrolateral funiculi on the ipsilateral side and the labeling in the contralateral white matter was very sparse. All 3 cases in which P H A - L was injected in the A7 cell group exhibited substantial labeling in the ipsilateral dorsal horn and was concentrated in the area that corresponds to laminae I - I V . Two of the A7 injections (Fig. 2B,C) produced dense labeling in the lateral part of the dorsal horn that approximately corresponds to laminae III and IV. Injections of P H A - L that did not include D B H - i r neurons in the A7 cell group produced only sparse labeling in the spinal cord. Fig. 3 shows the distribution ~f labeled axons produced by P H A - L injections that
to lamina X near the central canal and to the medial aspects of laminae VI-VII in the intermediate zone were more dense in the cervical and thoracic segments than those in the l u m b a r spinal cord segments. In addition, the descending A7 axons coursed through the lateral funiculus in cervical segments, while those in the l u m b a r or thoracic segments were located in the dorsolateral funiculus. Finally, there was only a minimal projection to the ipsilateral ventral horn or the contralateral spinal cord in all spinal cord segments. P H A - L - l a b e l e d axons and terminal segments in the spinal cord were short (usually less than 100/~m segments were seen in 40/~m sections), of fine diameter and seldom branched (Fig. 5). Estimates of the n u m b e r of doublelabeled axons revealed that nearly all of the axons and axon terminal segments which were anterogradely labeled with P H A - L also contained D B H - i r (Fig. 6). Only a few P H A - L - l a b e l e d axons did not contain DBH-ir.
Retrograde tracer experiments It is possible that some of the P H A - L - l a b e l e d terminals seen in the spinal cord following a P H A - L injection into the A7 cell group originate from spinally projecting
284
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Fig. 7. The location of neurons in the pontine tegmentum that were retrogradely labeled following bilateral injections of Fluoro-Gold into the lumbar segments of the spinal cord. The location of retrogradely labeled neurons and those that exhibited TH-ir are plotted on transverse sections from 3 different levels of the A7 cell group (Sections A, B and C). The injection site, represented by the solid area, is shown in Section D. Neurons labeled with Fluoro-Gold are represented by crosses, TH-ir somata are represented by open circles and double-labeled somata that contained both TH-ir and Fluoro-Goid are represented by filled circles. All of the labeled neurons in each section have been plotted. The numbers to the right of each section represent the distance (mm) rostral to the interaural line. n o n - c a t e c h o l a m i n e neurons in or near the A 7 cell group. To d e t e r m i n e the n u m b e r and distribution of spinally p r o j e c t i n g non-catecholamine neurons in the vicinity of the A 7 cell group, large bilateral injections of the r e t r o g r a d e tracer, F l u o r o - G o l d , were m a d e into the l u m b a r spinal cord and the distribution of neurons i n t h e pontine t e g m e n t u m that contained both retrogradely t r a n s p o r t e d F l u o r o - G o l d and tyrosine hydroxylase-immunoreactivity (TH-ir) was d e t e r m i n e d (Fig. 7). The m a j o r i t y ( > 8 0 % ) o f the T H - i r neurons in the A 7 cell group were labeled with retrogradely t r a n s p o r t e d Fluoro-
Fig. 8. Photomicrograph of a transverse bramstem section that shows a representative unilateral electrolytic lesion that included the A7 cell group. The calibration bar is t.0 mm G o l d (Figs. 1C,D and 7). Within the area in which the TH-ir neurons of the A 7 cell group are distributed, the vast majority ( > 9 0 % ) of r e t r o g r a d e l y filled neurons were also labeled with T H - i r (Figs. 1C,D and 7). These results suggest that most of the spinally projecting neurons within the i m m e d i a t e vicinity of the A 7 cell group contain catecholamines. H o w e v e r , there was a large cluster of spinally projecting non-catecholamine-containing neurons located just medial to the A 7 cell group that e x t e n d e d in a ventrolateral direction from the ventrolateral margin of the cerebral aqueduct to just dorsal to the superior olivary nucleus (Fig, 7). This cluster of neurons appears to correspond to the dorsal and ventral subcoeruleus nuclei ~.
Lesion experiments A n additional e x p e r i m e n t a l a p p r o a c h was used to examine the projections of n o r a d r e n e r g i c A 7 neurons to the spinal cord. The p u r p o s e of these experiments was to m a k e unilateral electrolytic lesions of the A 7 cell group and m e a s u r e the subsequent reduction in the density of catecholamine-containing axon terminals in the spinal cord. A representative A 7 lesion is shown in Fig. 8 and Fig. 9 shows the effects of 4 such lesions on the density of D B H - i r axons in the spinal cord. Each lesion p r o d u c e d a statistically significant (P < 0.05) reduction in the density of D B H - i r axon terminals in the dorsal horn, but only one in 4 animals d e m o n s t r a t e d a statistically significant density reduction in the ventral horn. The reduction in terminal density in the dorsal horn varied from 13,8% to 29.0% and was not c o r r e l a t e d with the size of the lesion. For e x a m p l e , Fig. 9 A shows that a relatively
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