Brain Research, 579 (1992) 67-73 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
67
BRES 17666
Spinal projections of the locus coeruleus and the nucleus subcoeruleus in the Harlan and the Sasco Sprague-Dawley rat K.A. Sluka and K.N. Westlund Department of Anatomy and Neurosciences and the Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, TX 77550 (USA)
(Accepted 10 December 1991) Key words: Descending system; Spinal cord; Retrograde tracing; Latex bead
The descending projections of the locus coeruleus (LC) and the nucleus subcoeruleus (SC) to the lumbar spinal cord were examined in rats from two vendors using retrograde transport of fluorescent latex beads. There was a vendor difference observed which agrees with previous findings. The differential dorsal horn and ventral horn projections of the Harlan and the Sasco Sprague-Dawley rats, reported by Fritschy and Grzanna, and Clark and Proudfit were confirmed. In the Harlan rat more cells were labeled in the LC following injections in the dorsal horn. In contrast, in the Sasco rat, more cells were labeled in the LC from injections in the ventral horn. Although, in all studies, the LC in rats from these vendors projected to some extent to both the dorsal and the ventral horn. A difference in labeling was noted also for the depth of placement of the tracer in the dorsal horn. When the site of injection was in the nucleus proprius, a predominately contralateral projection of the LC was noted. In contrast, when horseradish peroxidase (HRP) gel implants were placed to include the superficial laminae, the cells in the LC were labeled predominately ipsilaterally. The SC has a major projection to the dorsal horn in the Harlan rats while cells in the SC were predominately labeled following ventral horn injections in the Sasco rats. These cells send mostly ipsilateral projections to the dorsal and ventral horn of the spinal cord. Double labeled studies confirmed that 91% of LC and 86% of SC neurons projecting to the spinal cord were noradrenergic. The present results confirmed a difference in the descending catecholamine projections of rats purchased from different vendors. These strain differences may prove useful in studies of motor and sensory systems. INTRODUCTION Several of the n o r a d r e n e r g i c cell groups, n a m e l y those in the pons, have descending projections to the spinal cord. The locus coernleus (LC), the A 6 cell group, is 10cated in the dorsolateral pons and has been shown to provide n o r a d r e n e r g i c innervation of the spinal cord. The nucleus subcoeruleus (SC), located ventrolateral to the A 6 cell group in the rostral pons, also sends noradrenergic projections to the spinal cord. O t h e r regions of the pons containing n o r a d r e n e r g i c projections to the spinal cord include the ventrolaterally placed A 5 cell group, the A 7 cell group including the KSlliker-Fuse nucleus, the medial and lateral parabrachial nuclei and the ventrolateral reticular formation, The L C and the SC send fibers to the dorsal and ventral horn at all levels of the spinal cord l'2,4-1°A2A4-18,23, 25. A variety of m e t h o d s has b e e n used to d e t e r m i n e where the locus coeruleus projects in the spinal cord. These techniques include: electrophysiology 1'5'7'8'm17'18, a u t o r a d i o g r a p h y 1°'16'25, r e t r o g r a d e transport 4'8-1°A4-15' 23,25 (dopamine-fl-hydroxylase ( D B H ) , horseradish peroxidase ( H R P ) , fluorescent r e t r o g r a d e tracers), and m o r e
recently anterograde transport of the lectin, phaseolus vulgaris leucoagglutinin ( P H A - L ) 2'6. Classical studies have described autoradiographic labeling of the L C and SC projections into all laminae of the spinal gray. A n ipsilateral projection from the L C and SC was found with the densest portions of the projections terminating in the dorsal horn, the autonomic and somatic m o t o r cells, and the i n t e r m e d i a t e zone 10'16'25. The dorsal horn projections were less numerous and densest in laminae I and II with some labeling in the d e e p e r dorsal horn and the i n t e r m e d i a t e zone. Electron microscopy has shown direct noradrenergic termination on identified spinotha~ lamic tract neurons in the dorsal horn 24 and LC/SC projections on spinal m o t o r neurons using autoradiography 11. Westlund and Coulter 25 described bilateral spinal projections with an ipsilateral p r e d o m i n a n c e for the SC in the monkey. The SC terminates in the ventral horn, lamina X, and bilaterally in the dorsal horn (laminae I, II, IV-VI). Earlier anatomical work using a u t o r a d i o g r a p h y and lesions could not be confined totally to the L C or the SC. Recent studies using P H A - L have shown minimal spreading when injected and, therefore, placement of
Correspondence: K.N. Westlund, Marine Biomedical Institute, Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, TX, 77550, USA. Fax: (1) (409) 762-9382.
68 injections is m o r e precise. T h e r e is r e c e n t c o n t r o v e r s i a l
TABLE I
e v i d e n c e c o n c e r n i n g the d e s c e n d i n g p a t h w a y s and the spinal t e r m i n a t i o n s o f the L C in rats s u p p l i e d by two
Proportions o f labeled cells
d i f f e r e n t v e n d o r s . This d a t a is b a s e d o n P H A - L injeclions into the L C 2'6. Fritschy and G r z a n n a 6 i n j e c t e d PHA-L
into the L C of S p r a g u e - D a w l e y
Locus coeruleus Harlan (n = 2)
rats ( H a r l a n
distributors, F r e d e r i c k , M D ) and f o u n d the densest term i n a t i o n s in the superficial dorsal h o r n ( l a m i n a e I and II) and in the i n t e r m e d i a t e z o n e . F e w fibers w e r e s e e n in l a m i n a e V I I I and IX. T h e fibers travel t h r o u g h the gray m a t t e r to their t e r m i n a l s in the dorsal h o r n , lamin a e I and II. T h e SC and A 5 regions send p r o j e c t i o n s to the v e n t r a l h o r n with the p a t h w a y t r a v e l i n g t h r o u g h the
Cells/total
Dorsal horn inj. Ipsilateral Contralateral Ventral horn inj. Ipsilateral Contralateral
Sasco (n = 2) Av%
52/87 82/145 58% 12 25 27% 40 57 72% 35/87 63/145 42% 27 38 69% 8 25 31%
Cells/total
Av%
22/49 7 15 27/49 19 8
34% 35% 65% 66% 75% 25%
15/66 6 9 51/66 41 10
v e n t r o l a t e r a l funiculus ( V L F ) and the v e n t r a l funiculus (VF). C l a r k and Proudfit 2 r e p e a t e d the s a m e e x p e r i m e n t with P H A - L i n j e c t e d into the L C of S p r a g u e - D a w l e y rats s u p p l i e d by a different v e n d o r (Sasco distributors, O m a h a , N E ) . A n ipsilateral p r o j e c t i o n p r e d o m i n a t e l y to l a m i n a X and the v e n t r a l h o r n was s e e n in c o n t r a s t to the dorsal h o r n p r o j e c t i o n s seen in the H a r l a n rats. T h e s e t e r m i n a l s w e r e s h o w n to be 90% n o r a d r e n e r g i c in studies colocalizing D B H . C l a r k and Proudfit h a v e res o l v e d t h e s e d i f f e r e n c e s by confirming the d i f f e r e n c e in circuitry in rats f r o m the t w o suppliers in their o w n laboratory3, T h e p r e s e n t study was d e s i g n e d to f u r t h e r address the c o n t r o v e r s y c o n c e r n i n g differences in p o n t i n e projeclions to the spinal c o r d by c o m p a r i n g rats f r o m local sources o f the two suppliers used by Proudfit and G r z a n na's labs. R e t r o g r a d e t r a n s p o r t of e i t h e r r h o d a m i n e or fluorescein c o n j u g a t e d latex b e a d s f r o m injections confined to the dorsal o r v e n t r a l h o r n of the spinal c o r d o f S p r a g u e - D a w l e y rats f r o m two v e n d o r s was u s e d to det e r m i n e p o n t i n e spinal projections. F l u o r e s c e i n and r h o d a m i n e b e a d s s h o w m i n i m a l diffusion f r o m the injeclion site 13. T h e r e f o r e , dorsal h o r n and v e n t r a l h o r n inj e c t i o n s can be c o n f i n e d w h e r e p r e v i o u s l y this was difficult with substances such as H R P and a n t i b o d y to D B H .
