Results

II. Afferent projections to the locus coeruleus

A) Retrograde tracing experiments

Injection sites

As illustrated in Figs. 1A, 2 , 3A and 4C, CTb iontophoretic injection sites were characterized by a dense central area surrounded by a peripheral zone containing diffuse tracer and stained fibers . Usually, the histochemical reaction produced a crack at the center of the site. This crack did not correspond to necrosis of the tissue as determined on sections that were only counter stained with neutral red adjacent to those immunostained with CTb (Fig. 3A, 3B) as we previously observed in the cat 41. Indeed, on such sections, the tissue and the morphology of the LC cells localized in the injection site appeared normal (Fig. 3B).

We calculated that the homogeneous nuclear core of the LC is 170-230µm mediolaterally, 700 µm dorsoventrally and 400-500 µm long in the rostro-caudal direction. We obtained eight small CTb injection sites in the LC which appeared to have no or only limited extensions into surrounding areas. These sites had 200 to 350 µm diameters and were round or ovoid (Figs. 1A, 2B, 3A). Three sites were localized in the middle part of the LC (Fig. 2B) , four in its ventral part (Fig. 1A) and one in its dorsal part. Three of them appeared to be restricted to the nuclear core of the LC (Figs. 1A, 2B). Five of them slightly involved the MVe area just lateral to the LC (Fig. 3A). None of them appeared to involve the periaqueductal gray and the lamina medial to the LC, the Bar, the 5Me and the peri-5Me. We obtained also three large injections sites centered in the LC with 400-600µm diameters involving areas adjacent to the LC (peri-LC areas). These sites, which we termed LC+periLC injections, involved 1) the MVe areas just lateral and caudal to the LC and 2) the poor cell lamina and the periaqueductal gray medial to the LC (Fig. 2A). Three other large injections sites centered in the LC involved the same peri-LC areas but also partly the Bar, peri-5Me and surrounding periaqueductal gray.

Nine control injection sites 300 to 400µm in diameter were made in structures surrounding the LC. Three were centered in the Bar with only a moderate extension in the surrounding periaqueductal gray (Fig. 2C). Three other injections sites were centered in the peri-5Me with two of them slightly involving either the Bar or the rostral part of the LC. One these sites was smaller and appeared to be restricted to the peri-5Me (Fig. 2D). Two other control injection sites were localized in the 5Me lateral to the LC with no apparent involvement of the LC, the Bar, the peri-5Me or the MVe (Fig. 2E). The lateral extension of these two sites slightly involved the medial parabrachial nucleus. One control injection site was localized in the MVe region just lateral to the caudal part of the LC (Fig. 2F). This site did not appear to involve the 5Me.

Efferents from the locus coeruleus and surrounding areas

As previously described 41, after CTb injections, in addition to the retrograde labeling,we also observed anterograde labeling of the efferents of the nuclei injected with CTb. This anterograde labeling helped to corroborate the apparent specificity of the injection sites.
After LC or LC+periLC injections, we observed a large number of fibers localized in the lamina and the periaqueductal gray between the LC and the ventricle as well as in the MVe region close to the ventricle just caudal to the LC. In addition, many coarse fibers exited the site rostrally and caudally. One population of fibers was ventral to the 5Me forming a bundle at the location of the ascending noradrenergic bundle (Fig. 5D). In the rostral pons and the mesencephalon these fibers were localized ventrally to the periaqueductal gray (Fig. 6A). Rostrally in the forebrain, these fibers took a lateral position in the medial forebrain bundle (Fig. 7A). Scattered coarse fibers were visualized over the entire cerebral cortex (Fig. 21C, 21D).

A second bundle of fibers exited ventrally and caudally from the injection site descending vertically medially to the trigeminal motor nucleus at the location of the descending noradrenergic bundle, and then in the dorsal part of the parvocellular reticular nucleus in the medulla. Excepting the labeling of these ascending and descending bundles, only a few fibers were visible in other areas including the pontine and mesencephalic ventrolateral part of the periaqueductal gray.

