Sensory Coding and Sensitivity to Local Estrogens Shift during Critical Period Milestones in the Auditory Cortex of Male Songbirds

Critical period timeline, avian auditory circuit, and experimental paradigm. A, The critical period for song learning unfolds across a 3 month timespan. Whereas some songbird species begin song learning and recognition at embryonic stages of development (Colombelli-Négrel et al., 2012), zebra finch sensory learning begins at 25 dph (Clayton, 2013). Autogenous song production can occur as early as 35 dph (typically closer to 40 dph; personal observation), and initially overlaps with the sensory learning phase, until 65 dph when sensorimotor-only learning continues as birds begin to refine their developing subsong until eventual song crystallization (∼100 dph). Timeline adapted after Clayton (2013). B, Schematic of the avian ascending auditory neural circuit. After sounds are first processed in upstream peripheral and brainstem auditory regions, communication is encoded within the midbrain nucleus MLd (dorsal part of the lateral mesencephalic nucleus), which sends projections to the thalamic nucleus ovoidalis (Ov). Ov sends projections primarily to Field L, comparable to mammalian primary auditory cortex, as well as to NCM (Vates et al., 1996). Secondary auditory cortex regions NCM (caudomedial nidopallium) and CMM (caudomedial mesopallium) are reciprocally connected and receive afferent projections from Field L. C, Experimental setup and paradigm. Top: In vivo microdialysis and extracellular electrophysiology schematic. A microdialysis cannula was first descended into NCM (∼1.10 mm ventral; light gray circular region). Afterward, a carbon-fiber electrode was placed within the proximate region of perfusate diffusion. Bottom: Experimental timeline. aCSF, artificial cerebrospinal fluid; E₂, 17β-estradiol.

Multiunit shifts in NCM auditory responsiveness across development. A, Representative multiunit recordings from a 25-, 47-, and 95-dph subject (right, left, and left hemisphere, respectively). Top: Representative response to a single presentation of conspecific song (CON2) from a multiunit recording during Trial 1 (aCSF). Middle: Raster plot and corresponding peristimulus time histogram (6-s duration) across all CON2 presentations during Trial 1 (aCSF). Bottom: CON2 sonogram. B, 25–34 dph subjects have higher normalized auditory response than both 40–64 and 65–95 dph birds. Dotted-line in B is average CON z-score from adult male NCM recordings from a separate study (graphed for visual comparison; n = 4 birds [195–360 dph; average age = 267.7 dph]). C, D, Based on z-score results, we analyzed birds based on critical period phase (sensory [25–34 dph] vs. sensorimotor [40–95 dph]) and found that sensory-aged birds’ NCM have lower spontaneous firing rates (C) and elevated stimulus-evoked firing rates (D) compared with sensorimotor-aged subjects. ***p < 0.001 (z-score: 25–34 dph vs. 40–64 dph, and 25–34 dph vs. 65–95 dph; spontaneous and stimulus-evoked firing: sensory-aged versus sensorimotor-aged). MUA, multiunit activity; CON2, conspecific song 2.

Single-unit auditory response and encoding in NCM is elevated during sensory phase. A, Representative single neurons. Left: Two sorted single units distinctly clustered in principal components space; Middle: 100 sequential iterations from two separate single neurons overlaying their respective wave form template. Right: Interstimulus interval plots for top single unit. Each bin = 1 ms. Units derived from Trial 1 (aCSF) recording from a sensory-aged subject (30 dph; left NCM). B, Raster plot and peristimulus time histogram from representative single units from a sensory-aged and sensorimotor-aged bird (33 [right NCM] and 71 [left NCM] dph). C, D, Spontaneous firing rates are lower in sensory-aged subjects irrespective of hemisphere; however, there are no age-dependent differences in single-unit stimulus-evoked firing rates (D). E, F, Across hemispheres, single-unit auditory z-scores (E) and classification accuracy (F) are significantly higher in sensory-aged birds. Dotted-line in F is chance-level prediction for classifier (1 in 6 chance for accurately classifying a given stimulus = 16.67%). ***p < 0.001; **p < 0.01 (sensory-aged vs. sensorimotor-aged).

Estradiol (E₂) dampens auditory responsiveness in NCM. A–D, Relative to aCSF (Trial 1), E₂ treatment decreased z-scores (A), classification accuracy (B), and spontaneous (C) and stimulus-evoked (D) firing rates in the NCM of sensory-aged subjects. Hemisphere-specific averages are depicted for visual comparison and consistency, but there was no trial × hemisphere effect. Averaged measurements across hemispheres are depicted in the last set of columns (Both); **p < 0.01 (effect of trial; Trial 1 vs. Trial 2). Dotted-line in B is chance-level prediction for classifier (1 in 6 chance for accurately classifying a given stimulus = 16.67%). Inset in A, average z-score across trials in aCSF rundown experiment (p = 0.07; Trial 1 vs. Trial 2; n = 5 sensory-aged birds; 6 single units).