rescein beads were switched from dorsal to ventral horn in two animals to monitor the uptake of each. Each injection was 0.2 pl of beads and a total of 2-3/~1 was injected. Five to ten injections were made per side. In an additional two animals from each vendor, HRP gel implants (Type VI, Sigma Chemical Company, St. Louis, MO), were placed into the superficial dorsal horn. After 6-10 days, the animals were perfused transcardially with 4% paraformaldehyde (1 1) preceded by sterile saline with heparin (100 ml). A segment from the lumbar enlargement was placed in sucrose overnight and then cut on a freezing microtome (40-50 ~m). The sections of the lower midbrain, pons and upper medulla were cut and kept in serial order. The sections were mounted on gelatin coated slides and examined with a Zeiss microscope with an epifluorescence attachment. To quantify dorsal and ventral localization of cells, two animals from each vendor (Harlan and Sasco) with the most localized injections of fluorescent latex beads were used. In one Harlan animal receiving HRP gel implants into the dorsal horn, sections were mapped to demonstrate the distribution of the retrogradely labeled cells in the LC and SC. The nuclear boundaries used for the LC and SC were those depicted by Paxinos and Watson 2°. Retrogradely labeled cells from every 3-5 sections (150-250/~m) were mapped in animals injected with latex beads. The cells from these sections located within the borders of the LC bilaterally were counted. Cells labeled in the LC from dorsal and ventral horn injections were totalled. The percentage of labeled cells from the dorsal horn vs. ventral horn was determined. This procedure was repeated for the SC. These numbers are represented in Tables I and II. Five representative sections in two animals were stained immunocytochemically using an antibody directed against arterenol (norepinephrine; 1:100 for 1 h; Chemicon). Sections then were placed in IgG tagged with an AMCA (7-amino-4-methylcoumarin-
MATERIALS AND METHODS Male Sprague-Dawley rats (n = 7 Harlan, Houston; n = 3 Sasco, St. Louis) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and placed in a stereotaxie frame. A laminectomy was performed at the L3-L 5 level of the spinal cord. With a Hamilton syringe and a glass micropipette, retrograde fluorescent tracers were injected into the spinal cord. To maximize the data obtained from each of the animals used, two colors of latex beads were injected per animal. Dorsal horn injections of rhodamine labeled latex beads (Lumifluor, New Town, NY) were made 0.3-0.5 mm deep and 0.5-0.8 mm from the midline. Ventral horn injections of fluorescein labeled latex beads (Lumifluor, New Town, NY) were made at 1.0-1.3 mm from the midline and 1.0-1.5 mm deep on the opposite side of the spinal cord. Two animals'were injected 1.5 mm from the midline at a 30° angle and 1.5 mm deep to prevent any contamination in the dorsal horn. Rhodamine beads and fluo-
TABLE II Proportions o f labeled cells Nucleus subcoeruleus Harlan (n = 2)
Dorsal horn inj. Ipsilateral Contralateral Ventral horn inj. Ipsilateral Contralateral
Sasco (n = 2)
Cells/total
Av%
Cells/total
58/80 38 20 22/80 13 9
63% 76% 24% 37% 58% 41%
20/69 13 7 49/69 25 24
49/90 42 7 41/90 24 17
Av%
21/54 34% 17 73% 4 27% 33/54 66% 20 56% 13 44%
69 3-acetic acid) fluorophore (1:100 for 1 h; Jackson ImmunoResearch Lab.) for visualization using fluorescent microscopy. In an additional animal (Harlan) all sections were stained for tyrosine hydroxylase (TH; 1:400; 2 days, Pel-Freeze) followed by IgG tagged with an AMCA fluorophore (1:200, overnight). Either of these antibodies were used to define noradrenergic spinal projection neurons since no spinal projections from the LC contain dopamine4.
RESULTS T h e dorsal h o r n injections of latex b e a d s into the lumb a r spinal cord were c o n f i n e d to l a m i n a e I I I - V with m i n i m a l s p r e a d a l o n g the tract in l a m i n a e I a n d II a n d occasionally into l a m i n a e X. T h e p l a c e m e n t of the injec-
HARLAN
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Fig. 1. Maps depicting the distribution of the retrogradely labeled cells in the ports for individual sections using fluorescent latex beads from the lumbar ventral horn and the dorsal horn in the Harlan (left) and the Sasco (right) Sprague-Dawley rat. Cells labeled following injections in the ventral horn from one tissue section are illustrated by the closed circles and those from the dorsal horn by the x's. Each symbol represents one cell. The injection sites are shown below the labeled cells. The solid portion represents the densest area of injection. The diffuse spread of the tracer is represented by hatched lines. LC, locus coeruleus, SubCA, nucleus subcoeruleus alpha; SubCD, nucleus subcuruleus dorsal; SubCV, nucleus subcoeruleus ventral; MPB, medial parabrachial nucleus; LPBE, lateral parabrachial external nucleus; SuVe, superior vestibular nucleus; DPO, dorsal pontine olivary nucleus.
