Elsevier

Biological Psychiatry

Volume 46, Issue 10, 15 November 1999, Pages 1352-1363
Biological Psychiatry

A. E. Bennett Research Award
Anatomic basis for differential regulation of the rostrolateral peri–locus coeruleus region by limbic afferents

https://doi.org/10.1016/S0006-3223(99)00213-9Get rights and content

Abstract

Background: Neurochemical and electrophysiological studies indicate that the locus coeruleus (LC)-norepinephrine system is activated by physiological and external stressors. This activation is mediated in part by corticotropin-releasing factor (CRF), the hypothalamic neurohormone that initiates the endocrine response to stress. We have previously shown that the central nucleus of the amygdala (CNA) provides CRF afferents to noradrenergic processes in the peri-LC area that may serve to integrate emotional and cognitive responses to stress. The bed nucleus of the stria terminalis (BNST) shares many anatomical and neurochemical characteristics with the CNA, including a high density of CRF-immunoreactive cells and fibers; however, recent studies have suggested that the CNA and the BNST may differentially regulate responses to conditioned and unconditioned fear, respectively, suggesting divergent neuroanatomical circuits underlying these processes.

Methods: In the present study, neuroanatomical substrates subserving regulation of the LC by the BNST were examined. Anterograde tract-tracing was combined with immunoelectron microscopy to test the hypotheses that BNST efferents target noradrenergic neurons of the LC and that these efferents exhibit immunolabeling for CRF.

Results: Ultrastructural analysis of sections that were dually labeled for the anterograde tracer biotinylated dextran amine (BDA) injected into the BNST and tyrosine hydroxylase (TH)-immunoreactivity demonstrated that BDA-labeled axon terminals formed synaptic specializations (primarily inhibitory) with TH-labeled dendrites and dendrites that lacked TH immunoreactivity. In contrast to CNA efferents that exhibited substantial immunolabeling for CRF, far fewer BDA-labeled terminals from the BNST in the rostrolateral peri-LC contained CRF.

Conclusions: The present results indicate that the BNST may provide distinct neurochemical regulation of the peri-LC as compared to other limbic afferents such as the CNA. These data are interesting in light of behavioral studies showing that the CNA and BNST may be differentially involved in fear versus anxiety, respectively.

Introduction

Several lines of evidence suggest that neurons in the bed nucleus of the stria terminalis (BNST) and neurons in the central nucleus of the amygdala (CNA) exhibit similar morphology, neurochemistry and afferent/efferent pathways Alheid et al 1995, de Olmos et al 1985. Recent studies, however, suggest that although anatomically homogeneous, functional attributes of the CNA and BNST may be quite disparate. Specifically, Walker and Davis (1997) provided evidence suggesting that the CNA preferentially mediates the expression of conditioned fear whereas the BNST is involved in the expression of unconditioned fear.

Interestingly, both the CNA and BNST provide corticotropin releasing factor (CRF), a neuropeptide essential for integrated physiological responses to stress, to common hypothalamic and brainstem areas Alheid et al 1995, Alheid and Heimer 1988, de Olmos et al 1985. We have recently shown that the CNA provides CRF afferents to a selective portion of the dorsal pontine tegmentum that contains catecholaminergic dendrites of the locus coeruleus (LC) and may impact on activity of the LC-norepinephrine system through direct synaptic contacts Van Bockstaele et al 1996a, Van Bockstaele et al 1998. Previous studies have indicated that intracoerulear administration of CRF increases LC discharge rates Curtis et al 1997, Valentino et al 1983 and norepinephrine release in LC target regions Page and Abercrombie 1995, Smagin et al 1995 suggesting that CRF is an important mediator of noradrenergic function. The amygdaloid-CRF modulation of brain noradrenergic activity may serve as a mechanism for the integration of emotional and cognitive responses to stress (Redmond 1986).

