The noradrenergic projection from the locus coeruleus to the cochlear root neurons in rats

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The cochlear root neurons (CRNs) are key components of the primary acoustic startle circuit; mediating auditory alert and escape behaviors in rats. They receive a great variety of inputs which serve to elicit and modulate the acoustic startle reflex (ASR). Recently, our group has suggested that CRNs receive inputs from the locus coeruleus (LC), a noradrenergic nucleus which participates in attention and alertness. Here, we map the efferent projection patterns of LC neurons and confirm the existence of the LC-CRN projection using both anterograde and retrograde tract tracers. Our results show that each LC projects to the CRNs of both sides with a clear ipsilateral predominance. The LC axons terminate as small endings distributed preferentially on the cell body and primary dendrites of CRNs. Using light and confocal microscopy, we show a strong immunoreactivity for tyrosine hydroxylase and dopamine β-hydroxylase in these terminals, indicating noradrenaline release. We further studied the noradrenergic system using gene expression analysis (RT-qPCR) and immunohistochemistry to detect specific noradrenergic receptor subunits in the cochlear nerve root. Our results indicate that CRNs contain a noradrenergic receptor profile sufficient to modulate the ASR, and also show important gender-specific differences in their gene expression. 3D reconstruction analysis confirms the presence of sexual dimorphism in the density and distribution of LC neurons. Our study describes a coerulean noradrenergic projection to the CRNs that might contribute to neural processes underlying sensory gating of the ASR, and also provides an explanation for the gender differences observed in the behavioral paradigm.

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We thank the two anonymous reviewers for their constructive comments, which helped us to improve the manuscript. This research was supported by grants from the Spanish Ministry of Science and Innovation (MICINN, #BFU2010-17754) to Dr. Dolores E. López and the São Paulo State Research Foundation (FAPESP, #2008/02771-6) to Dr. José de Anchieta de Castro e Horta-Júnior.

Conflict of interest

The authors declare no competing financial interests.

Author information

Correspondence to Dolores E. López or José de Anchieta de Castro e Horta-Júnior.

Additional information

S. Hormigo and R. Gómez-Nieto contributed equally to this work.

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3D reconstruction with dynamic rotation of the locus coeruleus in a female and male rat. Each coerulean neuron is showed as a dot. Notice the gender-specific differences in distribution of LC neuronal density. This video corresponds to Fig. 10. 3D scale bars = 500 μm (WMV 13189 kb)

PCR primers of noradrenergic receptors (ADR α1A-C, ADR α2A-C, ADR β1-3). Primer location in the corresponding GenBank* sequences of rat origin is indicated (TIFF 10291 kb)

Retrogradely labeled neurons in the locus coeruleus (LC) after FluoroGold (FG) injections in the cochlear root nucleus. A and B, Micrographs of Nissl-counterstained coronal sections show FG-retrogradely labeled neurons in the ipsilateral (A) and contralateral (C) LC. This case corresponds to the injection site shown in Fig 3A. C and D, High magnification micrographs (corresponding to the frame in A and B) shows details of FG-labeled neurons in the LC. The number of FG-labeled neurons on the ipsilateral was considerably higher than on the contralateral side. The insets (positions denoted with arrows) are a higher magnification of the LC neurons retrogradely labeled with FG. PDTg, posterodorsal tegmental nucleus; scp, superior cerebellar peduncle; 4V, fourth ventricle. Scale bars = 300 μm in A and B; 100 μm in C and D (25 μm in the insets) (TIFF 17636 kb)

Tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) immunoreactivity in the cochlear root nucleus. A-B, High magnification micrographs show TH-immunolabeled endings on the cell bodies (arrowheads in A) and dendrites (arrows in B) of cochlear root neurons immunolabeled for CaBP. C-D, High magnification micrographs show DBH-immunolabeled endings on the cell body (arrowheads in C) and dendrites (arrows in D) of cochlear root neurons immunolabeled for CaBP. Scale bars = 25 μm in A, B, C and D (TIFF 15947 kb)

Tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) enzymes overlap in the cochlear root nucleus. A, Confocal image shows double immunolabeling for TH and DBH endings (in blue, Cy5, and in green, Cy2, respectively) in the cochlear root nucleus. B–C, Confocal images show TH (B) and DBH (C) immunolabeled endings corresponding to the merge in A. D-F, Colocalization of TH and DBH endings is confirmed by the 1 μm confocal image (D) and the orthogonal view (F). The arrow and arrowhead indicate the endings that were analyzed in the 1 μm confocal image and the orthogonal view, respectively. Scale bars = 20 μm in A, B, C, D and E (TIFF 19097 kb)

3D reconstruction of the locus coeruleus in a female rat. Each coerulean neuron is showed as a dot. The adobe PDF document contains an embedded 3D model of brainstem sections that were reconstructed using Neurolucida (Version 10). 3D scale bars = 500 μm (PDF 300 kb)

3D reconstruction of the locus coeruleus in a male rat. Each coerulean neuron is showed as a dot. The adobe PDF document contains an embedded 3D model of brainstem sections that were reconstructed using Neurolucida (Version 10). 3D scale bars = 500 μm (PDF 381 kb)

3D reconstruction with dynamic rotation of the locus coeruleus in a female and male rat. Each coerulean neuron is showed as a dot. Notice the gender-specific differences in distribution of LC neuronal density. This video corresponds to Fig. 10. 3D scale bars = 500 μm (WMV 13189 kb)

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Hormigo, S., Gómez-Nieto, R., Castellano, O. et al. The noradrenergic projection from the locus coeruleus to the cochlear root neurons in rats. Brain Struct Funct 220, 1477–1496 (2015).

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  • Acoustic startle reflex
  • Gender differences
  • Neuronal tracers
  • Noradrenergic receptors
  • Sensory gating
  • 3D reconstruction