70 tion was in the lateral portion of laminae III-IV, nucleus proprius (Fig. 1). In no case was spread found in laminae VIII-IX. Ventral horn injection sites were located in laminae VIII and laminae IX with occasional spread into the ventral funiculus or along the tract. In one animal, the injection site was in the VF bilaterally and the dorsolateral funiculus (DLF) unilaterally. The HRP gel implants were placed dorsally with the densest portion of the HRP labeling in laminae I-III (Fig. 2) and occa-
37% projected to the ventral horn (Fig. 3C, Table II). However, in the Sasco rat, 34% of the cells within the SC projected to the dorsal horn while 66% projected to the ventral horn. The ipsilaterally projecting cells, like
HARLAN ~
sionally spreading ventrally to laminae V. Locus coeruleus
The present studies demonstrated projections of the LC to the dorsal and the ventral horn in both Sasco and Harlan Sprague-Dawley rats (Figs. 1 and 2). In the Harlan rats, the cells retrogradely labeled from the dorsal horn were more numerous in LC (Fig. 3B) than those from the ventral horn (Fig. 3A). In contrast, cells of the LC were labeled relatively more often from the ventral horn in the Sasco rats. Within the boundaries of the LC, 58% of the cells labeled were from dorsal horn injections and 42% from the ventral horn in the Harlan rat. However, in the Sasco rat, 34% of the cells in the LC were labeled following dorsal horn injections and 66% from the ventral horn (Table I). Seventy-two percent of the cells that were labeled in the LC were located contralaterally following dorsal horn injections in the Harlan rat. Sixty-five percent of the cells located in the LC in the Sasco rat were located contralaterally. Ventral horn projections from the LC were also bilateral, but 69% of the retrogradely labeled neurons in the Harlan rat and 76% in the Sasco rat were labeled ipsilaterally. In one Harlan rat, when there was clearly no spread of fluorescent tracer into the dorsal horn along the needle tract, the LC projection was almost totally ipsilateral. As a result of the inability to confine latex beads to laminae I and II, HRP gel implants were placed into the superficial dorsal horn in two animals from each vendor. In all of these animals there was bilateral labeling with a clear tendency for the LC to project ipsilaterally to the dorsal horn (Fig. 2).
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As seen in Figs. 1 and 2, the SC nucleus was labeled bilaterally following dorsal horn injections with an ipsilateral predominance in both the Harlan and the Sasco rats for both fluorescent latex bead injections and HRP gel implants. Cells labeled ipsilaterally were localized more ventrally in the nucleus. Projections of the SC to the ventral horn were bilateral with an ipsilateral predominance. In the Harlan rat, 63% of the retrogradely labeled cells in the SC were from the dorsal horn and
HRP Fig. 2. Maps depicting the distribution of the retrogradely labeled cells in the pons using horseradish peroxidase (HRP) gel implants into the dorsal horn of a Harlan Sprague-Dawley rat. The Sasco rat, not depicted showed a similar distribution. Each symbol (x) representsone cell. The injection site is shown below the labeled cells. The solid portion represents the densest area of placement. The diffusespread of the HRP is represented by hatched lines. LC, locus coeruleus; SubCA, nucleus subcoeruleus alpha; SubCD, nucleus subcoernleus dorsal; SubCV, nucleus subcoeruleus ventral; MPB, medial parabrachial nucleus.
71 those in the LC, were found caudally in the nucleus.
Other nuclei Cells were also labeled in the medial and lateral parabrachial nuclei, and the K611iker-Fuse nucleus following injections of both the dorsal and the ventral horn. An occasional cell was found in the lateral wall of the fourth ventricle in the region of the A4 cell group following dorsal horn and ventral horn injections. The pontine reticular formation had numerous cells labeled from both dorsal and ventral horn. These nuclei included the superior central nucleus and the reticulotegmental nucleus. There was also labeling ipsilaterally in the superior vestibular nucleus from dorsal horn injections into laminae I I I - I V (Fig. 1) of the lumbar enlargement.
Tracts In one animal (Harlan), using fluorescein latex beads, the ventrally placed injection site in the lumbar enlargement was located in the VF bilaterally. In the same animal, a dorsal horn injection (rhodamine latex beads) was located in the DLF. The results from this animal indicated that the VF at the lumbar level carries only SC axons. The DLF injections retrogradely labeled both the LC and the SC. This data also indicates that latex beads were taken up by cut fibers of passage.
Immunocytochemistry In two animals, 5 representative sections were stained immunocytochemically for arterenol to visualize norepinephrine cells of the LC and SC. Many examples were found of retrogradely labeled cells within the nuclear borders of the LC or the SC that also were stained immunocytochemically for norepinephrine. In one additional animal (Harlan) the total number of noradrenergic projection neurons which were retrogradely labeled in the LC and the SC were counted. In this animal 91% (31/34) of retrogradely labeled cells in the LC were noradrenergic, with 94% (16/17) from dorsal horn injections and 88% (15/17) from ventral horn injections. In the SC 86% (17/20) of the retrogradely labeled cells were noradrenergic, with 83% (5/6) from dorsal horn injections and 88% (14/16) from ventral horn injections. DISCUSSION Fig. 3. Photomicrographs taken using epifluorescent optics illustrate retrogradely labeled cells following injection of fluorescent latex beads into the lumbar spinal cord of a Harlan Sprague-Dawley rat. A: a retrogradely labeled cell from the locus coeruleus following injection of fluorescent tracer into the ventral horn of the spinal cord. B: a group of labeled cells in the locus coeruleus following a dorsal horn injection of the retrograde tracer. C: two cells in the subcoeruleus nucleus labeled following injection of fluorescent latex beads into the ventral horn. V, fourth ventricle,
The results of the present study indicated a clear difference between rats from two vendors (Sasco and Harlan) for the spinal projection of the LC and the SC. In the present study, retrograde transport of fluorescent beads from the lumbar dorsal horn vs. the ventral horn was used to help clarify recent data obtained by others using the PHA-L method. The present experiment
72 agreed with the data of many earlier studies finding both a ventral horn and a dorsal horn projection of the LC in the Sasco and Harlan Sprague-Dawley rat. The ventral born projections were found to be ipsilateral in rats from both vendors. The dorsal horn projections using both techniques (fluorescent beads and HRP gel implants) were bilateral. The fluorescent beads retrogradely labeled cells primarily contralaterally and the HRP gel implants labeled cells primarily ipsilaterally. In addition, the projections to the SC were investigated and found to be in agreement with previous studies 3'21'23'25 which report ipsilateral projections to both the ventral and dotsal horn. Clark and Proudfit 2, as well as Westlund et al. 23, have demonstrated previously that the majority of cells retrogradely labeled in the LC and SC from the spinal cord are noradrenergic. In this study we demonstrated that 91% of the spinally projecting LC cells and 86% of the SC cells were noradrenergic. In addition, PHA-L injections into the LC and the SC result in up to 90% of the terminals in the spinal cord staining for DBH 2'6. Therefore the majority of spinally projecting cells within the nuclear borders of the LC and the SC were noradrenergic. Although the experiments presented are in agreement