Catecholaminergic Innervation of Central and Peripheral Auditory Circuitry Varies with Reproductive State in Female Midshipman Fish, Porichthys notatus

In seasonal breeding vertebrates, hormone regulation of catecholamines, which include dopamine and noradrenaline, may function, in part, to modulate behavioral responses to conspecific vocalizations. However, natural seasonal changes in catecholamine innervation of auditory nuclei is largely unexplored, especially in the peripheral auditory system, where encoding of social acoustic stimuli is initiated. The plainfin midshipman fish, Porichthys notatus, has proven to be an excellent model to explore mechanisms underlying seasonal peripheral auditory plasticity related to reproductive social behavior. Recently, we demonstrated robust catecholaminergic (CA) innervation throughout the auditory system in midshipman. Most notably, dopaminergic neurons in the diencephalon have widespread projections to auditory circuitry including direct innervation of the saccule, the main endorgan of hearing, and the cholinergic octavolateralis efferent nucleus (OE) which also projects to the inner ear. Here, we tested the hypothesis that gravid, reproductive summer females show differential CA innervation of the auditory system compared to non-reproductive winter females. We utilized quantitative immunofluorescence to measure tyrosine hydroxylase immunoreactive (TH-ir) fiber density throughout central auditory nuclei and the sensory epithelium of the saccule. Reproductive females exhibited greater density of TH-ir innervation in two forebrain areas including the auditory thalamus and greater density of TH-ir on somata and dendrites of the OE. In contrast, non-reproductive females had greater numbers of TH-ir terminals in the saccule and greater TH-ir fiber density in a region of the auditory hindbrain as well as greater numbers of TH-ir neurons in the preoptic area. These data provide evidence that catecholamines may function, in part, to seasonally modulate the sensitivity of the inner ear and, in turn, the appropriate behavioral response to reproductive acoustic signals

Seasonal difference in TH-ir innervation of the cholinergic octavolateralis efferent nucleus (OE).

<p>(A, B) Transverse sections through the rostral OE showing TH-ir fibers and terminals on somata and dendrites (d) of OE neurons labeled by choline acetyltransferase (ChAT)-ir. Data in C and D are expressed as the percent area of ChAT-ir in the OE that is covered by TH-ir fibers (mean ± SE). Mean percent area TH-ir overlap of OE somata and dendrites for animal in A = 22.4% and 33.4%; B = 17% and 29.7%, respectively. *<i>p</i> = 0.01, *** <i>p</i> = 0.0001. Abbreviations: MLF, medial longitudinal fasciculus; VIIm, facial motor nucleus. Scale bar = 100 μm.</p

Seasonal comparison of TH-ir fiber density in the auditory system.

<p>Arrows indicate direction of significant differences between reproductive (summer) and non-reproductive (winter) females.</p><p>TH-ir, tyrosine hydroxylase immunoreactivity.</p><p>*<i>P</i>-values represent results from 2-tailed independent groups t-test.</p><p>Seasonal comparison of TH-ir fiber density in the auditory system.</p

No seasonal differences in number of TH-ir cells in the noradrenergic locus coeruleus (LC).

<p>Examples of small numbers of clustered TH-ir neurons just lateral to the fourth ventricle (IV) characteristic of the LC in transverse sections through the isthmal brainstem in summer (A) and winter (B) animals. Mean ± SE for number of TH-ir cells/ section = 4.2 ± 0.37 (summer) and 4.7 ± 0.21 (winter). Scale bar = 100 μm.</p

Seasonal differences in dopaminergic but not cholinergic innervation of the saccule, the main endorgan of hearing.

<p>(A, B) Transverse sections through the saccular epithelium. TH-ir and ChAT-ir puncta are largely concentrated at the base of hair cells and within the support cell layer. HC and SC labels point to DAPI-stained nuclei of individual hair cells and support cells, respectively. The rest of the hair cell is unlabeled and is a light purple background. Stereocillia (unlabeled) are located at the apical end of the hair cells. Quantification of numbers (C) and size (D) of putative TH-ir and ChAT-ir terminals (puncta) in the saccule (mean ± SE). Arrowhead in B indicates an example of a thick, varicose TH-ir fiber along the base of the SC layer that was excluded from the puncta analysis. Colors in the graphs match TH-ir and ChAT-ir in the micrographs. Mean number of puncta per image for animal in A = 41.4; B = 68.7. Mean area per punctum for animal A = 0.63 μm²; B = 0.78 μm². *<i>p</i> = 0.017, **<i>p</i> = 0.001. Scale bar = 25 μm.</p

Dorsal view of an exposed midshipman brain and inner ear.

<p>Shaded areas indicate location of auditory nuclei analyzed in this study (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121914#pone.0121914.ref025" target="_blank">25</a>]). Vertical lines (A-H) indicate levels of transverse sections seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121914#pone.0121914.g002" target="_blank">Fig 2</a>. Abbreviations: AT, anterior tuberal nucleus; C, cerebellum; CP, central posterior nucleus of the thalamus; DO, descending octaval nucleus; M, midbrain; MED, medial octavolateralis nucleus; OB, olfactory bulb; OE, octavolateralis efferent nucleus; PGl, lateral division of nucleus preglomerulosus; SE, saccular epithelium of the inner ear; SO, secondary octaval nucleus; T, telencephalon; TS, torus semicircularis; VIII, eighth nerve. Scale bar = 1.5 mm.</p

Seasonal difference in TH-ir fiber density in the auditory hindbrain.

<p>(A, B) Transverse sections through the area that includes the dorsolateral division of the descending octaval nucleus (DOdl) and medial octavolateralis nucleus (MED). The left edge in A and B is the lateral part of the fourth ventricle. Data in C are represented as percent area of the analyzed region that contains TH-ir (mean ± SE). Mean TH-ir density for animal in A = 4.3%; B = 6.6%. *<i>p</i> = 0.03. Scale bar = 100 μm.</p

Seasonal differences in dopaminergic neuron number in the forebrain.

<p>(A, B) Clusters of small TH-ir cells (arrowheads) located in the lateral region of the anterior parvocellular preoptic nucleus (PPa). (D, E) Sparse numbers of TH-ir cells (arrowheads) in the anterior tuberal hypothalamus (AT). Data in C and D are expressed as number of TH-ir neurons per section (mean ± SE). Mean number of neurons per section for animal in A = 14; B = 32; D = 0; E = 0.6. **<i>p</i> = 0.001. Scale bar = 100 μm.</p

Seasonal comparison of catecholaminergic neuron number.

<p>Arrows indicate direction of significant differences between reproductive (summer) and non-reproductive (winter) females.</p><p>TH, tyrosine hydroxylase.</p><p>*<i>P</i>-values represent results from 2-tailed independent groups t-test.</p><p>^†Statistical difference is uncertain due to an unequal number of sections sampled between groups.</p><p>Seasonal comparison of catecholaminergic neuron number.</p

Example micrographs showing no seasonal differences in TH-ir fiber density in transverse sections through (A, B) anterior tuberal hypothalamus (AT), (C, D) torus semicircularis (TS) and (D, E) ventral division of secondary octaval nucleus (SOv).

<p>Mean ± SE TH-ir density for summer vs. winter: AT (15.22% ± 0.74/ 11.68% ± 2.7); TS (16.30% ± 1.5/ 14.28% ± 1.0); SOv (4.12% ± 0.44/ 3.94% ± 0.25). Scale bar = 100 μm.</p