In contrast, after all control injections around the LC, no or only a few anterogradely labeled fibers were visible in the two bundles labeled after LC injections. Instead, for each nucleus, a specific pattern of anterograde labeling appeared. After Bar or peri-5Me injections, a bundle of anterogradely labeled fibers was localized in the ventrolateral part of the pontine periaqueductal gray dorsal to the laterodorsal tegmental nucleus of Castaldi and proceeded close to the ventricle in the lateral part of the mesencephalic periaqueductal gray.

Only after Bar injections we saw also a descending bundle of fibers first medial to the trigeminal motor nucleus and then in the nucleus gigantocellularis up to the caudal medulla. Terminal like-labeling was also visible in the lateral paragigantocellular nucleus.

Specifically after CTb injections in the peri-5Me, anterogradely labeled varicose fibers were visible bilaterally in all posterior hypothalamic areas. The largest number of terminal-like fibers was clustered in the compact zone of the dorsomedial nucleus of the hypothalamus (Fig. 3D). Confirming this strong hypothalamic projection of the peri-5Me, we observed a cluster of retrogradely labeled cells specifically in the peri-5Me after CTb injections in the posterior hypothalamus, particularly those in the dorsomedial hypothalamic nucleus (Fig. 3C).

After the control MVe injection, specific anterograde labeling was visible bilaterally in the other vestibular nuclei as well as ipsilaterally in the oculomotor and trochlear nuclei and the interstitial nucleus of Cajal in the mesencephalon.

After 5Me injections, anterogradely labeled fibers were specifically observed in the motor trigeminal nucleus and the medullary parvocellular reticular nucleus.

After large injections centered in the LC, we observed anterograde labeling not only in the ascending and descending noradrenergic bundles but also additional anterograde labeling with a localization depending on the nucleus surrounding the LC included in the site.

Retrograde labeling

In the following part of the results, we describe the distribution of retrogradely labeled cells from rostral to caudal structures. To illustrate results of retrograde labeling, we made camera lucida drawings of 20µm sections from brains with representative LC, LC+periLC and Bar, injections (Figs. 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20) . A semiquantitative analysis of the retrograde labeling is also represented in Table 1.

The injection in the LC+periLC illustrated in Fig. 2A (rat RLC2) covered the entire LC and involved the periaqueductal gray and the cell poor lamina medial to it as well as part of the MVe region lateral to the LC. This site did not substantially encroach on the Bar, 5Me and peri-5Me. Strong anterograde labeling was present in the ascending and descending noradrenergic bundles and the terminal cortical field of the LC but also in the ventrolateral parts of the pontine and mesencephalic periaqueductal gray.

The representative LC injection illustrated in Fig. 2B (rat RLC15) was apparently restricted to the caudal and dorsal part of the LC. It did not appear to involve the lamina medial to the LC and the MVe region just lateral to the LC. After this injection, anterogradely labeled fibers were found only in the ascending and descending noradrenergic bundles.

The control site in Bar illustrated in Fig. 2C did not apparently involve the adjacent LC and peri-5Me. No fibers were present in the noradrenergic bundles. Instead, we saw a bundle of anterogradely labeled fibers in the ventrolateral part of the pontine periaqueductal gray. A descending bundle of fibers medial to the trigeminal motor nucleus and then in the nucleus gigantocellularis up to the caudal medulla was also present.

Examples of 250-400µm-diameter control injection sites localized in the peri-5Me, the 5Me, and the MVe are shown in Fig. 2D, 2E, 2F.

The cell counts given in the text for each area were made from one frontal section unilaterally only with the largest number of retrogradely labeled cells in the representative injections drawn in Figs. 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 . Therefore, note that the total number of cells labeled for each area depends upon the laterality and rostro-caudal extension of labeling in that area. The CTb labeling produced with the DAB-nickel histochemical procedure consisted of black punctate granules in the cell soma and dendrites of retrogradely labeled neurons (Figs 5C, 6C, 21A,B, 21C, 21D, 22C). Importantly, after LC or LC+periLC injections we observed that the intensity of the retrograde labeling was stronger in the lateral paragigantocellular, prepositus hypoglossi and hypothalamic paraventricular nuclei than in the other nuclei. This difference in intensity was most visible with shorter incubations in primary antibody (overnight, room temperature) or higher dilutions of the antibodies yielding relatively weak immunostaining of CTb. After the normal immunostaining procedure, retrogradely labeled cells were darkly stained in all structures.