with previous studies, the quantity of retrogradely labeled cells in the LC from the dorsal horn observed in the present study is greater than one would predict based on PHA-L studies of Fritschy and Grzanna 6, and Clark and Proudfit 2. In the Harlan Sprague-Dawley rat there was a small predominance for the dorsal horn projections over the ventral horn from the LC. In contrast, in the Sasco rat a greater number of cells were labeled retrogradely from ventral horn injections when compared to injections into the dorsal horn. This data confirmed a vendor difference existing between Sprague-Dawley rats which has been demonstrated previously in two separate studies using PHA-L 2'6. The differences in quantity of labeling revealed by the two methods, PHA-L and retrograde transport, can be explained by collateralization of axon terminals in the spinal cord. The collateralization of an axon from a single cell would provide a different picture using PHA-L than if the cell was retrogradely labeled. Alternatively, rats purchased from different farms from the same vendors may show slight variations, In the present study, differences were noted in the dorsal horn projections of the LC depending on whether the fluorescent beads or the HRP gel implants were used regardless of the vendors. Bilateral projections can be demonstrated with both techniques (fluorescent beads and HRP gel implants). Fluorescent beads retrogradely labeled cells primarily contralaterally while the HRP gel
implants labeled cells primarily ipsilaterally. In rats (Harlan and Sasco) with HRP gel implants, there was a bilateral projection, predominately ipsilateral, to the superficial dorsal horn which agrees with previous findings3' 6,10,23,25. The contralateral predominance of the LC projection in rats from both vendors to the dorsal horn from injections into the deeper laminae contradicts other previous work which found bilateral projections with an ipsilateral predominance 6'21'23'25. The difference between the results obtained using HRP gel implants vs. fluorescent latex beads can be attributed to the site of injections. The dorsal placement of the HRP gel implants to include lamina I and II indicated that the superficial dorsal horn has a predominately ipsilateral projection from the LC which agrees with Fritschy and Grzanna 6 and Clark et al. 3. The contralateral projection to the lumbar cord found with injection sites into the nucleus proprius also agrees with maps by Fritschy and Grzanna 6. The SC axons projected through the ventral funicuIUS6'21'22'25 and the DLF 21'22 to the dorsal and ventral horn bilaterally with strong ipsilateral predominance. This was not in agreement with the work of Fritschy and Grzanna 6 who found only ventral horn terminations and a ventral funiculus pathway for the SC in the Harlan rat. Previous investigators supported the DLF pathway 16'21'22 as well as the bilateral dorsal and ventral horn projections 1°'16'25. We found the LC projected through the DLF, which agrees with previous findings 16'21'25. Electrophysiological studies concur with the anatomical findings of a ventral horn and a dorsal horn noradrenergic projection from the LC. An enhancement of monosynaptic reflexes by stimulation of the LC in the rat and cat 1 was seen. The enhancement was blocked by prazosin, an a-1 adrenoreceptor antagonist, suggesting that the increased excitability of the motoneurons is me° diated through noradrenergic mechanisms. Fung and Barnes 7 electrically stimulated the LC and measured the membrane excitability of a motoneurons. A decrease in membrane resistance with an increase in motoneuron excitability was shown. LC/SC stimulation or glutamate microinjections inhibited nociceptive transmission as measured by response to heat stimuli (>50°C) 12. These multiresponsive neurons tested were located in laminae I-VI of the dorsal horn. The LC/SC stimulation was effective both ipsilateral and contralateral suggesting a strong bilateral input to the dorsal horn of the spinal cord. Thus, the electrophysiological studies support a projection to the ventral horn as well as the dorsal horn of the spinal cord. It is interesting to note that retrogradely labeled cells were localized in the superior vestibular nucleus from dorsal horn injections. This unexpected finding indicated the presence of a vestibulospinal tract through the lum-
73 bar spinal cord levels to the dorsal horn. Based on the current study using Harlan and Sasco Sprague-Dawley rats, a projection of the LC and the SC to both the ventral h o r n 1-4'7'16'18'19'23'25 and the dorsal h o r n 3'5-8'1°'12'16'17"19'23'25 exists and agrees with many other previous studies. The total number of cells labeled following a dorsal horn vs. the ventral horn injection is clearly different between vendors (Harlan vs. Sasco). The differences in spinal cord innervation between rats of these two vendors are, with the retrograde method, not as striking as was reported previously with the use
of the PHA-L method by Fritschy and Grzanna 6, Clark
REFERENCES 1 Chan, J.Y.H., Fung, S.J., Chan, S.H.H. and Barnes, C.D., Facilitation of lumbar monosynaptic reflexes by locus coeruleus in '~h¢ rat, Brain Res., 369 (1986) 103-109. 2 Clark, EM. and Proudfit, H.K., The projection of locus coeruleus neurons to the spinal cord in the rat determined by anterograde tracing combined with immunocytochemistry, Brain Res., 538 (1991) 231-245. 3 Clark, EM., Yoemans, D.C. and Proudfit, H.K., The noradrenergic innervation of the spinal cord: differences between two substrains of Sprague-Dawley rats determined using retrograde tracers combined with immunocytochemistry, Neurosci. Left., 125 (1991) 155-158. 4 Commissiong, J.W., Hellstrom, S.O. and Neff, N.H., A new projection from the locus coeruleus to the spinal ventral columns: histochemical and biochemical evidence, Brain Res., 148 (1978) 207-213. 5 Fleetwood-Walker, S.M., Mitchell, R., Hope, EJ., Molony, V. and Iggo, A., An a2-receptor mediates the selective inhibition by noradrenaline of nociceptive responses of identified dorsal horn neurons, Brain Res., 334 (1985) 243-254. 6 Fritschy, J.-M. and Grzanna, R., Demonstration of two separate descending noradrenergic pathways to the rat spinal cord: evidence for an intragriseal trajectory of locus coeruleus axons in the superficial layers of the dorsal horn, J. Comp. Neurol., 291 (1990) 553-582. 7 Fung, S.J. and Barnes, C.D., Membrane excitability changes in hindlimb motoneurons induced by stimulation of the locus coeruleus in cats, Brain Res., 402 (1987) 230-242. 8 Guyenet, P.G., The coeruleospinal noradrenergic neurons: anatomical and electrophysiological studies in the rat, Brain Res., 189 (1980) 121-133. 9 Hancock, M.B. and Fougerousse, C.L., Spinal projections from the nucleus locus coeruleus and nucleus subcoeruleus in the cat and monkey as demonstrated by the retrograde transport of horseradish peroxidase, Brain Res. Bull., 1 (1976) 229-234. 10 Holstege, G. and Kuypers, H.G.J.M., The anatomy of brainstem pathways to the spinal cord in the cat. A labelled amino acid tracing study, Prog. Brain Res., 57 (1982) 145-175. 11 Holstege, G. and Kuypers, H.G.J.M., Brainstem projections to spinal motorneuronal cell groups in rat by means of electron microscopy autoradiography, Prog. Brain Res., 57 (1982) 177183. 12 Jones, S.L. and Gebhart, G.E, Quantitative characterization of coeruleospinal inhibition of nociceptive transmission in the rat, J. Neurophysiol., 56 (1986) 1397-1410.
and Proudfit 2 and Clark et al. 3. Data reported previously from cats, opossums, and monkeys agrees more closely with the Sasco Sprague-Dawley rat. The Sasco rat may be more useful in studies of the involvement of the LC and SC in motor control. The Harlan rat, on the other hand, may be more useful in studies of the interactions of the LC and SC in sensory systems.
