Results

II. Afferent projections to the locus coeruleus

B) Anterograde tracing experiments

General remarks

In most rats, PHAL and CTb were iontophoretically deposited on opposite sides of the brain in the same structures and CTb or PHAL immunohistochemistry was performed on alternate sections. This procedure allowed us to increase results obtained with each animal, and to directly compare the anterograde labeling visualized by two different tracers. The CTb injection sites (1 µA, 15-30 min) with a diameter of about 600µm were darkly stained, round or ovoid and sharply delineated (Fig. 4C). In contrast, the delineation of the PHAL injection sites was more difficult. Indeed, PHAL sites (5 µA, 30 min) contained darkly labeled cells in the center of a light blue gray area about 1mm in diameter (Figs. 22E, 7B, 6D, 5B). Using PHAL, the anterogradely labeled fibers were strongly and completely labeled (Figs. 23A, 23B, 23C, 23D, 7E) . The CTb anterogradely labeled fibers were in general less completely labeled and sometimes showed only punctate labeling particularly when using stronger fixation (Figs. 23E, 1D). Moreover, in the case of CTb, in addition to the anterograde labeling of fibers, retrogradely labeled cells and dendrites were present in and around LC, in particular after CTb injections in the structures receiving an input from the LC or peri-LC such as the posterior hypothalamic areas (Figs. 7C) , the preoptic area and the Kölliker-Fuse nucleus (Fig. 1D). Only a few retrogradely labeled cells were seen in the LC after CTb injections in the periaqueductal gray, the laterodorsal tegmental nucleus, or the raphe dorsalis and magnus nuclei.

Despite the differences between the two tracers, after PHAL and CTb injections in the same contralateral structures we found comparable amounts of anterogradely labeled fibers in the efferent nuclei of the structure injected.

Frontal cortex

After PHAL injections involving area 1 of the frontal cortex and the adjacent lower limb region of the primary somatosensory area, only a few fibers were observed in the LC, Bar, Peri-5Me, 5Me, the lamina between the LC and the fourth ventricle and the periaqueductal gray region medial to the LC (Fig. 24C, 24D). In contrast, a large number of varicose fibers were clustered in the MVe regions located 1) between the LC and the 5Me (Fig. 24D) and 2) just lateral to the caudal pole of the LC and 3) caudally to the LC close to the ventricle. Many fibers were also observed in the periaqueductal gray and the pontine reticular formation ventromedial to the LC (Fig. 24C, 24D).

Infralimbic cortex

After a PHAL injection in the infralimbic cortex, only occasional fibers appeared in the nuclear core of the LC, the 5Me and the Bar. More fibers were observed in the lamina located between the LC and the fourth ventricle, the periaqueductal gray ventromedial to the LC, the Peri-5Me, the rostral part of the LC, the MVe regions 1) just lateral to the LC and 2) caudal to it close to the ventricle. The largest number of fibers was observed in the medial parabrachial nucleus.

Preoptic area dorsal to the supraoptic nucleus

After PHAL (Fig. 22E) or CTb injections in the preoptic area dorsal to the supraoptic nucleus, many anterogradely labeled fibers were localized in the nuclear core of the LC (Figs. 1B, 23B). A similar number of fibers was observed in the Peri-5Me (Fig. 23A). A larger number of fibers was found in 1) the periaqueductal gray medial or ventral to the LC, 2) the cell poor lamina and the ependyma separating the LC from the fourth ventricle (Figs. 1B, 23B), 3) the MVe region and the ependyma next to the ventricle caudal to the LC (Fig. 22D). The greatest innervation in the LC area, however was a very dense plexus of anterogradely labeled varicose fibers covering the Bar (Fig. 23A). A few or no fibers were found in the 5Me (Fig. 23A) and the MVe (Fig. 22D), respectively.

Posterior hypothalamic areas

The distribution of anterogradely labeled fibers in the LC area considerably varied depending on the localization of the injection sites in posterior hypothalamic areas.
PHAL or CTb injections in the dorsal hypothalamic area dorsal to the dorsomedial hypothalamic nucleus and caudal to it (CTb, n=2, PHAL, n=2) and in the perifornical area (CTb, n=2, PHAL, n=2)(Fig. 7B) gave rise to dense anterogradely labeled fibers in Bar (Fig. 7D), a large number of fibers in the lamina and the ependyma between the LC and the fourth ventricle and the periaqueductal gray medial to the LC (Fig. 7E), a substantial number of fibers in the peri-5Me (Fig. 7D), a small number of fibers in the LC (Fig. 7E), medial parabrachial nucleus, and 5Me (Fig. 7D). Some fibers were also observed in the ependyma caudal to the LC and the adjacent MVe region.

After PHAL or CTb injections in the lateral hypothalamic area just latero-dorsal to the fornix at the level of the dorsomedial hypothalamic nucleus (CTb, n=2, PHAL, n=2), we observed a moderate number of fibers in the LC and the 5Me (Figs. 23E, 7C). A much larger number of anterogradely labeled fibers was localized in the areas surrounding the LC, including Bar, Peri-5Me, medial parabrachial nucleus (Fig. 23E), the lamina and the ependyma between the LC and the fourth ventricle, the periaqueductal gray medial to the LC (Fig. 7C) and the MVe regions 1) just lateral to the LC and 2) caudal to the LC close to the ventricle including the ependyma. A few fibers were localized in the other parts of the MVe.

