Research reportEvidence for divergent projections to the brain noradrenergic system and the spinal parasympathetic system from Barrington's nucleus
Introduction
Recent investigations of the afferent projections to the major brain noradrenergic nucleus, locus coeruleus (LC), identified the medullary nucleus, paragigantocellularis (PGi), as a major source of afferent input to the LC 2, 25(see also [3]for a review). The PGi also projects to the intermediolateral column of the spinal cord 5, 23, 35, and several physiological studies have demonstrated parallel activation of the LC-norepinephrine system and peripheral sympathetic nervous system 1, 33, 39. Parallel projections from the PGi to the LC and sympathetic chain has been postulated to be a mechanism by which this coactivation may occur [44].
Retrograde tracing studies also suggested that neurons in nearby pericoerulear regions may provide afferent input to the LC, although the close proximity of these neurons to the LC made it difficult to determine whether labeling was due to retrograde transport 3, 48. Of these putative pericoerulear LC afferents, Barrington's nucleus, which lies just ventromedial to the rostral pole of the LC, is of interest. Recent preliminary studies revealed numerous retrogradely labeled neurons in Barrington's nucleus following restricted injections of WGA-Au-HRP into the LC [51]. Consistent with these retrograde tracing studies, injections of the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), into Barrington's nucleus labeled fibers in the LC [49].
Barrington's nucleus is thought to be important in the regulation of pelvic visceral function because it projects to the sacral parasympathetic nucleus 20, 22, 24, 36, 49. Consistent with this, physiological studies demonstrated that Barrington's nucleus is a critical component of the micturition reflex 6, 29, 36(see also [10]for a review). Its sacral innervation also implicates Barrington's nucleus in the regulation of gastrointestinal or genital function. Although its role in these functions has yet to be physiologically examined, recent studies demonstrated transynaptic labeling of Barrington's neurons by injections of pseudorabies virus into the distal colon, consistent with a role in gastrointestinal regulation [11]. Barrington's projections to the LC could serve as a mechanism for coordinating activity of the LC-noradrenergic system with the sacral parasympathetic system, and thereby forebrain activity with pelvic visceral function. Consistent with such coordination, the LC is activated by both bladder and colon distention, two pelvic visceral stimuli that would be predicted to activate Barrington's nucleus and its spinal projections [14].
It is also noteworthy than many Barrington's neurons are immunoreactive for corticotropin-releasing hormone (CRH) 38, 41, 47, 48, 49. CRH is the hypothalamic hormone responsible for initiation of the endocrine limb of the stress response [43], and has also been proposed to act as a neurotransmitter to initiate autonomic and behavioral limbs of the stress response 13, 30, 46. Substantial anatomical and physiological findings suggest that the LC is a target for CRH neurotransmission (see [46]for a review). A study combining retrograde tracing from the LC and CRH-immunohistochemistry indicated that CRH-IR fibers in the LC originate from the PGi and the dorsal cap of the paraventricular nucleus of the hypothalamus [48]. This study also suggested that CRH-IR fibers in the LC could arise from neurons in nearby pericoerulear regions, such as Barrington's nucleus. However, in this study, relatively large injections of the tracer were used, and it was difficult to discern whether labeling was due to retrograde transport or spread of the tracer. Moreover, this study used tissue from colchicine-treated rats, a pretreatment which we and others have found to interfere with retrograde transport 28, 49.
The present study used both anterograde and retrograde tract tracing in non-colchicine treated rats to investigate putative Barrington's projections to the LC, determine whether LC-projecting neurons were also spinal projecting, and whether these were CRH-containing projections.
Section snippets
Surgery
The subjects were adult male Sprague-Dawley rats (approximately 300–400 g; Taconic Farms). Rats were anesthetized with pentobarbital (50 mg/kg, i.p.) and positioned in a stereotaxic instrument with the snout lowered to place the skull at a 15° angle. A scalp incision was made and a hole, centered at 3.5 mm caudal to the intersection of λ and midline and 1.1 mm lateral, was drilled for injections of tract tracers into the LC or Barrington's nucleus. For injections in the ventral medulla, the rat
Anterograde labeling from Barrington's nucleus
Previous studies described the distribution of spinal-projecting and CRH-IR neurons of Barrington's nucleus with respect to other neurochemically identified neurons in the dorsal pons 34, 49. The distribution of both spinal-projecting and CRH-IR neurons is similar, and in coronal sections these cells form an oval cluster that lies just ventromedial to the rostral pole of the LC. Fig. 1A shows the cluster of CRH-IR Barrington's neurons with respect to TH-IR cells and processes of the LC in
Discussion
The present study demonstrated varicose fiber labeling within the LC after localized injections of biocytin in Barrington's nucleus. Consistent with these findings, retrograde tracing from the LC labeled a substantial number of Barrington's neurons that were identified by either their spinal projections, or CRH-IR. Moreover, a number of Barrington's neurons that were retrogradely labeled from both the LC and spinal cord were CRH-IR. These results implicate Barrington's nucleus as a
Acknowledgements
This work was supported by PHS Grants MH40008, MH42796, NS 24698 and a Research Scientist Development Award MH00840. The authors gratefully acknowledge Dr. Wylie Vale for the generous gift of CRH anti-serum, and the technical expertise of Mr. Bowen Kang and Ms. Wei Ping Pu.
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