Acknowledgements. The authors wish to thank Scott Richardson for excellent technical assistance. This work was supported by NIH Grants NS 11255 and NS 01445, and an unrestricted grant from the Bristol-Myers Squibb Company.
13 Katz, L.C. and Iarqvici, D.M., Green fluorescent latex microspheres: a new retrograde tracer, Neuroscience, 34 (1990) 511520. 14 Kuypers, H.G.J.M. and Maisky, V.A., Retrograde axonal transport of horseradish peroxidase from spinal cord to brainstem cell groups in the cat, Neurosci. Lett., 1 (1975) 9-14. 15 Lai, Y.-Y. and Barnes, C.D., A spinal projection of serotonergic neurons of the locus coeruleus in the cat, Neurosci. Lett., 58 (1985) 159-164. 16 Martin, G.E, Cabana, T., Ditirro, EJ., Ho, R.H. and Humbertson, A.O., Reticular and raphe projections to the spinal cord of the North American opossum. Evidence for connectional heterogeneity. In H.G.J.M. Kuypers and G.E Martin (Eds.), Anatomy of Descending Pathway to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 109-129. 17 Mohka, S.S., McMillan, J.A. and Iggo, A., Pathways mediating descending control of spinal nociceptive transmission from the nuclei locus coeruleus (LC) and raphe magnus (NRM) in the cat, Exp. Brain Res., 61 (1986) 597-606. 18 Nakazato, T., Locus coeruleus neurons projecting to the forebrain and spinal cord in the cat, Neuroscience, 23 (1987) 529538. 19 Nygren, L.-G. and Olson, L., A new major projection from locus coeruleus: the main source of noradrenergic nerve terminals in the ventral and dorsal columns of the spinal cord, Brain Res., 132 (1977) 85-93. 20 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd ed., Academic Press, Orlando, 1986. 21 Stevens, R.T., Aparkarian, A.V. and Hodge, C.J., Funicular course of catecholamine fibers innervating the lumbar cord of the cat, Brain Res., 336 (1985) 243-251. 22 Watkins, L.R., Griffin, G., Leinchnetz, G.R. and Mayer, D.J., Identification and somatotopic organization of nuclei projecting via the dorsolateral funiculus in rats: a retrograde tracing study using slow release gels, Brain Res., 223 (1981) 237-255. 23 Westlund, K.N., Bowker, R.M., Zeigler, M.G. and Coulter, J.D., Origins of descending noradrenergic projections to the spinal cord of monkey, Brain Res., 292 (1984) 1-16. 24 Westlund, K.N., Carlton, S.M., Zhang, D. and Willis, W.D., Direct catecholaminergic innervation of primate spinothalamic tract neurons, J. Comp. Neurol., 299 (1990) 178-186. 25 Westlund, K.N. and Coulter, J.D., Descending projections of the locus coeruleus and subcoernleus/medial parabrachial nuclei in monkey: axonal transport studies and dopamine-fl-hydroxylase immunocytochemistry, Brain Res. Rev., 2 (1980) 235-264.
67
BRES 17666
Spinal projections of the locus coeruleus and the nucleus subcoeruleus in the Harlan and the Sasco Sprague-Dawley rat K.A. Sluka and K.N. Westlund Department of Anatomy and Neurosciences and the Marine Biomedical Institute, The University of Texas Medical Branch, Galveston, TX 77550 (USA)
(Accepted 10 December 1991) Key words: Descending system; Spinal cord; Retrograde tracing; Latex bead
The descending projections of the locus coeruleus (LC) and the nucleus subcoeruleus (SC) to the lumbar spinal cord were examined in rats from two vendors using retrograde transport of fluorescent latex beads. There was a vendor difference observed which agrees with previous findings. The differential dorsal horn and ventral horn projections of the Harlan and the Sasco Sprague-Dawley rats, reported by Fritschy and Grzanna, and Clark and Proudfit were confirmed. In the Harlan rat more cells were labeled in the LC following injections in the dorsal horn. In contrast, in the Sasco rat, more cells were labeled in the LC from injections in the ventral horn. Although, in all studies, the LC in rats from these vendors projected to some extent to both the dorsal and the ventral horn. A difference in labeling was noted also for the depth of placement of the tracer in the dorsal horn. When the site of injection was in the nucleus proprius, a predominately contralateral projection of the LC was noted. In contrast, when horseradish peroxidase (HRP) gel implants were placed to include the superficial laminae, the cells in the LC were labeled predominately ipsilaterally. The SC has a major projection to the dorsal horn in the Harlan rats while cells in the SC were predominately labeled following ventral horn injections in the Sasco rats. These cells send mostly ipsilateral projections to the dorsal and ventral horn of the spinal cord. Double labeled studies confirmed that 91% of LC and 86% of SC neurons projecting to the spinal cord were noradrenergic. The present results confirmed a difference in the descending catecholamine projections of rats purchased from different vendors. These strain differences may prove useful in studies of motor and sensory systems. INTRODUCTION Several of the n o r a d r e n e r g i c cell groups, n a m e l y those in the pons, have descending projections to the spinal cord. The locus coernleus (LC), the A 6 cell group, is 10cated in the dorsolateral pons and has been shown to provide n o r a d r e n e r g i c innervation of the spinal cord. The nucleus subcoeruleus (SC), located ventrolateral to the A 6 cell group in the rostral pons, also sends noradrenergic projections to the spinal cord. O t h e r regions of the pons containing n o r a d r e n e r g i c projections to the spinal cord include the ventrolaterally placed A 5 cell group, the A 7 cell group including the KSlliker-Fuse nucleus, the medial and lateral parabrachial nuclei and the ventrolateral reticular formation, The L C and the SC send fibers to the dorsal and ventral horn at all levels of the spinal cord l'2,4-1°A2A4-18,23, 25. A variety of m e t h o d s has b e e n used to d e t e r m i n e where the locus coeruleus projects in the spinal cord. These techniques include: electrophysiology 1'5'7'8'm17'18, a u t o r a d i o g r a p h y 1°'16'25, r e t r o g r a d e transport 4'8-1°A4-15' 23,25 (dopamine-fl-hydroxylase ( D B H ) , horseradish peroxidase ( H R P ) , fluorescent r e t r o g r a d e tracers), and m o r e
recently anterograde transport of the lectin, phaseolus vulgaris leucoagglutinin ( P H A - L ) 2'6. Classical studies have described autoradiographic labeling of the L C and SC projections into all laminae of the spinal gray. A n ipsilateral projection from the L C and SC was found with the densest portions of the projections terminating in the dorsal horn, the autonomic and somatic m o t o r cells, and the i n t e r m e d i a t e zone 10'16'25. The dorsal horn projections were less numerous and densest in laminae I and II with some labeling in the d e e p e r dorsal horn and the i n t e r m e d i a t e zone. Electron microscopy has shown direct noradrenergic termination on identified spinotha~ lamic tract neurons in the dorsal horn 24 and LC/SC projections on spinal m o t o r neurons using autoradiography 11. Westlund and Coulter 25 described bilateral spinal projections with an ipsilateral p r e d o m i n a n c e for the SC in the monkey. The SC terminates in the ventral horn, lamina X, and bilaterally in the dorsal horn (laminae I, II, IV-VI). Earlier anatomical work using a u t o r a d i o g r a p h y and lesions could not be confined totally to the L C or the SC. Recent studies using P H A - L have shown minimal spreading when injected and, therefore, placement of
Correspondence: K.N. Westlund, Marine Biomedical Institute, Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, TX, 77550, USA. Fax: (1) (409) 762-9382.