Cortical areas

After LC+periLC injections, a substantial number of retrogradely labeled pyramidal cells were observed mainly ipsilaterally in the infralimbic cortex (IF) close to the tenia tecta (11-18 cells per section)(Fig. 21D), the caudal part of the area 1 of the frontal cortex (Fr) (Fig. 21A,21B) and the adjacent primary somatosensory area, particularly its lower limb region (4-12 cells per section). Smaller multipolar cells were also observed in the claustrum (Cl)(6-8 cells per section) (Fig. 21C). After LC or Bar injections, a much smaller number of retrogradely labeled cells were observed in these structures (1-2 cells per sections). More cells were found in the frontal and infralimbic cortices after LC injection sites involving the MVe region lateral to the LC and the control injection in the MVe region lateral to the LC. After this restricted MVe injection, no or only occasional cells were visible in the insular cortex and the claustrum. After 5Me injections, a large number of cells were localized in the claustrum and the insular cortex. Fewer appeared in the infralimbic cortex. The frontal cortex contained only occasional cells. After injections in the Peri-5Me, a substantial number of cells were observed in the infralimbic and insular cortex, and the claustrum. Only a few cells were visible in the frontal cortex.

Preoptic level

After LC+periLC injections, a dense collection of small cells (37-42 cells per section) was distributed with an ipsilateral predominance in the preoptic area localized dorsally to the supraoptic nucleus just rostral to and at the level of the rostral part of the suprachiasmatic nucleus (Figs. 8D, 9C, 22A, 22C). This group extended rostrally up to the rostral end of the third ventricle (Fig. 8C). After LC injections, a substantial number of neurons was still specifically found in this region of the preoptic area (8-12 cells per section) (Figs. 8A,8B, 9A).

After LC+periLC injections, additional small cells were demonstrated ipsilaterally in the medial part of the bed nucleus of the stria terminalis (BSTM) (Fig. 9C).
After LC or LC+periLC injections, no or only a few cells were observed in the medial preoptic nucleus (MPO) and the lateral part of the bed nucleus of the stria terminalis (BSTL) (Figs. 8B,8D, 9A,9C).

After Bar injections, many cells were localized in the preoptic area dorsal to the supraoptic nucleus and the medial part of the bed nucleus of the stria terminalis (Figs. 8F, 9E, 22B) . However, additional cell groups appeared in 1) the medial preoptic nucleus (Figs. 8F, 22B), 2) the lateral preoptic area dorsal to the horizontal limb of the diagonal band of Broca (Fig. 9E) and 3) the magnocellular subdivision of the medial part of the bed nucleus of the stria terminalis (Fig. 8F, 22B).

After injections in the 5Me or Peri-5Me, a large number of small cells were clustered ipsilaterally in the lateral part of the bed nucleus of the stria terminalis (21-24 cells per section). Only a moderate number of cells were observed in the medial part of the bed nucleus of the stria terminalis and the preoptic areas.

After the specific MVe injection, no cells were identified in these rostral forebrain areas.

Midhypothalamic level

At the level of the midhypothalamus, after LC, LC+periLC, Bar, 5Me or peri-5Me injections , a moderate number of small cells were localized mainly ipsilaterally in the parvocellular parts of the hypothalamic paraventricular nucleus and its lateral extension dorsal to the fornix (figs. 9B,9D,9F, 10A,10C,10E). After LC or LC+periLC injections, cells were distributed in the dorsal cap of the nucleus (Figs. 9B,9D, 10A,10C) while they were mainly localized in its medioventral part after Bar, 5Me or peri-5Me injections (Fig. 9F). Interestingly,the number of retrogradely labeled cells in this nucleus differed little after LC+periLC (5-7 cells per section) or LC (2-4 cells per sections) injections. At the same level, after any injection, only occasional cells were observed in the rostral part of the lateral hypothalamic area.