After PHAL or CTb injections in the lateral hypothalamic area located dorso-medially to the subthalamic nucleus (CTb, n=1, PHAL, n=1), the LC contained a small number of fibers (Fig. 23C). However, as found with other hypothalamic injections, many more fibers were seen in structures surrounding the LC, including the Bar, medial parabrachial nucleus, the Peri-5Me (Fig. 23D), the 5Me, the lamina and the ependyma between the LC and the fourthventricle and the periaqueductal gray medial to the LC (Fig. 23C, 23D).

Ventrolateral part of the periacqueducal gray

After CTb or PHAL injections in the lateral or ventrolateral part of the periaqueductal gray, a substantial to large number of anterogradely labeled fibers were observed in the nuclear core of the LC (Figs. 6B, 6E, 1C), the Peri-5Me, medial parabrachial nucleus and the rostral part of the 5Me. A much larger number of fibers were localized in peri-coerular regions, including the Bar, the lamina and the ependyma between the LC and the fourth ventricle (Fig. 6B, 6E), the periaqueductal gray medial to the LC, and the MVe region close to the ventricle caudal to the LC. Only few fibers were seen in the other parts of the MVe (Fig. 6B).

Area of the B9 serotoninergic cell group

After PHAL injections in the area of the mesencephalic reticular formation in and dorsal to the medial lemniscus just caudal to the red nucleus at the location of the B9 serotoninergic cell group, a few scattered fibers were observed in the LC, Bar, Peri-5Me and the lamina between the LC and the ventricle. Nearly no fibers were localized in the 5Me and the median parabriachial nucleus. A large number of fibers were distributed in the laterodorsal tegmental nucleus of Castaldi and the periaqueductal gray medial to the Bar and the LC.

Nucleus raphe dorsalis

After CTb injections in the nucleus raphe dorsalis, a substantial number of fibers were observed in the Bar and Peri-5Me as well as in the periaqueductal gray medial to the LC. Only a few anterogradely labeled fibers were observed in the nuclear core of the LC, the medial parabrachial nucleus, the 5Me, and the lamina and the ependyma separating the LC from the fourth ventricle. Few fibers were also localized caudal to the LC in the ependyma and the periventricular MVe region.

Laterodorsal tegmental nucleus of castaldi

After CTb injections in the laterodorsal tegmental nucleus of Castaldi, we observed a few varicose fibers in the LC and the median parabrachial nucleus. More fibers were located in the Bar, the Peri-5Me, the periaqueductal gray medial or ventral to the LC and the lamina between the LC and the fourth ventricle. The 5Me and the MVe only contained occasional labeled fibers.

Kölliker-Fuse nucleus

After CTb or PHAL injections in the nucleus Kölliker-Fuse, a substantial to large number of anterogradely labeled fibers covered the nuclear core of the LC (Figs. 1D, 5E, 24B) and the Bar (Fig. 24A). A greater number of labeled fibers were found in structures surrounding the LC, including 1) The lamina and the ependyma between the LC and the fourth ventricle (Figs. 5E, 24B), 2) the periaqueductal gray medial to the LC, 3) the MVe area just lateral to the LC (Fig. 5E) and 4) the MVe area and the ependyma close to the ventricle caudal to the LC. Few labeled fibers were found in the other areas of the MVe, the 5Me, peri-5Me and the median parabrachial nucleus (Fig. 24A, 24B).

Locus coeruleus

After LC+periLC injections of CTb, a few anterogradely labeled fibers were observed in the contralateral LC (Fig. 4A). Only occasional fibers were seen in the areas around the contralateral LC (Fig. 4A). After small LC injections, no fibers were seen contralaterally. Note that LC+periLC injections of CTb yielded a few retrogradely labeled contralateral LC neurons (Fig. 4A).

Nucleus raphe magnus

After CTb injections in the nucleus raphe magnus, a substantial number of varicose anterogradely labeled fibers were observed in the median parabrachial nucleus. In contrast, only a few fibers were visible in the LC, the Peri-5Me, the Bar, the 5Me and the lamina between the LC and the fourth ventricle.

Lateral paragigantocellular reticular nucleus

After CTb or PHAL injections in the retrofacial region of the lateral paragigantocellular reticular nucleus, a large number of anterogradely labeled fibers were observed in the nuclear core of the LC (Figs. 24E, 4F), the Bar, peri-5Me and the median parabrachial nucleus (Fig. 4E). A similar number of fibers were also observed in the ependyma and the lamina separating the LC from the ventricle, the periaqueductal gray medial or ventral to the LC and the periventricular MVe region and the ependyma caudal to the LC. Only occasional fibers were localized in the other MVe regions and the 5Me (Fig. 4F). Overall, anterograde labeling from the retrofacial lateral paragigantocellular reticular nucleus appeared to have a higher ratio of fibers in the core LC:periLC compared to other afferents studied.

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