68 injections is m o r e precise. T h e r e is r e c e n t c o n t r o v e r s i a l
TABLE I
e v i d e n c e c o n c e r n i n g the d e s c e n d i n g p a t h w a y s and the spinal t e r m i n a t i o n s o f the L C in rats s u p p l i e d by two
Proportions o f labeled cells
d i f f e r e n t v e n d o r s . This d a t a is b a s e d o n P H A - L injeclions into the L C 2'6. Fritschy and G r z a n n a 6 i n j e c t e d PHA-L
into the L C of S p r a g u e - D a w l e y
Locus coeruleus Harlan (n = 2)
rats ( H a r l a n
distributors, F r e d e r i c k , M D ) and f o u n d the densest term i n a t i o n s in the superficial dorsal h o r n ( l a m i n a e I and II) and in the i n t e r m e d i a t e z o n e . F e w fibers w e r e s e e n in l a m i n a e V I I I and IX. T h e fibers travel t h r o u g h the gray m a t t e r to their t e r m i n a l s in the dorsal h o r n , lamin a e I and II. T h e SC and A 5 regions send p r o j e c t i o n s to the v e n t r a l h o r n with the p a t h w a y t r a v e l i n g t h r o u g h the
Cells/total
Dorsal horn inj. Ipsilateral Contralateral Ventral horn inj. Ipsilateral Contralateral
Sasco (n = 2) Av%
52/87 82/145 58% 12 25 27% 40 57 72% 35/87 63/145 42% 27 38 69% 8 25 31%
Cells/total
Av%
22/49 7 15 27/49 19 8
34% 35% 65% 66% 75% 25%
15/66 6 9 51/66 41 10
v e n t r o l a t e r a l funiculus ( V L F ) and the v e n t r a l funiculus (VF). C l a r k and Proudfit 2 r e p e a t e d the s a m e e x p e r i m e n t with P H A - L i n j e c t e d into the L C of S p r a g u e - D a w l e y rats s u p p l i e d by a different v e n d o r (Sasco distributors, O m a h a , N E ) . A n ipsilateral p r o j e c t i o n p r e d o m i n a t e l y to l a m i n a X and the v e n t r a l h o r n was s e e n in c o n t r a s t to the dorsal h o r n p r o j e c t i o n s seen in the H a r l a n rats. T h e s e t e r m i n a l s w e r e s h o w n to be 90% n o r a d r e n e r g i c in studies colocalizing D B H . C l a r k and Proudfit h a v e res o l v e d t h e s e d i f f e r e n c e s by confirming the d i f f e r e n c e in circuitry in rats f r o m the t w o suppliers in their o w n laboratory3, T h e p r e s e n t study was d e s i g n e d to f u r t h e r address the c o n t r o v e r s y c o n c e r n i n g differences in p o n t i n e projeclions to the spinal c o r d by c o m p a r i n g rats f r o m local sources o f the two suppliers used by Proudfit and G r z a n na's labs. R e t r o g r a d e t r a n s p o r t of e i t h e r r h o d a m i n e or fluorescein c o n j u g a t e d latex b e a d s f r o m injections confined to the dorsal o r v e n t r a l h o r n of the spinal c o r d o f S p r a g u e - D a w l e y rats f r o m two v e n d o r s was u s e d to det e r m i n e p o n t i n e spinal projections. F l u o r e s c e i n and r h o d a m i n e b e a d s s h o w m i n i m a l diffusion f r o m the injeclion site 13. T h e r e f o r e , dorsal h o r n and v e n t r a l h o r n inj e c t i o n s can be c o n f i n e d w h e r e p r e v i o u s l y this was difficult with substances such as H R P and a n t i b o d y to D B H .
rescein beads were switched from dorsal to ventral horn in two animals to monitor the uptake of each. Each injection was 0.2 pl of beads and a total of 2-3/~1 was injected. Five to ten injections were made per side. In an additional two animals from each vendor, HRP gel implants (Type VI, Sigma Chemical Company, St. Louis, MO), were placed into the superficial dorsal horn. After 6-10 days, the animals were perfused transcardially with 4% paraformaldehyde (1 1) preceded by sterile saline with heparin (100 ml). A segment from the lumbar enlargement was placed in sucrose overnight and then cut on a freezing microtome (40-50 ~m). The sections of the lower midbrain, pons and upper medulla were cut and kept in serial order. The sections were mounted on gelatin coated slides and examined with a Zeiss microscope with an epifluorescence attachment. To quantify dorsal and ventral localization of cells, two animals from each vendor (Harlan and Sasco) with the most localized injections of fluorescent latex beads were used. In one Harlan animal receiving HRP gel implants into the dorsal horn, sections were mapped to demonstrate the distribution of the retrogradely labeled cells in the LC and SC. The nuclear boundaries used for the LC and SC were those depicted by Paxinos and Watson 2°. Retrogradely labeled cells from every 3-5 sections (150-250/~m) were mapped in animals injected with latex beads. The cells from these sections located within the borders of the LC bilaterally were counted. Cells labeled in the LC from dorsal and ventral horn injections were totalled. The percentage of labeled cells from the dorsal horn vs. ventral horn was determined. This procedure was repeated for the SC. These numbers are represented in Tables I and II. Five representative sections in two animals were stained immunocytochemically using an antibody directed against arterenol (norepinephrine; 1:100 for 1 h; Chemicon). Sections then were placed in IgG tagged with an AMCA (7-amino-4-methylcoumarin-
MATERIALS AND METHODS Male Sprague-Dawley rats (n = 7 Harlan, Houston; n = 3 Sasco, St. Louis) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and placed in a stereotaxie frame. A laminectomy was performed at the L3-L 5 level of the spinal cord. With a Hamilton syringe and a glass micropipette, retrograde fluorescent tracers were injected into the spinal cord. To maximize the data obtained from each of the animals used, two colors of latex beads were injected per animal. Dorsal horn injections of rhodamine labeled latex beads (Lumifluor, New Town, NY) were made 0.3-0.5 mm deep and 0.5-0.8 mm from the midline. Ventral horn injections of fluorescein labeled latex beads (Lumifluor, New Town, NY) were made at 1.0-1.3 mm from the midline and 1.0-1.5 mm deep on the opposite side of the spinal cord. Two animals'were injected 1.5 mm from the midline at a 30° angle and 1.5 mm deep to prevent any contamination in the dorsal horn. Rhodamine beads and fluo-
TABLE II Proportions o f labeled cells Nucleus subcoeruleus Harlan (n = 2)
Dorsal horn inj. Ipsilateral Contralateral Ventral horn inj. Ipsilateral Contralateral
Sasco (n = 2)
Cells/total
Av%
Cells/total
58/80 38 20 22/80 13 9
63% 76% 24% 37% 58% 41%
20/69 13 7 49/69 25 24
49/90 42 7 41/90 24 17
Av%
21/54 34% 17 73% 4 27% 33/54 66% 20 56% 13 44%
69 3-acetic acid) fluorophore (1:100 for 1 h; Jackson ImmunoResearch Lab.) for visualization using fluorescent microscopy. In an additional animal (Harlan) all sections were stained for tyrosine hydroxylase (TH; 1:400; 2 days, Pel-Freeze) followed by IgG tagged with an AMCA fluorophore (1:200, overnight). Either of these antibodies were used to define noradrenergic spinal projection neurons since no spinal projections from the LC contain dopamine4.