After 5Me or Peri-5Me injections, a large number of small cells were seen with an ipsilateral predominance in the central nucleus of the amygdala (25-72 cells per sections). In the case of LC, LC+periLC, Bar or MVe injections, no or only occasional cells were distributed in this nucleus or anywhere in the amygdala.

Posterior hypothalamus

After LC or LC+periLC injections, small retrogradely labeled cells were observed in the dorsal hypothalamic area located dorsal to the dorsomedial hypothalamic nucleus (Figs. 7A, 11A-D) . Intermingled small and medium-sized neurons were also found in the rostral part of the perifornical nucleus and the lateral hypothalamic area at all levels of the posterior hypothalamus (Figs. 7A, 10, 12A-D). In these posterior hypothalamic areas, there was a substantial difference in number of cells between LC+periLC injections (50-60 cells per section) and LC injections (13-21 cells per section).

After injections into the Bar, although neurons were found in the same areas as after LC or LC+periLC injections, additional groups of small cells appeared 1) in the caudal part of the dorsal hypothalamic area (Fig. 11F), and 2) around the fornix and ventromedial to it (Fig. 11E). In contrast to LC or LC+periLC injections, only a few cells appeared in the lateral hypothalamic area immediately medial to the internal capsule (Fig. 11E,11F).

After 5Me or Peri-5Me injections, the overall distribution of cells in the posterior hypothalamic areas was again different. A large number of small to medium-sized cells was found only in the lateral and caudal part of the lateral hypothalamic area located just medial to the internal capsule and the subthalamic nucleus. Fewer neurons were present in the perifornical nucleus and the dorsal hypothalamic area compared to LC, LC+periLC or Bar injections.

After the injection in the MVe, a substantial number of rather medium-sized neurons were diffusely distributed in the posterior hypothalamic areas with no clear topography.

After LC, LC+periLC, 5Me or MVe injections, no or only occasional cells were observed in the arcuate nucleus of the hypothalamus (Figs. 10, 11). After injections into the Bar (Fig. 10E) or peri-5Me, slightly more cells were localized in this nucleus.

In all cases, no or only a few cells were distributed in other structures such as the ventromedial hypothalamic nucleus, the tuberomamillary nucleus and the lateral habenula (Figs. 10, 11, 12).

After LC, LC+periLC or Bar injections, a small group of cells was consistently present in an undefined area located dorsal to the fasciculus retroflexus and ventral to the caudal tip of the lateral habenula (Fig. 12A).

Specifically after the MVe injection, cells were observed in the retrorubral field and the interstitial nucleus of Cajal.

Mesencephalon

After LC+periLC or Bar injections, a large number of small cells was labeled in the lateral and ventrolateral parts of the periaqueductal gray (Figs. 6A, 6C, 13, 14, 15, 16) (LC+periLC: 40-50 cells per section, Bar: 20-30 cells per section). After LC injections, a substantial number of cells were still observed in this area (Figs. 13, 14, 15, 16A,B) (10-15 cells per section). The distribution of the labeled cells appeared to be different for Bar and LC or LC+periLC injections. After Bar injections, the great majority of the CTb-positive cells were clustered in a medial area close to the ventricle (Figs. 14F, 15E, 15F, 16E), whereas they were more diffusely distributed laterally and ventrolaterally after LC or LC+periLC injections (Figs. 13, 14, 15C,15D). After Peri-5Me injections, a large number of cells were also seen in the same regions as after LC injections. After 5Me or MVe injections, only a few labeled cells were observed in the periaqueductal gray.

