Each recorded unit was driven by four songs (25 trials/song), in pseudo-random order, before, during and after pharmacological treatment. A) The sonogram for a song stimulus is shown in the top panel and a raster plot illustrating the discharge profile of a single-unit from NCM is shown in the bottom panel, along with its peri-stimulus time histogram. In the pre-drug session, data were split into a training set and a testing set, each including half of the stimulation trials per song. B) Spike counts from each trial were binned into 50 ms bins (a raw trace is shown for illustrative purposes and does not derive from the raster plot shown in A). The training set (n=13 trials/song) was used to train the classifier, and the remaining half of the trials (n=12/song) were used as testing vectors for cross-validation and calculation of classification accuracies. C) Activity vectors for each cell and song were plotted in hyper-dimensional space to generate templates for each song (song 1 is illustrated). D) After generating templates for each of the four songs used, we tested the ability of the classifier to decode stimulus identity based on the neural responses obtained from the testing set activity vectors (or trials obtained during drug, Off+2h and Off+4h sessions). Illustrated is an attempt to decode the stimulus identity (Sx) that originated the orange activity vector. The classifier measures Euclidean distance (d) between “inquiry” vectors and each of the song templates and assigns song identity based on the proximity between training and testing vectors. Classification accuracies are taken as the percentage of trials in which the classifier predicted the correct song based on the neural response.

A) The ability of the classifier to decode song identity based on the neuronal responses of NCM neurons is plotted as a function of the experimental session (before, during and after [2h and 4h] pharmacological treatment). Data for each pharmacological manipulation are grouped by color. Vehicle infusions in NCM (blue) do not alter classification accuracies over time. Local infusions of exogenous E2 (red) significantly enhance performance of the classifier, an effect that persists for at least 2h following termination of hormone infusion. Impairment of local E2 signaling either through blockade of estrogen receptors with ICI (green), or through local inhibition of aromatase with FAD (grey), significantly decreased auditory coding. These effects were also persistent (2 h), but returned to pre-drug levels 4h following the interruption of drug infusions. B) The impact of local E2’s actions in the effectiveness of NCM’s neuronal coding of songs was independently validated with estrogen receptor modulator TMX (green) and aromatase inhibitor ATD (grey). Note local blockade of estrogen receptors, or suppression of the local production of estradiol produces a significant and persistent decrease in the performance of the decoder. **p<0.01; ***p<0.001.

Two-dimensional confusion matrices and classifier’s performance for 3 representative NCM neurons treated with E2 (A–B), ICI (C–D) and FAD (E–F). Top panels show confusion matrices measured from classifications of the neural data before, during and after (2h and 4h) pharmacological treatment, and provide an index of the ability of NCM neurons to discriminate among the different songs tested. Each matrix plots the Euclidean distance, coded in grayscale values, between actual (y-axis) and predicted (x-axis) songs. A perfect decoder (100% accuracy) would yield a diagonal confusion matrix, with no off-diagonal values (song mis-indentifications). Note that the pattern classifier reliably decodes song identity in the Pre-drug condition. Bottom panels show classification accuracies for each representative neuron; dashed lines indicate data obtained for each song individually, and the solid black line shows data for all songs combined. A–B) Local infusions of E2 in NCM improve the neural discrimination of songs (A) and, consequently, significantly increase classification accuracies from pre-infusion levels for all neurons (B). These changes persist for at least 2 h after interruption of E2 infusions. C–D) Local blockade of estrogen receptors with ICI leads to a significant and persistent disruption in the ability of NCM neurons to discriminate across songs (C), and in the performance of the decoder (D). These changes persist for at least 2 h after interruption of E2 infusions, but return to pre-drug levels after 4 h. E–F) Suppression of the local production of estradiol with FAD drives a marked and lasting disruption in the structure of confusion matrices, indicating increased errors in song discrimination based on the neural responses (E). FAD significantly decreases the decoder’s performance for all representative neurons (F).