RESULTS T h e dorsal h o r n injections of latex b e a d s into the lumb a r spinal cord were c o n f i n e d to l a m i n a e I I I - V with m i n i m a l s p r e a d a l o n g the tract in l a m i n a e I a n d II a n d occasionally into l a m i n a e X. T h e p l a c e m e n t of the injec-
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Fig. 1. Maps depicting the distribution of the retrogradely labeled cells in the ports for individual sections using fluorescent latex beads from the lumbar ventral horn and the dorsal horn in the Harlan (left) and the Sasco (right) Sprague-Dawley rat. Cells labeled following injections in the ventral horn from one tissue section are illustrated by the closed circles and those from the dorsal horn by the x's. Each symbol represents one cell. The injection sites are shown below the labeled cells. The solid portion represents the densest area of injection. The diffuse spread of the tracer is represented by hatched lines. LC, locus coeruleus, SubCA, nucleus subcoeruleus alpha; SubCD, nucleus subcuruleus dorsal; SubCV, nucleus subcoeruleus ventral; MPB, medial parabrachial nucleus; LPBE, lateral parabrachial external nucleus; SuVe, superior vestibular nucleus; DPO, dorsal pontine olivary nucleus.
70 tion was in the lateral portion of laminae III-IV, nucleus proprius (Fig. 1). In no case was spread found in laminae VIII-IX. Ventral horn injection sites were located in laminae VIII and laminae IX with occasional spread into the ventral funiculus or along the tract. In one animal, the injection site was in the VF bilaterally and the dorsolateral funiculus (DLF) unilaterally. The HRP gel implants were placed dorsally with the densest portion of the HRP labeling in laminae I-III (Fig. 2) and occa-
37% projected to the ventral horn (Fig. 3C, Table II). However, in the Sasco rat, 34% of the cells within the SC projected to the dorsal horn while 66% projected to the ventral horn. The ipsilaterally projecting cells, like
HARLAN ~
sionally spreading ventrally to laminae V. Locus coeruleus
The present studies demonstrated projections of the LC to the dorsal and the ventral horn in both Sasco and Harlan Sprague-Dawley rats (Figs. 1 and 2). In the Harlan rats, the cells retrogradely labeled from the dorsal horn were more numerous in LC (Fig. 3B) than those from the ventral horn (Fig. 3A). In contrast, cells of the LC were labeled relatively more often from the ventral horn in the Sasco rats. Within the boundaries of the LC, 58% of the cells labeled were from dorsal horn injections and 42% from the ventral horn in the Harlan rat. However, in the Sasco rat, 34% of the cells in the LC were labeled following dorsal horn injections and 66% from the ventral horn (Table I). Seventy-two percent of the cells that were labeled in the LC were located contralaterally following dorsal horn injections in the Harlan rat. Sixty-five percent of the cells located in the LC in the Sasco rat were located contralaterally. Ventral horn projections from the LC were also bilateral, but 69% of the retrogradely labeled neurons in the Harlan rat and 76% in the Sasco rat were labeled ipsilaterally. In one Harlan rat, when there was clearly no spread of fluorescent tracer into the dorsal horn along the needle tract, the LC projection was almost totally ipsilateral. As a result of the inability to confine latex beads to laminae I and II, HRP gel implants were placed into the superficial dorsal horn in two animals from each vendor. In all of these animals there was bilateral labeling with a clear tendency for the LC to project ipsilaterally to the dorsal horn (Fig. 2).
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As seen in Figs. 1 and 2, the SC nucleus was labeled bilaterally following dorsal horn injections with an ipsilateral predominance in both the Harlan and the Sasco rats for both fluorescent latex bead injections and HRP gel implants. Cells labeled ipsilaterally were localized more ventrally in the nucleus. Projections of the SC to the ventral horn were bilateral with an ipsilateral predominance. In the Harlan rat, 63% of the retrogradely labeled cells in the SC were from the dorsal horn and
HRP Fig. 2. Maps depicting the distribution of the retrogradely labeled cells in the pons using horseradish peroxidase (HRP) gel implants into the dorsal horn of a Harlan Sprague-Dawley rat. The Sasco rat, not depicted showed a similar distribution. Each symbol (x) representsone cell. The injection site is shown below the labeled cells. The solid portion represents the densest area of placement. The diffusespread of the HRP is represented by hatched lines. LC, locus coeruleus; SubCA, nucleus subcoeruleus alpha; SubCD, nucleus subcoernleus dorsal; SubCV, nucleus subcoeruleus ventral; MPB, medial parabrachial nucleus.
71 those in the LC, were found caudally in the nucleus.
Other nuclei Cells were also labeled in the medial and lateral parabrachial nuclei, and the K611iker-Fuse nucleus following injections of both the dorsal and the ventral horn. An occasional cell was found in the lateral wall of the fourth ventricle in the region of the A4 cell group following dorsal horn and ventral horn injections. The pontine reticular formation had numerous cells labeled from both dorsal and ventral horn. These nuclei included the superior central nucleus and the reticulotegmental nucleus. There was also labeling ipsilaterally in the superior vestibular nucleus from dorsal horn injections into laminae I I I - I V (Fig. 1) of the lumbar enlargement.
Tracts In one animal (Harlan), using fluorescein latex beads, the ventrally placed injection site in the lumbar enlargement was located in the VF bilaterally. In the same animal, a dorsal horn injection (rhodamine latex beads) was located in the DLF. The results from this animal indicated that the VF at the lumbar level carries only SC axons. The DLF injections retrogradely labeled both the LC and the SC. This data also indicates that latex beads were taken up by cut fibers of passage.
