Although teleost fish have higher levels of brain aromatase activity than any other vertebrate group, its function remains speculative, and no study has identified its cellular basis. A previous study determined aromatase activity in a vocal fish, the plainfin midshipman (Porichthys notatus), and found highest levels in the telencephalon and lower levels in the sonic hindbrain, which was dimorphic between and within (males) sexes. We have now localized aromatase-containing cells in the midshipman brain both by immunocytochemistry using teleostspecific aromatase antibodies and by in situ hybridization using midshipman-specific aromatase probes. Aromatase-immunoreactivity and mRNA hybridization signal are consistent with relative levels of aromatase activity in different brain regions: concentrated in the dimorphic sonic motor nucleus, in a band just beneath the periaqueductal gray in the midbrain, in ventricular regions in the hypothalamus, and highest levels in the telencephalon especially in preoptic and ventricular areas. Surprisingly, double-label immunofluorescence does not show aromatase-immunoreactive colocalization in neurons, but instead in radial glia throughout the brain. This is the first study to identify aromatase expression mostly, if not entirely, in glial cells under normal rather than brain injury-dependent conditions. The abundance of aromatase in teleosts may represent an adaptation linked to continual neurogenesis that is known to occur throughout an individual's lifetime among fishes. The localization of aromatase within the intersexually and intrasexually dimorphic vocal-motor circuit further implies a function in the expression of alternative male reproductive phenotypes and, more generally, the development of natural, individual variation of specific brain nuclei.

For seasonally breeding vertebrates, reproductive cycling is often coupled with changes in vocalizations that function in courtship and territoriality. Less is known about changes in auditory sensitivity to those vocalizations. Here, we show that nonreproductive female midshipman fish treated with either testosterone or 17beta-estradiol exhibit an increase in the degree of temporal encoding of the frequency content of male vocalizations by the inner ear that mimics the reproductive female's auditory phenotype. This sensory plasticity provides an adaptable mechanism that enhances coupling between sender and receiver in vocal communication.

Among vertebrates, teleost fish have the greatest capacity for estrogen production in the brain. Previously, we characterized the distribution of the estrogen-synthesizing enzyme aromatase in the brain of the midshipman fish. Here, we investigated the distribution of estrogen receptor alpha (ERalpha). A partial cDNA of ERalpha was cloned and used to generate midshipman-specific primers for RT and real-time PCR which identified transcripts in liver and ovary, the CNS, and the sensory epithelium of the main auditory endorgan (sacculus). In situ hybridization revealed abundant expression throughout the preoptic area, a vocal-acoustic site in the hypothalamus, amygdala homologs of the dorsal pallium, the pineal organ, the inner ear, the pituitary, and the ovary. Weaker expression was found in the midbrain's nucleus of the medial longitudinal fasciculus and in the dimorphic vocal motor nucleus. ERalpha expression in the pineal, gonad, and pituitary axis may function to time seasonal abiotic cues to reproductive state, while expression in the vocal motor and auditory systems support neurophysiological evidence for estrogen as a modulator of vocal motor and auditory encoding mechanisms in midshipman fish. While ERalpha is restricted to specific nuclei, aromatase expression is abundant in glial cells throughout the entire forebrain, and high in midbrain and hindbrain - spinal vocal regions. The only site of aromatase-containing neurons is in the peripheral auditory system, where it is localized to ganglion cells in the auditory nerve. Estrogen production proximal to ERalpha-positive neurons may provide for focal sites of estrogen effects on reproductive-, vocal-, and auditory-related neurons.

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