Probing the structure and function of locus coeruleus projections to CNS motor centers

Abstract

The brainstem nucleus locus coeruleus (LC) sends projections to the forebrain, brainstem, cerebellum and spinal cord and is a source of the neurotransmitter norepinephrine (NE) in these areas. For more than 50 years, LC was considered to be homogeneous in structure and function such that NE would be released uniformly and act simultaneously on the cells and circuits that receive LC projections. However, recent studies have provided evidence that LC is modular in design, with segregated output channels and the potential for differential release and action of NE in its projection fields. These new findings have prompted a radical shift in our thinking about LC operations and demand revision of theoretical constructs regarding impact of the LC-NE system on behavioral outcomes in health and disease. Within this context, a major gap in our knowledge is the relationship between the LC-NE system and CNS motor control centers. While we know much about the organization of the LC-NE system with respect to sensory and cognitive circuitries and the impact of LC output on sensory guided behaviors and executive function, much less is known about the role of the LC-NE pathway in motor network operations and movement control. As a starting point for closing this gap in understanding, we propose using an intersectional recombinase-based viral-genetic strategy TrAC (Tracing Axon Collaterals) as well as established ex vivo electrophysiological assays to characterize efferent connectivity and physiological attributes of mouse LC-motor network projection neurons. The novel hypothesis to be tested is that LC cells with projections to CNS motor centers are scattered throughout the rostral-caudal extent of the nucleus but collectively display a common set of electrophysiological properties. Additionally, we expect to find these LC projection neurons maintain an organized network of axon collaterals capable of supporting selective, synchronous release of NE in motor circuitries for the purpose of coordinately regulating operations across networks that are responsible for balance and movement dynamics. Investigation of this hypothesis will advance our knowledge of the role of the LC-NE system in motor control and provide a basis for treating movement disorders resulting from disease, injury, or normal aging.

Keywords: TrAC; locus coeruleus; motor centers; norepinephrine; viral vector track tracing.

FIGURE 1

TrAC (Tracing Axon Collaterals) permits fluorescent labeling of genetically defined neuron populations based on axonal projections. (Top) Neurons with a history of En1 and Dbh expression constitute the locus coeruleus and an adjacent portion of dorsal subcoeruleus. (Center) In mice heterozygous for En1^Dre, Dbh^Flpo, and the RC:RFLTG indicator allele, locus coeruleus (LC)-norepinephrine (NE) neurons are labeled constitutively with tdTomato (red fluorescence) and switch to EGFP (green fluorescence) after Cre recombination. (Bottom) After injection of a retrograde CAV2-Cre virus (left schematic), EGFP labels LC-NE neurons projecting to the injection site (right image) as well as all their axon collaterals in other brain regions (middle image), scale bars = 100 μm. Adapted from Plummer et al. (2020).

FIGURE 2

Labeling of locus coeruleus neurons in TrAC-LC mice following CAV2-Cre injection in primary motor cortex (M1). (A) Coronal schematic of mouse forebrain section showing position of CAV2-Cre injection. (B) Bar graph showing percentage of EGFP-labeled LC neurons, ipsilateral and contralateral relative to the injection site (n = 4 mice). (C) Representative coronal sections through the rostrocaudal extent of the ipsilateral LC showing distribution of EGFP-labeled (green) and tdTomato-labeled (magenta) cells. Scale bar, 200 μm. Adapted from Plummer et al. (2020).

FIGURE 3

Locus coeruleus efferent fibers in TrAC-LC mice following CAV2-Cre injection in medial prefrontal cortex. tdTomato⁺ fibers (magenta) and EGFP⁺ fibers (green) are shown in primary motor cortex (M1), bed nuc stria terminalis (BNST), medial geniculate (MG). Note the paucity of labeled fibers in MG, i.e., axon collaterals of LC cells projecting to medial prefrontal cortex, scale bar = 100 μm. Adapted from Plummer et al. (2020).

FIGURE 4

Distribution of axon collaterals from LC neurons projecting to mPFC. The bar graph (n = 4 mice) indicates percentage of LC-NE axons at each brain region that originate from EGFP + mPFC-projecting LC-NE neurons. LC-NE neurons are represented as percent of total LC-NE inputs (sum of EGFP + and tdTomato +) in select brain regions. mPFC, medial prefrontal cortex; A24, ventral anterior cingulate; insular ctx, insular cortex; M1, primary motor cortex; piriform ctx, piriform cortex; BNST, bed nucleus of the stria terminalis; CA1, area CA1 of the hippocampus; BLA, basolateral amygdala; BMA, basomedial amygdala; LH, lateral hypothalamus; PVN, paraventricular hypothalamic nuc; AV, anteroventral thalamic nuc; PV, paraventricular thalamic nuc; VL, ventrolateral thalamic nuc; VM, ventromedial thalamic nuc; MG, medial geniculate nuc; Sup. coll., superior colliculus; SNr, substantia nigra. Adapted from Plummer et al. (2020).

FIGURE 5

Soma and dendritic fields (white arrows) of LC neurons labeled from viral vector injection in the ipsilateral VL thalamus. Rather than extend evenly into the peri-coerulear surround, the dendrites from these cells are concentrated in the dorsomedial (1), ventromedial (2), and lateral (3) zones of the peri-coerulear space, scale bar = 100 μm.

FIGURE 6

EGFP + NE-containing cells in the ipsi- (at right) and contralateral (at left) LC nucleus following unilateral injection of retrogradely transported viral vector in the cerebellar medial nuc, scale bar = 100 μm.

FIGURE 7

Locus coeruleus cells projecting to mPFC are physiologically distinct from those projecting to M1. (A) Representative traces of spontaneous action potentials indicate that cells projecting to mPFC (n = 19) fire three-fold faster than those projecting to M1 (n = 19, *p < 0.05). (B) The magnitude of afterhyperpolarization (AHP), as determined by the difference in voltage between action potential threshold and the lowest point of the AHP (dashed lines and arrows), was significantly lower in mPFC projection cells than those terminating in M1 (*p < 0.05). (C) Representative traces of AMPA mediated spontaneous excitatory post synaptic current (sEPSC) and the graph in panel (D) indicate that the amplitude of sEPSCs was significantly greater in mPFC vs. M1 projection cells (*p < 0.05).