Thirdly and finally multiple studies

Thirdly and finally, multiple studies have analyzed the activation of neurons in the auditory telencephalic areas of songbirds in response to various auditory stimuli. Neuronal activation was identified by the increased expression of immediate early genes such as fos or ZENK (also know as zif-268, egr-1, NIFI-A or krox-24)(Maney and Pinaud, 2011; Mello et al., 2004). These studies clearly demonstrate that the neuronal activation in these auditory area is specifically related to the nature of the sound stimuli ((Gentner et al., 2001; Leitner et al., 2005; Mello and Clayton, 1994; Mello and Ribeiro, 1998; Monbureau et al., 2015), reviewed in (Maney and Pinaud, 2011)). In addition, this approach has shown that estrogens modulate the immediate early gene response in telencephalic auditory areas mainly in NCM and in the caudomedial mesopallium, CMM. In females of a seasonally-breeding species, the white-throated sparrow (Zonotrichia albicollis), the systemic administration of estradiol resulted in a higher density of ZENK-immunopositive cells in NCM and CMM in response to song exposure as compared to pure tones but this stimulus specificity was absent in untreated GDC-0349 (Maney et al., 2006; Sanford et al., 2010). Similarly, species-specific songs and calls induced a higher density of ZENK–positive cells than heterospecific vocalizations in the NCM of male black-capped chickadees (Poecile atricapilus) during the breeding season but not in birds that were not in reproductive condition (Phillmore et al., 2011) Taken together, these studies indicate that estrogens modulate the auditory inputs reaching HVC. How much of this regulation mirrors changes in the inner ear that are reflected in the ABR versus central changes taking place in the telencephalic auditory areas remains somewhat unclear but recent work focusing on rapid effects of estrogens brings direct support to the latter of these options. This work has been reviewed multiple times (Krentzel and Remage-Healey, 2015; Remage-Healey, 2012, Remage-Healey, 2013; Remage-Healey et al., 2012) and is also considered in detail in this special issue (see Vahaba and Remage-Healey, this volume). It will thus be mentioned here only briefly. The development of in vivo dialysis and of ultrasensitive radioimmunoassays for estradiol recently allowed investigations of endogenous fluctuations of estradiol concentrations in the NCM of awake zebra finches. These studies revealed that local estradiol concentrations significantly increase within 30 min in NCM but not in adjacent areas when males engage in social interactions with females (Remage-Healey et al., 2008). Additional experiments showed that acoustic playback of male songs is sufficient to produce a rapid increase in NCM estradiol concentrations (Remage-Healey et al., 2008). Because these changes were not reflected in the periphery, they presumably reflect a local production of neuroestrogens. Retrodialysis of estradiol or of aromatase inhibitors was then combined with electrophysiology to analyze the functional significance of these rapid changes in estradiol concentration in NCM. Retrodialysis of estradiol in anesthetized males rapidly (within minutes) increased the auditory-evoked activity of NCM neurons and this response returned to baseline in the subsequent wash-out condition (Remage-Healey et al., 2010). This global increase was caused by the switch of some neurons from a tonic, isolated firing pattern to a burst firing pattern under the influence of estradiol. Conversely when the aromatase inhibitor Fadrozole™ was retrodialyzed, the rate of burst firing in neurons was significantly inhibited and again the effect rapidly disappeared at wash-out. Similar rapid electrophysiological effects of estradiol were observed in females although in this sex, elevated neuroestradiol concentrations were observed only in response to auditory stimuli whereas they were detected in response to visual and auditory stimuli in males (Remage-Healey et al., 2012).