The present study was designed to determine whether the BNST, like the CNA, targets similar portions of the peri-LC, whether the BNST contacts catecholaminergic dendrites and whether the BNST is an endogenous source of CRF here. Neuronal tract tracing was combined with immunohistochemistry and analyzed at both the light and electron microscopic level. A first set of studies combined anterograde tracing from the BNST with immunocytochemical detection of the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), in the rostrolateral peri-LC region to determine whether BNST efferents monosynaptically innervate LC neurons. Second, to determine whether BNST efferents were CRF-containing, sections were processed for the dual immunocytochemical identification of CRF and BDA. The results provide ultrastructural evidence for an impact on LC activity by projections from the BNST to the rostrolateral peri-LC dendrites many of which lack the neuropeptide, CRF.

Section snippets

Tract-tracing: surgery

For anterograde tracing experiments using injections of BDA into the BNST, adult male Sprague–Dawley rats (250–300 g; Harlan Sprague–Dawley, Inc., Indianapolis, IN) were initially anesthetized with sodium pentobarbital (45 mg/kg) and placed in a stereotaxic apparatus for surgery. Anesthesia was maintained throughout the surgical procedure by administering 0.5% isoflurane (at a rate of 1.0 liters per min; Viking Medical, Medford Lakes, NJ) via a specialized nose-cone affixed to the stereotaxic

Anterograde labeling in the rostrolateral peri-LC region from the BNST

By light microscopy, animals with injections of BDA into the BNST (Figure 1A) showed prominent anterograde transport to the dorsal pontine tegmentum (Figure 1B, C). BDA injection sites, defined by the location of BDA-labeled neuronal perikarya and processes, were located in either the medial or lateral aspects of the BNST and occasionally encroached on ventral aspects of the BNST Figure 1, Figure 2. BDA injections overlapped portions of the BNST known to contain dense CRF immunoreactivity

Discussion

The present study establishes the BNST as an additional source of limbic input to the rostrolateral peri-LC region. The similarity in innervation patterns of the peri-LC from both the CNA and BNST suggest that these limbic efferents may be equipotent in their influences on LC activity. Together these studies suggest that the rostrolateral LC is an important integrative center for limbic innervation of the LC-norepinephrine system. In contrast to results observed with CNA injections, however,

Acknowledgements

This work was supported by an Established Investigator Award from the American Heart Association to E.J.V.B. and a Research Scientist Developmental Award (MH00840) to R.J.V. and DA09082 and MH40008 from the National Institutes of Health. The authors gratefully acknowledge Mrs. Patti Telegan for expert assistance and Dr. Wylie Vale for providing the CRF antiserum.

References (36)

  • G.F. Alheid et al.

    Amygdala and extended amygdala

  • Carlin RK, Grab DJ, Cohen RS, Siekevitz P (1980): Isolation and characterization of postsynaptic densities from various...
  • S.Y. Chiu et al.

    Neurotransmitter-mediated signaling between axons and glial cells

    Glia

    (1994)
  • J. Ciriello et al.

    Effect of glutamate stimulation of bed nucleus of the stria terminalis on arterial pressure and heart rate

    Am J Physiol

    (1993)
  • A.L. Curtis et al.

    Activation of the locus coeruleus noradrenergic system by intracoerulear microinfusion of corticotropin-releasing factorEffects on discharge rate, cortical norepinephrine levels and cortical electroencephalographic activity

    J Pharmacol Exp Ther

    (1997)
  • de Olmos J, Alheid GF, Beltramino CA (1985): Amygdala. In: Paxinos G, editor. The Rat Nervous System, Vol. 1. New York:...
  • E.G. Gray

    Axosomatic and axo-dendritic synapses of the cerebral cortexAn electron microscopic study

    J Anat

    (1959)
  • T.S. Gray et al.

    Ibotenic acid lesions in the bed nucleus of the stria terminalis attenuate conditioned stress-induced increases in prolactin, ACTH and corticosterone

    Neuroendocrinology

    (1993)
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