After LC+periLC injections, a large number of small cells also appeared in the dorsal part of the periaqueductal gray (13-23 cells per section) (Figs. 13 , 14 , 15A,B) . After LC injections, the number of cells was much lower in this area (1-5 cells per section) (Figs. 8 , 9 , 10A,B) . After Bar (Figs. 8 , 9 , 10E,F) , peri-5Me, 5Me or MVe injections, only a few cells were visible in the dorsal periaqueductal gray.

After LC+periLC injections, a large number of small and medium-sized cells was found with an ipsisilateral predominance in the mesencephalic reticular formation, particularly 1) dorsal to the lateral part of the substantia nigra (Fig. 8D), 2) lateral to the periaqueductal gray (Fig. 9C,D), 3) lateral to the median raphe nucleus (Fig. 9C,D) and 4) in and just dorsal to the median lemniscus and lateral to the interpeduncular nucleus (Figs. 9, 10A,D). Fewer labeled cells were observed in these areas after LC (Fig. 8, 9A,B), Bar (Fig. 8, 9E,F), 5Me, Peri-5Me or MVe injections.

After LC or LC+periLC injections, a few medium-sized cells were seen with a slight ipsilateral predominance in the median raphe nucleus (Figs. 10, 11A-D). Only occasional cells were observed in this structure after Bar (Fig. 10, 11E,F), 5Me, Peri-5Me or MVe injections.

After LC+periLC injections, a large number of small cells was localized mainly in the rostral, dorsal and dorsolateral parts of the nucleus raphe dorsalis (Figs. 10D). Fewer cells were visible in the caudal part of the nucleus (Fig. 11C,D). Although many fewer in number, neurons were also seen in the dorsal and dorsolateral parts of the nucleus raphe dorsalis after LC or Bar injections (Figs. 10, 11, 12A,B,E,F). After 5Me, Peri-5Me or MVe injections, a substantial number of medium-sized cells was found in the medioventral part of the nucleus raphe dorsalis.

After LC+periLC injections, a few cells were also observed in the Edinger-Westphal nucleus (Figs. 8D, 9C). No such cells were observed after LC injections. A large number of CTb-positive cells were localized in the Edinger-Westphal nucleus after the specific MVe injection.

Pons

After LC or LC+periLC injections, small cells (LC+periLC, 40 cells; LC, 13 cells per section) were observed, bilaterally with a slight ipsilateral predominance, in the Kölliker-Fuse nucleus (K-F) (Figs. 16A-D, 17A,B). The neurons were localized just dorsally to the rubrospinal tract (rs) and ventral to the brachium conjunctivum at the caudal level of the ventral tegmental nucleus of Gudden (VTg) (Figs. 5A, 5C, 16, 17). After Bar or MVe injections, a limited number of cells were distributed in the Kölliker-Fuse nucleus (4-5 cells per section) (Fig. 11E,F). Only occasional cells were observed after 5Me or Peri-5Me injections.

After LC+periLC injections, a large number of small cells was seen, with a large ipsilateral predominance, in the lateral parabrachial nucleus (LPB) (Figs. 17C,D, 5D). The number of cells was limited after LC (Fig. 17A,B) , Bar (Fig. 17E,F) or 5Me injections. Few cells appeared in this nucleus after Peri-5Me or MVe injections.

After LC+periLC injections, the laterodorsal tegmental nucleus of Castaldi (LDT) also contained mainly ipsilaterally a small number of medium-sized retrogradely labeled cells (Figs. 16D, 17C,D) . After LC (Figs. 16B, 17A,B), Bar (Figs. 16, 17E,F) , 5Me, Peri-5Me or MVe injections, only a few cells were found in this nucleus. After LC, LC+periLC or the control injections, only a few medium-sized cells were demonstrated ipsilaterally in the pedunculopontine nucleus (PPT) (Figs. 15, 16).
At the caudal pontine level, after LC+periLC injections, medium-sized cells with an ipsilateral predominance were observed ventral to the facial nerve and laterodorsal to the superior olivary complex in the area of the A5 noradrenergic cell group 16 (Figs. 17, 18C,D). A few cells were still present in this area after LC injections (Fig. 18A,B). No or only occasional cells were seen there after Bar (Fig. 18E,F) , 5Me, Peri-5Me or MVe injections.