Immunocytochemistry In two animals, 5 representative sections were stained immunocytochemically for arterenol to visualize norepinephrine cells of the LC and SC. Many examples were found of retrogradely labeled cells within the nuclear borders of the LC or the SC that also were stained immunocytochemically for norepinephrine. In one additional animal (Harlan) the total number of noradrenergic projection neurons which were retrogradely labeled in the LC and the SC were counted. In this animal 91% (31/34) of retrogradely labeled cells in the LC were noradrenergic, with 94% (16/17) from dorsal horn injections and 88% (15/17) from ventral horn injections. In the SC 86% (17/20) of the retrogradely labeled cells were noradrenergic, with 83% (5/6) from dorsal horn injections and 88% (14/16) from ventral horn injections. DISCUSSION Fig. 3. Photomicrographs taken using epifluorescent optics illustrate retrogradely labeled cells following injection of fluorescent latex beads into the lumbar spinal cord of a Harlan Sprague-Dawley rat. A: a retrogradely labeled cell from the locus coeruleus following injection of fluorescent tracer into the ventral horn of the spinal cord. B: a group of labeled cells in the locus coeruleus following a dorsal horn injection of the retrograde tracer. C: two cells in the subcoeruleus nucleus labeled following injection of fluorescent latex beads into the ventral horn. V, fourth ventricle,
The results of the present study indicated a clear difference between rats from two vendors (Sasco and Harlan) for the spinal projection of the LC and the SC. In the present study, retrograde transport of fluorescent beads from the lumbar dorsal horn vs. the ventral horn was used to help clarify recent data obtained by others using the PHA-L method. The present experiment
72 agreed with the data of many earlier studies finding both a ventral horn and a dorsal horn projection of the LC in the Sasco and Harlan Sprague-Dawley rat. The ventral born projections were found to be ipsilateral in rats from both vendors. The dorsal horn projections using both techniques (fluorescent beads and HRP gel implants) were bilateral. The fluorescent beads retrogradely labeled cells primarily contralaterally and the HRP gel implants labeled cells primarily ipsilaterally. In addition, the projections to the SC were investigated and found to be in agreement with previous studies 3'21'23'25 which report ipsilateral projections to both the ventral and dotsal horn. Clark and Proudfit 2, as well as Westlund et al. 23, have demonstrated previously that the majority of cells retrogradely labeled in the LC and SC from the spinal cord are noradrenergic. In this study we demonstrated that 91% of the spinally projecting LC cells and 86% of the SC cells were noradrenergic. In addition, PHA-L injections into the LC and the SC result in up to 90% of the terminals in the spinal cord staining for DBH 2'6. Therefore the majority of spinally projecting cells within the nuclear borders of the LC and the SC were noradrenergic. Although the experiments presented are in agreement with previous studies, the quantity of retrogradely labeled cells in the LC from the dorsal horn observed in the present study is greater than one would predict based on PHA-L studies of Fritschy and Grzanna 6, and Clark and Proudfit 2. In the Harlan Sprague-Dawley rat there was a small predominance for the dorsal horn projections over the ventral horn from the LC. In contrast, in the Sasco rat a greater number of cells were labeled retrogradely from ventral horn injections when compared to injections into the dorsal horn. This data confirmed a vendor difference existing between Sprague-Dawley rats which has been demonstrated previously in two separate studies using PHA-L 2'6. The differences in quantity of labeling revealed by the two methods, PHA-L and retrograde transport, can be explained by collateralization of axon terminals in the spinal cord. The collateralization of an axon from a single cell would provide a different picture using PHA-L than if the cell was retrogradely labeled. Alternatively, rats purchased from different farms from the same vendors may show slight variations, In the present study, differences were noted in the dorsal horn projections of the LC depending on whether the fluorescent beads or the HRP gel implants were used regardless of the vendors. Bilateral projections can be demonstrated with both techniques (fluorescent beads and HRP gel implants). Fluorescent beads retrogradely labeled cells primarily contralaterally while the HRP gel
implants labeled cells primarily ipsilaterally. In rats (Harlan and Sasco) with HRP gel implants, there was a bilateral projection, predominately ipsilateral, to the superficial dorsal horn which agrees with previous findings3' 6,10,23,25. The contralateral predominance of the LC projection in rats from both vendors to the dorsal horn from injections into the deeper laminae contradicts other previous work which found bilateral projections with an ipsilateral predominance 6'21'23'25. The difference between the results obtained using HRP gel implants vs. fluorescent latex beads can be attributed to the site of injections. The dorsal placement of the HRP gel implants to include lamina I and II indicated that the superficial dorsal horn has a predominately ipsilateral projection from the LC which agrees with Fritschy and Grzanna 6 and Clark et al. 3. The contralateral projection to the lumbar cord found with injection sites into the nucleus proprius also agrees with maps by Fritschy and Grzanna 6. The SC axons projected through the ventral funicuIUS6'21'22'25 and the DLF 21'22 to the dorsal and ventral horn bilaterally with strong ipsilateral predominance. This was not in agreement with the work of Fritschy and Grzanna 6 who found only ventral horn terminations and a ventral funiculus pathway for the SC in the Harlan rat. Previous investigators supported the DLF pathway 16'21'22 as well as the bilateral dorsal and ventral horn projections 1°'16'25. We found the LC projected through the DLF, which agrees with previous findings 16'21'25. Electrophysiological studies concur with the anatomical findings of a ventral horn and a dorsal horn noradrenergic projection from the LC. An enhancement of monosynaptic reflexes by stimulation of the LC in the rat and cat 1 was seen. The enhancement was blocked by prazosin, an a-1 adrenoreceptor antagonist, suggesting that the increased excitability of the motoneurons is me° diated through noradrenergic mechanisms. Fung and Barnes 7 electrically stimulated the LC and measured the membrane excitability of a motoneurons. A decrease in membrane resistance with an increase in motoneuron excitability was shown. LC/SC stimulation or glutamate microinjections inhibited nociceptive transmission as measured by response to heat stimuli (>50°C) 12. These multiresponsive neurons tested were located in laminae I-VI of the dorsal horn. The LC/SC stimulation was effective both ipsilateral and contralateral suggesting a strong bilateral input to the dorsal horn of the spinal cord. Thus, the electrophysiological studies support a projection to the ventral horn as well as the dorsal horn of the spinal cord. It is interesting to note that retrogradely labeled cells were localized in the superior vestibular nucleus from dorsal horn injections. This unexpected finding indicated the presence of a vestibulospinal tract through the lum-
73 bar spinal cord levels to the dorsal horn. Based on the current study using Harlan and Sasco Sprague-Dawley rats, a projection of the LC and the SC to both the ventral h o r n 1-4'7'16'18'19'23'25 and the dorsal h o r n 3'5-8'1°'12'16'17"19'23'25 exists and agrees with many other previous studies. The total number of cells labeled following a dorsal horn vs. the ventral horn injection is clearly different between vendors (Harlan vs. Sasco). The differences in spinal cord innervation between rats of these two vendors are, with the retrograde method, not as striking as was reported previously with the use
of the PHA-L method by Fritschy and Grzanna 6, Clark
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Acknowledgements. The authors wish to thank Scott Richardson for excellent technical assistance. This work was supported by NIH Grants NS 11255 and NS 01445, and an unrestricted grant from the Bristol-Myers Squibb Company.
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