The ability of the decoder to identify stimulus identity based on the responses of NCM neurons was probed when the classifier was both trained and tested with trials obtained within each session of interest (e.g., trained with half of the trials within the E2 session, and tested with the remaining trials of that very same session; see Methods and Results). This study directly test whether drug-induced alterations in the responses of NCM neurons can occur in the absence of changes in their neural ability to discriminate songs, as assessed by the performance of our decoder. In each graph set, the decoder’s performance (y-axis) is plotted as a function of the recording session (pre-drug, drug, Off + 2h or Off + 4h; x-axis), for each pharmacological intervention of interest. We found that A) vehicle infusions did not influence the performance of the classifier in the short- or long-term (up to 4 h). B) In contrast, E2 treatment significantly increased classification accuracies. This effect was long-lasting, and persisted for at least 2 h after interruption of exogenous infusions of hormone. C–D) Blockade of estrogen receptors with ICI (C) or TMX (D) markedly suppressed the performance of the decoder. This effect persisted for 2 h, but not 4 h, after cessation of drug infusions. E–F) Inhibition of aromatase in NCM via local infusions of FAD (E) or ATD (F) markedly decreased the ability of NCM neurons to discriminate songs, as assessed by the classifier’s performance. Similarly to the findings obtained with estrogen receptor blockade, inhibition of aromatase drove long-lasting changes in NCM’s neuronal discrimination of songs, which persisted for at least 2 h. G–H) Infusions of 5β-DHT (G) or E1 (H) into NCM did not affect the short- or long-term performance of the decoder, indicating that estradiol selectively affected the neural discrimination of songs in NCM neurons. *p<0.05 **p<0.01; ***p<0.001.

A) Bilateral infusions of vehicle into the NCM do not impact the strong behavioral preference of adult birds to tutor over conspecific song. However, bilateral blockade of estrogen receptors with ICI or TMX, or suppression of the local production of E2, with FAD or ATD, largely abolish song preference. B) Bilateral infusions of vehicle, estrogen receptor antagonists ICI and TMX, and aromatase inhibitors FAD and ATD into NCM do not change call preference in freely-behaving animals. Horizontal grey lines indicate no preference. *p<0.05; **p<0.01; ***p<0.001.

After headposting and recording chamber implantation, all animals were subjected to several acclimation sessions (see Methods). In the day of the experiment, animals were exposed to two final acclimation sessions and were group based on the pharmacological manipulations to be carried out. A) For experiments with E2, ICI, TMX, 5β-DHT and E1, animals were exposed to the stimulation set, during which song-driven neural responses were obtained bilaterally from NCM (pre-drug session). Subsequently, animals were unilaterally injected with one of the drugs above, and contralaterally infused with vehicle; maintenance doses were applied during a second presentation of the stimulus set (drug session). Drug infusion was then interrupted and animals were left in silence for 2 h, with the electrodes in place. Subsequently, birds were exposed to the stimulation set a third time (Off+2h) and were left in silence for an additional 2 h. Animals were then stimulated a final time (Off+4h) before brains were obtained for histological analysis. B) In studies with FAD and ATD, the experimental configuration was identical to the one described above, except that the drug session was only initiated 30 min after the initial drug infusion. This approach was taken to ensure that aromatase inhibition was efficient and maximal (Wade et al., 1994; Tremere et al., 2009) and that endogenous E2 levels in NCM decayed to minimal levels (Remage-Healey et al., 2008).

A) Response profile of a representative NCM neuron before, during and after (2 h and 4 h) interruption of local vehicle infusion. The top panel (grey box) represents the spectrograms of the song stimulus, the middle panel represents a raster plot for the spiking behavior of the same neuron during the first 10 renditions of the stimulus, and the bottom panel illustrates the peristimulus time histograms (PSTHs). Vehicle infusions did not affect the short- or long-term discharge rates of NCM neurons, as determined by quantitative and statistical analyses (see Results). B) Local infusions of physiological E2 levels rapidly and significantly increase song-evoked firing rates of NCM neurons. Shown is a spike raster plot and associated PSTH of a representative single-unit in NCM, time-aligned with the stimulus sonogram. E2-mediated effects on firing rates remain detectable 2 h, but not 4 h, following interruption of E2 infusions. C) Local blockade of estrogen receptors with ICI significantly decreased firing rates of NCM neurons. This effect could be detected 2 h after interruption of drug infusions. Shown are raster plots and PSTHs of a single NCM neuron, time-aligned with the song stimulus, before, during and after (2 h and 4 h) local infusions of ICI 182780 (ICI), a high-affinity estrogen receptor antagonist. These effects were also detected when NCM neurons were treated with tamoxifen (TMX) an estrogen receptor modulator that acts as an antagonist in NCM (not shown, but detailed in Results). D) Local inhibition of aromatase with fadrozole (FAD), and suppression of the local production of estradiol in NCM, significantly decreased song-evoked discharge rates of NCM neurons. The effects of FAD on NCM discharge rates were detectable 2 h, but not 4 h, following interruption of drug infusions. Shown are the spectrograms of the stimulus (top panels), spike raster plots (middle panels) and PSTHs (bottom panels) of a single NCM neuron before, during and after (2 h and 4 h) stoppage of local FAD infusions. These effects were also observed when NCM neurons were treated with ATD, an additional aromatase inhibitor (not shown, but detailed in Results). These findings indicate that locally-produced estradiol exerts facilitatory effects in the song-evoked activity of NCM neurons.

