Elsevier

Brain Research

Volume 732, Issues 1–2, 2 September 1996, Pages 1-15
Brain Research

Research report
Evidence for divergent projections to the brain noradrenergic system and the spinal parasympathetic system from Barrington's nucleus

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Abstract

The present study was designed to determine whether Barrington's nucleus, which lies ventromedial to the locus coeruleus (LC) and projects to the sacral parasympathetic nucleus, is a source of afferent projections to the LC. Restricted injections of the anterograde tracer, biocytin, into Barrington's nucleus labeled varicose fibers that extended from the injection site into the LC. Consistent with this, injections of the retrograde tracers, wheatgerm agglutinin conjugated to horseradish peroxidase coupled to gold particles (WGA-Au-HRP) or fluorescein-conjugated latex beads, into the LC labeled numerous (approximately 10%) Barrington's neurons that were also retrogradely labeled by Fluoro-Gold (FG) injections in the spinal cord. Retrograde tracing from the LC combined with corticotropin-releasing hormone (CRH) immunohistochemistry revealed that at least one third of the retrogradely labeled neurons in Barrington's nucleus were CRH-immunoreactive (CRH-IR). Finally, in triple labeling studies, CRH-Barrington's neurons were consistently observed that were retrogradely labeled from both the LC and spinal cord. These findings implicate Barrington's nucleus as an LC afferent and a source of CRH-IR fibers in the LC. Additionally, the results suggest that some Barrington's neurons diverge to innervate both the spinal cord and the LC. This divergent innervation may serve to coregulate the sacral parasympathetic nervous system and brain noradrenergic system, thus providing a mechanism for coordinating pelvic visceral functions with forebrain activity.

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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