Rapamycin blocks the neuroprotective effects of sex steroids in the adult birdsong system

Abstract

In adult songbirds, the telencephalic song nucleus HVC and its efferent target RA undergo pronounced seasonal changes in morphology. In breeding birds, there are increases in HVC volume and total neuron number, and RA neuronal soma area compared to nonbreeding birds. At the end of breeding, HVC neurons die through caspase-dependent apoptosis and thus, RA neuron size decreases. Changes in HVC and RA are driven by seasonal changes in circulating testosterone (T) levels. Infusing T, or its metabolites 5α-dihydrotestosterone (DHT) and 17 β-estradiol (E2), intracerebrally into HVC (but not RA) protects HVC neurons from death, and RA neuron size, in nonbreeding birds. The phosphoinositide 3-kinase (PI3K)-Akt (a serine/threonine kinase)-mechanistic target of rapamycin (mTOR) signaling pathway is a point of convergence for neuroprotective effects of sex steroids and other trophic factors. We asked if mTOR activation is necessary for the protective effect of hormones in HVC and RA of adult male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii). We transferred sparrows from breeding to nonbreeding hormonal and photoperiod conditions to induce regression of HVC neurons by cell death and decrease of RA neuron size. We infused either DHT + E2, DHT + E2 plus the mTOR inhibitor rapamycin, or vehicle alone in HVC. Infusion of DHT + E2 protected both HVC and RA neurons. Coinfusion of rapamycin with DHT + E2, however, blocked the protective effect of hormones on HVC volume and neuron number, and RA neuron size. These results suggest that activation of mTOR is an essential downstream step in the neuroprotective cascade initiated by sex steroid hormones in the forebrain.

Keywords: birdsong; estrogen; hormone; mechanistic target of rapamycin; neuroprotection; neurotrophins; protein phosphorylation; rapamycin; signaling pathway; songbird; testosterone; trophic factors; trophic support.

Figure 1.

(a) Schematic sagittal drawing of the songbird brain showing projections of major nuclei in the song system and the distribution of androgen and estrogen receptors. Abbreviations: DLM, medial portion of the dorsolateral nucleus of the medial thalamus; HVC, acronym used as proper name; LMAN, lateral portion of the magnocellular nucleus of the anterior nidopallium; RA, robust nucleus of the acropallium; V, lateral ventricle; X, Area X of the medial striatum; nXIIts, tracheosyringeal part of the hypoglossal nucleus. (b) Schematic timeline of the experimental procedures. SD, short-day photoperiod; LD + T, long-day photoperiod plus systemic testosterone (T) implant. Birds were infused in HVC with 5-α dihydrotestosterone (DHT) and 17-β estradiol (E2), vehicle alone, or DHT+E2+rapamycin, starting on day 26 and were killed on day 35. (c) A representative image of a Nissl stained section showing the borders of HVC (black arrows) and cannula track (red asterisk). Cross-hairs indicate orientation. Scale bar = 500 μm. (d) Coronal view of brain at level of HVC (outlined in blue), showing the spread of Evan’s blue dye, contained in infused solution of treatment agent, from tip of in-dwelling cannulae. Note that the blue dye diffused throughout full extent of HVC.

Figure 2.

Infusing rapamycin in HVC decreased hormonal activation of mTOR. The ratio of phosphorylated mTOR (p-mTOR) to total mTOR in punches taken from HVC of birds infused with DHT+E2, DHT+E2 + rapamycin, or vehicle control. Data from individual birds represented as black dots, and bars show group mean ± SEM.

Figure 3.

(a) Coronal sections showing the borders of HVC (white arrows) in birds infused bilaterally in HVC with DHT+E2, DHT+E2 + rapamycin, or vehicle control. Scale bar, 250 μm. (b) HVC volume (mm³) in birds in each treatment group. (c) Total neuron number in HVC of birds in each treatment group. Data from individual birds represented as black dots, and bars show group mean ± SEM. Letters above bars indicate significant differences among treatment groups (one-way ANOVA, Holms-Sidak post hoc pairwise comparisons).

Figure 4.

a) Representative neurons within RA of birds infused bilaterally in HVC with DHT+E2, DHT+E2 + rapamycin, or vehicle control. Scale bar, 10 μm. b) Soma area (μm²) of neurons in RA of birds in each treatment group. Data from individual birds represented as black dots, and bars show group mean ± SEM. Letters above bars indicate significant differences among treatment groups (one-way ANOVA, Holms-Sidak post hoc pairwise comparisons).