The nuclei raphe pontis and magnus contained only a small number of medium-sized cells after LC, LC+periLC or Bar injections (Fig. 17, 18, 19). Nearly no cells were observed in these raphe nuclei after 5Me, Peri-5Me or MVe injections. The pontine reticular formation contained small to medium-sized dispersed cells after LC+periLC (Figs. 17, 18C,D), 5Me or MVe injections. After LC (Figs.17, 18A,D), Peri-5Me or Bar injections (Figs. 17, 18), only occasional cells were present in this area.

After LC or LC+periLC injections, only a small number (maximum 5 cells per section) of retrogradely labeled neurons were identified in the structures adjacent to the LC such as the Bar and the peri-5Me. Only after LC+periLC injections were a few labeled cells present in the contralateral LC (Figs. 4A, 18C,D). After Bar injections, darkly stained medium-sized cells were found in the ipsilateral LC caudal to the injection site.

Medulla

After LC or LC+periLC injections involving the MVe area just lateral or caudal to the LC, a number of cells was distributed with a slight ipsilateral predominance in the vestibular nuclei (Fig. 19C,D) . Only rare cells were observed in these nuclei after LC (Fig. 19A,B), Bar (Fig. 19E,F) or Peri-5Me injections. In contrast, a large number of retrogradely labeled neurons were found bilaterally in the vestibular nuclei after the control injection in the MVe.

The lateral paragigantocellular reticular nucleus (LPGi) contained a large number of small and medium-sized cells with a large ipsilateral predominance after LC+periLC injections (25-30 cells per section) (Figs. 4D, 19D). A substantial number of cells was still observed in this nucleus after LC injections (10-14 cells per section) (Fig. 19B). After Bar injections, a small number of cells was localized in this area (5 cells per section) (Fig. 19F). Fewer cells were visible after 5Me, Peri-5Me or MVe injections.

After LC or LC+periLC injections, the dorsomedial rostral medulla contained a substantial to large number of medium-sized retrogradely labeled cells (LC+periLC, 22 cells; LC, 7 cells per section) (Figs. 4B, 19A,C). More precisely, these cells were located in the ventromedial part of the nucleus prepositus hypoglossi (PrH) and the reticular formation ventral to it (dorsal paragigantocellular nucleus (DPGi)) 49. Note that the number of cells are based on unilateral counts; cells in the dorsomedial rostral medulla were bilaterally located.

After the MVe control injection, a large number of cells was also distributed in the dorsomedial rostral medulla (ventromedial part of the nucleus prepositus hypoglossi and dorsal paragigantocellular nucleus) with a distribution similar to LC or LC+periLC injections but with a strong contralateral predominance. After Bar (Fig. 19E,F), 5Me or Peri-5Me injections, only a few cells were observed in the same area.
After LC, LC+periLC or Bar injections, only a small number of cells were localized in the caudal part of the nucleus of the solitary tract (Fig. 20).

After 5Me injections, a large number of small cells was seen with a small ipsilateral predominance in the rostral and caudal part of the nucleus of the solitary tract (Sol). The caudal part of this nucleus also contained a large number of cells after Peri-5Me injections.

After LC+periLC injections, small cells were observed mainly ipsilaterally in the caudoventrolateral reticular nucleus (Fig. 20D). This area also contained a few cells after LC injections (Fig. 20B) and occasional cells after Bar (Fig. 20F), 5Me, Peri-5Me or MVe injections. Only after 5Me injections, a large number of small cells was seen with a small ipsilateral predominance in the parvocellular reticular nucleus (PCR).

Spinal cord

Even after LC+periLC injections, we observed only a limited number of retrogradely labeled cells mainly ipsilaterally in the intermediate layers at all levels of the spinal cord. No cells were observed in the superficial layers. After LC injections , only occasional cells were observed in the intermediate layers of the cord. The spinal cord was not examined in control experiments.

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