Abstract

The aim of this study was to examine the afferents to the rat locus coeruleus by means of retrograde and anterograde tracing experiments using cholera-toxin B subunit and phaseolus leucoagglutinin. To obtain reliable injections of cholera-toxin B in the locus coeruleus, electrophysiological recordings were made through glass micropipettes containing the tracer and the noradrenergic neurons of the locus coeruleus were identified by their characteristic discharge properties. After iontophoretic injections of cholera-toxin B into the nuclear core of the locus coeruleus, we observed a substantial number of retrogradely labeled cells in the lateral paragigantocellular nucleus and the dorsomedial rostral medulla (ventromedial prepositus hypoglossi and dorsal paragigantocellular nuclei) as previously described 6.

We also saw a substantial number of retrogradely labeled neurons in (1) the preoptic area dorsal to the supraoptic nucleus, (2) areas of the posterior hypothalamus, (3) the Kölliker-Fuse nucleus, (4) mesencephalic reticular formation. Fewer labeled cells were also observed in other regions including the hypothalamic paraventricular nucleus, nucleus raphe dorsalis, median raphe nucleus, dorsal part of the periaqueductal gray, the area of the noradrenergic A5 group, the lateral parabrachial nucleus and the caudoventrolateral reticular nucleus. No or only occasional cells were found in the cortex, the central nucleus of the amygdala, the lateral part of the bed nucleus of the stria terminalis, the vestibular nuclei, the nucleus of the solitary tract, or the spinal cord, structures which were previously reported as inputs to the locus coeruleus 10,13. Control injections of cholera-toxin B were made in areas surrounding the locus coeruleus, including 1) Barrington's nucleus, 2) the mesencephalic trigeminal nucleus, 3) a previously undefined area immediately rostral to the locus coeruleus and medial to the mesencephalic trigeminal nucleus that we named the peri-mesencephalic trigeminal nucleus, and 4) the medial vestibular nucleus lateral to the caudal tip of the locus coeruleus. These injections yielded patterns of retrograde labeling that differed from one another and also from that obtained with cholera-toxin B injection sites in the locus coeruleus.These results indicate that the area surrounding the locus coeruleus is divided into individual nuclei with distinct afferents.

These results were confirmed and extended with anterograde transport of cholera-toxin B or phaseolus leucoagglutinin. Injections of these tracers in the lateral paragigantocellular nucleus, preoptic area dorsal to the supraoptic nucleus, the ventrolateral part of the periaqueductal gray, the Kölliker-Fuse nucleus yielded a substantial to large number of labeled fibers in the nuclear core of the locus coeruleus. Anterograde transport of cholera-toxin B or phaseolus leucoagglutinin from the posterior hypothalamic areas yielded a moderate to small number of labeled fibers in the nuclear core of the locus coeruleus. These anterograde tracing experiments confirm that these areas send direct projections to the rat locus coeruleus. Importantly, fiber labeling from each of these areas was in most cases much denser in areas immediately surrounding the locus coeruleus than in the locus coeruleus proper. In particular, the lamina and the periaqueductal gray medial to the LC where many dendrites of LC noradrenergic cells are located contained a large number of fibers. These data might indicate that a large number of the afferents to the noradrenergic neurons of the locus coeruleus terminate on dendrites outside the dense core of the nucleus. Further electrophysiological as well as ultrastructural studies are necessary to test this hypothesis.

Introduction

The rat locus coeruleus (LC) is a nucleus of the pontine tegmentum composed of a homogeneous compact noradrenergic cell group innervating nearly all the neuroaxis 16. These neurons exhibit a regular tonic discharge during waking, decrease their activity during slow-wave sleep and almost stop firing during paradoxical sleep (PS) 4. During waking, these neurons are phasically activated by a variety of sensory stimuli 5. These and other data obtained by electrophysiological and pharmacological experiments suggest a crucial role of these cells in the control of behavioral state, including vigilance and attention, and a critical but permissive role in the generation of paradoxical sleep 4, 34, 43. For several years, several groups have sought to understand the afferent control of LC neurons to reveal brain circuits involved in state and attention functions.

In early anatomical studies, using the retrograde tracer HRP with diaminobenzidine (DAB) as a chromogen, it was reported that many CNS structures project to the LC 10, 13, 45,57. In contrast, using the more sensitive retrograde tracers Fluoro-Gold and WGA-HRP with tetramethylbenzidine (TMB) as a chromogen, Aston-Jones et al. 6 recently found that major inputs to the LC as indicated by numerous strongly labeled neurons emanate from two nuclei, the paragigantocellularis lateralis and prepositus hypoglossi, both in the rostral medulla. This conclusion was substantiated by confirmatory anterograde tracing as well as by electrophysiological studies showing prominent functional influences of these two nuclei on LC neurons. We recently reported in cats that subunit B from the cholera-toxin (CTb), when iontophoretically applied and visualized with streptavidin-HRP, is a highly sensitive retrograde and anterograde tracer 41. Therefore, here we applied our method to rats in order to examine the afferents to the LC. Single cell recordings through the injection pipette were used to accurately place injections in the LC. We confirmed the specificity of the retrogradely labeled afferents to the LC by means of 1) CTb control injections in the regions surrounding the LC and 2) injections of the anterograde tracers CTb and Phaseolus vulgaris leucoagglutinin (PHAL) in the areas projecting to the LC. Preliminary results of this work have been reported 39.

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