The mutual information (MI) between stimulus and neural responses is plotted as a function of the experimental session (before, during and after [2h and 4h] pharmacological treatment). Each graph set shows mean MI data obtained under different pharmacological conditions that included vehicle (A), E2 (B), estrogen receptor antagonists ICI 182780 (C) and tamoxifen (D), as well as inhibitors of aromatase activity fadrozole (E) and ATD (F). Note that information rates are not impacted by local vehicle infusions into NCM (A). Significant and persistent increases in MI rates are observed as a function of local E2 infusions (B). In contrast, these values are markedly decreased below pre-drug levels after blockade of estrogen receptors (C–D) or inhibition of the local production of E2 in NCM (E–F). Similar to estrogen receptor antagonism, local inhibition of aromatase yield effects on information rates that persist for several hours after interruption of drug treatment. *p<0.05; **p<0.01; ***p<0.001.

A) The ability of the decoder to uncover song identity based on the neural responses in NCM is plotted as a function of the experimental session (before, during and after [2h and 4h] 5β-DHT treatment). No differences in the performance of the classifier were detected during or after 5β-DHT treatment. B) Mutual information (MI) between neural responses and stimulus is plotted as a function of experimental session (before, during and after [2h and 4h] 5β-DHT infusions into NCM). MI rates did not change as a result of the local treatment of NCM neurons with 5β-DHT. C) No changes in the performance of the classifier were detected during or after treatment of NCM neurons with E1. D) MI rates were also unaffected by E1 treatment, indicating that coding properties of NCM neurons are not broadly modulated by steroidal hormones, but rather are selectively shaped by estradiol.

A–B) Illustration of spike-extraction and -insertion manipulations. A) Schematic representation of song-evoked spike trains obtained from an NCM neuron during infusions of vehicle (blue), estradiol (red), estrogen receptor antagonists ICI or TMX (green) and aromatase inhibitors FAD or ATD (grey). Illustrated are the typical effects of these agents: vehicle does not affect firing rates. E2 robustly increases discharge rates of NCM neurons. In contrast, blockade of estrogen receptors or inhibition of aromatase activity decrease NCM neuronal firing rates. For illustrative purposes, we suggest that the number of action potentials in the spike train increases from 10 to 20 during E2 treatment, and decreases from 10 to 6 during ICI/TMX or FAD/ATD treatment (see n values on the right side of spike trains). B) We normalized the firing rates of NCM neurons during pharmacological treatment by obtaining neurophysiological responses and computationally processing individual spike trains to match the number of spikes in the drug session, with that in the pre-drug session (see Methods). The cartoon illustrates the two types of manipulations implemented. For E2 infusions (red), which increase NCM neuronal spike rates, we randomly eliminated spikes from the train (asterisks), to match the spike numbers of the average pre-drug session. For ICI/TMX or FAD/ATD infusions (green and grey, respectively), which decrease NCM neuronal spike rates, we inserted spikes at random spike-times in the train (asterisks) to match the spike numbers of the average pre-drug session (see Methods). Note the n values on the right side of spike trains, and compare them against the n values in (A). This spike normalization process eliminates the effects of pharmacological agents on NCM neuronal firing rates. These manipulations were also carried out in the same manner for Off + 2h and Off + 4h sessions (not illustrated). C–H) The performance of the classifier in decoding the identity of the song stimulus based on the neuronal responses of NCM neurons is plotted as a function of the experimental session (before, during and after [2 h and 4 h] pharmacological treatment). Data obtained with intact (not normalized) and processed (normalized) spike trains are shown for each drug group in solid and dashed lines, respectively. Note that although the normalization process (and consequently elimination of firing rate effects), diminishes the effects of all pharmacological agents on the decoding performance, this manipulation does not eliminate drug effects on NCM’s neuronal coding of songs. These findings indicate that E2 affects both rate and temporal coding of auditory signals in NCM neurons. *p<0.05; **p<0.01; ***p<0.001.