We initially established using fMRI that auditory processing

We initially established using fMRI that auditory processing by the caudomedial nidopallium (NCM) of male European starlings (Sturnus vulgaris) of species-specific aspects of songs (whistles and warblings i.e. high-pitched trills coming from the end of the warbling that are found in the repertoire of all male starlings; SPEC) significantly differs between breeding and non-breeding seasons, while processing of individual-specific aspects of songs (individual motifs taken from the initial part of the warbling that are specific to each individual; INDIV) and pure-tones (control) remain unaffected (De Groof et al., 2013b) (see (Hausberger et al., 1997) for detailed description of these vocalizations). Seasonal songbirds are exposed to extremely different concentrations of testosterone (T) and its RWJ 56110 metabolite estradiol (E2) in the breeding and non-breeding seasons (Fusani et al., 2000; Riters et al., 2001; Soma et al., 2003). In follow-up studies, we therefore asked whether these seasonal differences in activity of auditory regions are controlled by changes in steroid concentrations. Furthermore, since E2 has been shown to rapidly modulate neuronal activity in the auditory cortex of zebra finches (Remage-Healey et al., 2010; Tremere and Pinaud, 2011), we asked whether rapid changes in brain estrogens concentrations would also affect auditory processing and which brain regions would be involved. Availability of estrogens in the brain was affected in these experiments by acutely blocking aromatase activity by an i.p. injection of the aromatase inhibitor Vorozole™ (VOR). Finally, because studies in a variety of models showed that rapid behavioral effects of E2 are modulated by the photoperiod (in California mice, Peromyscus californicus; (Trainor et al., 2008)) or by seasons (in a songbird, the song sparrow, Melospiza melodia; (Heimovics et al., 2012; Heimovics et al., 2015)), we wondered whether rapid effects of manipulations of E2 availability would be similar at different times of the year. Therefore, fMRI imaging sessions assessing acute effects of estrogen depletion were performed at three different times of the year, specifically in December (birds exposed to a 8 h light/16 h dark cycle), in early March (11 h light/13 h dark) and again in May/June (16 h light/8 h dark). Because we wanted to assess the effect of seasonal changes on rapid effects of E2 depletion without the confounding effect of the seasonal changes in circulating testosterone (T) concentrations, males were chronically treated with T for 3 weeks before each imaging session. In each set of studies, fMRI was used to assess auditory brain responses to the presentation of song stimuli conveying either species-specific (SPEC songs) or individual information (INDIV songs)(see (De Groof et al., 2017)) or to synthetic pure tones (PT). fMRI responses were first quantified during a control session and then 10 min after an intraperitoneal injection of the aromatase inhibitor Vorozole (6-[(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-methyl-1H-benzotriazole; 30 mg/kg) (VOR; (Wouters et al., 1994)) (See Fig. 1). fMRI data were acquired in a Magnetic Resonance system (Bruker Biospin) as previously described (De Groof et al., 2013b). Subjects were anesthetized throughout data acquisition and their body temperature was constantly monitored and maintained at 41.5 ± 0.5 °C by a feedback heating system, a critical feature to ensure reliability and reproducibility of measures of brain activity. Anesthesia was first induced by an intramuscular injection of a standard amount of medetomidine and ketamine and then maintained during the entire period of image acquisition by continuous infusion of this mixture at doses that were adapted to maintain a stable respiration rate and amplitude. fMRI data were acquired using a T2-weighted fast-spin echo sequence. In addition, anatomical three-dimensional (3D) images were obtained using a RARE T2-weighted sequence to provide accurate localization of functional data.